Discovery of N2-(4-Amino-cyclohexyl)-9-cyclopentyl- N6-(4-morpholin-4-ylmethyl-phenyl)- 9H-purine-2,6-diamine as a Potent FLT3 Kinase Inhibitor for Acute Myeloid Leukemia with FLT3 Mutations

Article abstract of DOI:10.1021/acs.jmedchem.7b01529

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.

Full text of DOI:10.1021/acs.jmedchem.7b01529

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2
6
Discovery of N-(4-Amino-cyclohexyl)-9-cyclopentyl-N-(4-morpholin-4-
ylmethyl-phenyl)-9H-purine-2,6-diamine as a Potent FLT3 Kinase
Inhibitor for Acute Myeloid Leukemia with FLT3 mutations
Tomas Gucky, Eva #ezní#ková, Tereza Radosova Muchova, Radek Jorda, Zuzana Klejova, Veronika
Malinkova, Karel Berka, Vaclav Bazgier, HARESH AJANI, Martin Lepsik, Vladimir Divoky, and Vladimir Krystof
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01529 • Publication Date (Web): 19 Apr 2018
Downloaded from http://pubs.acs.org on April 19, 2018
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Discovery of N ꢀ(4ꢀAminoꢀcyclohexyl)ꢀ9ꢀcyclopentylꢀN ꢀ(4ꢀmorpholinꢀ4ꢀylmethylꢀphenyl)ꢀ9Hꢀ
purineꢀ2,6ꢀdiamine as a Potent FLT3 Kinase Inhibitor for Acute Myeloid Leukemia with FLT3
mutations
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a#
b#
c
b
c
Tomáš Gucký , Eva Řezníčková , Tereza Radošová Muchová , Radek Jorda , Zuzana Klejová ,
a
d
d
d,e
e
Veronika Malínková , Karel Berka , Václav Bazgier , Haresh Ajani, Martin Lepšík , Vladimír
c
b*
Divoký , Vladimír Kryštof
a
Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and
Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech
Republic
b
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural
Research, Palacký University and Institute of Experimental Botany AS CR, Šlechtitelů 27, 783 71
Olomouc, Czech Republic
c
Department of Biology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775
1
5 Olomouc, Czech Republic
d
Department of Physical Chemistry, Regional Centre of Advanced Technologies and Materials,
Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic
e
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo
nám. 2, 166 10 Prague 6, Czech Republic
#
These authors contributed equally.
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Abstract
FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients
with FLT3ꢀITD mutations respond poorly to standard cytotoxic agents and there is a clear link
between the disease and the oncogenic properties of FLT3. We present novel 2,6,9ꢀtrisubstituted
purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar
activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3ꢀ
ITD mutations, whereas other transformed and normal human cells are several orders of magnitude
less sensitive. The MV4ꢀ11 cells treated with 7d suppressed the phosphorylation of FLT3 and its
downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a
single dose of 7d in mice with subcutaneous MV4ꢀ11 xenografts caused sustained inhibition of FLT3
and STAT5 phosphorylation over 48 hours, in contrast to the shorter effect observed after
administration of the reference FLT3 inhibitor quizartinib.
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Keywords
acute myeloid leukemia, FLT3, kinase inhibitor, docking, drug design
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Introduction
Acute myeloid leukemia (AML) is a cancer for which current therapies are inadequate. Recent
advances in molecular cancer biology have enhanced our understanding of AML biology and led to
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the discovery of many cancerꢀrelated genes, including potential targets for treatment. The most
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common mutations in AML occur in the FLT3 gene; the mutations are observed in approximately 30
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%
of AML patients and are tightly linked to poor prognosis. The FLT3 gene encodes the membraneꢀ
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bound receptor tyrosine kinase FLT3, which is closely related to KIT, FMS and PDGFR. Upon
binding to an extracellular ligand, FLT3 dimerizes, gets autophosphorylated, and activates
downstream signaling pathways including RAS/MAPK, JAK/STAT5 and PI3K/AKT. These pathways
collectively promote growth, proliferation, survival and differentiation of myeloid cells.
The most common group of FLT3 mutations (found in 23% of all AML patients) are internal
tandem duplications (ITD) of variable length and position that promote ligandꢀindependent
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dimerization. The length of ITD mutations influences prognosis: patients with longer ITDs have
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worse outcomes and lower overall survival. Point mutations within the activation loop of FLT3 are
also somewhat common, occurring in approximately 7 % of all AML cases. These mutations stabilize
the kinase in its active conformation and promote constitutive activation.
FLT3 is regarded as a potential drug target because patients with FLT3ꢀITD mutations respond
poorly to standard cytotoxic agents and there is a clear link between AML and the oncogenic
6
properties of FLT3. Consequently, many groups have attempted to develop specific FLT3 inhibitors
6ꢀ8
for AML therapy and a variety of drugs have entered clinical trials. Early trials used general receptor
tyrosine kinase inhibitors such as sunitinib, sorafenib or lestaurtinib that exhibit activity against FLT3
7
but were originally developed for other indications. These compounds were quite potent in preclinical
experiments, but generally failed in phase I/II clinical trials with AML patients.
The toxicity problems and limited efficacy of these drugs are probably due to their broad
7,8
specificity towards receptor tyrosine kinases. Therefore, new and more specific FLT3 inhibitors
have been developed. These soꢀcalled secondꢀgeneration compounds include quizartinib, crenolanib,
pexidartinib (PLX3397), tandutinib (MLN518) and gilteritinib (ASP2215). These compounds
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demonstrated significant numbers of complete and partial responses in AML patients and are often
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,10
also active in quizartinibꢀresistant AML cells with secondary FLT3 point mutations.
Some of the developed compounds are multiꢀtargeted antiꢀFLT3 drugs effective against both
AML and other indications. For example, the doublyꢀselective JAK2/FLT3 inhibitor pacritinib was
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successful in phase 2 clinical trials against myelofibrosis and lymphoma. Similarly, the clinically
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tested compound gilteritinib targets FLT3 and AXL, while TG02 (SB1317) inhibits FLT3, JAK2,
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and all cyclinꢀdependent kinases (CDKs). The effectiveness of simultaneously targeting FLT3 and
CDKs is further demonstrated by the example of AMG 925 (FLX925), which selectively targets FLT3
1
3,14
and CDK4.
continues to present a major challenge in drug development.
We have previously prepared nanomolar CDK inhibitors with a 6ꢀbenzylaminopurine scaffold,
Despite these promising results, acquired resistance to specific FLT3 inhibitors
1
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but we found that modified analogs (in which the 6ꢀbenzylaminopurine core was replaced with a 6ꢀ
anilinopurine) exhibited substantially reduced cytotoxic activity in most cancer cell lines other than
MV4ꢀ11 bearing FLT3ꢀITD mutation. Cells from this line rapidly stopped proliferating and entered
G1 phase arrest upon treatment with nanomolar concentrations, whereas the other tested cell lines did
not respond to subꢀmicromolar concentrations. Here we report the preparation of a new set of
compounds in this series and demonstrate that their target in the MV4ꢀ11 cell line is FLT3.
Results and discussion
Chemistry
The synthesis started from commercially available 2,6ꢀdichloropurine, which was initially alkylated
with isopropanol, cyclopentanol or benzyl alcohol via the Mitsunobu reaction to obtain 9ꢀsubstitutedꢀ
16
15
17
2,6ꢀdichloroꢀ9Hꢀpurines 1a,
1b
and 1c
with good regioselectivity. 2,6ꢀdichloroꢀ9ꢀ(2ꢀ
tetrahydropyranyl)ꢀ9Hꢀpurine (1d) was prepared using the previously described reaction of 2,6ꢀ
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dichloroꢀ9Hꢀpurine with 3,4ꢀdihydroꢀ2Hꢀpyran.
Most of the used 4ꢀsubstitutedꢀphenylamines were commercially available, but 6ꢀmorpholinꢀ4ꢀ
ylpyridinꢀ3ꢀamine (4a) and 4ꢀ(2ꢀoxaꢀ6ꢀazaspiro[3.3]heptꢀ6ꢀyl)aniline (4b) were prepared by the
reaction of 4ꢀbromonitrobenzene (2a) or 2ꢀbromoꢀ5ꢀnitropyridine (2b) with the corresponding cyclic
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secondary amine to yield nitro derivatives (3a, 3b) that were reduced with hydrogen on palladium at
atmospheric pressure (see Scheme 1).
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Scheme 1.
2 3 2
Reaction conditions: a) appropriate secondary amine, K CO , ethanol, reflux; b) H /PdꢀC 5%, rt,
atmospheric pressure.
The nucleophilic substitution at position 6 of the purine moiety with appropriate 4ꢀsubstitutedꢀ
aniline or 2ꢀsubstitutedꢀ5ꢀaminopyridine derivatives was performed in the presence of Hünig’s base to
obtain compounds (5aꢀ5m). The substitution of the chlorine at position 2 with a primary or secondary
amine was performed with a large excess of the appropriate amine in 1,2ꢀethandiol at 160 °C. These
reactions proceeded smoothly, providing the desired compounds with good yields and high purities
(
see Scheme 2). We used transꢀ1,4ꢀdiaminocyclohexane for the synthesis of compounds 6a, 6c, 6d,
6
h, 6l, 7aꢀ7f and 8l. The 2ꢀaminopropanꢀ1ꢀol used for the synthesis of compounds 6b, 6e, 6f, 6i and
m was racemic and for the synthesis of compound 8l 2ꢀaminobutanꢀ1ꢀol of Rꢀ configuration was
6
used. The collection of newly synthesized compounds (6aꢀ6o, 7aꢀ7f, and 8aꢀ8o) is presented in Tables
ꢀ3.
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Scheme 2.
Reaction conditions: a) appropriate 4ꢀsubstituted aniline or 6ꢀsubstitutedꢀpyridineꢀ3ꢀamine, DIPEA,
nꢀpropanol, 120 °C, 4ꢀ20 hours, sealed tube; b) appropriate primary or secondary amine, 1,2ꢀethandiol,
160 °C, 4ꢀ20 hours, sealed tube.
In Vitro Structure−Activity Relationships
The newly prepared 6ꢀ(4ꢀsubstitutedꢀanilino)purine (6aꢀ6o, 7aꢀ7f, 8aꢀ8o) and 6ꢀ(5ꢀsubstitutedꢀ3ꢀ
aminopyridine)purine (8l, 8m) compounds were tested to determine their ability to inhibit the
recombinant FLT3ꢀITD kinase and to evaluate their antiproliferative activity in the myeloid leukemia
MV4ꢀ11 (acute; FLT3ꢀITDꢀpositive) and K562 (chronic; FLT3ꢀnegative) cell lines. Tables 1ꢀ3
summarize the results obtained.
We began our investigation into the compounds’ structureꢀactivity relationships using
molecules 6c and 6d, which exhibited high selectivity towards FLT3ꢀITDꢀpositive cells and only weak
activity in K562 cancer cell line. Both these compounds have aminocyclohexylamino and 4ꢀ
halogenophenylamine groups at positions 2 and 6, respectively, with a cyclopentyl group at position 9.
To study the impact of substituting position 9 of the purine ring, we compared these initial compounds
to a series of analogs with the same substituents at positions 2 and 6 but isopropyl, tetrahydropyranꢀ2ꢀ
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yl, or benzyl groups at position 9. The lowest GI50 values in MV4ꢀ11 cells were obtained with
compounds bearing isopropyl (6a) and cyclopentyl (6c and 6d) substituents (7, 10 and 21 nM,
respectively); these compounds were also very potent inhibitors of FLT3ꢀITD. Introducing the
tetrahydropyranꢀ2ꢀyl group (6l) reduced both the cellular and kinaseꢀinhibitory activities of the
compounds, but the lowest activities were observed for compound 6h, which has a benzyl group in
position 9. To verify the positive impact of substituent 9, we also modified position 2 by replacing the
aminocyclohexylamino group with various branched alkylamines (6eꢀg) or bulky morpholino (6j) or
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4ꢀmethylꢀpiperazinꢀ1ꢀyl (6k) moieties. All the analogs substituted in this way had weaker
antiproliferative and FLT3ꢀinhibitory activities than the 4ꢀaminocyclohexylamineꢀsubstituted
compounds, and substantially higher IC50 and GI50 values (Table 1).
We next explored the effect of extended substituents at position 6. All the compounds tested in
these experiments had a cyclopentyl group at position 9 and an aminocyclohexylamino group at
position 2 (7aꢀ7f, Table 2). The halogen in the phenylamine substituent of the lead molecule was
replaced with pyrrolidinꢀ1ꢀyl (7a), morpholinꢀ4ꢀyl (7b), 4ꢀethylꢀpiperazinꢀ1ꢀyl (7c), morpholinꢀ4ꢀ
ylmethyl (7d), 2ꢀoxaꢀ6ꢀazaꢀspiro[3.3]heptꢀ6ꢀyl (7e) or Nꢀ4ꢀbenzylꢀpiperazinꢀ1ꢀyl (7f) moieties. These
compounds were among the most active in the entire series: their GI50 values in FLT3ꢀITD positive
MV4ꢀ11 cells were in the singleꢀdigit nanomolar range, and approximately 100 times lower than the
corresponding values for K562 cells. They also strongly inhibited the enzymatic activity of FLT3ꢀITD
at low nanomolar concentrations. These results indicate that position 6 can host various substitutions
without significant loss of in vitro activity (Table 2).
Since the most active compounds of the series (7bꢀ7d) have a cyclopentyl group in position 9
and extended 6ꢀ(4ꢀsubstitutedꢀphenylamino) groups, we investigated analogs of these compounds with
modifications at position 2 to determine whether the aminocyclohexylamine moiety is the optimal
substituent with respect to in vitro activity. We investigated linear aminoalkylamines (8a, 8b, 8c, 8n,
8
o), hydroxyalkylamines (8i, 8j, 8k), bulky saturated heterocyclic moieties (8f, 8g, 8h, 8m) or
aromatic substituents (8d, 8e). Compounds with an 4ꢀaminobutylamine group in position 2 (8a, 8n,
o) showed comparable potency against FLT3ꢀITD to starting molecules 7d, 7b, 7c and inhibited
8
MV4ꢀ11 proliferation at low nanomolar concentrations, with GI50 values of about 11 nM. Longer
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aminoalkyl side chains yielded weaker antiproliferative and kinaseꢀinhibitory activities: the FLT3ꢀITD
IC50 values for the butylꢀ (8a), pentylꢀ (8b), and hexylꢀ (8c) substituted compounds were 1, 5, and 10
nM, respectively, and the corresponding MV4ꢀ11 GI values were 11, 35, and 86 nM, respectively.
50
Compounds with hydroxyalkylamines in position 2 (8i, 8j, 8k) exhibited weaker biological activity:
the most potent of these derivatives (8k) was 6 times less potent than 7d with respect to FLT3ꢀITD
inhibition, and had a 40ꢀfold higher GI value in MV4ꢀ11 cells. Shortening of the hydroxyalkyl chain
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of 8k (hydroxyethylamine 8i and hydroxypropylamine 8j) further reduced their in vitro activity.
Similarly low activities were achieved with compounds bearing bulky morpholine (8f) or
methylpiperazine (8g) substituents in position 2: the least active compounds in the group were those
bearing piperidine (8h), benzylamine (8d), or 4ꢀmethoxybenzylamine (8e) moieties (Table 3).
Our synthetic approach allowed us to further modify the first aromatic ring in position 6; we
replaced the phenylamine with pyridinamine and extended it with morpholine (8l, 8m). This
modification did not improve the molecules’ biological activity ꢀ for example, compound 8l displayed
similar activity to its phenylamine counterpart 7b (Tables 2 and 3).
To investigate the cellular selectivity of the new compounds, the antiproliferative activities of
the most potent derivatives were tested in cancer cell lines with different histological origins (MOLMꢀ
13, Kasumiꢀ1, THPꢀ1, U937, MCFꢀ7, HCCꢀ827) and in noncancerous cells (MRCꢀ5, HUVEC, BJ;
Table 4). The compounds exhibited nanomolar activity in the MOLMꢀ13 line (which is FLT3ꢀITDꢀ
positive, like MV4ꢀ11), but their GI50 values in other cancer cell lines were in the micromolar range,
confirming their high selectivity towards FLT3ꢀITDꢀpositive cells. Notably, Kasumiꢀ1 and HCCꢀ827
cells bearing an activating mutation in cꢀKIT (Asn822Lys) and an activating deletion in EGFR,
respectively, displayed submicromolar sensitivity to compounds 7aꢀ7e, and compounds from series 8
(
8aꢀ8d, 8o, 8n) seemed to be even more selective for FLT3ꢀITDꢀpositive cell lines. Importantly,
nontransformed human MRCꢀ5 and HUVEC cells, as well as nonproliferating BJ cells, were not
affected by these compounds even when treated with concentrations 1000ꢀfold higher than those toxic
against the FLT3ꢀITD positive MV4ꢀ11 and MOLMꢀ13 cell lines.
To further confirm the FLT3ꢀinhibitory effects of our compounds, we evaluated the
antiproliferative activity of 7d in murine Ba/F3 proꢀB cells stably transfected with FLT3ꢀITD. While
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the Ba/F3 FLT3ꢀITD cells were highly sensitive to the 7d treatment and the GI50 values reached low
nanomolar concentrations (GI50 = 0.034 ± 0.015 µM), in parental Ba/F3 cells the GI50 values were
higher than 1 µM (GI50 = 1.136 ± 0.389). The similar results were obtained using quizartinib as a
control (GI50 = 0.007 ± 0.003 µM in Ba/F3 FLT3ꢀITD and 1.452 ± 0.166 µM in parental Ba/F3 cells).
Experiments were performed at least in triplicates; for representative graphs see Figure S1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. 2ꢀsubstitutedꢀaminoꢀ6ꢀ(4ꢀhalogenophenyl)aminoꢀ9ꢀsubstitutedꢀ9Hꢀpurines 6aꢀ6o and their
biochemical (recombinant FLT3ꢀITD) and cellular activities.
a
a
Substituents
R1
IC50 (µM)
GI50 (µM)
FLT3
ITD
R2
R3
Q
MV4ꢀ11
K562
6
a
Isopropyl
Isopropyl
Br
CH
0.010
0.591
0.007
1.173
6b
Br
CH
1.498
17.800
6
c
Cyclopentyl
Cyclopentyl
Br
Cl
CH
CH
0.004
0.005
0.010
0.021
0.670
0.840
6d
6
e
f
Cyclopentyl
Cyclopentyl
Cl
Br
CH
CH
1.723
0.628
0.235
0.444
10.910
>25
6
6g
6h
6i
Cyclopentyl
Benzyl
Br
Br
Br
CH
CH
CH
0.281
0.798
9.848
1.307
2.287
>4
7.630
3.067
9.447
Benzyl
6j
Benzyl
Br
Br
Br
CH
CH
CH
>20
>20
>4
>4
>12.5
3.800
5.170
6k
Benzyl
6l
Tetrahydropyranꢀ2ꢀyl
0.070
0.181
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3
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4
4
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4
4
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5
5
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6
m
Tetrahydropyranꢀ2ꢀyl
Br
CH
1.141
2.430
27.900
6
n
o
Tetrahydropyranꢀ2ꢀyl
Tetrahydropyranꢀ2ꢀyl
Br
Br
CH
CH
1.195
4.207
3.884
3.204
3.745
7.130
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
For SD values see Supporting information (Table S1).
Table 2. 2ꢀ(4ꢀaminocyclohexylamino)ꢀ6ꢀ(4ꢀsubstitutedꢀphenyl)aminoꢀ9ꢀcyclopentylꢀ9Hꢀpurines 7aꢀ7f
their biochemical (recombinant FLT3ꢀITD) and cellular activities.
a
a
Substituents
R1
IC50 (µM)
FLT3
GI50 (µM)
R2
R3
Q
MV4ꢀ11
K562
ITD
7a
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
CH
CH
CH
CH
CH
0.003
0.002
0.003
0.003
0.004
0.018
0.002
0.001
0.002
0.003
0.940
0.965
2.070
0.380
1.220
7b
7c
7d
7e
7f
Cyclopentyl
CH
0.006
0.017
0.700
a
For SD values see Supporting information (Table S1).
Table 3. 9ꢀcyclopentylꢀ2ꢀsubstitutedꢀaminoꢀ6ꢀarylaminoꢀ9Hꢀpurines 7d, 8aꢀ8o and their biochemical
(
recombinant FLT3ꢀITD) and cellular activities.
a
a
Substituents
IC50 (µM)
FLT3
GI50 (µM)
R1
R2
R3
Q
MV4ꢀ11
K562
ITD
7
d
a
Cyclopentyl
Cyclopentyl
CH
CH
0.003
0.001
0.002
0.011
0.380
2.095
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b
c
Cyclopentyl
Cyclopentyl
CH
0.005
0.010
0.035
0.086
1.530
3.787
NH2
8
N
CH
H
8d
Cyclopentyl
Cyclopentyl
CH
CH
0.026
0.209
0.196
0.892
3.597
6.693
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
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5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8e
8f
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
CH
CH
CH
CH
CH
CH
N
0.019
0.053
0.050
0.029
0.047
0.018
0.004
0.071
0.004
0.061
0.066
0.701
0.110
0.157
0.083
0.004
0.070
0.011
4.470
2.745
11.023
2.005
2.348
1.452
1.297
>25
8g
8h
8
8
8
i
j
k
8l
8
8
m
N
n
o
CH
3.490
8
Cyclopentyl
CH
0.001
0.010
0.012
0.003
1.335
>20
quizartinib
a
For SD values see Supporting information (Table S1).
Table 4. Biochemical (recombinant enzymes) and cellular activities of the most potent compounds.
a
a
IC50 (µM)
GI50 (µM)
FLT3
WT
FLT3
MOLMꢀ Kasumiꢀ
HCCꢀ
827
THPꢀ1
U937
MCFꢀ7
MRCꢀ5 HUVEC
BJ
D835Y
13
1
6
c
0.016
0.021
0.021
0.010
0.013
0.013
0.006
0.025
0.021
0.004
0.011
0.007
0.002
0.008
0.028
0.005
0.065
0.069
0.015
0.481
2.280
3.114
1.159
0.514
0.713
0.693
3.739
2.418
3.590
1.797
3.171
1.572
0.605
0.664
0.638
1.341
3.629
2.920
0.520
0.487
0.320
1.050
0.197
1.063
2.105
2.120
5.004
1.275
1.068
0.521
0.592
0.327
0.456
1.664
1.120
2.248
20.628
14.046
23.265
21.964
20.565
9.797
10.062
12.077
4.531
4.439
5.866
5.947
6.265
14.165
10.321
14.688
15.894
12.249
12.932
12.663
14.580
10.351
11.174
7.265
7a
0.024
0.004
0.004
0.001
0.004
0.019
0.020
0.054
0.569
0.816
0.839
0.513
0.734
2.241
1.283
1.024
7b
7c
7d
7e
8a
3.878
8b
5.883
8c
4.095
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8
d
0.083
0.023
0.013
0.624
0.007
0.003
0.136
0.122
0.017
0.004
0.002
4.757
2.016
2.071
0.064
>10
6.906
4.355
>20
9.793
4.070
3.419
>20
8.600
3.255
2.150
>20
>5
1.408
0.976
>5
23.932
7.559
10.312
n.t.
10.105
8.058
11.421
n.t.
>25
12.976
15.389
n.t.
8n
8
o
quiz
0.036
a
For SD values see Supporting information (Table S2). n.t. – not tested.
0
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8
9
0
Binding mode in FLT3 active site
Kinase inhibitors bind via several types of binding modes, type I and II inhibitors were described for
19, 20
FLT3.
Type I inhibitors bind to the ATP binding site, forming hydrogen bonds with the hinge
region and presenting substituents in the hydrophobic region near the gatekeeper residue in the active
kinase conformation (activation loopꢀout; DFGꢀin). In contrast, type II inhibitors bind to the allosteric
site in the inactive kinase conformation (DFGꢀout). Type II FLT3 kinase inhibitors (e.g. quizartinib)
19
were previously structurally characterized in complex with the inactive state of FLT3. The inhibitors
in the present study, however, are suggested to have type I binding because of their similar inhibition
21
of WT and D835Yꢀmutated FLT3 enzymes (Table 4). Due to the unavailability of the structure of the
active conformation of FLT3 kinase, we prepared its homology model and docked the lead compound
7d (see Supporting information). The binding mode is consistent with type I binding, i.e. hingeꢀregion
hydrogen bonds and 2,6,9ꢀsubstituents pointing to their respective pockets (Figure 1). In the hinge
region, Cys694 backbone forms two classical Hꢀbonds with 7d (Figure 1) as expected for type I
inhibitors. The cyclopentyl ring of 7d makes hydrophobic contacts with several hydrophobic residues
including the Phe691 gatekeeper. The 1,4ꢀdiaminocyclohexyl moiety uses its protonated distal 4ꢀ
amino group to make a salt bridge with Asp829. Further modeling showed that many of the nonꢀ
hingeꢀregion interactions would be lost in compounds such as 6j, resulting in a loss of activity. The N
atom of the Nꢀmethyl morpholino group (pKa of 7.38) loses the proton upon making Hꢀbond with
Lys614.
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Figure 1. Docked binding pose of 7d (orange sticks) in FLT3. The kinase (cyan cartoon) is modeled
in active conformation; the Pꢀloop (green cartoon), helixꢀC (magenta) and activation loop (AL;
yellow) are highlighted. FLT3 interacting residues are shown as sticks colored grey for carbon, blue
for nitrogen, red for oxygen, yellow for sulfur and white for hydrogen. Figure prepared with Maestro,
Schrodinger, LLC.
Protein Kinase Selectivity
In addition to FLT3ꢀITD, most of the active compounds were also potent inhibitors of recombinant
wildꢀtype FLT3 and, even more importantly, of a recombinant FLT3 variant bearing the D835Y point
mutation, which contributes to the development of resistance (Table 4). Both FLT3ꢀITD and FLT3ꢀ
D835Y display similar sensitivity to majority of our compounds, with GI50 values in a low nanomolar
range. In contrast, quizartinib was at least 10× less potent on D835Y mutant, which is in agreement
2
1
with published data. Compound 7d, the most active candidate, was then screened for its activity
against 309 protein kinases (Carna Biosciences). This screening confirmed 7d as a potent inhibitor of
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2
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2
2
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FLT3. It also achieved significant inhibition (>90%) against several other kinases at a concentration of
10 nM, notably PDGFRα/β, CLK1, TRK, QIK, CaMK2δ, SIK, YES, FMS, CAMK2γ, KIT (D816V),
MNK2, ACK and SRC (For more details, see the Supporting information Table S3).
0
1
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8
9
0
1
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1
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8
9
0
Cellular activity of 7d is related to FLT3ꢀinhibition
Because compound 7d inhibited the proliferation of FLT3ꢀITD positive MV4ꢀ11 and MOLMꢀ13 cell
lines very effectively at low nanomolar concentrations (GI50 values 2 and 1 nM, respectively), we
investigated its molecular mechanism of action in cells. MV4ꢀ11 cells were treated for 1 hour with
increasing concentrations of the compound and analyzed by immunoblotting, revealing that
concentrations as low as 1 nM were sufficient to block the autophosphorylation of the FLT3 receptor
tyrosine kinase at three different tyrosine residues (589, 591 and 842). Moreover, this inhibition
suppressed phosphorylation of several downstream targets of FLT3. Notably, 7d abolished
phosphorylation of STAT5 at Y694, which is a direct substrate of the oncogenic FLT3ꢀITD variant.
The second pathway affected was the mitogenꢀactivated protein kinase (MAPK) cascade: two key
components of this signaling pathway, ERK1/2 (T202/Y204) and MEK1/2 (S217/221), exhibited
reduced phosphorylation upon treatment with our compound. 7d also interfered with PI3K/AKT
pathway which was confirmed by reduced phosphorylation of AKT at S473 (Figure 2). Similar results
were obtained in MV4ꢀ11 cells after treatment with clinicallyꢀtested FLT3 inhibitor quizartinib (Figure
S3). Because the MAPK and STAT pathways are crucial regulators of cell proliferation, their
inhibition induces cell cycle arrest in the G1 phase. To confirm this, MV4ꢀ11 cells were treated for 24
hours with increasing doses of compound 7d and quizartinib, stained with propidium iodide, and
analyzed by flow cytometry. Even a 1 nM concentration induced massive G1 arrest. In addition,
higher doses increased the number of cells in the subꢀG1 phase of the cell cycle, corresponding to an
increased number of apoptotic cells (Figure 3A, Figure S4). This result was verified by
immunoblotting, which revealed increased cleavage of the apoptotic marker protein PARPꢀ1 (89 kDa
fragment) upon treatment with 7d at 10 or 100 nM for 24 hours. This cleavage was accompanied by
reduced levels of the antiapoptotic protein Mclꢀ1 (Figure 3B). To confirm that the proapoptotic effect
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of our compound is closely related to the presence of oncogenic FLT3 variants, we studied the
activation of caspases, which play key roles in apoptotic cascades, in MV4ꢀ11 (FLT3ꢀITD) and K562
(
no expression of FLT3) cells after treatment with 7d for 24 hours. Whereas even subnanomolar
concentrations of 7d induced caspase activation in MV4ꢀ11, K562 cells were much less sensitive and
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0
required vastly greater concentrations for caspase induction (Figure 3C).
Figure 2. Effect of 7d on FLT3 and some of its downstream signaling pathways. PCNA was used as a
control for protein loading. Densitometric analysis is available from the Supporting information Figure
S2.
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Figure 3. Effect of 7d on cell cycle (A) and apoptosis (B, C) in the MV4ꢀ11 and K562 cell lines.
Activity in MV4ꢀ11 xenograft
To determine the efficacy of compound 7d in vivo, we compared its effects to that of quizartinib using
human tumor xenografts grown in immunocompromised mice. First, we determined that treatment
with 7d caused no gross toxicity in rats (at doses up to 100 mg/kg i.v. or p.o.; for more details see
Supporting information). Next, a single dose of 10 mg/kg of 7d or quizartinib was administered i.p. to
mice with subcutaneously implanted MV4ꢀ11 xenografts. Control mice were injected with an equal
volume of diluent alone. Tumors were harvested at 4 postꢀtreatment time points: 2, 24, 36, and 48
hours after 7d, quizartinib, or vehicle injection. The protein lysates were analyzed to detect
phosphorylation of the target oncogenic tyrosine kinase FLT3ꢀITD and its downstream signaling
molecules (Figure 4, Figure S5). Compound 7d effectively inhibited FLT3ꢀITD autophosphorylation
22ꢀ24
in MV4ꢀ11 xenografts (Figure 4A), and unlike quizartinib (Figure 4B),
it sustained this inhibition
of phosphorylation at all analyzed timeꢀpoints. Next, we investigated the inhibition of the key
downstream effectors of FLT3ꢀITD, namely pꢀSTAT5 and pꢀERK1/2 (Figure 4A, B). Treatment with
7d reduced STAT5 phosphorylation by over 95% after 24 hours, which was a slightly stronger effect
than that of quizartinib (Figure S5). Mirroring the results observed for pꢀFLT3, the suppression of pꢀ
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STAT5 phosphorylation induced by 7d was sustained over 48 hours (Figure 4A), whereas a rebound
in STAT5 phosphorylation was detected in lysates from quizartinibꢀtreated tumors (Figure 4B).
In contrast to the results observed for pꢀSTAT5, pꢀERK1/2 was only transiently inhibited in
the tumor tissue, exhibiting similar rebound kinetics for both tested inhibitors in all animals (Figure
1
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4A, B). Two hours after treatment with 7d or quizartinib, the suppression of ERK1/2 was reduced by
95% relative to controls (Figure S5). However, its phosphorylation was restored within 24 hours of
treatment with either compound, suggesting that there is an adaptive feedback mechanism capable of
ERK reactivation in response to FLT3ꢀITD inhibition. Similar behavior was observed in earlier studies
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using quizartinib and sorafenib.
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Figure 4. Compound 7d induces sustained suppression of FLT3ꢀITD signaling in MV4ꢀ11 xenograft
tumors. Subcutaneous mouse xenografts were treated with 10 mg/kg 7d (A) or quizartinib (B), and
the phosphorylation status of FLT3 and its downstream signaling molecules was analyzed by
immunoblotting at the indicated times. GAPDH was used as a control for protein loading. Each line
represents a separate animal.
Conclusion
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We have presented the synthesis of a new generation of highly potent FLT3 inhibitors with a 2,6,9ꢀ
trisubstutituted purine scaffold. A focused library of 49 compounds was prepared, some members of
which exhibited selective nanomolar activity against acute myeloid leukemia cell lines expressing an
oncogenic variant of FLT3. The lead compound 7d inhibited FLT3 autophosphorylation and
deactivated its downstream signaling pathways, leading to cell cycle arrest and apoptosis in MV4ꢀ11
cells. Docking of 7d to FLT3 suggests a type I binding mode and explains the structural determinants
of its potency. Experiments with subcutaneously implanted MV4ꢀ11 xenografts confirmed that a
single dose of the tested compound induced sustained inhibition of FLT3 in vivo. In conclusion, we
suggest this series to be followed for development of potent and specific FLT3 inhibitors for use as
drug candidates for treating AML.
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Experimental Section
Reagents and General Methods
Chemistry
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Melting points were determined on Büchi Melting Point Bꢀ540 apparatus and are uncorrected. H and
13
1
CꢀNMR spectra were recorded on a Jeol 500 ECA instrument operating at 500 MHz for H and 126
13
MHz for C or on a Bruker Avance 300 spectrometer (300 MHz) at ambient temperature in DMSOꢀd6
or CDCl . Chemical shifts are reported in ppm. Coupling constants (J) are reported in Hertz (Hz), and
the following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), quintet (qui), sextet
sex), septet (sep), multiplet (m). Mass spectra were recorded using an LCQ ion trap mass
3
(
spectrometer (Finnigan MAT, San Jose, CA, USA). The chromatographic purity of the compounds
was determined using HPLCꢀDADꢀMS. An Alliance 2695 separations module (Waters) linked
simultaneously to a PDA 996 (Waters) and a QꢀTof micro (Waters) benchtop quadrupole orthogonal
acceleration timeꢀofꢀflight tandem mass spectrometer were used. Samples were dissolved in methanol
–
1
and diluted to a concentration of 10 ꢁg·mL in the mobile phase (initial conditions). Then, 10 ꢁL of
the solution were injected on a RPꢀcolumn (150 mm × 2.1 mm; 3.5 ꢁ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
–
1
of 0.2 mL·min , 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 reꢀ
equilibrated to the initial conditions for 10 min. The effluent was introduced into the DAD (scanning
range 210–400 nm, with 1.2 nm resolution) and an electrospray source was applied (source
temperature 110 °C, capillary voltage +3.0 kV, cone voltage +20 V, desolvation temperature 250 °C).
–
1
–1
Nitrogen was used as both the desolvation gas (500 L·h ) and the cone gas (50 L·h ). The mass
spectrometer was operated in positive (ESI+) ionization mode, and data were acquired in the 50–1000
m/z range. Elemental analyses were performed using an EA1112 CHN analyser (Thermo Finnigan);
the results obtained for C, H, and N were within acceptable limits of the expected values. Merck silica
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gel Kieselgel 60 (230–400 mesh) was used for column chromatography. The purity of biologically
evaluated compounds was >95% as determined by HPLCꢀDADꢀMS and elemental analysis.
General procedure for the preparation of derivatives 4
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4ꢀFluoronitrobenzene (2a) or 5ꢀbromoꢀ2ꢀnitropyridine (2b) (1.00 mmol), the appropriate secondary
amine (1.05 mmol), and potassium carbonate (2.00 mmol) in ethanol (10 mL) were heated under an
argon atmosphere in a sealed tube at 100 °C for 4 hours. The completion of the reaction was checked
with TLC on silica (chloroformꢀmethanol, 19:1). After cooling to room temperature, the reaction
mixture was evaporated under reduced pressure. The residue was partitioned between dichloromethane
(
25 mL) and water (25 mL). The water phase was extracted twice with dichloromethane (25 mL). The
combined organic phases were washed with water, brine, dried over sodium sulphate, and
concentrated under reduced pressure. The crude product (3) was then used directly without further
purification.
The crude product (3) from the previous step (0.75 mmol) was hydrogenated under atmospheric
pressure in methanol (50 mL) with 5% wt. palladium on a charcoal (50 mg). After consumption of
hydrogen, the reaction mixture was filtered through celite, washed with methanol and evaporated
under reduced pressure. The crude product was dissolved in 2 M hydrochloric acid (50 mL) and
extracted with dichloromethane (25 mL). The water phase was neutralised with 5 % sodium hydrogen
carbonate, then the precipitate was filtered off and washed with water. The crude product was dried in
a vacuum dessicator.
4ꢀ(5ꢀNitroꢀpyridinꢀ2ꢀyl)ꢀmorpholine (3a)
Yield: 85%. Elemental analysis: Calcd.for C H N O (209.21): C, 51.67; H, 5.30; N, 20.09. Found: C,
9
11
3
3
51.57; H, 5.48; N, 19.74. HPLCꢀMS (ESI+): 210.31 (99.8%). GCꢀMS (EI, M+ (rel.int. m/z)): 209
1
(18), 124 (100), 87 (28). H NMR (500 MHz, CDCl
3
) δ ppm 2.92ꢀ2.98 (m, 4H), 3.71ꢀ3.75 (m, 4H),
6.52 (d, J = 9.5, 1H), 8.15 (s, 1H), 9.02 (d, J = 9.5, 1H).
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ꢀ(4ꢀNitroꢀphenyl)ꢀ2ꢀoxaꢀ6ꢀazaꢀspiro[3.3]heptane (3b)
Yield: 72%. Elemental analysis: Calcd.for C11 (220.22): C, 59.99; H, 5.49; N, 12.72. Found:
12 2 3
H N O
C, 59.66; H, 5.12; N, 12.19. HPLCꢀMS (ESI+): 221.36 (99.7%). GCꢀMS (EI, M+ (rel.int. m/z)): 220
1
(
21), 150 (100), 120 (57). H NMR (500 MHz, DMSOꢀd
6
) δ ppm 4.19ꢀ4.21(m, 4H), 4.70ꢀ4.72 (m,
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4H), 6.44 (d, J=9.15, 2H), 8.04 (d, J 9.15, 2H).
6ꢀMorpholinꢀ4ꢀylꢀpyridinꢀ3ꢀylamine (4a)
Yield: 98%. Elemental analysis: Calcd.for C
9 13 3
H N O (179.22): C, 60.32; H, 7.31; N, 23.45. Found: C,
59.96; H, 6.83; N, 23.19. HPLCꢀMS (ESI+): 180.28 (99.8%). GCꢀMS (EI, M+ (rel.int. m/z)): 179
1
(
22), 124 (100). H NMR (500 MHz, CDCl
3
) δ ppm 3.05 (s(br, 2H), 3.30ꢀ3.34 (m, 4H), 3.79ꢀ3.82 (m,
4H), 6.55 (d, J = 8.5, 1H), 6.98 (s, 1H), 7.78 (d, J = 8.5, 1H).
4
ꢀ(2ꢀOxaꢀ6ꢀazaꢀspiro[3.3]heptꢀ6ꢀyl)ꢀphenylamine (4b)
O (190.24): C, 69.45; H, 7.42; N, 14.73. Found: C,
9.13; H, 7.12; N, 14.96. HPLCꢀMS (ESI+): 191.5 (99.7%). GCꢀMS (EI, M+ (rel.int. m/z): 190 (11),
14 2
Yield: 58%. Elemental analysis: Calcd.for C11H N
6
1
4
1
32 (100). H NMR (500 MHz, DMSOꢀd
6
) δ ppm 3.72ꢀ3.75(m, 4H), 4.40 (s (br), 2H), 4.65ꢀ4.68 (m,
H), 6.21 (d, J= 8.16, 2H), 6.46 (d, J= 8.15, 2H)
General procedure for the preparation of compounds 5a – 5m:
To a suspension of 9ꢀcyclopentylꢀ2,6ꢀdichloroꢀ9Hꢀpurine (13) (1.98 mmol) in a mixture of nꢀpropanol
(
10 mL) and N,NꢀdiisopropylꢀNꢀethylamine (8.72 mmol), appropriate amine (2.18 mmol) was added.
The suspension was heated with stirring in a sealed tube under an argon atmosphere at 100 °C for 2ꢀ6
hours. The reaction was checked by TLC. After completion of the reaction, the reaction mixture was
cooled to room temperature and evaporated under reduced pressure. The residue was partitioned
between water (50 mL) and dichloromethane (50 mL), and the water phase was extracted two times
more with the same volume of dichloromethane. The combined organic phases were washed with
water and brine and evaporated under reduced pressure. The crude product was crystallized from
petroleum ether:ethyl acetate 3:1.
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ꢀ(Bromoꢀphenyl)ꢀ(2ꢀchloroꢀ9ꢀisopropylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀamine (5a)
Yield: 89 % m.p.: 154ꢀ156 °C. Elemental analysis: Calcd.for C H BrClN (366.64): C, 45.86; H,
14
13
5
1
3
.57; N, 19.10. Found: C, 45.94; H, 3.81; N, 18.82. HPLCꢀMS (ESI+): 367.6 (97.8%). H NMR (500
MHz, DMSOꢀd ) δ ppm 1.49 (d, J=7.0, 6H), 4.70 (sep, J=7.0, 1H), 7.49 (d, J=9.00, 2H), 7.80 (d,
J=9.00, 2H), 8.44 (s, 1H), 10.39 (s, 1H). C NMR (DMSOꢀd , 126 MHz) δ ppm 22.63, 47.49, 115.61,
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119.74, 123.45, 131.84, 137.88, 141.17, 150.92, 152.38, 152.69.
4ꢀ(Bromoꢀphenyl)ꢀ(2ꢀchloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀamine (5b)
Yield: 85 % m.p.: 193ꢀ194 °C. Elemental analysis: Calcd.for C16
H15BrClN
5
(392.69): C, 48.94; H,
1
3.85; N, 17.83. Found: C, 49.06; H, 3.94; N, 17.49. HPLCꢀMS (ESI+): 393.7 (98.4%). H NMR (300
6
MHz, DMSOꢀd ) δ ppm 1.87ꢀ1.92 (m, 2H), 1.92ꢀ1.99 (m, 4H), 2.01ꢀ2.23 (m, 2H), 4.85 (qui, J=7.17,
1H), 7.54 (d, J=8.73, 2H), 7.84 (d, J=8.73, 2H), 8.45 (s, 1H), 10.42 (s, 1H).
(
2ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀchloroꢀphenyl)ꢀamine (5c)
Yield: 78 % m.p.: 212ꢀ214 °C. Elemental analysis: Calcd.for C16
H15Cl
2
N
5
(348.24): C, 55.19; H, 4.34;
1
N, 20.11. Found: C, 55.56; H, 4.05; N, 19.89. HPLCꢀMS (ESI+): 349.4 (96.4%). H NMR (300 MHz,
DMSOꢀd
6
) δ ppm 1.67ꢀ1.71(m, 2H), 1.72ꢀ1.76(m, 4H), 1.89ꢀ2.20 (m, 2H), 4.84 (sep, J= 7.17, 1H),
7.40 (d, J= 8.85, 2H), 7.89(d, J= 8.85, 2H), 8,44 (s, 1H), 10.43 (s, 1H).
(
9ꢀBenzylꢀ2ꢀchloroꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀbromoꢀphenyl)ꢀamine (5d)
Yield: 54 % m.p.: 231ꢀ233 °C. Elemental analysis: Calcd.for C18
H13BrClN
5
(414.69): C, 52.13; H,
1
3.16; N, 16.89. Found: C, 52.02; H, 3.45; N, 16.93. HPLCꢀMS (ESI+): 415.8 (97.8%). H NMR (500
6
MHz, DMSOꢀd ) δ ppm 5.53 (s, 2H), 7.31ꢀ7.42 (m, 5H), 7.56 (d, J=8.43, 2H), 8.03 (d, J=8.43, 2H),
8.87 (s, 1H), 9.25 (s(br), 1H).
(
9ꢀBenzylꢀ2ꢀchloroꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀmorpholinꢀ4ꢀylmethylꢀphenyl)ꢀamine (5e)
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Yield: 76 %. m.p.: 194ꢀ196 °C. Elemental analysis: Calcd.for C23
H
23ClN
6
O (434.93): C, 63.52; H,
1
5.33; N, 19.32. Found: C, 63.82; H, 5.12; N, 19.01. HPLCꢀMS (ESI+): 435.9 (96.2%). H NMR (300
MHz, DMSOꢀd ) δ ppm 2.35ꢀ2.41 (m, 4H), 3.43 (s, 2H), 3.60 (t, J=7.85, 4H), 5.41 (s, 2H), 7.26ꢀ7.37
6
1
3
(
m, 7H), 7.76 (d, J=8.40, 2H), 8.43 (s, 1H), 10.34 (s, 1H). C NMR (76 MHz, DMSOꢀd
6
) δ ppm
6.95, 53.64, 62.51, 66.69, 119.26, 121.68, 127.98, 128.43, 129.34, 129.69, 133.32, 137.09, 138.11,
42.85, 151.23, 152.94, 153.08.
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(
4ꢀBromoꢀphenyl)ꢀ[2ꢀchloroꢀ9ꢀ(tetrahydroꢀpyranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀyl]ꢀamine (5f)
Yield: 76 %. m.p.: 143ꢀ144 °C. Elemental analysis: Calcd.for C H BrClN O (408.69): C, 47.02; H,
1
6
15
5
1
3
.70; N, 17.14. Found: C, 47.13; H, 3.44; N, 17.11. HPLCꢀMS ESI+): 410.2 (97.5%). H NMR (300
MHz, DMSOꢀd ) δ ppm 1.55ꢀ1.58(m, 2H), 1.73ꢀ1.76 (m, 1H), 1.95ꢀ1.99 (m, 2H), 2.20 (t, J=5.3, 1H),
.67ꢀ3.75 (m, 1H), 4.00 (d, J=5.3, 1H), 5.63 (d, J=10.71, 1H), 7.54 (d, J=8.79, 2H), 7.82 (d, J=8.79,
6
3
2
8
1
3
H), 8.58 (s, 1H), 10.49 (s(br), 1H). C NMR (76 MHz, DMSOꢀd
6
) δ ppm 22.19, 24.39, 29.81, 67.61,
0.90, 115.19, 118.78, 122.95, 131.25, 138.09, 140.53, 150.22, 152.09, 152.34
(
2ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀpyrrolidinꢀ1ꢀylꢀphenyl)ꢀamine (5g)
Yield: 92%. m.p.: 168ꢀ169 °C. Elemental analysis: Calcd.for C20
H23ClN
6
(382.89): C, 62.74; H, 6.05;
1
N, 21.95. Found: C, 62.54; H, 5.82; N, 21.83. HPLCꢀMS (ESI+): 384 (99.9%). H NMR (500 MHz,
DMSOꢀd
6
) δ ppm 1.63ꢀ1.81(m, 2H), 1.86ꢀ1.99 (m, 8H), 2.14ꢀ2.21 (m, 2H), 3.19ꢀ3.23 (m, 4H), 4.81
13
(
(
qui, J=7.17, 1H), 6.49 (d, J=8.73, 2H), 7.49 (d, J=8.73, 2H), 8.28 (s, 1H), 9.89(s, 1H). C NMR
6
126 MHz, DMSOꢀd ) δ ppm 24.03, 25.47, 32.57, 48.02, 55.97, 111.85, 119.27, 123.95, 127.55,
140.56, 145.38, 150.70, 152.90, 153.19.
(
2ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀmorpholinꢀ4ꢀylꢀphenyl)ꢀamine (5h)
Yield: 72%. m.p.: 145ꢀ148 °C. Elemental analysis: Calcd.for C20
H
23ClN
6
O (398.89): C, 60.22; H,
1
5.81; N, 21.07. Found: C, 60.25; H, 5.88; N, 21.19. HPLCꢀMS (ESI+): 400.7 (98.5%). H NMR (300
3
MHz, CDCl ) δ ppm 1.79ꢀ1.83 (m, 2H), 1.85ꢀ1.96 (m, 4H), 2.27ꢀ2.36 (m, 2H), 3.14ꢀ3.18 (m, 4H),
3.88ꢀ3.90 (m, 4H), 4.94 (qui, J=7.32, 1H), 6.97 (d, J=8.94, 2H), 7.65 (d, J=8.94, 2H), 7.80 (s (br),
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H), 7.83 (s, 1H). C NMR (126 MHz, DMSOꢀd ) δ ppm 23.91, 33.04, 49.89, 55.97, 66.96, 116.54,
1
19.28, 121.89, 130.84, 138.78, 148.11, 150.70, 152.48, 154.03.
2
ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ[4ꢀ(4ꢀethylꢀpiperazinꢀ1ꢀyl)ꢀphenyl]ꢀamine (5i)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Yield: 82%. m.p.: 128ꢀ131 °C. Elemental analysis: Calcd.for C22
H28ClN
7
(425.96): C, 62.03; H, 6.63;
1
N, 23.02. Found: C, 61.93; H, 6.79; N, 23.22. HPLCꢀMS (ESI+): 427.3 (99.5%). H NMR (300 MHz,
CDCl
3
) δ ppm 1.16 (t, J=7.2, 3H), 1.65ꢀ1.68 (m, 2H), 1.83ꢀ1.95 (m, 4H), 2.26ꢀ2.32 (m, 2H), 3.05ꢀ3.11
(
m, 4H), 3.27ꢀ3.35 (m, 4H), 4.78 (qui, J=6.9, 1H), 6.89 (d, J=9.00, 2H), 7.57 (d, J=9.00, 2H), 8.32 (s,
1
3
1
H), 10.02 (s, 1H). C NMR (300 MHz, CDCl ) δ ppm 11.27, 24.24, 24.45, 32.19, 32.32, 54.84,
3
5
5.48, 63.33, 113.59, 114.68, 122.36, 131.50, 137.37, 140.41, 152.24, 159.16.
(
2ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀmorpholinꢀ4ꢀylmethylꢀphenyl)ꢀamine (5j)
Yield: 69%. m.p.: 123ꢀ124 °C. Elemental analysis: Calcd.for C21
H25ClN
6
O (412.92): C, 61.08; H,
1
6
.10; N, 20.35. Found: C, 61.33; H, 5.99; N, 20.02. HPLCꢀMS (ESI+): 414.2 (99.5%). H NMR (300
3
MHz, CDCl ) δ ppm 1.78ꢀ1.82 (m, 2H), 1.84ꢀ1.99 (m, 4H), 2.28ꢀ2.36 (m, 2H), 2.47ꢀ2.52 (m, 4H),
3
.53 (s, 2H), 3.70ꢀ3.74 (m, 4H), 4.95 (qui, J=5.4, 1H), 7.37(d, J=8.22, 2H), 7.74 (d, J=8.22, 2H), 7.86
13
(
s, 1H). C NMR (300 MHz, CDCl
3
) δ ppm 23.93, 33.04, 53.51, 56.04, 62.92, 67.06, 119.52, 120.01,
1
29.42, 132.12, 137.29, 139.07, 150.87, 152.30, 153.87.
2
ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ[4ꢀ(2ꢀoxaꢀ6ꢀazaꢀspiro[3.3]heptꢀ6ꢀyl)ꢀphenyl]ꢀamine (5k)
Yield: 74%. m.p.: 119ꢀ120 °C. Elemental analysis: Calcd.for C21
H23ClN
6
O (410.90): C, 61.38; H,
1
5
.64; N, 20.45. Found: C, 61.14; H, 5.39; N, 20.19. HPLCꢀMS (ESI+): 411.9 (97.7%). H NMR (300
MHz, DMSOꢀd ) δ ppm 1.67ꢀ1.72 (m, 2H), 1.84ꢀ1.99 (m, 4H), 2.11ꢀ2.18 (m, 2H), 3.92ꢀ3.94 (m, 4H),
6
4
.70ꢀ4.72(m, 4H), 4.81 (qui, J= 5.4, 1H), 6.43 (d, J=8.70, 2H), 7.53 (d, J=8.70, 2H), 8.33 (s, 1H),
9.99 (s, 1H).
[
4ꢀ(4ꢀBenzylꢀpiperazinꢀ1ꢀyl)ꢀphenyl]ꢀ(2ꢀchloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀamine (5l)
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5
5
6
Yield: 79 %. m.p. 181ꢀ182 °C. Elemental analysis: Calcd.for C27
H
30ClN
7
(488.03): C, 66.45; H, 6.20;
1
N, 20.09. Found: C, 66.71; H, 6.41; N, 19.84. HPLCꢀMS (ESI+): 490.6 (99.9%). H NMR (DMSOꢀd
6
,
5
00 MHz) δ ppm 1.62ꢀ1.70 (m, 2H), 1.79ꢀ1.86 (m, 2H), 1.89ꢀ1.97 (m, 2H), 2.10ꢀ2.16 (m, 2H), 3.06ꢀ
.09 (m, 4H), 3.29ꢀ3.31 (m, 4H), 3.49 (s, 2H), 4.78 (qui, J=7.5, 1H), 6.88 (d, J=9.0, 2H), 7.21ꢀ7.25
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
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9
0
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7
8
9
0
1
3
(
m, 1H), 7.27ꢀ7.31 (m, 4H), 7.57 (d, J=9.0, 2H), 8.32 (s, 1H), 10.03 (s, 1H). C NMR (126 MHz,
DMSOꢀd ) δ ppm 24.03, 32.56, 49.14, 53.09, 56.01, 62.60, 115.99, 119.38, 123.13, 127.51, 128.74,
6
129.46, 130.91, 138.61, 140.84, 148.10, 150.85, 152.76, 152.99.
2ꢀChloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(6ꢀmorpholinꢀ4ꢀylꢀpyridinꢀ3ꢀyl)ꢀamine (5m)
Yield: 88%. m.p. 113ꢀ115 °C. Elemental analysis: Calcd.for C19
H
22ClN
7
O (399.88): C, 57.07; H, 5.55;
1
N, 24.52. Found: C, 57.23; H, 5.21; N, 24.14. HPLCꢀMS (ESI+): 400.9 (99.5%). H NMR (500 MHz,
6
DMSOꢀd ) δ ppm 1.79ꢀ1.83 (m, 2H), 1.85ꢀ1.96 (m, 4H), 2.27ꢀ2.36 (m, 2H), 3.14ꢀ3.18 (m, 2H), 3.88ꢀ
3.90 (m, 2H), 4.91 (qui, J=6.55, 1H), 6.93 (s, 1H), 7.41ꢀ7.47 (m, 2H), 7.98 (s, 1H), 8.25 (s(br), 1H).
General procedure for the preparation of compounds 6aꢀ6o, 7aꢀ7f and 8aꢀ8o:
The mixture of 2ꢀchloroꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ6ꢀsubst.amino derivative 15 (1.00 mmol) and transꢀ
1
,4ꢀdiaminocyclohexane (10.0 mmol) in 1,2ꢀethandiole (5,0 mL) was heated with stirring at 160 °C for
hours in an argon atmosphere. After cooling to room temperature, the mixture was diluted with ethyl
4
acetate (40 mL) and washed with water (40 mL). The organic phase was washed with brine, dried over
sodium sulphate and concentrated under reduced pressure. The crude product was purified by column
chromatography on silica using mobile phase chloroform – methanol (4:1, v/v).
2
6
N ꢀ(4ꢀAminoꢀcyclohexyl)ꢀN ꢀ(4ꢀbromoꢀphenyl)ꢀ9ꢀisopropylꢀ9Hꢀpurineꢀ2,6ꢀdiamine (6a)
Yield: 85 %. m.p.: 224ꢀ226 °C. Elemental analysis: Calcd. for C20
H
26BrN
7
(444.37): C, 54.06; H, 5.90;
1
N, 22.06. Found: C, 53.99; H, 5.73; N, 22.23. HPLCꢀMS (ESI+): 446.0 (97.9%). H NMR (500 MHz,
DMSOꢀd ) δ ppm 1.07 – 1.29 (m, 4H), 1.45 (d, J = 6.4, 6H), 1.77 (d, J = 11.8, 2H), 1.93 (d, J = 11.5,
6
2H), 3.64 – 3.52 (m, 2H), 4.59 – 4.49 (sep, J=6.50, 1H), 6.47 (s(br), 1H), 7.39 (d, J = 8.7, 2H), 7.91 (s,
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H), 7.99(d, J=8.7, 2H), 9.55 (s(br), 1H). C NMR (126 MHz, DMSOꢀd ) δ ppm 22.57, 31.84, 35.76,
4
6.29, 50.56, 113.53, 114.40, 122.17, 131.42, 136.69, 140.54, 152.20, 158.59.
1
ꢀ[6ꢀ(4ꢀBromoꢀphenylamino)ꢀ9ꢀisopropylꢀ9Hꢀpurinꢀ2ꢀylamino]ꢀpropanꢀ2ꢀol (6b)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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5
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0
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0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Yield: 79 %. m.p.: 178ꢀ180 °C. Elemental analysis: Calcd. For C17
H21BrN
6
O (405.30): C, 50.38; H,
1
5
.22; N, 20.74. Found: C, 50.59; H, 5.49; N, 20.58. HPLCꢀMS (ESI+): 407.0 (98.1%). H NMR (500
6
MHz, DMSOꢀd ) δ ppm 1.06 (d, J = 6.2, 3H), 1.46 (d, J = 6.8, 6H), 3.20 – 3.12 (m, 1H), 3.30 – 3.23
(
m, 2H), 3.81(sep, J = 5.5, 1H), 4.55 (sep, J = 7.0, 1H), 4.70 (s, 1H), 6.50 (t, J = 5.8, 1H), 7.39 (d, J =
1
3
8
2
1
.9, 2H), 7.93 (s, 1H), 7.97 (d, J = 8.7, 2H), 9.55 (s(br), 1H). C NMR (126 MHz, DMSOꢀd ) δ ppm
6
1.93, 22.56, 46.40, 49.82, 65.92, 113.66, 114.68, 122.40, 131.49, 136.78, 140.38, 151.87, 152.24,
59.30.
2
6
N ꢀ(4ꢀAminoꢀcyclohexyl)ꢀN ꢀ(4ꢀbromoꢀphenyl)ꢀ9ꢀcyclopentylꢀ9Hꢀpurineꢀ2,6ꢀdiamine (6c)
Yield: 79 %. m.p.: 162ꢀ164 °C. Elemental analysis: Calcd. For C22
H28BrN
7
(470.42): C, 56.17; H,
1
6
.00; N, 20.84. Found: C, 56.39; H, 5.63; N, 20.51. HPLCꢀMS (ESI+): 473.0 (99.8%). H NMR (300
6
MHz, DMSOꢀd ) δ ppm 1.09ꢀ1.32 (m, 4H), 1.64ꢀ1.68 (m, 2H), 1.78ꢀ2.08 (m, 12H), 2.57 (m, 1H),
3
8
3
.58ꢀ3.61 (m, 1H), 4.69 (qui, J=7.11, 1H), 6.50 (d, J=7.41, 1H), 7.42 (d, J=8.85, 2H), 7.90 (s, 1H),
13
.04 (d, J=8.85, 2H), 9.58 (s (br), 1H). C NMR (76 MHz, DMSOꢀd
6
) δ ppm 24.18, 24.48, 31.77,
2.28, 35.67, 49.12, 50.63, 133.53, 122.22, 123.80, 131.42, 137.46, 140.55, 150.85, 152.26, 158.52.
2
6
N ꢀ(4ꢀAminoꢀcyclohexyl)ꢀN ꢀ(4ꢀchloroꢀphenyl)ꢀ9ꢀcyclopentylꢀ9Hꢀpurineꢀ2,6ꢀdiamine (6d)
Yield: 84 % m.p.: 113ꢀ115 °C. Elemental analysis: Calcd. for C22
H28ClN
7
(425.96): C, 62.03; H, 6.63;
1
N, 23.02. Found: C, 61.87; H, 6.89 N, 21.31. HPLCꢀMS (ESI+): 426.2 (98.7%). H NMR (500 MHz,
DMSOꢀd
6
) δ ppm 1.29–1.04 (m, 4H), 1.68–1.56 (m, 2H), 2.11 – 1.72(m, 10H), 2.56–2.48 (m, 1H),
3
8
3
.62 – 3.52 (m, 1H), 4.65 (qui, J = 6.3, 1H), 6.48 (d, J = 4.9, 1H), 7.26 (d, J = 8.5, 2H), 7.87 (s, 1H),
13
.06 (d, J = 8.8, 2H), 9.54 (s(br), 1H). C NMR (126 MHz, DMSOꢀd ) δ ppm 24.29, 31.77, 32.28,
6
5.67, 50.56, 50.78, 55.50, 114.48, 121.80, 125.55, 128.51, 137.40, 140.13, 152.27, 158.52.
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ꢀ[6ꢀ(4ꢀChloroꢀphenylamino)ꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ2ꢀylamino]ꢀpropanꢀ2ꢀol (6e)
Yield: 79 % m.p.: 128ꢀ129 °C. Elemental analysis: Calcd. for C19H23ClN
6
O (386.88): C, 58.99; H,
1
5.99; N, 21.72. Found: C, 58.63; H, 6.22 N, 21.94. HPLCꢀMS (ESI+): 387.3 (98.7%). H NMR (300
6
MHz, DMSOꢀd ) δ ppm 1.09 (d, J= 6.18, 3H), 1.63ꢀ1.67 (m, 2H), 1.87ꢀ2.00 (m, 4H), 2.09ꢀ2.21 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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9
0
2H), 3.14ꢀ3.22 (m, 2H), 3.75ꢀ3.89 (m, 1H), 4.67 (sep, J= 7.41, 1H), 4.72 (s(br), 1H), 6.54 (t, J= 5.19,
1
3
1H), 7.30 (d, J= 8.67, 2H), 7.89 (s, 1H), 8.06 (d, J=8.67, 2H), 9.58 (s, 1H). C NMR (DMSOꢀd , 76
6
MHz) δ ppm 21.29, 23.63, 31.66, 49.20, 54.81, 65.28, 114.12, 121.36, 125.07, 127.96, 136.74, 139.33,
151.67, 158.67.
1ꢀ[6ꢀ(4ꢀBromoꢀphenylamino)ꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ2ꢀylamino]ꢀpropanꢀ2ꢀol (6f)
Yield: 94 %. m.p.: 133ꢀ134 °C. Elemental analysis: Calcd. for C19
H23BrN
6
O (431.33): C, 52.91; H,
1
5.37; N, 19.48. Found: C, 52.84; H, 5.19 N, 19.56. HPLCꢀMS (ESI+): 433.0 (98.0%). H NMR (500
6
MHz, DMSOꢀd ) δ ppm 1.05 (d, J= 6.23, 3H), 1.62ꢀ1.67 (m, 2H), 1.80ꢀ1.86 (m, 2H), 1.92ꢀ1.97 (m,
2H), 2.04ꢀ2.09 (m, 2H), 3.11ꢀ3.17 (m, 1H), 3.25ꢀ3.29 (m, 1H), 3.81 (sep, J=5.8, 1H), 4.63ꢀ4.68 (m,
13
2
H), 6.51 (t, J= 5.28, 1H), 7.39 (d, J= 8.85, 2H), 7.89 (s, 1H), 7.99 (d, J=8.85, 2H), 9.56 (s, 1H). C
, 126 MHz) δ ppm 21.95, 24.27, 32.31, 49.83, 55.42, 60.25, 65.88, 113.64, 114.78,
22.43, 131.48, 137.40, 140.41, 152.28, 159.27.
NMR (DMSOꢀd
6
1
(
R)ꢀ3ꢀ[6ꢀ(4ꢀBromoꢀphenylamino)ꢀ9ꢀcyclopentylꢀ9Hꢀpurinꢀ2ꢀylamino]ꢀ2ꢀmethylꢀpropanꢀ1ꢀol (6g)
Yield: 85 %. m.p.: 156ꢀ158 °C. Elemental analysis: Calcd. for C H BrN O (445.36): C, 53.94; H,
20
25
6
1
5
.66; N, 18.87. Found: C, 53.67; H, 5.49 N, 18.41. HPLCꢀMS (ESI+): 447.07 (97.2%). H NMR (500
MHz, DMSOꢀd ) δ ppm 0.86 (t, J = 7.4, 3H), 1.46 (qui, J = 7.0, 1H), 1.68 – 1.57 (m, 3H), 1.88 – 1.79
m, 2H), 2.00 – 1.90 (m, 2H), 2.11 – 2.02 (m, 2H), 3.52 – 3.45 (m, 1H), 3.83 – 3.74 (m, 1H), 4.60 (t, J
6
(
=
5.2, 1H), 4.66 (qui, J = 7.7, 1H), 6.23 (d, J = 8.1, 1H), 7.39 (d, J = 8.9, 2H), 7.88 (s,1H), 7.98 (d, J =
13
7.7, 2H), 9.52 (s, 1H). C NMR (126 MHz, DMSOꢀd
6
) δ ppm 11.26, 24.24, 24.44, 32.19, 32.31,
54.84, 55.47, 63.32, 113.59, 114.67, 122.35, 131.49, 137.37, 140.41, 152.24, 159.15.
2
6
N ꢀ(4ꢀAminoꢀcyclohexyl)ꢀ9ꢀbenzylꢀN ꢀ(4ꢀbromoꢀphenyl)ꢀ9Hꢀpurineꢀ2,6ꢀdiamine (6h)
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7
8
9
Yield: 82 %. m.p.: 129ꢀ131 °C. Elemental analysis: Calcd. for C24
H
26BrN
7
(492.41): C, 58.54; H, 5.32;
1
N, 19.91. Found: C, 58.66; H, 5.39 N, 19.82. HPLCꢀMS (ESI+): 493.5 (98.1%). H NMR (500 MHz,
DMSOꢀd ) δ ppm 1.15ꢀ1.26 (m, 4H), 1.80 (d, J=9.0, 2H), 1.92 (d, J=9.0, 2H), 2.57ꢀ2.63 (m, 1H),
6
3
.55ꢀ3.62 (m, 1H), 5.19 (s, 2H), 6.60 (s(br), 1H), 7.23ꢀ7.32 (m, 5H), 7.39 (d, J=8.0, 2H), 7.94 (s, 1H),
.00 (d, J=8.0, 2H), 9.67 (s(br), 1H).
1
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8
9
0
8
1ꢀ[9ꢀBenzylꢀ6ꢀ(4ꢀbromoꢀphenylamino)ꢀ9Hꢀpurinꢀ2ꢀylamino]ꢀpropanꢀ2ꢀol (6i)
Yield: 96 % m.p.: 148ꢀ149 °C. Elemental analysis: Calcd. for C21
H
21BrN
6
O (453.34): C, 55.64; H,
1
4.67; N, 18.54. Found: C, 55.41; H, 5.02 N, 18.20. HPLCꢀMS (ESI+): 454.5 (97.8%). H NMR (300
6
MHz, DMSOꢀd ) δ ppm 1.08 (d, J=6.21, 3H), 3.13ꢀ3.22 (m, 2H), 3.81 (sep, J=5.37, 1H), 4.67 (d,
J=5.26, 1H), 5.24 (s, 2H), 6.61 (t, J=5.46, 1H), 7.24ꢀ7.44 (m, 5H), 7.42 (d, J=8.91, 2H), 7.98 (s, 1H),
1
3
8
.01 (d, J=8.91, 2H), 9.64 (s(br), 1H). C NMR (126 MHz, DMSOꢀd
6
) δ ppm 21.92, 46.27, 49.84,
65.80, 113.74, 114.21, 122.48, 128.16, 129.15, 131.49, 137.89, 138.74, 140.33, 152.30, 159.69.
(
9ꢀBenzylꢀ2ꢀmorpholinꢀ4ꢀylꢀ9Hꢀpurinꢀ6ꢀyl)ꢀ(4ꢀbromoꢀphenyl)ꢀamine (6j)
Yield: 87 % m.p.: 171ꢀ172 °C. Elemental analysis: Calcd. for C22
H
21BrN
6
O (465.36): C, 56.78; H,
1
4.55; N, 18.06. Found: C, 56.53; H, 4.71 N, 17.83. HPLCꢀMS (ESI+): 466.5 (97.6%). H NMR (300
MHz, DMSOꢀd
6
) δ ppm 3.63ꢀ3.68 (m, 8H), 5.28 (s, 2H), 7.28ꢀ7.36 (m, 5H), 7.47 (d, J=8.82, 2H), 7.84
13
(
d, J=8.82, 2H), 8.07 (s, 1H), 9.79 (s, 1H). C NMR (76 MHz, DMSOꢀd
6
) δ ppm 44.77, 45.77, 65.94,
113.52, 113.76, 122.22, 127.61, 127.71, 128.56, 131.01, 137.16, 139.02, 139.22, 151.33, 158.44.
[
9ꢀBenzylꢀ2ꢀ(4ꢀmethylꢀpiperazinꢀ1ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀyl]ꢀ(4ꢀbromoꢀphenyl)ꢀamine (6k)
Yield: 88 % m.p.: 162ꢀ164 °C. Elemental analysis: Calcd. for C H BrN (480.40): C, 57.50; H, 5.46;
23
26
7
1
N, 20.41. Found: C, 57.38; H, 5.14 N, 20.11. HPLCꢀMS (ESI+): 481.8 (96.2%). H NMR (300 MHz,
DMSOꢀd
6
) δ ppm 2.17 (s, 3H), 2.32ꢀ2.36 (m, 4H), 3.69ꢀ3.72 (m, 4H), 5.26 (s, 2H), 7.26ꢀ7.34 (m, 5H),
13
7.47 (d, J=8.79, 2H), 7.85 (d, J=8.79, 2H), 8.03 (s, 1H), 9.76 (s (br), 1H). C NMR (76 MHz,
DMSOꢀd
6
) δ ppm 44.16, 45.78, 54.37, 113.45, 113.54, 122.11, 127.57, 127.69, 128.52, 130.98,
137.16, 138.80, 139.30, 151.31, 151.74, 158.35.
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