Synthesis and evaluation of 18F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

Article abstract of DOI:10.1016/j.ejmech.2014.09.019

Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the respo

Full text of DOI:10.1016/j.ejmech.2014.09.019

European Journal of Medicinal Chemistry 86 (2014) 769e781
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
18
Synthesis and evaluation of F-labeled ATP competitive inhibitors of
topoisomerase II as probes for imaging topoisomerase II expression
Pierre Daumar, Brian M. Zeglis, Nicholas Ramos, Vadim Divilov, Kuntal Kumar Sevak,
NagaVaraKishore Pillarsetty*, Jason S. Lewis*
Department of Radiology and the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription,
replication, and chromosome segregation processes and, as such, represents an attractive target for
cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is
highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay
to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders.
With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET)
imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the
structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of
Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and
MCF-7 cell lines. Based on these results, 18F-labeled analogs of these two compounds were synthesized
and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts.
Received 21 July 2014
Received in revised form
2 September 2014
Accepted 6 September 2014
Available online 8 September 2014
Keywords:
Topoisomerase II
Catalytic inhibitors
Purine derivatives
Cytotoxicity
[
18F]-18 and [18F]-19b were synthesized from their corresponding protected tosylated derivatives by
PET imaging
18F-labeling
fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both com-
pounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool
very rapidly and getting metabolized over. The insights gained from the current study will surely aid in
the design and construction of future generations of PET agents for the non-invasive delineation of Topo-
II expression.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
segregation processes, and its accurate functioning is a prereq-
uisite for the development of normal mitosis [1e3]. In mammals,
Type II topoisomerase (Topo-II) is an ATPase that unravels
DNA supercoiling in a very precise fashion through transient
double-strand DNA cleavage followed by re-ligation. The enzyme
is essential in the transcription, replication, and chromosome
two isoforms of Topo-II exist: Topo-IIa (170 kDa) and Topo-IIb
(180 kDa) [4]. Despite a high degree of homology, recent studies
have suggested that the two isoforms might play different roles
in cellular processes and are regulated very differently. Topo-II
a
is expressed in proliferating cells only. This isoform over-
expression, a specific and sensitive marker for cell proliferation,
has been observed in many types of cancer [5,6] and presents an
attractive target for cancer therapy. Topo-IIb, on the other hand,
is expressed at lower levels at steady state levels during all stages
of cell cycle.
Overall, inhibitors of Topo-II can be divided into two broad
classes: poisons and catalytic inhibitors. Members of the first
class interfere with Topo-II function by binding to and stabilizing
the DNAeTopo-II complex, ultimately promoting the formation
of extremely toxic double-strand breaks. The discovery of topo-
isomerase poisons has been tapped effectively in the develop-
ment of several clinically successful chemotherapeutics [4,7e9].
Abbreviations: DAST, diethylaminosulfur trifluoride; DCM, dichloromethane;
DHP, dihydroyrane; DMF, dimethylformamide; DMSO, dimethylsulfoxide; ESI,
electrospray ionization; GI, gastrointestinal; MS, mass spectrometry; NMR, nuclear
magnetic resonance; PET, positron emission tomography; p.i, post-injection; PTSA,
para-toluenesulfonic acid; QAP1, quinoline aminopurine 1; RP-HPLC, reversed
phase high-performance liquid chromatography; RT, room temperature; SARs,
structureeactivity relationships; SNAr, aromatic nucleophilic substitution; TBAF,
tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid; THF, tetrahydrofurane;
THP, tetrahydropyrane; TLC, thin layer chromatography; Topo-II, type II topo-
isomerase; UV, ultra-violet.
* Corresponding authors. Department of Radiology, Memorial Sloan-Kettering
Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
E-mail addresses: pillarsn@mskcc.org (N. Pillarsetty), lewisj2@mskcc.org
(J.S. Lewis).
http://dx.doi.org/10.1016/j.ejmech.2014.09.019
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
Table 1
Anthracyclines, for example, represent one of the most successful
classes of topoisomerase poisons and are widely used in the
treatment of breast cancer. Catalytic inhibitors of Topo-II, on the
other hand, are a heterogeneous group of compounds with
antineoplastic activity that act on various steps in the catalytic
cycle [10]. These compounds might interfere with the binding of
Topo-II to DNA, stabilize non-covalent DNAetopoisomerase II
complexes, or prevent the catalytic turnover by inhibiting ATP
binding [10,11]. The currently used Topo-II-targeted anticancer
Structures of fluorinated purine derivatives.
drugs are not specific to an isoform and inhibit both Topo-II
Topo-II
a and
b
.
While inhibitors of Topo-II d both poisons and catalytic in-
hibitors d have proven to be effective anti-neoplastic drugs,
several independent studies have established that the sensitivity of
tumors to Topo-II inhibitors is highly dependent on both the
expression and activity levels of the enzyme, and that lower Topo-II
levels in tumors minimizes drug efficacy [5,12e17]. Currently,
however, this information can be obtained only through biopsies.
Therefore, it follows that there is an urgent need to develop non-
invasive imaging methods that can provide this vital information
without the risk, discomfort, and unreliability of invasive pro-
cedures. Indeed, in this regard, positron emission tomography
(PET) represents an extremely attractive route. Over the past
twenty years, PET and single photon emission computer tomogra-
phy (SPECT) have revolutionized the field of diagnostic imaging by
allowing clinicians to visualize biomarkers that provide functional
information about tumor biology. In the case at hand, a PET
radiotracer capable of the non-invasive delineation of the levels of
Topo-II expression or activity in a tumor would be a powerful tool
as a prognostic indicator of response to the Topo-II inhibitor
therapy.
We have been interested in evaluating the efficacy of topo-
isomerase II targeted probes as non-invasive markers for imaging
Topo-II expression levels. Previously, we investigated the potential
of 64Cu-radiolabeled thiosemicarbazone complexes as Topo-II
expression probes and provided proof-of-concept that Topo-II-
rich tumors could be visualized via PET using these radiotracers
[18]. To date, this remains the only successful report of the use of
PET agents for Topo-II delineation in tumor. However, the phar-
macokinetic properties of the lipophilic copper complexes were
suboptimal and led to high non-target tissue uptake. Additionally
the exact mechanism of these complexes in targeting the Topo-II
enzyme remains unclear and therefore complicates the interpre-
tation of images.
Compound
R2
R6
R8
18
19b
20
21b
22b
23b
24b
25b
A
B
B
B
C
C
D
E
tBuNHe
tBuNHe
tBuNHe
CYNHe
tBuNHe
H
H
Et
H
H
Et
H
H
tBuNHe
F(CH2)2Oe
purine derivatives as a novel structural class of catalytic topo-
isomerase II inhibitors, Furet et al. recently used a structure-
based approach to rationally design a purine scaffold that acts
as a ATP competitive inhibitor of Topo-II [11,19,20]. Quinoline
aminopurine 1 (QAP1, Fig. 1) and its benzothiazole derivatives
were described as first representatives of this new class of ATP
competitive inhibitors. QAP1 was reported to have micromolar
affinity for the enzyme with a fairly good selectivity over various
protein kinases, and represents a promising starting point for
lead optimization campaigns and further drug discovery efforts
[20]. QAP1 inhibits topoisomerase ATPase activity as well as the
decatenation reaction and targets both alpha and beta isoforms
in cell-free assays. The ability of QAP1 to antagonize doxorubicin
induced DNA damage and aberrant chromosome segregation
further provides evidence that QAP1 is a catalytic inhibitor of
Topo-II rather than a poison. [20,21]
The aim of this work is to identify fluorinated purine analogues
that function as potent Topo-II inhibitors and have potential to be
developed as radiotracers for the non-invasive assessment of Topo-
More recently, as part of our continuing efforts toward the
noninvasive delineation of Topo-II expression in vivo, we
reasoned that a radiolabeled agent that is rationally designed to
specifically bind to the ATP-binding pocket of Topo-II has po-
tential to provide a direct measurement of Topo-II expression
levels in the tumor. Following the identification of substituted
IIa overexpression levels in the tumors. In addition, these probes
have the potential to provide information on the pharmacokinetics
and tissue biodistribution of these new Topo-II catalytic inhibitors.
Herein, we describe the radiosynthesis and in vitro studies on
fluorinated purine derivatives as Topo-II ATP competitive inhibitors
and their in vivo evaluation as PET tracers for imaging Topo-II
expression.
2. Results and discussion
2.1. Design of the compounds
The 2,6-diaminopurine moiety is critical for optimal binding of
the molecule to the ATP binding site of Topo-II, whereas the aryl
moiety sits outside the binding pocket and is amenable for modi-
fication [11]. Therefore this aryl group was chosen for derivatization
and installation of fluorine substituent [20]. The aryl group is ideal
for substitution because it does not disrupt the binding pocket in-
teractions and is oriented away from important amino acid residues
Fig. 1. The structure of QAP1.

P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
771
of the enzyme. As a consequence, all compounds (with the excep-
tion of compound 25b) were designed to follow these salient
design features (Table 1). Another strategy for maximizing favor-
able interactions between the inhibitor and its target was used in
the case of compound 25b, as we introduced a hydrophobic fluo-
roethoxy group in position 6 of the purine ring in order to make
favorable hydrophobic contact with various residues and occupy a
hydrophobic pocket. It was also important to anticipate the feasi-
bility of 18F labeling and to consider synthetic routes amenable to
incorporation of a suitable precursor for radiolabeling, which is
developed in the radiochemistry section (vide infra).
Scheme 2. Synthesis of 2-chloropurine derivatives 2a, 2b, 3 and 4. Reagents and
conditions: (i) 3,4-DHP, PTSA cat., EtOAc, 3.5 h, 50 ꢀC; (ii) t-BuNH2 (compound 2a) or
cyclohexylamine (compound 3), EtOH, 2e4 h, reflux; (iii) fluoroethanol, NaH, THF, 4 h,
60 ꢀC; TFA/MeOH, 12 h, RT.
2.2. Chemical synthesis
Our synthetic strategy involved utilization of C-6 functionalized
2-chloropurines as key intermediates and
a synthetic route
wherein these derivatives were obtained first and then coupled to
the appropriate aryl- or heteroarylamine at a late stage was
preferred. Two methods were used for the formation of the CeN
bond linking the purine ring and the aryl/heteroaryl moiety
(Scheme 1): reaction of an N-9 protected 2-chloropurine (2a, 3, 4 or
6b) with an aryl-/heteroarylamine in BuchwaldeHartwig Pd-
catalyzed coupling conditions (method A) or from a non-protected
purine (2b, 6a) and an aryl-/heteroarylamine via an acid-
catalyzed SNAr chlorine displacement (method B). Both methods
were adapted from previously reported procedures [11,22]. A syn-
thetic approach in which no protection and deprotection steps are
required might seem more attractive, but 6-aminoquinolines and
the 2-amino-6-fluoropyridine proved either unreactive or unstable
in harsh acidic conditions at high temperature. In the end, both
methods were tried for all compounds but compound 18, which
was obtained in a satisfactory 73% yield using method B. For all
other compounds, the highest-yielding procedure d method A in
most cases d is reported. The BuchwaldeHartwig coupling step
provided the products, but the yields were inconsistent. Removal of
the THP protective group was achieved using trifluoroacetic acid in
methanol.
The synthesis of 2-chloropurine derivatives was accomplished
as follows: 2,6-dichloropurine 1a is commercially available and
slightly modified reported methods for N-9 protection and selec-
tive C-6 functionalization led us to compounds 2a and 2b, 3 and 4
(Scheme 2) [22e24]. Notably, the order in which the two steps were
performed had a limited impact on the two-step overall yield. In
the case of compound 2b however, we found that a three-step
procedure (protection, functionalization, and deprotection) was
more favorable than the one-step introduction of the tert-butyla-
mino group in position 6 from 2,6-dichloropurine. On the other
hand, access to a C-6 functionalized 2-chloropurine with a small
alkyl group in position 8 requires more complicated preparation
procedures where the purine ring is built from barbituric acid de-
rivative via the intermediate 2,4-dichloro-5,6-diaminopyrimidine 5
[20]. The ring closure step which led to compound 6a from 5 was
accomplished in a moderate yield (44%, Scheme 3). It is important
Scheme 3. Synthesis of 2-chloropurine derivatives 6a and 6b. Reagents and condi-
tions: (i) EtC(OEt)3, 1 h, MW 160 ꢀC; (ii) t-BuNH2, NMP, 2 h, MW 160 ꢀC; (iii) 3,4-DHP,
PTSA cat., EtOAc, 6 h, 60 ꢀC.
to note that while the introduction of a THP group on 2,6-
dichloropurine proceeded in high yield (90%), the presence of an
ethyl group in position 8 affected the product yield in the synthesis
of compound 6b (67%).
Apart from commercially available 2-amino-6-fluoropyridine
and 6-aminobenzothiazole, all aryl- or heteroarylamines were ob-
tained in multi-step syntheses. Aniline 9 was obtained in two steps
starting from commercially available compound 7 (Scheme 4):
following a DAST/CH2Cl2 fluorination step, the nitro group was
reduced to the desired compound in near quantitative yield using
Fe/AcOH. The synthesis of 6-aminobenzothiazole 12 was accom-
plished in two steps from compound 10 in accordance with Scheme
5. The intermediate 10 was obtained after regioselective nitration of
2-chlorobenzothiazole according to previous report [25]. Subse-
quent SNAr displacement with in situ generated fluoroethanoate
and reduction of the nitro group led to desired compound 12 in a
good three-step overall yield. The synthesis of 6-aminoquinolines
16 and 17 (Scheme 6) began with chloroquinoline 13, an interme-
diate that was synthesized in two steps according to the literature
[26]. Reaction of compound 13 with fluoroethanol and 2-
morpholinoethanol afforded nitro compounds 14 and 15, respec-
tively. The nitro group was subsequently reduced to the desired
aminoquinolines 16 and 17 using Fe/AcOH.
Full synthetic details and characterization data are given in the
experimental section (vide infra). All intermediates and final purine
derivatives were purified via flash column chromatography and
identified and characterized by 1H NMR, 13C NMR, and ESIeMS.
Scheme 1. Reaction scheme for purine compounds 18, 20, 19b, 21b-25b and QAP 1.
Reagents and conditions: (i) Cs2CO3, binap, Pd(OAc)2, 45 min, MW 160 ꢀC, N-9 pro-
tected purine; (ii) TFA, MeOH, 1e3 h, RT (Method A) or (iii) HCl cat., n-butanol, 24 h,
130 ꢀC, non-protected purine (Method B).
Scheme 4. Synthesis of 4-(2-fluoroethyl)aniline 9. Reagents and conditions: (i) DAST,
DCM, 6 h, 0 ꢀC to RT; (ii) EtOH/H2O/AcOH, Fe powder, 1 h, reflux.

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P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
previously reported results that indicated that QAP1 is less potent
in the cellular context as compared to other catalytic inhibitors [19].
Nevertheless, in addition to the fact that QAP1 targets topoisom-
erase II in cells, the slight differential antiproliferative effect that is
observed on high versus low Topo-IIa expressing cell line suggests
that the inhibition of the enzyme may play a role in the anti-
proliferative effects of this derivative.
Scheme 5. Synthesis of 2-(2-fluoroethoxy)benzo[d]thiazol-6-amine 12. Reagents and
conditions: (i) fluoroethanol, NaH, THF, 2 h, RT; (iii) EtOH/H2O/AcOH, Fe powder, 1 h,
reflux..
Most importantly, the MTT assay reveals that compounds 21b
Scheme 6. Synthesis of 2-(2-fluoroethoxy)quinolin-6-amine 16 and 2-(2-morpholinoethoxy)quinolin-6-amine 17. Reagents and conditions: (i) fluoroethanol (compound 14) or 2-
morpholinoethanol (compound 15), NaH, DMF, 12 h, RT; (iv) H2, Pd/C, THF (compound 16) or DCM/EtOH (1:1) (compound 17), 20 h, RT..
2.3. Topoisomerase IIa inhibition assay
After successful synthesis and isolation of QAP1 and fluorinated
purine derivatives 18, 19b, 20, 21b-25b, we investigated their Topo-
IIa inhibition activity with an agarose gel electrophoresis experi-
ment. Their ability to inhibit the relaxation of a supercoiled DNA
substrate (plasmid pHOT-1) was tested in a qualitative assay, which
was performed using a commercially available kit (TopoGen, Port
Orange, FL). The DNA substrate was incubated with Topo-II
presence of the derivatives in question at a concentration of
100 M. QAP1 and the well-known Topo-II poison etoposide were
a in the
Fig. 2. Agarose gel assay for Topo-II
19b, 20, 21be25b.
a inhibition in the presence of 100 mM QAP1, 18,
m
used as positive controls, and pHOT-1 DNA and DMSO vehicle were
used as negative controls.
Table 2
Topo-II
This experiment indicated that compounds 21b and 24b are not
a inhibition activity and antiproliferative activity (GI50/mM) of the fluori-
nated purine derivatives and QAP1.
active towards Topo-II
fluorinated compounds were found to be active Topo-II
at 100 M. Indeed, the absence of relaxed DNA in lanes 21b and 24b
a
inhibition (Fig. 2). In contrast, all other
a
inhibitors
Compound
Topo-II
inhibition/
100
a
SK-BR-3 cells
GI50
MCF-7 cells
GI50
m
/
m
M
R2
/
m
M
R2
reveals that the enzyme is not inhibited as opposed to other lanes
and positive controls in which supercoiled DNA, the initial sub-
strate, is present. A wider range of fluorinated compounds would be
necessary to discern strong structureeactivity relationships (SARs)
and possibly discover new important structural features, leading
the way for optimizing the purine-based reported model. Now, in
any case, given the relatively high tested concentration, it seems
reasonable to conclude that neither an aminopyridyl group in po-
sition 2 of the purine scaffold nor an aminocyclohexyl group in
m
M
18
19b
20
þ
þ
þ
ꢁ
þ
þ
ꢁ
þ
þ
6.8 0.8
8.0 0.8
13.6 0.9
>100
2.0 0.9
7.0 0.9
>100
0.975
0.936
0.954
N/Aa
0.988
0.981
N/A
14.5 0.5
46.3 0.9
4.9 0.3
>100
13.6 0.7
3.2 0.7
>100
0.903
0.958
0.966
N/A
0.954
0.966
N/A
21b
22b
23b
24b
25b
QAP1
11.4 0.8
10.2 0.9
0.965
0.977
>100
N/A
0.961
32
2
position 6 allows for efficient Topo-IIa inhibition.
a
Not applicable.
2.4. Cytotoxicity assay
and 24b d both inactive in the Topo-IIa inhibition assays d dis-
QAP1 and fluorinated purine derivatives were tested in vitro to
played no antiproliferative activity in the tested cell lines, even at
concentrations where a general cytotoxic effect is usually observed.
Many cell lineage dependent factors, like the cellular uptake, can
modify the activity of a drug, but the antiproliferative activity
observed in our MTT assay relates relatively well with the in vitro
Topo-II inhibition assay results, which again suggests that Topo-II
inhibition is a possible cause for the antiproliferative effect. The
other fluorinated purines displayed antiproliferative activity at
evaluate their anti-proliferative activities and how the anti-
proliferative activities related to Topo-II
a expression level. This
was accomplished using cytotoxicity assays (MTT) in two breast
cancer cell lines. SK-BR-3 and MCF-7 cell lines, that express high
and low levels (10-fold) of the enzyme, respectively, were chosen as
positive and negative controls, respectively [27]. MTT assays were
performed using 72 h drug incubations. These cell proliferation
assays were aimed at the evaluation of the cytotoxic properties of
our novel purine derivatives towards breast cancer cell lines, and
provide a leading candidate for labeling with fluorine-18 and
evaluate its efficacy in relevant in vivo models. Growth-inhibition
values obtained for both cell lines are compiled in Table 2.
concentrations ranging from 2 to 46 mM, in general with higher
potency than QAP1 in both cell lines. Compound 22b was the most
active towards growth inhibition of SK-BR-3 cells, with a GI50 value
of 2.0 0.9
the ethyl group on position 8, was most active towards growth
inhibition of MCF-7 cells, with a GI50 value of 3.2 0.7 M. With
regard to the in vitro potency, no general trend is observed, but GI50
mM. Compound 23b, which only differs from 22b with
The MTT data indicates that after 72 h incubation, QAP1 has GI50
m
values of 10.2 0.9
mM and 32
2 mM with SK-BR-3 and MCF-7 cell
lines, respectively. These rather high values are in accordance with

P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
773
values are lower in the Topo-II
a
overexpressing SK-BR-3 cells than
in the MCF-7 cells for compounds 18, 19b, 22b and 25b, with a
highly marked difference in some cases. More data would be
necessary to support the hypothesis that Topo-IIa inhibition is the
major mechanism involved in growth inhibition and to discern
further SARs. However, at present, some of the fluorinated com-
pounds reported herein as new purine based Topo-II catalytic in-
hibitors showed a higher potency than the parent QAP1.
2.5. Radiolabeling precursors synthesis and radiochemistry
After demonstrating that the fluorinated purines analogues
were comparable to QAP1 in cell growth inhibition and potency
towards Topo-IIa, our next objective was to use the corresponding
Scheme 8. Synthesis of precursor 34 and radiosynthesis of [18F]-19b. Reagents and
conditions: (i) 2-((triisopropylsilyl)oxy)ethanol, NaH, THF/DMF, 48 h, 90 ꢀC; (ii) 2a,
Cs2CO3, binap, Pd(OAc)2, 45 min, MW 160 ꢀC; (iii) TBAF, THF, 1 h, RT; (iv) TsCl, pyridine,
12 h, RT; (v) K[18F]F/K222/K2CO3, MeCN, 12 min, 115 ꢀC; (vi) TFA, MeOH, MeCN, 10 min,
RT.
18F-labeled derivatives for in vivo studies. Our ultimate goal is the
design and development of PET imaging agents to provide infor-
mation on Topo-II expression level in vivo non-invasively. Further,
to date, no information exists in the published scientific literature
on the pharmacokinetic and tissue distribution of QAP1 or other
members of this promising new class of purine based Topo-II in-
hibitors in murine models. PET imaging gives access to such
knowledge, which will be critical in the further development and
potential clinical applications as imaging agents of our fluorinated
purines.
Based on the biological assay results discussed above, two
compounds d 18 and 19b d were chosen for radiolabeling with
18F. The feasibility of both the synthesis of a suitable radiolabeling
precursor and the radiolabeling reaction itself were also taken into
account for the selection of these two compounds. Indeed, we
initially hoped to 18F-radiolabel the active, quinoline-containing
compound 22b to complete our studies, but the lack of access to
a precursor that can be directly radiolabeled hindered our efforts,
and this idea was not pursued further.
Typically, a one-step synthesis is the ideal scenario for radio-
chemical synthesis with short-lived isotopes, as it reduces the
overall synthesis time and allows for higher real overall (non-decay
corrected) yields. However, we anticipated that protecting the free
amine in position 9 of the purine scaffold was necessary with our
compounds. Our efforts to synthesize [18F]-18 from a precursor that
was not protected at position-9 proved unsuccessful (data not
shown), strongly supporting the importance of protecting group.
Therefore, the need for a suitable fluorination precursor led us to
design new synthetic routes to tosylated compounds 29 and 34
(Schemes 7 and 8). Tosylate is one of the most common precursors
used for 18F labeling of organic molecules as it provides a good
balance between the stability and its affinity to undergo nucleo-
philic substitution, which is also the reason why we chose to
introduce the tosyl group at a late stage of the syntheses.
Fig. 3. Analytical radio-HPLC chromatograms.
The synthesis of compound 29, the immediate precursor to
[
18F]-18, was accomplished in two steps from commercially avail-
able aniline 27. First, conditions B (see Chemical Synthesis) were
used for the Pd-catalyzed coupling step with purine 2a. Subsequent
tosylation of intermediate 27 with TsCl/Et3N led to the desired
compound in a 63% two-step overall yield.
The synthesis of the radiolabeling precursor for [18F]-19b was
less straightforward, as it was found that the hydroxyl group that
was to be tosylated had to be introduced and appropriately pro-
tected early in the synthetic route. The synthesis of 6-
aminobenzothiazole 31 was accomplished via SNAr displacement
with 2-((triisopropylsilyl)oxy)ethanoate generated in situ. Subse-
quent coupling with purine 2a and the removal of the TIPS pro-
tecting group led to intermediate 33 in a 35% three-step overall
yield. Before TIPS was identified as a suitable protecting group,
other synthetic strategies involving protection with Bz, or TBDPS
groups were attempted with little success, the failures occurring
either at the coupling or at the deprotection stage. The final tosy-
lation of compound 33 with TsCl/pyridine gave precursor 34 in a
68% yield. Both precursors, like all other intermediates, were
identified and characterized by 1H NMR, 13C NMR, and ESIeMS, as
detailed in the experimental section.
Scheme 7. Synthesis of precursor 29 and radiosynthesis of [18F]-18. Reagents and
conditions: (i) 2a, Cs2CO3, binap, Pd(OAc)2, 45 min, MW 160 ꢀC; (ii) TsCl, Et3N, DCM,
72 h, RT; (iii) K[18F]F/K222/K2CO3, MeCN, 12 min, 115 ꢀC; (iv) 1 N HCl in MeOH, MeCN,
10 min, 115 ꢀC.

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P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
The 18F radiosyntheses proceeded from 29 or 34 in one-pot,
to improve their in vivo behavior and lead to better uptake in Topo-
II-expressing tumors.
two-step reactions to [18F]-18 and [18F]-19b, respectively. First,
precursors were treated with [18F]-KF/Kryptofix 2.2.2 in CH3CN at
110 ꢀC to produce the N9-THP protected fluorinated intermediates.
Subsequently, the 18F-labeled purines were converted to the
desired compounds by removal of the THP protecting groups.
Notably, acidic conditions used for the synthesis of [18F]-18 (HCl in
MeOH at RT) were too harsh and could not be used for the suc-
cessful synthesis of [18F]-19b. In the latter case, a mixture of MeOH
and TFA (50% v/v) at RT were later found to be suitable conditions.
Radiosyntheses were carried out in just over 2 h including puri-
fication and formulation in saline for injection. Both compounds
were isolated in radiochemical purity greater than 98% (Fig. 3)
with dcRCY of 26% ([18F]-18) and 6% ([18F]-19b). As both radio-
labeled purines were synthesized for preliminary in vivo studies,
RCYs were not optimized and specific activities were not deter-
mined. Notably, these two-step radiolabelings were remarkably
clean, as very minor (if any) fluorinated side-products were
formed in both steps.
3. Conclusion
Herein, we have presented the synthesis of ATP competitive
Topo-II inhibitor QAP1 and its fluorinated derivatives. Structural
modifications were well tolerated, as most of the derivatives
retained fairly high potency toward Topo-II inhibition in vitro and
showed similar antiproliferative activity to QAP1 in two breast
cancer cell lines. Some SARs were found, and the library of purine
based Topo-II catalytic inhibitors was broadened. The successful
radiosynthesis of two 18F-labeled purine derivatives is also re-
ported. Both agents were isolated in high radiochemical purity;
however, in vivo PET imaging experiments revealed suboptimal
biodistribution profiles. It is clear from our in vivo studies that
improvements of the pharmacokinetic profile of the QAP1 analogs
is needed to make them suitable PET imaging agents. Additionally,
the pharmacokinetic data also indicates that the compounds might
fare poorly in the in vivo tumor regression studies, because of
suboptimal bioavailability resulting from rapid clearance. Indeed,
the question remains whether purine based derivatives could be
efficient imaging agents for the delineation of high Topo-II
expressing tumors, but further pharmacomodulation studies are
currently underway. It is our hope that the work presented here
will encourage further drug discovery efforts and provide useful
information towards the identification of Topo-II imaging agents
and the development of much-needed novel catalytic inhibitors.
2.6. Small animal PET imaging
Following successful radiosyntheses of [18F]-18 and [18F]-19b,
small animal PET imaging experiments were performed in mouse
bearing SK-BR-3 xenografts to evaluate the in vivo pharmacokinetic
behavior of the two radiotracers. [18F]-18 or [18F]-19b in 150
mL
saline was administered to athymic nude mice (n ¼ 4) bearing SK-
BR-3 xenografts on the right shoulder via tail vein injection. The
animals were subsequently imaged at 30 min, 1, 2 and 4 h post
administration. PET images (showed for [18F]-18 only, Fig. 4) clearly
indicate a very fast clearance from blood circulation with excretion
mainly into the liver and the gastrointestinal (GI) tract and also into
the bladder. Subsequently, we observe as soon as 1 h p.i. that strong
activity remains principally in the GI tract, revealing a rapid hepatic
clearance. However, intense focal uptake in the GI tract regions
suggests activity accumulation in the gallbladder that is visible at
early time point and remain the most prominent feature at later
time points. Notably, there was some uptake observed in bone at
late time points, suggesting that our tracers did undergo a slow
metabolic defluorination. A similar biodistribution profile was
observed for the two tracers starting from 30 min p.i. Unfortu-
nately, we observed that the tumor uptake was extremely low
which demonstrates a non-favorable pharmacological profile. This
PET study indicates that the pharmacokinetic properties of these
potent QAP1 analogs render them unsuitable as imaging agents.
Indeed, significant modifications to their design will be necessary
4. Experimental section
4.1. General chemistry
All chemicals and solvents were obtained from SigmaeAldrich
Co., Fisher Scientific or TCI America and, unless stated otherwise,
were used as received without further purification. All DMSO used
for biological assays was of molecular biology grade (>99.9%;
Sigma, D8418). Water (>18.2 MU
cmꢁ1 at 25 ꢀC) was obtained from
an Alpha-Q Ultrapure water system (Millipore). All MTT assay kit
materials were purchased from American Tissue Culture Collection
(ATCC, Manassas, VA, USA). All Topo-IIa inhibition assay materials
were purchased from TopoGEN, Inc. (Port Orange, FL, USA). All in-
struments were calibrated and maintained in accordance with
standard quality-control procedures. 1H and 13C NMR spectra were
recorded on Bruker instruments (500 MHz or 600 MHz). Chemical
shifts (d) are determined relative to CDCl3 (referenced to 7.26 ppm
for 1H NMR and 77.16 ppm for 13C NMR) or DMSO-d6 (referenced to
2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR). Coupling
constants (J) are given in hertz and spectral splitting patterns are
designated as singlet (s), doublet (d), triplet (t), quadruplet (q),
multiplet or overlapped (m), doublet of doublets (dd), and broad
(br). Low resolution mass spectra (LRMS) were acquired on a Wa-
ters Acquity Ultra Performance LC with electrospray ionization and
SQ detector (ESI). No MS data is given for compounds 1b, 4,11,14,15
and 17, which did not respond in either negative or positive ioni-
zation mode. Thin-layer chromatography (TLC) was performed on
E. Merck (Darmstadt, Germany) silica gel 60 F-254 aluminum-
backed plates with visualization under UV (254 nm). Flash chro-
matography was performed using an Isco (Lincoln, NE) Combiflash
Companion with RediSep Rf Gold Silica™ columns and Fisher Op-
tima™ grade solvents. Microwave reactions were performed in a
Biotage® Initiator microwave reaction system. All compounds
employed were >98% pure, as determined by 1H NMR and/or
analytical RP-HPLC.
Fig. 4. Representative coronal PET images (Maximum Intensity Projection) of SCID
mice bearing SK-BR-3 xenografts on the right shoulder. Images A, B and C are the
results of 15-min static scans following iv injection of the mouse with [18F]-18. Images
A, B and C were obtained at 30, 60 and 240 min after injection, respectively.

P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
775
4.2. Chemistry
4.2.5. 2-Chloro-6-(2-fluoroethoxy)-9-(tetrahydro-2H-pyran-2-yl)-
9H-purine 4
To a solution of fluoroethanol (117
4.2.1. 2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine 1b
To a solution of 2,6-dichloropurine 1a (2 g, 10.58 mmol) and
PTSA (200 mg, 0.11 mmol) in EtOAc (35 mL) at RT was added 3,4-
DHP (2.4 mL, 26.45 mmol). The mixture was stirred at 50 ꢀC for
3.5 h. After cooling to RT, saturated aqueous NaHCO3 (35 mL) was
added and layers were separated. The aqueous layer was extracted
with EtOAc (2 ꢂ 30 mL). The combined organic layers were dried
over MgSO4 and concentrated under vacuum. The oily residue was
triturated with Et2O (20 mL) and subsequent filtration gave com-
pound 1b (2.60 g, 90%) as a white solid. 1H NMR (500 MHz, CDCl3)
mL, 2.02 mmol) in dry THF
(20 mL) at 0 ꢀC under argon was added a 60% dispersion of NaH in
mineral oil (88 mg, 2.21 mmol). The mixture was allowed to react
for 30 min at 0 ꢀC before a solution of compound 1b (500 mg,
1.84 mmol) in dry THF (4 mL) was added. The reaction mixture was
stirred at 60 ꢀC for 4 h. After cooling to RT, water (25 mL) and EtOAc
(25 mL) were added and layers were separated. The aqueous layer
was extracted with EtOAc (2 ꢂ 25 mL). The combined organic layers
were washed with brine (20 mL), dried over MgSO4 and concen-
trated under vacuum to an oily residue. Purification by gradient
flash chromatography (SiO2, Hexane/EtOAc) yielded compound 4
d
8.33 (s, 1H), 5.76 (dd, J ¼ 2.4, 10.8 Hz, 1H), 4.19 (dt, J ¼ 2.1, 11.9 Hz,
1H), 3.78 (td, J ¼ 2.6, 11.7 Hz, 1H), 2.22e2.14 (m, 1H), 2.13e2.06 (m,
1H), 1.97 (qd, J ¼ 3.9, 12.4 Hz, 1H), 1.87e1.71 (m, 2H), 1.71e1.65 (m,
(500 mg, 90%) as a colorless oil. 1H NMR (500 MHz, CDCl3)
d 8.15 (s,
1H), 5.74 (dd, J ¼ 2.1, 10.7 Hz, 1H), 4.96e4.75 (m, 4H), 4.20e4.14 (m,
1H), 3.78 (dd, J ¼ 2.4, 11.7 Hz, 1H), 2.17e2.05 (m, 2H), 2.02e1.93 (m,
1H), 1.84e1.71 (m, 2H), 1.69e1.62 (m, 1H). 13C NMR (126 MHz,
1H). 13C NMR (126 MHz, CDCl3)
d 153.08, 152.29, 151.87, 143.81,
130.91, 82.62, 69.07, 32.17, 24.84, 22.64.
CDCl3)
d 160.62, 152.94, 152.77, 140.82, 120.35, 82.12, 81.10 (d,
2
1JCeF ¼ 171.5 Hz), 68.97, 66.81 (d, JCeF ¼ 20.6 Hz), 32.22, 24.93,
4.2.2. N-(tert-butyl)-2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-amine 2a
22.78.
A solution of compound 1b (500 mg, 1.83 mmol) and t-BuNH2
(1.93 mL, 18.32 mmol) in EtOH (10 mL) was refluxed for 4 h. After
cooling to RT, the solvent was stripped under vacuum and saturated
aqueous NaHCO3 (60 mL) was added. The aqueous mixture was
extracted with EtOAc (3 ꢂ 30 mL). The combined organic layers
were dried over MgSO4 and concentrated under vacuum. Purifica-
tion by gradient flash chromatography (SiO2, Hexane/EtOAc) yiel-
ded compound 2a (483 mg, 85%) as a colorless oil. 1H NMR
4.2.6. N-(tert-butyl)-2-chloro-8-ethyl-9H-purin-6-amine 6a
This compound was synthesized in two steps starting from 2,6-
dichloropyrimidine-4,5-diamine 5 (Axon MedChem BV, Groningen,
Netherlands), according to the procedure described by Baenteli R.
et al. [22]. Briefly, a sealed microwave vial was charged with a so-
lution of 5 (400 mg, 2.25 mmol) in EtC(OEt)3 (8 mL) and heated at
160 ꢀC for 1 h. After cooling to RT, Et2O (10 mL) was added and the
mixture was placed in an ice bath for 1 h. The intermediate product
was filtered off and rinsed with ice-cold Et2O (10 mL). The solid was
added to a sealed microwave vial charged with a mixture of NMP
and t-BuNH2 (10 mL; 1/4 v:v), and the mixture was heated at 160 ꢀC
for 2 h. After cooling to RT, the reaction mixture was partitioned
between a saturated NaHCO3 aqueous solution (10 mL) and EtOAc
(10 mL). Layers were separated and the aqueous layer was extracted
with EtOAc (2 ꢂ 20 mL). The combined organic layers were washed
with brine (10 mL), dried over MgSO4 and concentrated under
vacuum to an oily residue. Purification by flash chromatography
(SiO2, DCM/MeOH, 98/2) yielded compound 6a (250 mg, 44%) as a
(500 MHz, CDCl3)
d
7.90 (s, 1H), 5.82 (s, 1H), 5.67 (dd, J ¼ 2.2, 10.
8 Hz, 1H), 4.19e4.12 (m, 1H), 3.76 (td, J ¼ 2.3, 11.6 Hz, 1H), 2.14e2.06
(m, 1H), 2.03e2.00 (m, 1H), 1.92 (qd, J ¼ 3.7, 12.3 Hz, 1H), 1.82e1.67
(m, 2H), 1.66e1.59 (m, 1H), 1.55 (s, 9H). 13C NMR (126 MHz, CDCl3)
d
154.93, 154.07, 148.98, 137.44, 118.94, 81.57, 68.87, 52.86, 32.35,
29.09, 24.99, 22.85. ESIeMS m/z 310.23, 312.22 [MþH]þ, 332.07,
334.08 [MþNa]þ.
4.2.3. N-(tert-butyl)-2-chloro-9H-purin-6-amine 2b
Compound 2a (122 mg, 0.39 mmol) was stirred at RT for 12 h in
a MeOH/TFA (3/1) mixture (20 mL). When no more starting ma-
terial was detected on TLC, the mixture was concentrated under
vacuum. The residue was partitioned between a saturated NaHCO3
aqueous solution and EtOAc. Layers were separated and the
aqueous layer was extracted with EtOAc (2e3ꢂ). The combined
organic layers were washed with brine (20 mL), dried over MgSO4
and concentrated under vacuum. Compound 2b (77 mg, 87%) was
isolated after purification by gradient flash chromatography (SiO2,
white solid. 1H NMR (500 MHz, CDCl3)
d 13.13 (bs, 1H), 5.83 (s, 1H),
3.01 (q, J ¼ 7.6 Hz, 2H), 1.57 (s, 9H), 1.44 (t, J ¼ 7.6 Hz, 3H). 13C NMR
(126 MHz, CDCl3)
d 154.28, 151.94, 136.58, 130.09, 118.91, 52.95,
29.23, 23.09, 13.06. ESIeMS m/z 254.24, 256.06 [MþH]þ, 276.05,
278.06 [MþNa]þ.
4.2.7. N-(tert-butyl)-2-chloro-8-ethyl-9-(tetrahydro-2H-pyran-2-
yl)-9H-purin-6-amine 6b
Hexane/EtOAc) as a white solid. 1H NMR (600 MHz, CDCl3)
d
7.90 (s,
155.07,
1H), 6.04 (s, 1H), 1.58 (s, 9H). 13C NMR (151 MHz, CDCl3)
d
To a solution of compound 6a (240 mg, 0.95 mmol) and PTSA
(10 mg, 0.05 mmol) in EtOAc (50 mL) at RT was added 3,4-DHP
153.10, 150.05, 137.99, 118.84, 53.14, 29.11. ESIeMS m/z 226.17,
228.18 [MþH]þ, 248.14, 250.16 [MþNa]þ.
(170 m
L, 1.90 mmol). The mixture was stirred at 60 ꢀC for 6 h. Af-
ter cooling to RT, saturated aqueous NaHCO3 (35 mL) was added
and layers were separated. The aqueous layer was extracted with
EtOAc (2 ꢂ 40 mL). The combined organic layers were dried over
MgSO4 and concentrated under vacuum. Purification by gradient
flash chromatography (SiO2, Hexane/EtOAc) yielded compound 6b
4.2.4. 2-Chloro-N-cyclohexyl-9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-6-amine 3
Compound 3 was synthesized according to the procedure
described for compound 2a using cyclohexylamine (reflux: 2 h).
Compound 3 (231 mg, 94%) was isolated as a colorless oil. 1H NMR
(215 mg, 67%) as a colorless oil. 1H NMR (600 MHz, CDCl3)
d 5.76 (s,
1H), 5.68 (dd, J ¼ 2.5, 11.4 Hz, 1H), 4.18e4.12 (m, 1H), 3.71 (t,
J ¼ 10.9 Hz, 1H), 3.06 (dq, J ¼ 7.5, 15.2 Hz, 1H), 2.94 (dq, J ¼ 7.4,
15.1 Hz, 1H), 2.33 (qd, J ¼ 4.3, 12.1 Hz, 1H), 2.04 (dd, J ¼ 2.7, 8.4 Hz,
1H), 1.89e1.82 (m, 1H), 1.77e1.69 (m, 2H), 1.63e1.58 (m, 1H), 1.54 (s,
(600 MHz, CDCl3)
d
7.92 (s, 1H), 5.77 (s, 1H), 5.67 (dd, J ¼ 6.0, 9.4 Hz,
1H), 4.17e4.09 (m, 2H), 3.80e3.70 (m, 1H), 2.11e2.00 (m, 4H),
1.97e1.86 (m, 1H), 1.80e1.69 (m, 4H), 1.68e1.58 (m, 2H), 1.53e1.40
(m, 2H), 1.32e1.16 (m, 3H). 13C NMR (151 MHz, CDCl3)
d
154.87,
9H), 1.40 (t, J ¼ 7.6 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d 154.18,
154.66, 149.22, 137.73, 118.47, 81.58, 68.94, 49.31, 33.26, 32.38,
153.78, 152.88, 150.25, 117.67, 82.52, 69.12, 52.70, 30.63, 29.15,
25.64, 25.02, 24.88, 22.90. ESIeMS m/z 336.06, 338.20 [MþH]þ.
25.06, 23.24, 22.77, 12.24. ESIeMS m/z 338.20, 340.22 [MþH]þ.

776
P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
4.2.8. 1-(2-Fluoroethyl)-4-nitrobenzene 8
4.2.12. 2-(2-Fluoroethoxy)-6-nitroquinoline 14
To a solution of fluoroethanol (278 L, 4.80 mmol) in dry DMF
To a solution of 2-(4-nitrophenyl)ethanol 7 (800 mg, 4.79 mmol)
m
in DCM (20 mL) under argon at 0 ꢀC was added DAST (940
mL,
(30 mL) at 0 ꢀC under argon was added a 60% dispersion of NaH in
mineral oil (212 mg, 5.28 mmol). The mixture was allowed to react
for 30 min at 0 ꢀC before a solution of compound 13 (500 mg,
2.40 mmol) in dry DMF (10 mL) was added. The reaction mixture
was stirred at RT for 12 h before water (100 mL) was added. The
aqueous mixture was extracted with EtOAc (3 ꢂ 50 mL). The
combined organic layers were washed with brine (60 mL), dried
over MgSO4, and concentrated under vacuum to a brown solid.
Stirring in EtOH at 50 ꢀC and filtration of the warm suspension
afforded compound 14 (391 mg, 69%) as a pure beige solid. 1H NMR
7.18 mmol). The mixture was allowed to warm to RT and stirred
under argon for 7 h. The reaction mixture was partitioned between
a saturated Na2CO3 aqueous solution (60 mL) and EtOAc (120 mL).
The organic layer was washed with water (60 mL) and brine
(60 mL), dried over MgSO4, and concentrated under vacuum. Pu-
rification by gradient flash chromatography (SiO2, Hexane/EtOAc)
yielded compound 8 (466 mg, 58%) as a solid. 1H NMR (500 MHz,
CDCl3)
d
8.19 (d, J ¼ 8.6 Hz, 1H), 7.42 (d, J ¼ 8. 6 Hz, 1H), 4.69 (dt,
J ¼ 6.1, 46.9 Hz, 1H), 3.12 (dt, J ¼ 6.0, 25.7 Hz, 1H). 13C NMR
(126 MHz, CDCl3)
d
147.11, 145.26 (d, 3JCeF ¼ 3.9 Hz), 129.95, 123.88,
(500 MHz, CDCl3)
1H), 8.16 (d, J ¼ 8.8 Hz,1H), 7.90 (d, J ¼ 9.2 Hz,1H), 7.12 (d, J ¼ 8.8 Hz,
1H), 4.95e4.71 (m, 4H). 13C NMR (126 MHz, CDCl3)
163.97, 149.71,
d
8.69 (d, J ¼ 2.5 Hz, 1H), 8.41 (dd, J ¼ 2.5, 9.2 Hz,
83.15 (d, 1JCeF ¼ 170.3 Hz), 36.84 (d, JCeF ¼ 20.3 Hz). ESIeMS m/z
2
192.07 [MþNa]þ.
d
144.11, 140.29, 128.71, 124.26, 124.10, 123.59, 115.57, 81.78 (d,
2
1JCeF ¼ 169.8 Hz), 65.67 (d, JCeF ¼ 20.0 Hz).
4.2.9. 4-(2-Fluoroethyl)aniline 9
Compound 8 (90 mg, 0.53 mmol) was added to a suspension of
10% Pd/C (18 mg) in EtOH (15 mL) and stirred 5 h under hydrogen
atmosphere (1 atm) at RT. The mixture was filtered through Celite®
and evaporated under reduced pressure to yield compound 9
(72 mg, 97%) as a brown oil which was used without further pu-
4.2.13. 4-(2-((6-Nitroquinolin-2-yl)oxy)ethyl)morpholine 15
Compound 15 was synthesized according to the procedure
described for compound 14 using 2-morpholinoethanol. Purifica-
tion of the crude compound by stirring in EtOAc at 50 ꢀC and
filtration of the warm suspension afforded compound 15 (136 mg,
rification. 1H NMR (500 MHz, CDCl3)
d
6.99 (d, J ¼ 8.2 Hz, 1H), 6.60
31%) as a pure orange solid. 1H NMR (500 MHz, CDCl3)
d 8.67 (d,
(d, J ¼ 8.2 Hz, 1H), 4.53 (dt, J ¼ 6.7, 47.2 Hz, 1H), 3.50 (s, 1H), 2.88 (dt,
J ¼ 2.6 Hz, 1H), 8.40 (dd, J ¼ 2.6, 9.0 Hz, 1H), 8.12 (d, J ¼ 8.8 Hz, 1H),
7.89 (d, J ¼ 9.3 Hz,1H), 7.06 (d, J ¼ 8.8 Hz,1H), 4.67 (t, J ¼ 5.8 Hz, 2H),
3.74 (t, J ¼ 4.5 Hz, 4H), 2.87 (t, J ¼ 5.8 Hz, 2H), 2.61 (t, J ¼ 4.5 Hz, 4H).
J ¼ 6.7, 22.3 Hz, 1H). 13C NMR (126 MHz, CDCl3)
d 145.11, 129.83,
126.78 (d, 3JCeF ¼ 7.2 Hz), 115.34, 84.56 (d, 1JCeF ¼ 167.2 Hz), 36.09
(d, 2JCeF ¼ 20.3 Hz). ESIeMS m/z 139.90 [MþH]þ.
13C NMR (126 MHz, CDCl3)
d 164.39, 149.94, 143.93, 139.95, 128.62,
124.23, 123.93, 123.49, 115.76, 67.07, 63.98, 57.53, 54.16.
4.2.10. 2-(2-Fluoroethoxy)-6-nitrobenzo[d]thiazole 11
4.2.14. 2-(2-Fluoroethoxy)quinolin-6-amine 16
To a solution of fluoroethanol (354 mL, 6.11 mmol) in dry THF
Compound 14 (334 mg,1.41 mmol) was added to a suspension of
10% Pd/C (72 mg) in THF (25 mL) and stirred 20 h under hydrogen
atmosphere (1 atm) at RT. The mixture was filtered through Celite®
and concentrated under vacuum. Purification of the residue by
gradient flash chromatography (SiO2, Hexane/EtOAc) yielded
compound 16 (246 mg, 86%) as an orange solid. 1H NMR (500 MHz,
(20 mL) at 0 ꢀC under argon was added a 60% dispersion of NaH in
mineral oil (270 mg, 6.72 mmol). The mixture was allowed to react
for 15 min at 0 ꢀC before a solution of compound 10 (1.31 g,
6.11 mmol) in dry THF (5 mL) was added. The reaction mixture was
stirred at RT for 2 h before dilution with EtOAc (40 mL). The mixture
was washed with water (30 mL), brine (30 mL), dried over MgSO4,
and concentrated under vacuum to a beige solid. Stirring in MeOH
for 30 min at RT and filtration afforded compound 11 (1.33 mg, 90%)
CDCl3)
d
7.79 (d, J ¼ 8.8 Hz, 1H), 7.63 (d, J ¼ 8.8 Hz, 1H), 7.07 (dd,
J ¼ 2.2, 8.8 Hz, 1H), 6.94e6.82 (m, 2H), 4.80 (dt, J ¼ 3.8, 47.6 Hz, 2H),
4.69 (dt, J ¼ 4.2, 29.2 Hz, 2H), 3.78 (s, 2H). 13C NMR (126 MHz,
as a white solid. 1H NMR (500 MHz, DMSO-d6)
1H), 8.25 (dd, J ¼ 2.4, 8.9 Hz, 1H), 7.84 (d, J ¼ 8.9 Hz, 1H), 4.99e4.71
d
8.97 (d, J ¼ 2.3 Hz,
CDCl3)
d 159.68, 142.94, 140.73, 137.43, 128.32, 126.42, 121.06,
113.33, 109.16, 82.35 (d, 1JCeF ¼ 168.8 Hz), 64.69 (d, 2JCeF ¼ 20.0 Hz).
(m, 5H). 13C NMR (126 MHz, DMSO-d6)
d 176.72, 153.59, 143.28,
ESIeMS m/z 205.01 [MꢁH]ꢁ, 207.15 [MþH]þ, 229.16 [MþNa]þ.
132.19, 121.95, 120.72, 119.19, 81.36 (d, 1JCeF ¼ 167.0 Hz), 71.84 (d,
2JCeF ¼ 18.7 Hz).
4.2.15. 2-(2-Morpholinoethoxy)quinolin-6-amine 17
Compound 17 was synthesized according to the procedure
described for compound 16 using a DCM/EtOH (1:1) mixture as
solvent. Compound 17 (81 mg, 67%) was isolated as a brown solid.
4.2.11. 2-(2-Fluoroethoxy)benzo[d]thiazol-6-amine 12
To a solution of compound 11 (1.33 g, 5.49 mmol) in a mixture of
EtOH (25 mL), H2O (4 mL) and AcOH (2.5 mL) at RT was added Fe
powder (1.50 g). The reaction mixture was refluxed for 1 h. After
cooling to RT the mixture was filtered through Celite® and
concentrated under vacuum. The residue was partitioned between
a saturated NaHCO3 aqueous solution (50 mL) and EtOAc (70 mL).
Layers were separated and the aqueous layer was extracted with
EtOAc (2 ꢂ 50 mL). The combined organic layers were washed with
water (50 mL) and brine (50 mL), dried over MgSO4, and concen-
trated under vacuum to a brown solid. Purification by column
chromatography (SiO2, Hexane/EtOAc, 3/1) yielded compound 12
(930 mg, 80%) as an off-white solid. 1H NMR (500 MHz, CDCl3)
1H NMR (500 MHz, CDCl3)
d
7.75 (d, J ¼ 8.8 Hz, 1H), 7.63 (d,
J ¼ 8.8 Hz, 1H), 7.06 (dd, J ¼ 2.7, 8.9 Hz, 1H), 6.87 (d, J ¼ 2.6 Hz, 1H),
6.83 (d, J ¼ 8.8 Hz, 1H), 4.57 (t, J ¼ 5.8 Hz, 2H), 3.79 (bs, 1H), 3.73 (t,
J ¼ 4.6 Hz, 4H), 2.83 (t, J ¼ 5.8 Hz, 2H), 2.59 (t, J ¼ 4.6 Hz, 4H). 13
C
NMR (126 MHz, CDCl3)
d 160.07, 142.79, 140.87, 137.12, 128.25,
126.23, 120.95, 113.45, 109.12, 67.06, 62.86, 57.78, 54.13.
4.3. General synthetic procedures for the synthesis of final
derivatives
Two methods were employed for the synthesis of final de-
rivatives. In cases where a compound was obtained via both
methods, only the higher yielding one was reported. Compounds
19b, 21be25b and QAP1 were obtained according to method A via
THP protected intermediates 19a, 21ae25a and 26, respectively.
Compounds 18 and 20 were obtained according to method B.
d
7.45 (d, J ¼ 8.5 Hz, 1H), 6.94 (d, J ¼ 2. 3 Hz, 1H), 6.73 (dd, J ¼ 2.4,
8.5 Hz, 1H), 4.91e4.68 (m, 4H), 3.68 (s, 2H). 13C NMR (126 MHz,
CDCl3)
d 169.72, 143.25, 141.95, 133.50, 121.45, 114.88, 106.84, 81.39
(d, 1JCeF ¼ 171.0 Hz), 70.04 (d, 2JCeF ¼ 20.2 Hz). ESIeMS m/z 213.08
[MþH]þ, 235.08 [MþNa]þ.

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777
1
4.3.1. Method A (two-step procedure)
121.16, 120.17, 115.53, 113.57, 81.32 (d, JCeF ¼ 171.4 Hz), 70.30 (d,
2JCeF ¼ 20.4 Hz), 52.21, 29.32. ESIeMS m/z 402.02 [MþH]þ.
A microwave vial was charged under argon with a solution of
the appropriate 2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
derivative (2a, 3, 4 or 6b, 1 eq.) in dry degassed dioxane. To this
solution were added Cs2CO3 (5 eq.), BINAP (0.2 eq.), Pd(OAc)2
(0.1 eq.) and the appropriate arylamine (1.2 eq.). The mixture was
heated under microwave irradiation for 45 min at 160 ꢀC. After
cooling to RT, water and EtOAc were added and layers were sepa-
rated. The aqueous layer was extracted with EtOAc (2ꢂ). The
combined organic layers were washed with brine, dried over
MgSO4 and concentrated under vacuum. Pure 9N-THP protected
derivatives were isolated after purification by gradient flash chro-
matography (SiO2, Hexane/EtOAc) in moderate to good yields.
For the removal of the THP group, 9N-THP protected compounds
were stirred at RT for 1e3 h in a MeOH/TFA (3/1) mixture. When no
more starting material was detected on TLC, the mixture was
concentrated under vacuum. The residue was partitioned between
a saturated NaHCO3 aqueous solution and EtOAc. Layers were
separated and the aqueous layer was extracted with EtOAc (2e3ꢂ).
The combined organic layers were washed with brine (20 mL),
dried over MgSO4 and concentrated under vacuum. Pure com-
pounds were isolated after purification by gradient flash chroma-
tography (SiO2, Hexane/EtOAc) in moderate to good yields.
4.3.6. N6-(tert-butyl)-8-ethyl-N2-(2-(2-fluoroethoxy)benzo[d]
thiazol-6-yl)-9H-purine-2,6-diamine 20
Starting from 12 and 6a. Yield 50%. 1H NMR (500 MHz, CDCl3)
d
10.46 (s, 1H), 8.26 (d, J ¼ 2.0 Hz, 1H), 7.56 (d, J ¼ 8.6 Hz, 1H), 7.32
(dd, J ¼ 2.2, 8.7 Hz, 1H), 6.9 (s, 1H), 5.52 (s, 1H), 4.98e4.61 (m, 4H),
2.59 (q, J ¼ 7.6 Hz, 2H), 1.54 (s, 9H), 1.25 (t, J ¼ 7.6 Hz, 3H). 13C NMR
(126 MHz, CDCl3)
d 171.10, 155.66, 154.22, 150.83, 150.29, 143.98,
136.96, 132.91, 120.87, 118.65, 115.12, 111.83, 81.35 (d,
1JCeF ¼ 171.5 Hz), 70.19 (d, JCeF ¼ 20.4 Hz), 52.13, 29.40, 22.64,
2
12.49. ESIeMS m/z 428.12 [MþH]þ, 430.15 [MꢁH]ꢁ.
4.3.7. N6-cyclohexyl-N2-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-
9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 21a
Starting from 12 and 3. Yield 100%. 1H NMR (500 MHz, CDCl3)
d
8.48 (s, 1H), 7.73 (s, 1H), 7.56 (d, J ¼ 8.7 Hz, 1H), 7.28 (dd, J ¼ 2.0,
8.7 Hz, 1H), 7.06 (s, 1H), 5.61e5.49 (m, 2H), 4.94e4.66 (m, 4H), 4.16
(d, J ¼ 14.92 Hz, 1H), 3.75 (td, J ¼ 2.23, 11.51 Hz, 1H), 2.21e2.03 (m,
5H), 1.90e1.59 (m, 7H), 1.45 (q, J ¼ 12.50 Hz, 2H), 1.35e1.21 (m, 3H).
13C NMR (126 MHz, CDCl3)
d 170.66, 156.45, 154.20, 143.41, 137.24,
135.39, 132.73, 120.56, 117.82, 110.75, 81.70, 81.39 (d,
4.3.2. Method B (one-step procedure)
2
1JCeF ¼ 171.4 Hz), 70.02 (d, JCeF ¼ 20.3 Hz), 68.65, 53.43, 33.36,
To a solution of purine derivative 2b or 6a (1 eq.) in n-butanol
was added the appropriate arylamine (1.1 eq.) and a catalytic
amount of concentrated 37% HCl (0.1 eq.). The mixture was heated
at 117 ꢀC until no more starting material was detected on TLC
(typically 12e15 h). After cooling to RT, water and EtOAc were
added and layers were separated. The aqueous layer was extracted
with EtOAc (3ꢂ). The combined organic layers were washed with
brine, dried over MgSO4 and concentrated under vacuum. Pure
compounds were isolated after purification by gradient flash
chromatography (SiO2, Hexane/EtOAc) in moderate yields.
31.39, 25.71, 25.23, 25.03, 23.01. ESIeMS m/z 510.4 [MꢁH]ꢁ.
4.3.8. N6-cyclohexyl-N2-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-
9H-purine-2,6-diamine 21b
Yield 50%. 1H NMR (600 MHz, DMSO-d6)
d 12.40 (s, 1H), 9.05 (s,
1H), 8.63 (s, 1H), 7.83 (s, 1H), 7.60 (dd, J ¼ 2.2, 8.8 Hz, 1H), 7.54 (d,
J ¼ 8.8 Hz, 1H), 7.25 (s, 1H), 4.88e4.72 (m, 4H), 4.08 (s, 1H),
2.00e1.92 (m, 2H), 1.85e1.75 (m, 2H), 1.67 (d, J ¼ 13.0 Hz, 1H),
1.44e1.30 (m, 4H), 1.24e1.13 (m, 1H). 13C NMR (151 MHz, DMSO-d6)
d
169.21,156.05,153.54,150.90,141.91,138.65,136.15,131.53,120.19,
4.3.3. N6-(tert-butyl)-N2-(4-(2-fluoroethyl)phenyl)-9H-purine-2,6-
diamine 18
1
117.68, 113.93, 109.49, 81.54 (d, JCeF ¼ 165.4 Hz), 70.55 (d,
2JCeF ¼ 18.8 Hz), 64.90, 32.51, 25.30, 15.14. ESIeMS m/z 428.06
[MþH]þ, 426.16 [MꢁH]ꢁ.
Starting from 9 and 2b. Yield 73%. 1H NMR (600 MHz, CDCl3)
d
13.10 (s, 1H), 7.48 (d, J ¼ 8.3 Hz, 2H), 7.19 (d, J ¼ 8.1 Hz, 2H), 6.87 (s,
1H), 6.56 (s, 1H), 5.65 (s, 1H), 4.62 (dt, J ¼ 6.5, 47.0 Hz, 2H), 2.97 (dt,
J ¼ 6.5, 23.3 Hz, 2H), 1.52 (s, 9H). 13C NMR (151 MHz, CDCl3)
d 156.17,
4.3.9. N6-(tert-butyl)-N2-(2-(2-fluoroethoxy)quinolin-6-yl)-9-
(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6 diamine 22a
Starting from 16 and 2a. Yield 76%. 1H NMR (500 MHz, CDCl3)
3
155.03, 149.78, 138.53, 135.68, 132.00 (d, JCeF ¼ 6.2 Hz), 129.87,
1
121.88, 115.50, 84.29 (d, JCeF ¼ 169.0 Hz), 52.15, 36.38 (d,
2JCeF ¼ 20.3 Hz), 29.28. ESIeMS m/z 329.25 [MþH]þ, 351.23
[MþNa]þ, 327.31 [MꢁH]ꢁ.
d
8.34 (d, J ¼ 2.4 Hz, 1H), 7.91 (d, J ¼ 8.9 Hz, 1H), 7.74 (m, 2H), 7.67
(dd, J ¼ 2.4, 9.0 Hz, 1H), 7.04 (s, 1H), 6.94 (d, J ¼ 8.8 Hz, 1H),
5.64e5.56 (m, 2H), 4.92e4.66 (m, 4H), 4.18 (d, J ¼ 9.9 Hz, 1H), 3.78
(t, J ¼ 11.5 Hz,1H), 2.12e2.08 (m, 3H),1.85e1.61 (m, 3H),1.57 (s, 9H).
4.3.4. N6-(tert-butyl)-N2-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-
9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 19a
Starting from 12 and 2a. Yield 84%. 1H NMR (500 MHz, CDCl3)
13C NMR (126 MHz, CDCl3)
d 160.43, 156.27, 154.83, 149.48, 142.16,
138.38, 137.03, 135.23, 127.41, 125.85, 123.75, 115.89, 114.53, 113.26,
82.17 (d, 1JCeF ¼ 169.3 Hz), 81.90, 68.79, 64.80 (d, 2JCeF ¼ 20.2 Hz),
52.09, 31.56, 29.38, 25.17, 23.15. ESIeMS m/z 480.4 [MþH]þ.
d
8.38 (s, 1H), 7.70 (s, 1H), 7.57 (d, J ¼ 8.6 Hz, 1H), 7.31 (d, J ¼ 6.9 Hz,
1H), 6.93 (s, 1H), 5.62e5.50 (m, 2H), 4.96e4.70 (m, 4H), 4.17 (d,
J ¼ 10.4 Hz, 1H), 3.76 (t, J ¼ 10.3 Hz, 1H), 2.15e2.04 (m, 3H),
1.88e1.60 (m, 3H), 1.53 (s, 9H). 13C NMR (126 MHz, CDCl3)
d 170.92,
156.29, 154.81, 149.48, 143.76, 137.18, 135.23, 132.74, 120.65, 118.55,
4.3.10. N6-(tert-butyl)-N2-(2-(2-fluoroethoxy)quinolin-6-yl)-9H-
purine-2,6-diamine 22b
1
115.88, 111.75, 81.93, 81.36 (d, JCeF
¼
171.5 Hz), 70.17 (d,
2JCeF ¼ 20.2 Hz), 68.76, 52.09, 31.48, 29.33, 25.18, 23.13. ESIeMS m/z
486.13 [MþH]þ, 484.11 [MꢁH]ꢁ.
Yield 73%. 1H NMR (600 MHz, DMSO-d6)
d 12.47 (bs, 1H), 9.08 (s,
1H), 8.46 (s, 1H), 8.04 (d, J ¼ 8.8 Hz, 1H), 7.93 (dd, J ¼ 1.9, 9.0 Hz, 1H),
7.82 (s, 1H), 7.65 (d, J ¼ 8.9 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 1H), 6.35 (s,
1H), 4.81 (dt, J ¼ 3.8 Hz, 1JHeF ¼ 47.9 Hz, 2H), 4.62 (dt, J ¼ 4.0 Hz,
3JHeF ¼ 30.5 Hz, 2H), 1.56 (s, 9H). 13C NMR (151 MHz, DMSO-d6)
4.3.5. N6-(tert-butyl)-N2-(2-(2-fluoroethoxy)benzo[d]thiazol-6-yl)-
9H-purine-2,6-diamine 19b
Yield 83%. 1H NMR (500 MHz, CDCl3)
d
12.02 (s, 1H), 8.09 (s, 1H),
d 159.54, 155.68, 154.15, 150.33, 140.72, 138.41, 138.05, 136.17,
7.60 (d, J ¼ 8.6 Hz, 1H), 7.38 (d, J ¼ 8.5 Hz, 1H), 6.89 (s, 1H), 6.80 (s,
126.67, 126.47, 125.23, 123.81, 114.91, 114.70, 113.21, 112.68, 82.07 (d,
1JCeF ¼ 165.7 Hz), 64.60 (d, 2JCeF ¼ 18.8 Hz), 51.12, 29.00. ESIeMS m/
z 396.15 [MþH]þ, 394.13 [MꢁH]ꢁ.
1H), 5.58 (s, 1H), 4.92e4.68 (m, 4H), 1.53 (s, 9H). 13C NMR (126 MHz,
CDCl3) d 171.48,156.37,155.01,149.88,144.79,136.34,135.18,133.02,

778
P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
4.3.11. N6-(tert-butyl)-8-ethyl-N2-(2-(2-fluoroethoxy)quinolin-6-
yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine 23a
Starting from 16 and 6b. Yield 26%. 1H NMR (500 MHz, CDCl3)
J ¼ 2.15, 8.88 Hz,1H), 4.93e4.76 (m, 4H). 13C NMR (151 MHz, DMSO-
d6)
d 159.48, 155.06, 154.33, 152.80, 147.61, 139.34, 139.03, 134.31,
1
122.60, 118.63, 114.81, 109.6, 81.91 (d, JCeF ¼ 166.2 Hz), 65.38 (d,
2JCeF ¼ 19.2 Hz). ESIeMS m/z 331.06 [MþH]þ, 353.05 [MþNa]þ,
328.99 [MꢁH]ꢁ.
d
8.34 (d, J ¼ 2.2 Hz, 1H), 7.90 (d, J ¼ 8.8 Hz, 1H), 7.73 (d, J ¼ 8.9 Hz,
1H), 7.68 (dd, J ¼ 2.3, 8.9 Hz, 1H), 6.99e6.88 (m, 2H), 5.58 (s, 1H),
4.93e4.65 (m, 4H), 4.21 (d, J ¼ 11.8 Hz, 1H), 3.71 (t, J ¼ 11.7 Hz, 1H),
3.01e2.86 (m, 2H), 2.81 (qd, J ¼ 3.9,12.8 Hz,1H), 2.12 (d, J ¼ 12.5 Hz,
1H), 1.89 (d, J ¼ 13.1 Hz, 1H), 1.85e1.62 (m, 4H), 1.57 (s, 9H), 1.39 (t,
4.3.17. N6-(tert-butyl)-8-ethyl-N2-(2-(2-morpholinoethoxy)
quinolin-6-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-
diamine 26
J ¼ 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d 160.33, 155.36, 154.18,
151.01, 150.75, 141.98, 138.30, 137.35, 127.34, 125.91, 123.63, 114.66,
Starting from 17 and 6b. Yield 43%. 1H NMR (500 MHz, CDCl3)
1
114.06, 113.22, 83.19, 82.34 (d, JCeF ¼ 168.8 Hz), 69.20, 64.79 (d,
d
8.29 (s, 1H), 7.89 (d, J ¼ 8.8 Hz, 1H), 7.77e7.67 (m, 2H), 6.88 (d,
2JCeF ¼ 20.0 Hz), 52.00, 30.07, 29.46, 25.33, 23.78, 22.38, 12.58.
ESIeMS m/z 507.8 [MþH]þ.
J ¼ 8.8 Hz, 1H), 5.75 (s, 1H), 5.54 (d, J ¼ 11.0 Hz, 1H), 4.82e4.60 (m,
2H), 4.21 (d, J ¼ 10.4 Hz, 1H), 3.87e3.79 (m, 4H), 3.71 (t, J ¼ 11.7 Hz,
1H), 3.18e3.06 (m, 2H), 3.04e2.80 (m, 6H), 2.78e2.64 (m, 2H), 2.11
(d, J ¼ 11.5 Hz, 1H), 1.89 (d, J ¼ 13.0 Hz, 1H), 1.84e1.70 (m, 2H), 1.66
(d, J ¼ 12.3 Hz, 1H), 1.57 (s, 9H), 1.40 (t, J ¼ 7.5 Hz, 3H). 13C NMR
4.3.12. N6-(tert-butyl)-8-ethyl-N2-(2-(2-fluoroethoxy)quinolin-6-
yl)-9H-purine-2,6-diamine 23b
Yield 80%. 1H NMR (600 MHz, CDCl3)
d
10.34 (bs, 1H), 8.25 (d,
(126 MHz, CDCl3) d 160.25, 151.05, 148.15, 144.25, 142.20, 138.33,
J ¼ 2.4 Hz, 1H), 7.87 (d, J ¼ 8.8 Hz, 1H), 7.73 (d, J ¼ 8.9 Hz, 1H), 7.66
137.01, 133.84, 131.44, 127.37, 125.82, 123.87, 114.49, 113.14, 83.27,
69.25, 65.96, 61.70, 57.21, 53.45, 52.25, 30.15, 29.38, 25.23, 23.65,
22.30, 12.44. ESIeMS m/z 575.45 [MþH]þ, 597.42 [MþNa]þ.
(dd, J ¼ 2.4, 9.0 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J ¼ 8.8 Hz, 1H), 5.58 (s,
2
1H), 4.82 (dt, J ¼ 4.2 Hz, JCeF ¼ 47.6 Hz, 2H), 4.72 (dt, J ¼ 4.12 Hz,
3JCeF ¼ 29.26 Hz, 2H), 2.61 (q, J ¼ 7.6 Hz, 2H), 1.58 (s, 9H), 1.26 (t,
J ¼ 7.6 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d
160.50, 155.63, 154.16,
4.3.18. N6-(tert-butyl)-8-ethyl-N2-(2-(2-morpholinoethoxy)
150.78, 150.27, 142.21, 139.70, 138.33, 136.80, 127.63, 125.86, 123.77,
114.67, 113.39, 82.31 (d, 1JCeF ¼ 169.2 Hz), 64.82 (d, 2JCeF ¼ 20.0 Hz),
52.12, 29.42, 22.69, 14.35. ESIeMS m/z 424.27 [MþH]þ, 422.18
[MꢁH]ꢁ.
quinolin-6-yl)-9H-purine-2,6-diamine QAP1
Yield 82%. 1H NMR (600 MHz, CDCl3)
d 11.07 (bs,1H), 8.22 (s,1H),
7.80 (d, J ¼ 8.6 Hz, 1H), 7.73 (d, J ¼ 8.9 Hz, 1H), 7.65 (dd, J ¼ 2.4,
9.0 Hz, 1H), 7.02 (bs, 1H), 6.84 (d, J ¼ 8.8 Hz, 1H), 5.63 (bs, 1H), 4.60
(t, J ¼ 5.8 Hz, 2H), 3.76 (t, J ¼ 4.7 Hz, 4H), 2.85 (t, J ¼ 5.8 Hz, 2H),
2.66e2.58 (m, 4H), 2.48 (q, J ¼ 7.7 Hz, 2H), 1.57 (s, 9H), 1.18 (t,
4.3.13. N6-(tert-butyl)-N2-(6-fluoropyridin-2-yl)-9-(tetrahydro-
2H-pyran-2-yl)-9H-purine-2,6-diamine 24a
J ¼ 7.6 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d 160.96, 155.44, 154.16,
Yield 40%. Starting from 2-amino-6-fluoropyridine (Sigma) and
150.76, 150.56, 142.44, 137.97, 136.52, 127.65, 125.67, 123.64, 115.06,
114.72, 113.63, 67.07, 62.95, 57.77, 54.13, 52.15, 29.41, 22.57, 12.42.
ESIeMS m/z 491.35 [MþH]þ, 489.38 [MꢁH]ꢁ.
2a. 1H NMR (500 MHz, CDCl3)
d
8.33 (dd, J ¼ 2.2, 8.1 Hz, 1H),
7.82e7.69 (m, 2H), 7.59 (s, 1H), 6.47 (dd, J ¼ 2.4, 7.8 Hz, 1H),
5.69e5.52 (m, 2H), 4.17 (m, J ¼ 11.5 Hz, 1H), 3.78 (td, J ¼ 2.4, 11.6 Hz,
1H), 2.18e2.06 (m, 3H), 1.89e1.62 (m, 3H), 1.54 (s, 9H). 13C NMR
4.4. Synthetic procedures for radiolabeling precursors
1
(126 MHz, CDCl3)
d
162.28 (d, JCeF ¼ 237.9 Hz), 154.78, 154.48,
152.22 (d, 3JCeF ¼ 15.0 Hz), 148.98, 142.42 (d, 3JCeF ¼ 8.0 Hz), 135.78,
116.40, 108.20, 100.54 (d, 2JCeF ¼ 35.6 Hz), 81.98, 68.77, 52.25, 31.64,
29.26, 25.15, 23.09. ESIeMS m/z 386.2 [MþH]þ.
4.4.1. 2-(4-((6-(tert-butylamino)-9-(tetrahydro-2H-pyran-2-yl)-
9H-purin-2-yl)amino)phenyl)ethanol 28
Starting from 4-aminophenethyl alcohol 27 (74 mg) and 2a
(140 mg), compound 28 (155 mg, 83%) was synthesized according
to the general procedure described for compounds 19a-26 (method
A) and was isolated as an off-white solid. 1H NMR (500 MHz, CDCl3)
4.3.14. N6-(tert-butyl)-N2-(6-fluoropyridin-2-yl)-9H-purine-2,6-
diamine 24b
Yield 61%. 1H NMR (600 MHz, DMSO-d6)
d
12.62 (s, 1H), 9.35 (s,
d
7.66 (s, 1H), 7.61 (d, J ¼ 8.4 Hz, 2H), 7.17 (d, J ¼ 8.5 Hz, 2H), 6.85 (s,
1H), 8.25 (d, J ¼ 7.4 Hz, 1H), 7.88 (s, 1H), 7.85 (q, J ¼ 8.2 Hz, 1H), 6.58
1H), 5.69 (s, 1H), 5.59e5.50 (m, 1H), 4.23e4.08 (m, 1H), 3.85 (t,
J ¼ 6.8 Hz, 2H), 3.75 (td, J ¼ 2.6, 11.6 Hz, 1H), 2.84 (t, J ¼ 6.5 Hz, 2H),
2.04 (m, 2H), 1.96 (s, 1H), 1.86 (s, 1H), 1.78e1.59 (m, 3H), 1.54 (s, 9H).
(d, J ¼ 7.5 Hz, 1H), 6.50 (s, 1H), 1.51 (s, 9H). 13C NMR (151 MHz,
DMSO-d6)
d
161.54 (d, 1JCeF ¼ 233.6 Hz), 154.20, 153.99, 152.68 (d,
3JCeF ¼ 15.9 Hz), 149.89, 142.64 (d, JCeF ¼ 7.9 Hz), 136.87, 115.34,
108.52, 99.50 (d, 2JCeF ¼ 36.2 Hz), 51.32, 28.99. ESIeMS m/z 302.07
[MþH]þ, 324.06 [MþNa]þ, 300.12 [MꢁH]ꢁ.
13C NMR (126 MHz, CDCl3)
d 156.36, 154.77, 149.42, 139.29, 134.97,
3
131.39, 129.36, 119.27, 115.57, 81.67, 68.72, 63.87, 52.02, 38.76, 31.65,
29.34, 25.14, 23.10. ESIeMS m/z 410.9 [MþH]þ.
4.3.15. N-(6-(2-fluoroethoxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-2-yl)benzo[d]thiazol-6-amine 25a
4.4.2. 4-((6-(tert-butylamino)-9-(tetrahydro-2H-pyran-2-yl)-9H-
purin-2-yl)amino)phenethyl 4-methylbenzenesulfonate 29
To a solution of compound 28 (50 mg, 0.12 mmol) and Et3N
Starting from 6-aminobenzothiazole and 4. Yield 46%. 1H NMR
(500 MHz, CDCl3)
d
8.87 (s, 1H), 8.64 (d, J ¼ 2.1 Hz, 1H), 8.06 (d,
(75 m
L, 0.54 mmol) in dry DCM (10 mL) at 0 ꢀC under argon was
J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.51 (dd, J ¼ 2.2, 8.8 Hz, 1H), 7.22 (s, 1H),
5.62 (dd, J ¼ 2.3, 10.5 Hz, 1H), 4.98e4.75 (m, 4H), 4.20 (dd, J ¼ 3.0,
10.7 Hz, 1H), 3.79 (td, J ¼ 2.5, 11.6 Hz, 1H), 2.29e2.02 (m, 3H),
added TsCl (116 mg, 0.60 mmol). The mixture was allowed to warm
up and stirred at RT for 72 h before water (15 mL) was added. The
layers were separated and the aqueous layer was extracted with
DCM (2 ꢂ 10 mL). The combined organic layers were dried over
MgSO4 and concentrated under vacuum to a yellow oil. Purification
by gradient flash chromatography (SiO2, DCM/MeOH, 98/2) yielded
compound 29 (46 mg, 68%) as an off-white solid. 1H NMR
1.87e1.60 (m, 3H). 13C NMR (126 MHz, CDCl3)
d 160.60, 155.37,
153.20, 152.01, 148.81, 138.33, 138.04, 135.03, 123.54, 119.00, 116.56,
110.65, 82.41, 81.47 (d, J ¼ 170.9 Hz), 68.84, 65.83 (d, J ¼ 21.6 Hz),
31.34, 25.13, 23.07. ESIeMS m/z 437.2 [MþNa]þ.
(600 MHz, CDCl3)
d
7.71 (s, 1H), 7.70 (d, J ¼ 8.0 Hz, 2H), 7.56 (d,
4.3.16. N-(6-(2-fluoroethoxy)-9H-purin-2-yl)benzo[d]thiazol-6-
amine 25b
J ¼ 8.2 Hz, 2H), 7.28 (d, J ¼ 8.1 Hz, 2H), 7.05 (d, J ¼ 8.2 Hz, 2H), 6.83
(s, 1H), 5.59 (s, 1H), 5.56 (dd, J ¼ 3.7, 8.9 Hz, 1H), 4.19 (t, J ¼ 7.2 Hz,
2H), 4.18e4.14 (m,1H), 3.8 (td, J ¼ 2.4,11.6 Hz,1H), 2.92 (t, J ¼ 7.2 Hz,
2H), 2.41 (s, 3H), 2.10e2.05 (m, 2H), 1.80e1.72 (m, 3H), 1.67e1.63
Yield 53%. 1H NMR (600 MHz, DMSO-d6)
d
12.92 (s, 1H), 9.68 (s,
1H), 9.17 (s, 1H), 8.77 (s, 1H), 7.97 (d, J ¼ 8.83 Hz, 1H), 7.79 (dd,

P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
779
(m, 1H), 1.54 (s, 9H). 13C NMR (151 MHz, CDCl3)
d
156.19, 154.75,
argon. The reaction mixture was neutralized with a 2% aqueous HCl
solution and extracted with EtOAc (3 ꢂ 15 mL). The combined
organic layers were washed with brine (15 mL), dried over MgSO4,
and concentrated under vacuum. Purification by gradient flash
chromatography (SiO2, Hexane/EtOAc, 9/1) yielded compound 34
149.38, 144.72, 139.66, 135.08, 133.10, 129.89, 129.27, 128.87, 127.95,
118.98, 115.65, 81.65, 71.12, 68.76, 52.04, 34.88, 31.64, 29.33, 25.13,
23.10, 21.76. ESIeMS m/z 587.2 [MþNa]þ.
4.4.3. 2-(2-((triisopropylsilyl)oxy)ethoxy)benzo[d]thiazol-6-amine
(65 mg, 68%) as a solid. 1H NMR (500 MHz, CDCl3)
d 8.36 (d,
31
J ¼ 2.2 Hz,1H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.71 (s,1H), 7.50 (d, J ¼ 8.6 Hz,
1H), 7.32e7.25 (m, 3H), 6.98 (s, 1H), 5.59 (s, 1H), 5.55 (dd, J ¼ 2.3,
10.4 Hz, 1H), 4.69 (t, J ¼ 4.5 Hz, 2H), 4.45 (t, J ¼ 4.4 Hz, 2H),
4.19e4.14 (m, 1H), 3.76 (td, J ¼ 4.9, 8.8, 9.7 Hz, 1H), 2.38 (s, 3H),
2.19e2.05 (m, 3H), 1.79e1.71 (m, 2H), 1.68e1.63 (m, 1H), 1.54 (s,
To a solution of 2-((triisopropylsilyl)oxy)ethanol (303 mg,
1.39 mmol) in a mixture of dry THF (1 mL) and dry DMF (128 mL) at
0 ꢀC under argon was added a 60% dispersion of NaH in mineral oil
(67 mg, 2.77 mmol). The mixture was allowed to react for 1 h at 0 ꢀC
before a solution of compound 30 (150 mg, 0.82 mmol) in a mixture
9H). 13C NMR (126 MHz, CDCl3)
d 156.22, 154.77, 149.43, 145.13,
of dry THF (1 mL) and dry DMF (128
m
L) was added. The reaction
143.51, 137.21, 135.23, 132.80, 132.67, 130.06, 129.97, 128.10, 120.64,
118.47, 115.85, 111.55, 81.90, 68.74, 68.30, 67.52, 52.06, 31.45, 29.31,
25.16, 23.12, 21.74. ESIeMS m/z 636.22 [MꢁH]ꢁ, 638.19 [MþH]þ,
660.19 [MþNa]þ.
mixture allowed to reach RT and refluxed for 48 h before water
(20 mL) was added. The aqueous layer was extracted with EtOAc
(3 ꢂ 15 mL). The combined organic layers were washed with brine
(15 mL), dried over MgSO4, and concentrated under vacuum to a
brown oil. Purification by column chromatography (SiO2, Hexane/
EtOAc, 9/1) yielded compound 31 (169 mg, 56%) as a yellow oil. 1H
NMR (500 MHz, CDCl3)
d
7.44 (d, J ¼ 8.5 Hz, 1H), 6.91 (d, J ¼ 2.4 Hz,
4.5. Topoisomerase IIa inhibition assays
1H), 6.70 (dd, J ¼ 2.4, 8.5 Hz, 1H), 4.58 (d, J ¼ 4.9 Hz, 2H), 4.06 (d,
J ¼ 4.9 Hz, 2H), 3.58 (s, 2H), 1.24e0.96 (m, 21H). 13C NMR (126 MHz,
Topoisomerase inhibition assay were performed using
a
CDCl3)
d
170.25, 142.99, 142.23, 133.34, 121.25, 114.71, 106.80, 72.80,
eukaryotic topoisomerase II drug screening kit purchased from
a
61.74, 18.04, 12.11. ESIeMS m/z 367.15 [MþH]þ, 389.30 [MþNa]þ.
TopoGen (Port Orange, FL). All complementary materials (including
topoisomerase inhibitor etoposide) were also purchased from
TopoGen, and all experiments were performed according to the
manufacturer's instructions and specifications. In a representative
4.4.4. N6-(tert-butyl)-9-(tetrahydro-2H-pyran-2-yl)-N2-(2-(2-
((triisopropylsilyl)oxy)ethoxy)benzo[d]thiazol-6-yl)-9H-purine-2,6-
diamine 32
inhibition experiment, 14
buffer, 1 L of supercoiled DNA substrate, and 1
topoisomerase-II (~2 units of activity, as described by the manu-
facturer) were combined in a 1.7 mL microcentrifuge tube. In ex-
periments involving added drug, 2 L of DMSO stock solution (10%
v/v) of compound were added, and the initial volume of water was
adjusted to allow for a final reaction volume of 20 L. The reaction
mixture was vortexed gently, centrifuged briefly, and incubated at
37 ꢀC for 30 min. After 30 min, 2
L of 10% SDS was added to quench
the reaction, 2 L of proteinase K was added to digest remaining
proteins, and the reaction mixture was again incubated at 37 ꢀC for
15 min. After this incubation, 2.5
mL of H2O, 4
m
L of topoisomerase reaction
Starting from compounds 31 (189 mg) and 2a (133 mg), com-
pound 32 (187 mg, 68%) was synthesized according to the general
procedure described for compounds 19ae26 (Method A) and was
m
m
L of eukaryotic
a
isolated as a white solid. 1H NMR (500 MHz, CDCl3)
d
8.34 (d,
m
J ¼ 2.3 Hz, 1H), 7.71 (s, 1H), 7.57 (d, J ¼ 8.7 Hz, 1H), 7.31 (dd, J ¼ 2.3,
8.7 Hz, 1H), 7.07 (s, 1H), 5.60 (s, 1H), 5.54 (dd, J ¼ 2.5, 10.3 Hz, 1H),
4.63 (t, J ¼ 5.1 Hz, 2H), 4.19e4.12 (m,1H), 4.09 (t, J ¼ 5.1 Hz, 2H), 3.74
(td, J ¼ 2.5, 11.3 Hz, 1H), 2.19e2.00 (m, 3H), 1.79e1.70 (m, 2H),
1.66e1.61 (m, 1H), 1.52 (s, 9H), 1.08 (m, 21H). 13C NMR (126 MHz,
m
m
m
CDCl3) d 171.50,156.31, 154.75, 149.41, 144.09,136.87, 135.12,132.55,
120.47, 118.47, 115.75, 111.78, 81.86, 72.96, 68.70, 61.75, 52.04, 31.47,
29.28, 25.14, 23.08,18.04,12.11. ESIeMS m/z 638.42 [MꢁH]ꢁ, 640.50
[MþH]þ.
m
L of 10ꢂ loading dye was added
to the reaction mixture, and the tube was subsequently vortexed
lightly and centrifuged. The sample was then loaded onto a 1%
agarose gel and electrophoresed for 5 h at 5 V/cm. Gels were
4.4.5. 2-((6-((6-(tert-Butylamino)-9-(tetrahydro-2H-pyran-2-yl)-
9H-purin-2-yl)amino)benzo[d]thiazol-2-yl)oxy)ethanol 33
To a solution of compound 32 (187 mg, 0.29 mmol) in THF (7 mL)
stained using incubation in a 1ꢂ TAE solution containing 5
mg/mL
ethidium bromide. Gels were visualized with a BioRad chem-
iluminescence detector, and the data were processed with Quantity
One software (Bio-Rad, version 4.3.0).
at RT was added TBAF (880 mL, 1 M solution in THF). After stirring
for 1 h at RT, the mixture was concentrated under vacuum and the
crude residue was directly purified by gradient flash chromatog-
raphy (SiO2, Hexane/EtOAc) to yield compound 33 (127 mg, 91%) as
a white solid. 1H NMR (500 MHz, CDCl3)
d
8.33 (d, J ¼ 2.3 Hz, 1H),
4.6. Cell culture
7.71 (s, 1H), 7.56 (d, J ¼ 8.7 Hz, 1H), 7.32 (dd, J ¼ 2.3, 8.7 Hz, 1H), 7.00
(s, 1H), 5.70 (s, 1H), 5.54 (dd, J ¼ 2.4, 10.3 Hz, 1H), 4.7 (t, J ¼ 4.1 Hz,
2H), 4.18e4.13 (m, 1H), 4.03 (t, J ¼ 4.2 Hz, 2H), 3.91 (s, 1H), 3.75 (td,
J ¼ 2.6, 11.4 Hz, 1H), 2.16e2.00 (m, 3H), 1.79e1.68 (m, 2H), 1.67e1.60
Human breast cancer cell lines SK-BR-3 and MCF-7 were ob-
tained from the American Tissue Culture Collection (ATCC, Mana-
ssas, VA) and maintained by weekly serial passage in a 5% CO2(g)
atmosphere at 37 ꢀC. Cells were harvested using a formulation of
0.25% trypsin and 0.53 mM EDTA in Hank's buffered salt solution
(HBSS) without calcium or magnesium. SK-BR-3 cells were grown
in a 1:1 mixture of Dulbecco's modified Eagle medium: F-12 me-
(m, 1H), 1.53 (s, 9H). 13C NMR (126 MHz, CDCl3)
d 171.83, 156.28,
154.73, 149.39, 143.48, 137.14, 135.13, 132.52, 120.54, 118.61, 115.70,
111.71, 81.86, 73.95, 68.72, 61.60, 52.05, 31.45, 29.27, 25.14, 23.09.
ESIeMS m/z 484.28 [MþH]þ.
dium, supplemented with 10% fetal calf serum, 2 mM L-glutamine,
4.4.6. 2-((6-((6-(tert-Butylamino)-9-(tetrahydro-2H-pyran-2-yl)-
9H-purin-2-yl)amino)benzo[d]thiazol-2-yl)oxy)ethyl 4-
methylbenzenesulfonate 34
nonessential amino acids, and 100 units/mL penicillin and strep-
tomycin. MCF-7 cells were grown in minimum essential medium,
supplemented with 10% fetal calf serum, 0.01 mg/mL bovine insulin
To a solution of compound 33 (73 mg, 0.15 mmol) in dry pyri-
dine (3 mL) at 0 ꢀC under argon was added TsCl (43 mg, 0.23 mmol).
The mixture was allowed to reach RT and stirred for 12 h under
(Sigma Aldrich, St. Louis, MO), non-essential amino acids, 2 mM L-
glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and
100 units/mL penicillin and streptomycin.

780
P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
4.7. Cell proliferation assay
residual solution was diluted with water (5 mL). The solution was
passed through a C18 light Sep-Pak® cartridge preconditioned with
EtOH (5 mL) and water (5 mL). The cartridge was washed with
water (3 mL) and [18F]-18 was eluted with EtOH (1 mL). EtOH was
evaporated at 70 ꢀC with a gentle flow of argon and the product was
formulated in 0.9% saline for PET studies. The final isolated decay
corrected radiochemical yield (dcRCY) after formulation was
around 26% with radiochemical purity (RCP) superior to 98% as
determined by analytical HPLC (gradient A; tR ¼ 12.7 min). The total
radiosynthesis time was about 125 min.
The cellular proliferation of MCF-7 and SK-BR-3 cells upon
treatment drugs was quantified using an MTT assay obtained from
ATCC. For the assay, MCF-7 (50 000 cells/well) and SK-BR-3
(20 000 cells/well) were seeded in 96-well plates (Costar, 3596)
overnight (18e24 h). Then, after overnight incubation, the old
media was aspirated and discarded, and new media mixed (via 1:20
dilution) with different drug concentrations were added to each
well in 100
m
L aliquots. The drug-treated plates were then incu-
bated for 72 h at 37 ꢀC/5% CO2. After drug treatment, 10
mL of MTT
reagent was added to each well to yield a final concentration of
5 mg MTT/mL and the plates were then incubated at 37 ꢀC/5% CO2
for 3 h more. After this incubation, cells were solubilized overnight
(18e24 h) at room temperature with the kit-provided detergent
reagent. After this solubilization step, the absorbance of each well
at 570 nm was measured using a SpectraMax M5 plate reader
(Molecular Devices, New Orleans, LA), and the amount of prolifer-
ation in each well was normalized to 1 using the untreated, control
well.
4.10. Radiosynthesis of [18F]-19b
[
18F]-19b was synthesized in two steps from tosylate precursor
34 using the same radiolabelling conditions as for compound [18F]-
18. Briefly, to anhydrous K[18F]FeK222 complex was added a solu-
tion of the tosylate precursor 34 (3.8 mg) in dry MeCN (400
the mixture was heated at 110 ꢀC for 15 min and then allowed to
cool to RT and a 10 L aliquot was removed and diluted with water
mL), and
m
for HPLC analysis (gradient B). The radio-chromatogram showed
single peak corresponding to the [18F]THP-protected intermediate
(tR ¼ 19.9 min). For the removal of the THP group, the reaction
mixture was transferred to a vial containing a mix of MeOH and TFA
4.8. General radiochemistry
QMA light ion-exchange cartridges and C-18 light Sep-Pak®
cartridges were obtained from Waters (Milford, MA). High-
performance liquid chromatography (HPLC) was performed on a
Shimadzu system equipped with a DGU-20A degasser, two LC-
20AB pumps, a SPD-M20A photodiode array detector, and a Flow
Count PIN diode radiodetector from BioScan. HPLC solvents (A: H2O
with 0.1% v/v TFA, and B: MeCN with 0.1% v/v TFA) were filtered
before use. Semi-preparative HPLC purification was performed on a
C-18 Phenomenex Luna reversed-phase column (10 mm ꢂ 250 mm,
(120
15 min and a 10
m
L, 50% v/v MeOH) and incubated at room temperature for
mL aliquot was removed and diluted with water for
HPLC analysis (gradient B). The radio-chromatogram showed a
major peak corresponding to [18F]-19b (tR ¼ 15.5 min). The reaction
mixture was injected into semi-prep HPLC for purification (gradient
A). The fraction containing [18F]-19b was collected, MeCN was
evaporated under vacuum, and the residual solution was diluted
with water (5 mL). The solution was passed through a C18 light Sep-
Pak® cartridge preconditioned with EtOH (5 mL) and water (5 mL).
The cartridge was washed with water (3 mL) and [18F]-19b was
eluted with EtOH (1 mL). EtOH was evaporated at 70 ꢀC with a
gentle flow of argon and the product was formulated in 0.9% saline
for PET studies. The final isolated dcRCY after formulation was
around 6% with radiochemical purity (RCP) greater than 98% as
determined by analytical HPLC (gradient B; tR ¼ 15.6 min). The total
radiosynthesis time was about 135 min.
5
mm; 5 mL/min) employing gradient A (0e13 min, 20e80% B;
13e17 min, 80% B; 17e20 min, 80e20% B; 20e30 min, 20%) or
gradient B (0e15 min, 20e80% B; 15e17 min, 80% B; 17e22 min,
80e20% B; 22e25 min, 20%). Analytical HPLC was performed on a
C-18
Phenomenex
Luna
reversed-phase
column
(4.6 mm ꢂ 250 mm, 5
m
m; 1 mL/min) employing gradients A or B.
Radioactivity was assayed using a Capintec CRC1243 Dose Cali-
brator (Capintec, Ramsey, NJ). No-carrier-added 18F-fluoride was
obtained via the 18O(p,n)18F nuclear reaction of 11-MeV protons in
an EBCO TR-19/9 cyclotron using enriched 18O-water.
4.11. Xenografts models
All animal studies were conducted in compliance with Institu-
tional Animal Care and Use Committee (IACUC) guidelines. Female
severe combined immunodeficient (SCID, Restricted Flora, CB17SC-
F) mice (6e8 weeks old) were obtained from Taconic Farms, Inc.
(Hudson, NY, USA) and were allowed to acclimatize at the MSKCC
vivarium for 1 week prior to implanting tumors. Mice were pro-
vided with food and water ad libitum. SK-BR-3 tumors were
induced on the right shoulder via subcutaneous injection of
4.9. Radiosynthesis of [18F]-18
[
18F]-fluoride in water was trapped on a QMA light cartridge
preconditioned with 0.5 M K2CO3 (5 mL) and water (5 mL). The
activity was eluted from the cartridge into a sealed reaction vial
with 1 mL of a solution of Kryptofix K222 (2.5 mg) and K2CO3
(0.5 mg) in MeCN (5 mL) and water (2.5 mL). Water was removed
azeotropically with acetonitrile (3 ꢂ 1 mL) at 110 ꢀC under a slow
stream of argon. To the dry K[18F]FeK222 complex was added a
solution of the tosylate precursor 29 (4.0 mg) in dry MeCN (400
and the mixture was heated at 110 ꢀC for 15 min. The reaction vial
was then cooled to RT and a 10 L aliquot was removed and diluted
1.0 ꢂ 106 cells in a 200-
mL cell suspension of a 1:1 v/v mixture of
media with reconstituted basement membrane (BD Matrigel,
Collaborative Biomedical Products, Inc., Bedford, MA, USA) under
2% isoflurane anesthesia. Palpable tumors developed after a period
of 25e30 days.
mL),
m
with water for HPLC analysis (gradient A). The radio-chromatogram
showed one peak corresponding to the [18F]THP-protected inter-
mediate (tR ¼ 14.9 min). The reaction mixture was transferred to a
4.12. Small-animal positron emission tomography imaging
vial containing 0.5 N HCl in MeOH (120
m
L). The mixture was
L aliquot was removed
and diluted with water for HPLC analysis (gradient A). The radio-
chromatogram showed one peak corresponding to
18F]-18
PET imaging experiments were conducted either on a microPET
Focus 120 or on a microPET R4 rodent scanner (Concorde Micro-
systems). Mice bearing subcutaneous SK-BR-3 tumors
allowed to react for 15 min at RT and a 10
m
[
(100e200 mm3) (n ¼ 4) were administered [18F]-Topo II
a
inhibitors
(tR ¼ 12.7 min). The reaction mixture was injected into semi-prep
HPLC for purification (gradient A). The fraction containing [18F]-
18 was collected, MeCN was evaporated under vacuum, and the
formulations [5.2e7.2 MBq (140e194 mCi) in 200 mL 0.9% sterile
saline] via tail vein injection. Approximately 5 min prior to the PET
images, mice were anesthetized by inhalation of 2% isoflurane

P. Daumar et al. / European Journal of Medicinal Chemistry 86 (2014) 769e781
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filtered back-projection into
a
128
ꢂ
128
ꢂ
63
(0.72 ꢂ 0.72 ꢂ 1.3 mm) matrix. The image data were normalized to
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Funding sources
This work was supported by the U.S. Department of Energy
(Award DE-SC0002456; JSL). Partial funding from the Core Grant
(CCSG P30 CA008748) in supporting Center of Comparative Medi-
cine & Pathology and Radiochemistry and Molecular Imaging
Probes Cores is also acknowledged.
[16] M.M. Heck, W.C. Earnshaw, Topoisomerase II: a specific marker for cell pro-
liferation, J. Cell Biol. 103 (1986) 2569e2581.
Author contributions
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G. Lazar, L. Thurzo, T. Brodowicz, Z. Kahan, Tumor topoisomerase II alpha
status and response to anthracycline-based neoadjuvant chemotherapy in
breast cancer, Oncology 80 (2011) 269e277.
[18] L. Wei, J. Easmon, R.K. Nagi, B.D. Muegge, L.A. Meyer, J.S. Lewis, 64Cu-azabi-
cyclo[3.2.2]nonane thiosemicarbazone complexes: radiopharmaceuticals for
PET of topoisomerase II expression in tumors, J. Nucl. Med. 47 (2006)
2034e2041.
PD, NR, VD, KS, NP conducted the experiments and analyzed the
data. PD, BMZ, NP, JSL designed the experiments. PD, BMZ, NP, JSL
wrote the manuscript. All authors have given approval to the final
version of the manuscript.
[19] L.H. Jensen, A.V. Thougaard, M. Grauslund, B. Sokilde, E.V. Carstensen,
H.K. Dvinge, D.A. Scudiero, P.B. Jensen, R.H. Shoemaker, M. Sehested,
Acknowledgments
Substituted purine analogues define
a novel structural class of catalytic
topoisomerase II inhibitors, Cancer Res. 65 (2005) 7470e7477.
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R. Schmitz, T. Radimerski, Catalytic inhibition of topoisomerase II by a novel
rationally designed ATP-competitive purine analogue, BMC Chem. Biol. 9
(2009) 1.
The authors thank the MSKCC Radiochemistry and Cyclotron
Core and the MSKCC Small-Animal Imaging Core for technical ser-
vices, Drs. Athanasios Glekas, Darren R. Veach and Tony Taldone for
helpful discussions and advice regarding the chemical synthesis.
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[22] R. Baenteli, P. Chene, S.P. Collingwood, P. Furet, P. Meier, J. Schoepfer, Use of
9h-purine-2,6-diamine Derivatives in the Treatment of Proliferative Diseases
and Novel 9h-purine-2,6-diamine Derivatives, in, Google Patents, 2005.
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S. Gorzalka, M. Knospe, A.C. Schiedel, G. Cristalli, C.E. Muller, Structure-activity
relationships of adenine and deazaadenine derivatives as ligands for adenine
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.019.
receptors,
5974e5989.
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