Synthesis of 6,8,9 poly-substituted purine analogue libraries as pro-apoptotic inducers of human leukemic lymphocytes and DAPK-1 inhibitors

Article abstract of DOI:10.1039/c5ob00230c

A 18-member library of 6,8,9-poly-substituted purines was prepared from pyrimidines, primary alcohols, and N,N-dimethylamides under basic conditions via a novel one-pot synthetic pathway controlled by amide sizes and the novel analogues were tested against two leukemia cell lines: Jurkat (acute T cell leukemia) and K562 (chronic erythroleukemia) cells. Compounds having a benzoxy group at C6 position of the aromatic ring exhibited antiproliferative activity in Jurkat cells whereas all compounds induced a lower effect on K562 cells. Analysis of cell cycle, Annexin-V staining, and cleavage of initiator caspases assays showed that the active purine analogues induce cell death by apoptosis. Based on these results, a new purine derivative was synthesized, 6-benzyloxy-9-tert-butyl-8-phenyl-9H-purine (6d), which displayed the highest activity of the series against Jurkat cell lines. Finally, ³³P-radiolabeled kinase assays using 96 recombinant human kinases known to be involved in apoptotic events were performed. Just one of the kinases tested, DAPK-1, was inhibited 50% or more by the phenotypic hits at 10 μM, suggesting that the inhibition of this target could be responsible for the induction of cell death by apoptosis. In agreement with the phenotypic results, the most active antiproliferative agent, 6d, displayed also the lowest IC₅₀ value against recombinant DAPK1 (2.5 μM), further supporting the potential role of this protein on the observed functional response. DAPK-1 inhibition led by 6d together with its pro-apoptotic properties against the Jurkat line makes it an interesting candidate to further investigate the role of DAPK1 kinase in triggering apoptosis in cancer cells, a role which is attracting recent interest.

Full text of DOI:10.1039/c5ob00230c

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of 6,8,9 poly-substituted purine
analogue libraries as pro-apoptotic inducers of
human leukemic lymphocytes and DAPK-1
inhibitors†
Cite this: Org. Biomol. Chem., 2015,
13, 5224
Maria J. Pineda de las Infantas,*a Sara Torres-Rusillo,b Juan Diego Unciti-Broceta,c
Pablo Fernandez-Rubio,b Maria Angelica Luque-Gonzalez,a,c Miguel A. Gallo,a
Asier Unciti-Broceta,d Ignacio J. Molina*‡b and Juan J. Diaz-Mochon*‡a,c
A 18-member library of 6,8,9-poly-substituted purines was prepared from pyrimidines, primary alcohols,
and N,N-dimethylamides under basic conditions via a novel one-pot synthetic pathway controlled by
amide sizes and the novel analogues were tested against two leukemia cell lines: Jurkat (acute T cell
leukemia) and K562 (chronic erythroleukemia) cells. Compounds having a benzoxy group at C6 position
of the aromatic ring exhibited antiproliferative activity in Jurkat cells whereas all compounds induced a
lower effect on K562 cells. Analysis of cell cycle, Annexin-V staining, and cleavage of initiator caspases
assays showed that the active purine analogues induce cell death by apoptosis. Based on these results, a
new purine derivative was synthesized, 6-benzyloxy-9-tert-butyl-8-phenyl-9H-purine (6d), which dis-
played the highest activity of the series against Jurkat cell lines. Finally, 33P-radiolabeled kinase assays
using 96 recombinant human kinases known to be involved in apoptotic events were performed. Just
one of the kinases tested, DAPK-1, was inhibited 50% or more by the phenotypic hits at 10 µM, suggesting
that the inhibition of this target could be responsible for the induction of cell death by apoptosis. In
agreement with the phenotypic results, the most active antiproliferative agent, 6d, displayed also the
lowest IC50 value against recombinant DAPK1 (2.5 µM), further supporting the potential role of this protein
on the observed functional response. DAPK-1 inhibition led by 6d together with its pro-apoptotic pro-
perties against the Jurkat line makes it an interesting candidate to further investigate the role of DAPK1
kinase in triggering apoptosis in cancer cells, a role which is attracting recent interest.
Received 3rd February 2015,
Accepted 25th March 2015
DOI: 10.1039/c5ob00230c
www.rsc.org/obc
1. Introduction
The new paradigm of anticancer therapy is the development of
new drugs that enable personalized medicine. Targeted
aDepartamento de Química Farmacéutica y Orgánica. Facultad de Farmacia,
Universidad de Granada, Campus de Cartuja s/n, 18011 Granada, Spain.
E-mail: juanjose.diaz@genyo.es, mjpineda@ugr.es
bInstituto de Biopatología y Medicina Regenerativa, Centro de Investigación
Biomédica, Universidad de Granada, Parque Tecnológico de Ciencias de la Salud
(PTS), Avda. del Conocimiento s/n, 18016 Armilla, Granada, Spain.
E-mail: imolina@ugr.es
cCentro Pfizer - Universidad de Granada - Junta de Andalucía de Genómica e
Investigación Oncológica (GENYO), Parque Tecnológico de Ciencias de la Salud
therapy, which encompasses therapies with antibodies and
with small-molecule drugs, has that idyllic aim. In contrast to
classic cytotoxic chemotherapies, which killed rapidly dividing
cells, targeted therapies are focused to tackle essential bio-
chemical pathways, acting against the progression and pro-
liferation of tumour cells.8 Biomolecules, which participate in
these essential pathways, are also present in normal tissues,
but they are either mutated or overexpressed in tumour tissues
if compared with normal ones. This makes them excellent
molecular candidates to be targeted. In this particular area,
protein kinases can be considered cornerstones of many
of these cancer molecular mechanisms.26 Protein kinase
networks control cellular signalling pathways and are key
elements in tumour development and progression which use
ATP, a purine derivative, to transfer phosphate groups.14
(PTS), Avenida de la Ilustración 114, 18016 Granada, Spain
dEdinburgh Cancer Research UK Centre, MRC Institute of Genetics and Molecular
Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, UK
†Electronic supplementary information (ESI) available: Experimental synthetic
protocols and characterization data, including NMR data (1H and 13C) and mass
spectrometry data (HRMS) of compounds. Raw data of the 33P radiolabeled
kinase assay containing the list of the 96 kinases was used for IC50
_
DAPK1
graphs. See DOI: 10.1039/c5ob00230c
‡IJM and JJD-M share credit for senior authorship.
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Purine derivatives are synthetic compounds that mimic their
endogenous counterparts acting as antimetabolites. These
kinds of antimetabolites interrupt cellular processes by inter-
fering with essential enzymes involved in the intracellular
signalling, the tyrosine kinases.10
Tackling kinases by small-molecules is therefore a very
attractive way to both develop new antitumor drugs and to elu-
cidate new molecular pathways involved in cancer.25 In fact, at
least fourteen small-molecules which act as kinase inhibitors
have been approved over the last few years by the FDA for use
as antitumor drugs in humans.19
A total of 518 proteins have been characterized up to date
as kinases, forming the so-called human kinome.15 This
means that there is still room for finding both drug targets
and biological pathways related to cancer which remain
unknown. To confront this challenge, new small-molecule
libraries need to be prepared and used to interrogate kinase
activity in cellular and biochemical systems. As most small-
molecule kinase inhibitors target the conserved adenosine
triphosphate (ATP)-binding site, purine libraries decorated
with different substituents are expected to have a high prob-
ability of yielding bioactive compounds and kinase inhibitors,9
and consequently, purine-derived compounds have been
subject to vast exploration in both heterocyclic and medicinal
chemistry.
The aim of our research was to develop a drug discovery
workflow process divided step-wise into: (i) synthesis of poly-
substituted purine libraries via novel synthetic pathways deve-
loped by our group; (ii) in vitro cell-based assays using human
erythroleukemic cells and leukemic T lymphocytes to select
hits based on their antiproliferative activity and to determine
the mechanism(s) by which cell proliferation is affected; and
(iii) target deconvolution by searching potential molecular
targets known to be involved in pathways leading to the
observed antiproliferative effects.
Scheme 1 Synthetic routes to generate purine types “A” or “B”. Size of
R1 controls which synthetic mechanism is followed under the same
reaction conditions.
dimethylacetamide, respectively. When steric hindrance of
amides increase with the use of N,N-dimethylbenzamide or
N,N-dimethylpropionamide, Route A is then impeded and a
metal-free tandem alcohol oxidation/annulation reaction
occurs giving rise to Route B products. In this case, the
purine’s C8 substituent originates from the primary alcohols
used in the reactions. Therefore, competition between alkoxy-
iminium formation and metal-free oxidative coupling of
primary alkoxides and diaminopyrimidines with Schiff base
formation and subsequent annulation can be controlled. In
both routes, SNAr of the chloro atom at C6 by alkoxides takes
place and, along with the R3 group of the starting pyrimidine,
implements structural diversity in a one-pot reaction that over-
comes the low yields found in some of the reactions. Fig. 1
shows the set of 18 purines presented here which were pre-
pared by either Route A or B. Therefore, this synthetic platform
allows creating a diversity of purine analogues in a parallel
and straightforward fashion using a variety of amides and
alcohols with different pyrimidines under the same reaction
conditions.
2. Results and discussion
2.1. Chemistry
Our group has developed new one-pot synthetic ways to
prepare 6,8,9-substituted purines from 6-chloro-4,5-diamino-
pyrimidines using alcohols and N,N-dimethylamides under
basic and metal-free conditions (see ref. 2 and Pineda de las
Infantas et al., submitted). Depending on the size of the R1
group of the amides employed to carry out the chemical reac-
tions (Scheme 1), two different synthetic pathways can take
2.2. Biological activity
2.2.1. Antiproliferative activity of compounds 5a–n and
place, resulting in purine analogues whose substituent at C8 6a–c. The panel of small molecules was incubated with two
comes either from the amide (Route A) or the alcohol’s carbon haematopoietic tumour cell lines: Jurkat (acute T cell leukae-
chains (Route B). We have just reported these findings else- mia) and K562 (chronic erythroleukaemia). Cells were cultured
where to explain these competing reactions (Pineda de las for 48 h in the presence of increasing concentrations of each
Infantas et al., submitted).
compound: 50 µM and 100 µM (ESI Fig. 1†) and 25 µM and
Briefly, Route A proceeds via in situ generation of alkoxy- 200 µM (not shown) and cell viability assessed by the MTT col-
iminium species, which are created by reaction between orimetric screening assay. Results shown in ESI Fig. 1† are
amides and alkoxides, leading to purine derivatives with either expressed as percentage of inhibition of cell proliferation cal-
H or methyl in C8 when using N,N-dimethylformamide or N,N- culated over the values obtained with untreated cells. Prolifer-
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Table 1 IC50 values of indicated compounds assayed by inhibition of
cell proliferationa
Compound
Jurkat (µM)
K562 (µM)
5e
5i
6a
205
152
63
>500
>500
154
a IC50 values were calculated over a lineal regression line of specific
inhibition of proliferation values in a MTT assay of cells treated for
48 h with increasing amounts of compounds.
activity of the assayed compounds, thus confirming the
general tendency of this library.
2.2.2. Study of the mechanism responsible for inhibiting
cell proliferation. Cell cycle analyses were performed to deter-
mine whether the antiproliferative effect was a consequence of
cell cycle arrest, induction of apoptosis, or death by necrosis.
Jurkat and K562 cells were treated for 48 h with each com-
pound (5e, 5i and 6a) at different concentrations and the cell
cycle profile was determined by flow cytometry after staining
of nuclear DNA with propidium iodide. Treatment of Jurkat
cells with the chosen sub-library of compounds resulted
in accumulation of cells within the sub-G1 region18 due to
DNA fragmentation and, consequently, loss of nuclear DNA
content, which is consistent with apoptotic populations. The
three compounds induced cell death in Jurkat cells in a dose-
dependent manner (Fig. 3). Interestingly, the G0/G1 peak was
greatly diminished after treatment of cells with our com-
pounds, thus suggesting that no cell cycle arrest was involved.
Flow cytometry profiles of K562 cells (ESI Fig. 2†) showed a
lower cell population accumulating within the sub-G1 region
when comparing the effect induced by each compound to
Fig. 1 Set of 18 purines prepared through either Route A or B (see
Scheme 1).
ation of Jurkat T cells was inhibited at levels close or superior
to 50% by a number of the analogues (ESI Fig. 1†), whereas
the K562 erythroleukaemic cells were significantly more resist-
ant to the action of the compounds (not shown). From this
initial set of data, a sub-pool of compounds which inhibited
cell proliferation by 50% or more were chosen for further pheno-
typic characterization (5e, 5i, and 6a; Fig. 2). Interestingly,
the three of them presented a benzoxy group at purine’s C6
position.
Dose–response curves in Jurkat and K562 cell lines were
determined for the chosen derivatives and their IC50 values
calculated (Table 1), highlighting compound 6a as the stron-
gest antiproliferative hit. These IC50 demonstrate that K562
cells are more resistant than Jurkat cells to the antiproliferative
Fig. 3 Effect of compounds 5e, 5i and 6a on cell cycle. A representative
experiment showing Jurkat cells incubated for 48 h in the presence of
indicated compounds and doses. Cell cycle subpopulations were ana-
lyzed by flow cytometry after permeabilisation and staining of cells with
propidium iodide. Additional controls containing equivalent amounts of
DMSO used as drug vehicle were included, showing no toxicity in any
case (not shown). Percentage of cells within the sub-G1 region are indi-
cated in the plots.
Fig. 2 Structure of purine analogues 5e, 6a and 5i, which were chosen
for detailed functional screening.
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Fig. 5 Effect of indicated compounds on primary lymphocytes and
Jurkat cells. Primary cell line N1 and Jurkat cells were treated for 48 h at
the indicated doses of compounds 5e, 5i and 6a and cell cycles were
analysed by flow cytometry after staining with propidium Iodide. Graphs
represent the mean
S.D. of cells gated within the sub-G1 region
obtained from three independent experiments. UT: Untreated.
Fig. 4 Membrane phosphatidylserine expression after treatment of
Jurkat cells with compounds 5e, 5i and 6a. Jurkat cells were treated for
48 h with the indicated compounds and doses and the percentages of
apoptotic cells was measured by flow cytometry after double staining
with propidium iodide and Annexin-V-Fluos. Dot plots represent the
results of a typical experiment. Untreated cells and cells exposed to Eto-
poside were included as negative and positive controls.
mediate doses, this could be of importance in the event of a
future therapeutic development of this family of compounds.
To gather further direct evidence that treatment of Jurkat
cells with the selected compounds resulted in programmed
cell death by apoptosis, caspase activation events were ana-
lysed by Western Blot.
Caspases are a series of cysteine-proteases that cleave
certain proteins after specific aspartic acid residues.23
Caspases are expressed as inactive proenzymes that become
activated after being cleaved by their preceding caspase in a
cascade-like mechanism.16 Initiator caspases (8 and 9) trigger
effector caspases (3, 6 and 7) and, therefore, cleavage of
initiator caspases is an early event of the biochemical events
leading to apoptosis. The appearance of cleavage products
(Fig. 6) of caspases 8 and 9 indicates that apoptosis was trig-
those observed on Jurkat cells, suggesting that the former are
more resistant to death induced by compounds 5e, 5i and 6a.
To further analyse the cell death mechanism(s) we used
Annexin V, a calcium-dependent phospholipid-binding protein
with a high affinity for phosphatidylserine (PS), which is a dis-
tinct feature of early stages of apoptosis.12 Fig. 4 shows a repre-
sentative flow cytometry dot plot of double stained cells with
propidium iodide and Annexin-V. As expected, untreated
Jurkat cells (upper left-hand panel) presented a negligible
apoptotic cell population (right-hand gates).
Etoposide was used as a positive inductor of apoptosis
(lower left-hand panel), and two populations of cells arise after
exposure to this agent: early apoptotic cells (Annexin-V+/PI−)
and late apoptotic cells (Annexin-V+/PI+). In Jurkat cells treated
with 5e, 5i and 6a at the indicated doses, transition behaviour
from early to late apoptotic stages was observed, confirming
that these compounds induce death cell by apoptosis in a
dose-dependent manner (Fig. 4).
To address whether or not our compounds had a preferen-
tial activity on tumour cells, we directly compared their action
on two cell lines belonging to the same lineage: Jurkat and N1,
both CD4+ T cells. The latter cells are a long-term allospecific
primary cell line established in our laboratory by weekly allosti-
mulation of peripheral blood lymphocytes with the Class II+
Raji B cell line in the presence of rIL-2.24 We treated both cells
with our compounds and assessed their effect on the cell
cycle. Although these compounds, particularly 6a, have a dis-
creet cytotoxic effect on primary T lymphocytes at high doses,
the overall activity of our compounds is significantly lower on
primary lymphocytes than that exerted over tumour cells
(Fig. 5). Since primary T cells remain viable at low and inter-
Fig. 6 Study of early signaling events of apoptosis induced by com-
pound 6a in Jurkat cells. Western Blot analysis of total lysates of Jurkat
cells after treatment with either compound 6a (200 µM, left-hand lanes)
or equivalent amount of vehicle (DMSO, right-hand lanes) for 5, 6 or 7 h.
Membranes were probed with antibodies against initiator caspases 8 or
9, using actin as loading control. Cells treated with Etoposide were
included as positive control. Arrows indicate cleavage products (43/41
kD bands for caspase 8 and 37/< kD bands for caspase 9), which are
detected after caspase activation whereas upper bands correspond to
native proteins. Similar results were obtained after treating cells with
compounds 5e and 5i (not shown). NT: not treated.
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Fig. 7 Structure of compound 6-benzyloxy-9-tert-butyl-8-phenyl-9H-
purine 6d.
gered in cells treated with compound 6a. Similar effects were
observed with compounds 5e and 5i (not shown).
2.2.3. Synthesis and biological evaluation of compound
6d. The pro-apoptotic activity of compound 6a prompted us to
design and synthesize a new derivative, compound 6d (Fig. 7),
by replacing the isopropyl group found at N9 position of the
aromatic ring for a tert-butyl group. These groups are known to
bind lipophilic pockets of enzymes, improving generally inhi-
biting activities of small-molecules. With this structure in
mind, we decided to apply the novel one-pot synthetic route
developed (Scheme 1, Route B) using benzyl alcohol, N,N-di-
methylpropanamide and 5-amino-4-tert-butylamino-6-chloro-
pyrimidine in the presence of NaH. Following synthesis,
purification and characterization, Jurkat and K562 cells were
incubated for 48 h with compound 6d and cell viability
assessed by MTT. Evident dose-dependent curves of inhibition
of cell proliferation were detected after exposure of cells to
purine 6d, allowing calculation of its IC50 values against Jurkat
(Fig. 8, solid points) and K562 cells on a 11-point 1 : 2
serial dilution, yielding values of 29 2.4 µM for Jurkat and
120 10 µM for K562.
Fig. 9 (A) Effect of compound 6d on the cell cycle of Jurkat cells.
Jurkat cells were treated for 48 h with 6d at indicated concentrations or
their equivalent amounts of DMSO used as vehicle. Cells gated on the
sub-G1 region after staining with propidium iodide are indicated. (B)
Membrane phosphatidylserine expression after treatment of Jurkat cells
with compound 6d. Jurkat cells were treated for 24 h with compound
6d and the indicated doses and the percentages of apoptotic cells was
measured by flow cytometry after double staining with propidium iodide
and Annexin-V-Fluos. Dot plots represent the results of a typical experi-
ment. Untreated cells and cells exposed to Etoposide were included as
negative and positive controls. (C) Percentages of apoptotic cells after
treatment of Jurkat cells with compound 6d. Graph summarizes values
of percentages of apoptotic cells and SEM obtained from three indepen-
dent experiments. UT: Untreated. (D) Activation of initiator caspases 8
and 9 after cell treatment with compound 6d. Western Blot analysis of
total lysates from Jurkat cells after treatment with either compound 6d
(200 µM, right-hand lanes) or vehicle (DMSO, left-hand lines) for 2, 4
and 6 h. Membranes were probed with antibodies against initiator cas-
pases 8 or 9, using actin as loading control. Arrows indicate cleavage
products (43/41 kD bands for caspase 8 products and 37/< kD bands for
caspase 9 ones) after caspase activation whereas upper bands corres-
pond to native proteins (caspase 8 correspond to 55/57 kD band and
caspase 9 to 45 kD band).
To determine the effects of the new compound, cell cycle
analyses of Jurkat (Fig. 9, panel A) and K562 cells (ESI Fig. 3†)
were carried out after exposure of cells to purine 6d. Treatment
of Jurkat cells for 24 h resulted in cell death induction which
was determined by the percentage of cells accumulated in the
sub-G1 fraction (Fig. 9, panel A). Consistent with results
obtained with compounds 5e, 6a and 5i, K562 cells showed
increased resistance to induction of cell death after treatment
with 6d (ESI Fig. 3†), which also agrees with the lower anti-
proliferative effect observed for this cell line.
As described above for other compounds, the mechanism
by which 6d induces death in Jurkat cells was further studied.
Again, Annexin-V flow cytometric analysis of cells treated with
compound 6d (Fig. 9, panel B) showed a stronger apoptotic-
induced effect than those triggered by compounds 5e, 5i and
6a (Fig. 4).
The capacity of compound 6d to induce activation of cas-
pases was then assessed by Western Blotting (Fig. 9, panel C).
Treatment of Jurkat cells with 6d for 2, 4 and 6 h yielded
similar results to those shown in Fig. 5, clearly showing the
Fig. 8 IC50 value of compound 6d in Jurkat cells as determined by inhi-
bition of cell proliferation. A 11-point 1 : 2 serial dilution of compound
6d, starting at 800 µM, was used to determine its IC50 value by the MTT
assay after treatment of Jurkat cells for 48 h (solid circles). Equivalent
amounts of DMSO used as vehicle in each dilution were set as negative
controls (open circles). Results indicate percentages of cell proliferation
over the results obtained with untreated cells. Data are expressed as
mean SEM.
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expected cleavage products of both caspases 8 and 9 as early
as 2 h of treatment.
Table 2 IC50 values of indicated compounds calculated as inhibition of
DAPK1 enzymatic activity
To further confirm that the cytotoxic effect exerted by
compound 6d was strictly dependent upon caspase activation,
we pre-treated Jurkat cells with the pan-caspase inhibitor
Q-VD-OPh, which is known to be a potent and irreversible
blocker of cell death mediated by apoptosis with reduced tox-
icity.3 Fig. 10 shows that pre-treatment of Jurkat cells with the
pan-caspase inhibitor inhibits almost completely apoptosis of
Jurkat cells induced by compound 6d, as analysed by cell cycle
staining with propidium iodide (Fig. 10 A, bottom panels). Fur-
thermore, this effect is clearly specific as Western Blot analysis
shows that the caspase-8 cleavage products strongly observed
after treatment of Jurkat cells with compound 6d are absent in
those cells pre-treated with the pan-caspase inhibitor (Fig. 10,
panel B). As expected, cells treated with the Q-VD-OPh alone
showed no toxicity (Fig. 10A, upper right panel).
Compound
DAPK1 (µM)
5e
5i
6a
6d
23
5.9
NDa
2.5
a The IC50 curve for 6a against DAPK1 presented a Gaussian profile,
which suggested inconsistent solubility of this compound and
therefore impeded a precise calculation of the IC50 dose in this system
(see ESI Fig. 4–7). Whereas both 6a and 6d present very similar c log P
values, the added steric behavior of tert-butyl groups was expected to
also influence its solubility properties. Furthermore, the sigmoid curve
obtained on the MTT assay (Fig. 6) is a strong indication of the
improved solubility of compound 6d.
2.2.4. Kinase screening assay. Since both cell cycle studies
and caspase 8/9 activation demonstrated that the chosen sub-
family of compounds triggered apoptosis, a panel of kinases
from the human kinome were selected. The selection criteria
were based on their known involvement in apoptotic pro-
cesses. Both 5e and 6a purines were chosen as representative
compounds to perform the assay in order to see if this family
of purines, presenting a benzoxy group at C6 position, were
capable of inhibiting kinases. Hence, 96 recombinant kinases
were used to run a 33P-radiolabeled kinase assay profiling
using a single-dose duplicate mode at a 10 µM purine concen-
tration with 10 μM ATP. Surprisingly, just one kinase out of the
96 tested showed a significant reduction in kinase activity by
both compounds. This kinase was Death Associated Protein
Kinase-1 (DAPK1) which presented a percentage of activity of
50.9% and 44.3% when incubated with compounds 5e and 6a,
respectively. Compounds 5e, 5i, 6a and 6d purines were then
tested against DAPK1 via a 33P-radiolabeled kinase assay using
10-doses to determine their IC50 (DAPK1) values (Table 2).
This assay revealed that our compounds inhibited DAPK1
at the low micromolar level. This result is remarkable since an
elegant analysis where 178 small-molecules kinase inhibitors
were assayed against 300 recombinant protein kinases1
showed that DAPK1 has 0.03 as Selective Score 50, one of the
lowest for any of the kinases included in that study. This
meant that just 5 out of the 178 compounds tested at 10 µM by
this group against DAPK1 inhibited this kinase more than
50%, evidence of how unusual it is to hit this target. Moreover,
these 5 compounds were very promiscuous, targeting many
other kinases. While more studies are needed in order to link
their pro-apoptotic features with their DAPK1 inhibition, our
results indicate that these derivatives do not inhibit other
kinases known to be involved in apoptotic events. Further-
more, our data supports recent findings by Schneider–Stocke-
mail4 that for the first time reported that selective inhibition
of DAPK1, an enzyme that plays a critical role in interferon-
γ-mediated apoptosis,5–7,27 might impinge upon the biology of
tumour cells via STAT3 activation. The relevant role of DAPK1
in pro-apoptotic pathways is underlined by the fact that many
These results are clear evidence that activation of caspases
is an absolute requirement for the cytotoxic effect mediated by
compound 6d, thereby confirming that cell death occurs by
apoptosis.
Fig. 10 Pre-treatment of Jurkat cells with the pan-caspase inhibitor
Q-VD-OPh abrogates cell dead mediated by compound 6d. (A) Jurkat
cells were pre-treated for 1 h with 50 µM of the Q-VD-OPh pan-caspase
inhibitor and cultured thereafter for 24 h in the presence of 200 µM of
compound 6d (bottom left panel). Controls include untreated cells
(upper left panel); cells treated with Q-VD-OPh alone (upper right
panel) or only with compound 6d (bottom left panel). Cells were stained
with propidium iodide and cell cycles analysed by flow cytometry. Per-
centages of cells within the sub-G1 region are indicated in the plots. (B)
Western Blot analysis of caspase-8 activation. The same cultures of cells
examined in Panel A were collected 8 h after treatment with compound
6d and cytosolic lysates were resolved by SDS-PAGE and membranes
probes with the anti-caspase 8 mAb. Actin was used as loading control.
UT: Untreated.
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chronic lymphocytic leukaemia cells down-regulate the could shed light on the exact participation of DAPK1 in tumor
expression of the protein to favour tumour progression.11,20 development. Furthermore, the fact that primary T lympho-
Our findings, as well as those of Schneider–Stockemail,4 may cytes are relatively refractory to cytotoxicity induced by our
thus be surprising if we only view DAPK1 as a pro-apoptotic compounds makes it worthwhile to pursue further studies
kinase. The pro-apoptotic activity of the enzyme requires the aiming to explore DAPK1 as a potential therapeutic target in
integrity of the Death Domain,6 but growing evidence indicates certain tumors.
that post-transcriptional modifications may confer opposing
functions to the protein. For instance, autophosphorylation
restrains the apoptotic activity of members of the DAPK
family22 and disruption of the cathepsin B-DAPK1 interaction
4. Experimental procedures
4.1. Synthesis and characterization of compounds
breaks a multiprotein complex whose perturbation sensitizes
cells to apoptosis.13 Since our compounds have a preferential
activity on Jurkat cells when compared to K562, a cell line with
low expression of DAPK-1, it can be speculated that the mech-
anism of action for the observed pro-apoptotic effects after
inhibiting DAPK1 activity may be related to posttranscriptional
mechanisms resulting in sensitizing treated cells to death
by apoptosis. The precise mechanisms involved, however, need
further investigations which will answer the question of
whether targeting DAPK1 is a sound strategy for the treatment
of certain types of tumours.
4.1.1. General. Reaction courses and products mixtures
were routinely monitored by TLC on silica gel (Merck
60–200 mesh). Melting points were determined on a Stuart
Scientific SMP3 apparatus and are uncorrected. 1H-NMR
spectra were obtained in CDCl3, CD3OD solutions on a Varian
Inova Unity (300 MHz) and Varian Direct Drive (400 MHz and
500 MHz). Chemical shifts (δ) are given in ppm upfield from
tetramethylsilane. 13C-NMR spectra were obtained in CDCl3,
CD3OD solutions on a Varian Direct Drive (125 MHz). All pro-
ducts reported showed 1H-NMR and 13C-NMR spectra in agree-
ment with the assigned structures. Mass spectra were obtained
by electrospray (ESY) with a LCT Premier XE Micromass Instru-
ment (high resolution mass spectrometry).
3. Conclusions
To overcome the disadvantages of target-centric drug discovery
4.1.2. General procedure for the preparation of compounds
4(a–c). To commercially available 4,6-dichloro-5-nitropyr-
imidine (10 mmol, 1 equiv.), in anhyd. THF (15 ml) was added
NaHCO3 (11 mmol, 1.1 equiv.). The mixture was then heated
to 55 °C and at this temperature the appropriate amine
(11 mmol, 1.1 equiv.) dissolved in THF was added dropwise.
After 1 h, the mixture was cooled at room temperature, filtered
and evaporated to give a crude product, which was directly
used for the next step without purification.
This crude solution (5 mmol, 1 equiv.) in EtOH (10 ml) con-
taining SnCl2·2H2O (25 mmol, 5 equiv.), was refluxed for 1 h,
monitoring the reaction by TLC. The mixture was then cooled
at room temperature and NaHCO3 was added until pH 8 was
reached. After two extractions with EtOAc (10 ml for each one),
the organic phase was washed with an aq. saturated solution
of NaCl (2 × 25 mL) and dried with Na2SO4. Evaporation under
vacuum gave a solid that was purified by column chromato-
graphy eluting with ethyl acetate–petroleum ether solutions.
4.1.2.1. 5-Amino-6-chloro-4-isopropylaminopyrimidine (4a).
White solid, yield 73%; mp 185–186 °C. δH (400.58 MHz,
CDCl3): 8.08 (1H, s, NCHN), 4.68 (1H, bs, NH), 4.29 (1H, m,
CH(CH3)2), 3.33 (2H, bs, NH2), 1.27 (6H, d, J = 8, CH(CH3)2.
4.1.2.2. 5-Amino-4-tert-butylamino-6-chloropyrimidine (4b).
White solid; yield 75%; mp 135 °C. δH (499.79 MHz, CDCl3):
8.03 (1H, s, NCHN), 4.87 (1H, bs, NH), 3.45 (2H, bs, NH2), 1.47
(9H, s, C(CH3)3). ES+HRMS: Calculated M + H = 201.0907.
C8H14N4Cl. Obtained: 201.0909.
strategies and promote
a more empirical, evidence-led,
approach to anticancer drug discovery, novel derivatives were
directly tested in disease relevant leukemia cell lines. In order
to obtain purine analogues, novel chemistry pathways develo-
ped by our group and based on one-pot synthesis were carried
out. The novel chemistry allows obtaining 6,8,9 poly-substi-
tuted purines starting from diamino-pyrimidines and a range
of different primary alcohols and amides. Following this
approach, a series of 18 purine analogues exhibiting antiproli-
ferative activity and pro-apoptotic properties on the acute T
cell leukemia Jurkat cell line were identified, all of them
having a benzyloxy group at C6 position and 9-tert-butyl-
6-(benzyloxy)-8-phenyl-9H-purine (6d) being the most active
one displaying a IC50 value of 29 µM on Jurkat cells. Cell cycle
analyses, Annexin-V staining, cleavage of initiator caspases
and blocking of the cytotoxic effects after pre-incubation of
cells with a pan-caspase inhibitor are cumulative evidence
clearly showing that cell death was induced by apoptosis.
Finally, to assess if their pro-apoptotic activities were due
to inhibition of multiple kinases, 96 kinases known to be
involved in apoptosis processes were assayed against these
purines. Surprisingly, just one kinase, DAPK1, was inhibited at
significant levels, with the 6d purine compound being the one
showing the lowest IC50_DAPK1 (2.5 µM). Compound 6d is there-
fore the first selective inhibitor of DAPK1. Since all other avail-
able inhibitors target multiple protein kinases, this makes it
difficult to dissect precisely the role of DAPK1 on certain bio-
4.1.2.3. 5-Amino-4-benzilamino-6-chloropyrimidine
(4c).
logical processes. Among these, growing evidence of the role White solid; yield 65%; mp 205–206 °C. δH (499.79 MHz,
of DAPK128 on cancer processes is appearing and our results CD3OD): 7.78 (1H, s, NCHN), 7.35–7.22 (5H, m, Ph), 4.69 (2H,
tend to support this. Therefore, the use of a selective inhibitor s, CH2Ph). δC (125.68 MHz, CDCl3): 150.13, 142.76, 137.79,
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132.02, 131.18, 130.72, 127.49, 48.51. ES+HRMS: Calculated NCHN), 7.88 (1H, s, NHCHN), 7.34–7.28 (5H, m, Ph), 5.41 (2H,
M + H = 235.0750. C11H12N4Cl. Obtained: 235.0753. s, CH2Ph), 4.66 (2H, q, J = 10 Hz, OCH2CH3), 1.51 (3H, t, J = 10
4.1.3. General procedure for the preparation of compounds Hz, OCH2CH3). δC(125.68 MHz, CDCl3): 160.93, 152.33, 141.81,
5(a–n). Synthesis A. A suspension of NaH (50%) in mineral oil 135.34, 129.08, 128.27, 127.75, 121.39, 63.16, 47.42, 14.53.
(3.7 mmol, 10 equiv.) was dissolved in a mixture cooled at 0 °C ES+HRMS: Calculated M + H = 255.1246 C14H15N4O. Obtained:
constituted both from the appropriate alcohol (3.7 mmol, 255.1252.
4.1.3.7. 9-Benzyl-6-ethoxy-8-methyl-9H-purine
(5g). White
10 equiv.) and amide (18.5 mmol, 50 equiv.). The solution was
stirred at room temperature for 30 min and then at 90 °C for
the same time. At 90 °C compounds 4(a–c) (0.37 mmol,
1 equiv.), dissolved in the appropriate amide (18.5 mmol,
50 equiv.) were added dropwise and the mixture heated for
24 h. The reaction mixture, quenched to pH 7 with an aq. satu-
rated NH4Cl solution, was then extracted with CH2Cl2 (3 ×
15 mL), the combined organic extracts dried (Na2SO4) and the
solvent evaporated. The residue was chromatographed on
silica gel using ethyl acetate–petroleum ether solutions.
4.1.3.1. 6-Ethoxy-9-isopropyl-9H-purine (5a). Light yellow
solid; yield 44%. δH(499.79 MHz, CDCl3): 8.49 (1H, s, NCHN),
7.96 (1H, s, NHCHN), 4.88 (1H, m, CH(CH3)2), 4.64 (2H, q, J =
5 Hz, OCH2CH3), 1.60 (6H, d, J = 5 Hz, NCH(CH3)2), 1.49 (3H, t,
J = 5 Hz, OCH2CH3). δC(125.68 MHz, CDCl3): 160.81, 151.70,
139.53, 121.83, 62.98, 47.33, 22.60, 14.51. ES+HRMS: Calcu-
lated M + H = 207.1246. C10H15N4O. Obtained: 207.1245.
4.1.3.2. 6-Ethoxy-8-ethyl-9-isopropyl-9H-purine (5b). Yellow
viscous oil; yield 10%. δH(300.20 MHz, CDCl3): 8.47 (1H, s,
NCHN), 4.72 (1H, m, CH(CH3)2), 4.67 (2H, q, J = 6 Hz,
OCH2CH3), 2.96 (2H, q, J = 6, CH2CH3), 1.74 (6H, d, J = 6 Hz,
NCH(CH3)2), 1.53 (3H, t, J = 6 Hz, OCH2CH3), 1.47 (3H, t, J =
6 Hz, –CH2CH3). ES+HRMS: Calculated M + H = 235.1559.
C12H19N4O. Obtained: 235.1557.
solid; yield 41%; mp 56–58 °C. δH(499.79 MHz, CDCl3): 8.50
(1H, s, NCHN), 7.32–7.13 (5H, m, Ph), 5.40 (2H, s, CH2Ph), 4.65
(2H, q, J = 10 Hz, OCH2CH3), 2.51 (3H, s, CH3), 1.51 (3H, t, J =
10 Hz, OCH2CH3). δC(125.68 MHz, CDCl3): 159.83, 153.55,
151.51, 151.42, 135.49, 128.98, 128.10, 126.92, 120.33, 62.95,
46.08, 14.58, 14.39. ES+HRMS: Calculated M + H = 269.1402
C15H17N4O. Obtained: 269.1396.
4.1.3.8. 9-Benzyl-6-ethoxy-8-ethyl-9H-purine
(5h). White
viscous oil; yield 2%. δH(499.79 MHz, CDCl3): 8.50 (1H, s,
NCHN), 7.31–7.12 (5H, m, Ph), 5.43 (2H, s, CH2Ph), 4.68 (2H,
q, J = 10 Hz, OCH2CH3), 2.80 (2H, q, J = 10 Hz, CH2CH3), 1.53
(3H, t, J = 10 Hz, OCH2 CH3), 1.27 (3H, t, J = 10 Hz, CH2CH3).
ES+HRMS: Calculated M + H = 283.1559 C16H19N4O. Obtained:
283.1560.
4.1.3.9. 9-Benzyl-6-(benzyloxy)-9H-purine (5i). White solid;
yield 30%. δH(499.79 MHz, CDCl3): 8.59 (1H, s, NCHN), 7.90
(1H, s, NHCHN), 7.57–7.28 (10H, m, Ph), 5.70 (2H, s, OCH2Ph),
5.43 (2H, s, CH2Ph). δC(125.68 MHz, CDCl3): 161.51, 153.09,
142.91, 137.06, 136.17, 129.98, 129.72, 129.40, 129.31, 129.23,
128.99, 128.65, 127.84, 122.32, 69.31, 48.35. ES+HRMS: Calcu-
lated M + H = 317.1402. C19H17N4O. Obtained: 317.1407.
4.1.3.10. 6-Isopropoxy-9-isopropyl-9H-purine
(5j). Yellow
viscous oil; yield 10%. δH(300.20 MHz, CDCl3): 8.54 (1H, s,
NCHN), 7.99 (1H, s, NHCHN), 5.70 (1H, m, OCH(CH3)2), 4.91
(1H, m, NCH(CH3)2), 1.66 (6H, d, J = 6 Hz, OCH(CH3)2), 1.51
(6H, d, J = 6 Hz, NCH(CH3)2). δC(125.68 MHz, CDCl3): 160.84,
151.87, 150.40, 139.60, 121.83, 70.68, 47.55, 22.89, 22.05.
4.1.3.3. 6-Ethoxy-9-isopropyl-8-methyl-9H-purine (5c). Yellow
viscous oil; yield 10%. δH(499.79 MHz, CDCl3): 8.44 (1H, s,
NCHN), 4.72 (1H, m, CH(CH3)2), 4.62 (2H, q, J = 10 Hz,
OCH2CH3), 2.64 (3H, s, CH3), 1.68 (6H, d, J = 5 Hz, NCH
(CH3)2), 1.50 (3H, t, J = 10 Hz, OCH2CH3). δC(125.68 MHz,
CDCl3): 159.74, 153.15, 151.75, 150.53, 120.71, 62.67, 48.31,
21.24, 15.22, 14.58. ES+HRMS: Calculated M + H = 221.1402.
C11H17N4O. Obtained: 221.1396.
ES+HRMS: Calculated
Obtained: 221.1402.
M
+
H
=
221.1402.
C
11H17N4O.
4.1.3.11. 6-Isopropoxy-9-isopropyl-8-methyl-9H-purine
(5k).
Yellow viscous oil; yield 12%. δH(300.20 MHz, CDCl3): 7.99
(1H, s, NCHN), 5.70 (1H, m, OCH(CH3)2), 4.91 (1H, m, NCH-
(CH3)2), 2.07 (3H, s, CH3), 1.71 (6H, d, J = 6 Hz, OCH(CH3)2),
1.49 (6H, d, J = 6 Hz, NCH(CH3)2). ES+HRMS: Calculated M +
H = 235.1559.C12H19N4O. Obtained: 235.1553.
4.1.3.4. 6-(Benzyloxy)-9-isopropyl-9H-purine
(5d). White
solid; yield 38%; mp 167–169 °C. δH(499.79 MHz, CDCl3): 8.56
(1H, s, NCHN), 8.00 (1H, s, NHCHN), 7.56–7.30 (5H, m, Ph),
5.69 (2H, s, OCH2Ph), 4.91 (1H, m, CH(CH3)2), 1.64 (6H, d, J =
10 Hz, NCH(CH3)2). δC(125.68 MHz, CDCl3): 160.53, 151.63,
139.77, 136.27, 130.03, 128.41, 128.29, 128.05, 68.29, 47.43,
22.63. ES+HRMS: Calculated M + H = 269.1402. C15H17N4O.
Obtained: 269.1402.
4.1.3.12. 6-tert-butoxy-9-isopropyl-9H-purine
(5l). Yellow
viscous oil; yield 15%. δH(300.20 MHz, CDCl3): 8.51 (1H, s,
NCHN), 7.95 (1H, s, NHCHN), 4.90 (1H, m, NCH(CH3)2), 1.78
(9H, s, C(CH3)3), 1.65 (6H, d, J = 6 Hz, OCH(CH3)2). ES+HRMS:
Calculated M + H = 235.1559. C12H19N4O. Obtained: 235.1563.
4.1.3.5. 6-(Benzyloxy)-9-isopropyl-8-methyl-9H-purine
(5e).
4.1.3.13. 9-Isopropyl-6-methoxy-9H-purine
viscous oil; yield 10%. δH(400.57 MHz, CDCl3): 8.53 (1H, s,
NCHN), 7.98 (1H, s, NHCHN), 4.89 (1H, m, CH(CH3)2), 4.18
(5m). White
White solid; yield 16%. δH(499.79 MHz, CDCl3): 8.47 (1H, s,
NCHN), 7.54–7.30 (5H, m, Ph), 5.65 (2H, s, OCH2Ph), 4.75 (1H,
m, CH(CH3)2), 2.65 (3H, s, CH3), 1.69 (6H, d, J = 10 Hz, NCH-
(CH3)2). δC(125.68 MHz, CDCl3): 159.41, 153.57, 150.69, 150.41,
136.42, 128.43 128.35, 127.98, 120.73, 68.06, 48.35, 21.23,
15.22. ES+HRMS: Calculated M + H = 283.1559. C16H19N4O.
Obtained: 283.1558.
(3H, s, OCH3), 1.63 (6H, d,
J = 4 Hz, NCH(CH3)2).
δC(125.68 MHz, CDCl3): 161.19, 152.25, 151.86, 139.89, 122.07,
54.26, 47.63, 22.78. ES+HRMS: Calculated M + H = 193.1089.
C9H13N4O. Obtained: 193.1087.
4.1.3.14. 9-tert-butyl-6-ethoxy-9H-purine (5n). White solid;
yield 57%. δH(300.20 MHz, CDCl3): 8.49 (1H, s, NCHN), 7.97
4.1.3.6. 9-Benzyl-6-ethoxy-9H-purine (5f). White solid; yield
62%; mp 133–135 °C. δH(499.79 MHz, CDCl3): 8.55 (1H, s,
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(1H, s, NHCHN), 4.64 (2H, q, OCH2CH3), 1.80 (9H, s, NC- 4.2. Cells and culture
(CH3)3), 1.50 (3H, t, OCH2CH3). δC(75.49 MHz, CDCl3):
Jurkat (acute T cell leukemia) and K562 (chronic erythro-
leukaemia) cell lines were cultured at 37 °C in a humidified
atmosphere of 5% CO2 in RPMI 1640 medium (BioWhittaker,
Verviers, Belgium) supplemented with 10% Fetal Bovine
Serum (Gibco, Auckland, New Zealand) and 1% Glutamax
(Gibco) and Penicillin-Streptomycin (BioWhittaker). The N1
cell line are long-term primary CD4+ T lymphocytes derived
from a normal individual24 and expanded by weekly stimu-
lation of Mytomicin-C treated Class II+ Raji B lymphoma cells
in the presence of 50 U.I. ml−1 of rIL-2 (kindly provided by the
National Institutes of Health’s AIDS Reagent Program,
Bethesda, MD) as described in detail elsewhere.17
4.2.1 Cell proliferation assay. To analyze 5e, 5i and 6a
activity, cells were cultured in 96-well microtiter plates (50 000
cells per well) and treated for 48 h with the library of com-
pounds at increasing concentrations (25, 50, 100 and 200 μM).
Cell proliferation was measured with the MTT (3-[4-5 dimethyl-
thiazol-2-7P]-2,5-diphenyl tetrazolium bromide: thiazolyl blue)
(Sigma) colorimetric assay as described in detail elsewhere,21
and results are expressed as percentage of inhibition calcu-
lated over the values obtained with untreated cells and cor-
rected for the non-specific effect of the amounts of DMSO
contained in each sample of the compounds. Data are rep-
161.22, 151.20, 139.87, 123.16, 63.10, 29.30, 14.80. ES+-
HRMS: Calculated M + H = 221.1402. C11H17N4O. Obtained:
221.1400.
4.1.4. General procedure for the preparation of compounds
6(a–d). Synthesis B. A suspension of NaH (50%) in mineral oil
(3.7 mmol, 10 equiv.) was dissolved in a mixture cooled at 0 °C
constituted both for appropriate alcohol (3.7 mmol, 10 equiv.)
and
appropriate
corresponding
N,N-dimethylamide
(18.5 mmol, 50 equiv.), when the amide is liquid, or the
mixture is dissolved in dioxane, when the amide is solid. The
solution was stirred at room temperature for 30 min and then
at 90 °C for the same time. At 90 °C compound 4(a–b)
(0.37 mmol, 1 equiv.), dissolved in the amide (18.5 mmol, 50
equiv.) or in dioxane was added dropwise and the mixture
heated for 24 h. The reaction mixture, quenched to pH 7 with
an aq. saturated NH4Cl solution, was then extracted with
CH2Cl2 (3 × 15 mL), the combined organic extracts dried
(Na2SO4) and the solvent evaporated. The residue was
chromatographed on silica gel using ethyl acetate–petroleum
ether solutions.
4.1.4.1. 6-Benzyloxy-9-isopropyl-8-phenyl-9H-purine
(6a).
White solid; yield 24%. δH(300.20 MHz, CDCl3): 8.57 (1H, s,
NCHN), 7.70–7.38 (10H, m, Ph, CH2Ph), 5.72 (2H, s, OCH2Ph),
4.80 (1H, m, CH(CH3)2), 1.76 (6H, d, J = 6 Hz, NCH(CH3)2).
δC(125.68 MHz, CDCl3): 160.54, 153.14, 151.01, 136.66, 130.40,
129.80, 128.95, 128.76, 128.61, 128.27, 128.05, 122.14, 68.42,
50.04, 21.51. ES+HRMS: Calculated M + H = 345.1715.
C21H21N4O. Obtained: 345.1716.
4.1.4.2. 8-Ethyl-9-isopropyl-6-propoxy-9H-purine (6b). Yellow
viscous oil; yield 20%. δH(300.20 MHz, CDCl3): 8.43 (1H, s,
NCHN), 4.69 (1H, m, CH(CH3)2), 4.52 (2H, t, J = 9 Hz,
OCH2CH2CH3), 2.94 (2H, q, J = 6 CH2CH3), 1.92 (2H, m,
OCH2CH2CH3), 1.70 (6H, d, J = 10 Hz, NCH(CH3)2), 1.43 (3H, t,
J = 6 Hz, CH2CH3), 1.05 (3H, t, J = 9, OCH2CH2CH3).
δC(125.68 MHz, CDCl3): 160.28, 155.28, 150.65, 150.31, 124.41,
68.65, 48.57, 21.24, 23.13, 22.49, 21.51, 12.49, 10.66.
resented as mean
experiments.
standard deviation of the different
To analyze 6d activity, each well of a 96-well tissue culture
plate containing 50 µL from serial doubling dilutions of 6d
compound was inoculated with 50 µL of previously prepared
cell culture at 20 000 cells mL−1, with the exception of two
rows, which received medium only. Eleven different final con-
centrations of each compound, ranging from 0.8–800 µM, were
tested in the assay. Each concentration point was assayed in
three replicates, and experiments were repeated at least three
times. The same volume of DMSO, 6d solvent, used in each
concentration point was assayed in parallel as control. The
IC50 value was calculated using GraphPad Prism5 software.
ES+HRMS: Calculated
Obtained: 249.1721.
M
+
H
=
249.1715.
C
13H21N4O.
4.3. Analysis of cell cycle and Western Blotting
For the analysis of cell cycle, 250 000 cells per well were
cultured in 24-well plates and incubated with the chosen com-
pounds at the indicated doses for 48 h, as described else-
where.21 After treatment with the compounds, cells were
collected, fixed with 70% cold ethanol for 5 min at 4 °C and
then incubated with DNA extraction buffer (0.2 M Na2HPO4
and 0.004 M citric acid) at 37 °C for 10 min. Subsequently,
cells were re-suspended in PBS containing 100 µg ml−1 RNase
4.1.4.3. 6-Isobutoxy-8,9-diisopropyl-9H-purine
(6c). White
solid; yield 22%. δH(300.20 MHz, CDCl3): 8.44 (1H, s,
NCHN), 4.74 (1H, m, NCH(CH3)2), 4.36 (2H, d, OCH2Pri, J =
9 Hz), 3.28 (1H, m, CCH(CH3)2), 2.29 (1H, m, CH2CH-
(CH3)2), 1.72 (6H, d, J = 9 Hz, NCH(CH3)2), 1.47 (6H, d, J =
9 Hz, CCH(CH3)2), 1.06 (6H, d, J = 9 Hz, CH2CH(CH3)2).
ES+HRMS: Calculated
Obtained: 277.2036.
M + H = 277.2028. C15H25N4O.
4.1.4.4. 9-tert-butyl-6-(benzyloxy)-8-phenyl-9H-purine
(6d). and 40 µg ml−1 propidium iodide (PI) and incubated at 37 °C
White solid; yield 45%. δH(300.20 MHz, CDCl3): 8.59 (1H, s, for 30 min in the dark. Quantitative analysis of cells with sub-
NCHN), 7.58–7.34 (10H, m, Ph, CH2Ph), 5.69 (2H, s, OCH2Ph), G1 DNA content was carried out in a FACScan flow cytometer
1.70 (9H, s, NC(CH3)3). δC(125.68 MHz, CDCl3): 160.62, using the Cell Quest software (BD Biosciences). Data are
154.52, 153.37, 150.46, 136.58, 134.97, 130.11, 129.72, expressed as mean
Standard Error Mean (SEM) of three
128.85, 128.59, 128.27, 128.09, 121.76, 68.43, 61.06, 31.15. different experiments. For the analysis of caspase activation,
ES+HRMS: Calculated
Obtained: 359.1880.
M
+
H
=
359.1872.
C
22H23N4O. cells were treated with compounds at 200 μM for the indicated
times and lysed in 150 mM NaCl, 50 mM Tris-Cl pH 8.0, 1%
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NP-40 and protease inhibitors for 30 min. Lysates were
resolved by SDS-PAGE as described, blotted onto PVDF mem-
Acknowledgements
branes, blocked for 1 h and hybridized overnight with pre- We thank undergraduate students Alvaro Lorente Macias and
determined optimal concentrations of monoclonal antibodies Pilar Aguilar Martinez for their help with basic laboratory tech-
against human caspase-9 (R&D Systems; Minneapolis, MN) niques. JJDM thanks the Spanish Ministerio de Economia y
and caspase-8 (Alexis Biochemicals; San Diego, CA). Mem- Competitividad for funding a Ramon y Cajal Fellowship. AUB
branes were washed, incubated for 1 h with an HRP-labelled thanks Medical Research Council UK – Institute of Genetics
goat anti-mouse antibody and revealed by chemiluminescence and Molecular Medicine for funding an Academic Fellowship.
(ECL Advance Western Blotting Detection Kit, Amersham, GE We acknowledge the generous supply of rIL-2 provided by the
Healthcare UK). Light emission was detected with a digital National Institutes of Health AIDS Reagent Program. IJM is
imaging system (Fujifilm Image Analyzer LAS-4000, Tokyo, grateful for the support provided to his laboratory by grant
Japan) and analyzed with the Multi Gauge software. Where 12UDG01-ATF from Sparks, the children’s medical charity,
indicated, cells were pre-treated with the Pan Caspase London, UK.
OPH Inhibitor Q-VD (Q-Val-Asp(non-omethylated)-OPh) (R&D
systems, Minneapolis, MN) for 1 h prior to adding compound
6d and maintained throughout the culture.
References
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1020.
For the analysis of phosphatidylserine exposure, 250 000 cells
per well were cultured in 24-well plates and incubated with the
chosen compounds at the indicated doses for 48 h, as
described above. Etoposide was used as positive control and
untreated cells were used as controls of live cells. After treat-
ment, cells were collected and washed with annexin V binding
buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2)
and re-suspended in 100 µl of this buffer. Then, 2.5 µl of
Annexin V-FLUOS solution (Roche, Mannheim, Germany) was
added to each sample, followed by incubation for 20 min in
the dark at room temperature. After a final wash, the pellet
was re-suspended in annexin V binding buffer containing
10 µl of PI at 10 µg ml−1. Stained samples were immediately
5 O. Cohen, E. Feinstein and A. Kimchi, EMBO J., 1997, 16,
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analyzed by flow cytometry using
cytometer. Data were analyzed with the FlowJo software.
a FACSCalibur flow
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4.5. In vitro kinase screening assay
A panel of 96 recombinant kinases (see ESI†) was used to run
a
33P-radiolabeled kinase assay profiling against compounds
5e, 6a and 5i by Reaction Biology Corp. (Malvern, PA) accord-
ing to their established procedures. Compounds were tested in
a single-dose duplicate mode at a concentration of 10 µM.
Reactions were carried out at 10 μM ATP. Compounds were
tested in a 10-dose IC50 mode with 2-fold serial dilution start-
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before to determine their IC50 values against DAPK1 and
DAPK2.
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biological experiments. MAG contributed with reagents. 15 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and
MALG, MPI, AUB and JJDM analyzed the chemical data. AUB,
S. Sudarsanam, Science, 2002, 298, 1912–1934.
JJD and IJM interpreted the biological data and wrote the 16 D. R. McIlwain, T. Berger and T. W. Mak, Cold Spring
paper. IJM and JJDM directed the work.
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