Synthesis and screening of 6-alkoxy purine analogs as cell type-selective apoptotic inducers in Jurkat cells

Article abstract of DOI:10.1002/ardp.202100095

Purines are ubiquitous structures in cell biology involved in a multitude of cellular processes, because of which substituted purines and analogs are considered excellent scaffolds in drug design. In this study, we explored the key structural features of a purine-based proapoptotic hit, 8-tert-butyl-9-phenyl-6-benzyloxy-9H-purine (1), by setting up a library of 6-alkoxy purines with the aim of elucidating the structural requirements that govern its biological activity and to study the cell selectivity of this chemotype. This was done by a phenotypic screening approach based on cell cycle analysis of a panel of six human cancer cell lines, including T cell leukemia Jurkat cells. From this study, two derivatives (12 and 13) were identified as Jurkat-selective proapoptotic compounds, displaying superior potency and cell selectivity than hit 1.

Full text of DOI:10.1002/ardp.202100095

Received: 8 March 2021
Revised: 23 April 2021
Accepted: 22 May 2021
|
|
DOI: 10.1002/ardp.202100095
F U L L P A P E R
Synthesis and screening of 6‐alkoxy purine analogs as cell
type‐selective apoptotic inducers in Jurkat cells
1
,2
2
2
Álvaro Lorente‐Macías
| Inmaculada Iañez | M. Carmen Jiménez‐López
|
1
2
1
Manuel Benítez‐Quesada | Sara Torres‐Rusillo | Juan J. Díaz‐Mochón
Ignacio J. Molina | María J. Pineda de las Infantas
|
2
1
1
Department of Medicinal & Organic
Chemistry and Excellence Research Unit of
Chemistry Applied to Biomedicine and the
Abstract
“
Purines are ubiquitous structures in cell biology involved in a multitude of cellular
processes, because of which substituted purines and analogs are considered
excellent scaffolds in drug design. In this study, we explored the key structural
features of a purine‐based proapoptotic hit, 8‐tert‐butyl‐9‐phenyl‐6‐benzyloxy‐9H‐
purine (1), by setting up a library of 6‐alkoxy purines with the aim of elucidating the
structural requirements that govern its biological activity and to study the cell
selectivity of this chemotype. This was done by a phenotypic screening approach
based on cell cycle analysis of a panel of six human cancer cell lines, including T cell
leukemia Jurkat cells. From this study, two derivatives (12 and 13) were identified
as Jurkat‐selective proapoptotic compounds, displaying superior potency and cell
selectivity than hit 1.
Environment”, Faculty of Pharmacy,
University of Granada, Granada, Spain
2
Institute of Biopathology and Regenerative
Medicine, University of Granada, Granada,
Spain
Correspondence
Sara Torres‐Rusillo, Institute of Biopathology
and Regenerative Medicine, Centre for
Biomedical Research, University of Granada,
Avda. Del Conocimiento s/n, 18100 Granada,
Spain.
Email: storres@ugr.es
María J. Pineda de las Infantas, Department of
Medicinal & Organic Chemistry and
Excellence Research Unit of “Chemistry
applied to Biomedicine and the Environment”,
Faculty of Pharmacy, University of Granada,
Campus de Cartuja s/n, 18071 Granada,
Spain.
K E Y W O R D S
anticancer drugs, apoptosis, leukemia, phenotypic screening, purine analogs
Email: mjpineda@ugr.es
Funding information
Ministerio de Ciencia e Innovación,
Grant/Award Number: RTC‐2017‐6620;
Ministerio de Educación, Cultura y Deporte,
Grant/Award Number: FPU 14/00818
1
|
INTRODUCTION
cellular energy and intracellular signaling processes (ATP, GTP) and en-
[3]
zyme cofactors (NADH, coenzyme A). The biological ubiquity of this
heterocyclic ring makes it an excellent scaffold in drug design. Hence,
several purine analogs have been developed for the treatment of dif-
ferent diseases such as leukemias, viral infections, cancer or immune
The purine ring system (imidazo[4,5‐d]pyrimidine) is the most abundant
[1,2]
nitrogen‐based heterocycle in nature.
This scaffold is present in
adenine and guanine, and, therefore, it is part of nucleic acids and nu-
[4–6]
cleotides (AMP, GMP), which are essential molecules that regulate
disorders, and many others are currently being studied.
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
©
2021 The Authors. Archiv der Pharmazie published by Wiley‐VCH Verlag GmbH on behalf of Deutsche Pharmazeutische Gesellschaft
Arch Pharm. 2021;e2100095.
wileyonlinelibrary.com/journal/ardp
|
1 of 13
https://doi.org/10.1002/ardp.202100095

2
of 13
LORENTE‐MACÍAS ET AL.
|
Typically, purine analogs are generated by the introduction of
substituents at positions C2, C6, C8, and/or N9 using different
nonsubstituted purines, whereas sterically hindered N,N‐dimethyl‐
amides, such as dimethylbenzamide (DMB), led the synthesis through
route B, where the alcohol provided radicals at both C6 and C8. For
compounds containing the 2,5‐diamino pyrimidine core, microwave
irradiation at higher temperatures was employed due to the lower
reactivity of this core compared with the 5‐amino‐2‐
chloropyrimidine one. In addition to the proposed library, in those
cases where DMB was used together with bulky alcohols (benzyl
alcohol or 4‐bromobenzyl alcohol), the intermediate imines 12i–16i
were also isolated (Table 1). Interestingly, in the preparation of the
aminopurine 16, only the corresponding imine 16i was isolated.
[
7,8]
synthetic methodologies.
In this respect, our group developed a
two‐step synthetic methodology that allows the preparation of
[
9]
purine derivatives substituted at positions C6, C8, and N9. This is
achieved using different 6‐chloro‐4,5‐diaminopyrimidines, alcohols,
[
10,11]
and N,N‐dimethylamides under strong basic conditions.
Ad-
ditionally, the compounds generated with this procedure present an
ether group attached at C6, which is a less explored structural fea-
ture in purine libraries, where C6‐aminated analogs are predominant.
The initial compound series generated with this strategy led to the
identification of purine 1, an antiproliferative compound that exerts
its activity by strong apoptotic induction on Jurkat (acute T cell
[12]
leukemia) cells.
2.2 Pharmacology/biology
|
Cell cycle analysis was chosen as the primary screening output due
to the capability of this method to identify active compounds and
discriminate whether the new analogs maintain or do not maintain
the proapoptotic activity of the lead molecule 1. Thus, the final
compound library was tested against a panel of six human cancer cell
lines (hematopoietic: Jurkat, acute T cell leukemia; K562, chronic
erythroleukemia; nonhematopoietic: HeLa, cervix adenocarcinoma;
G361, malignant melanoma; MDA‐MB‐231, breast adenocarcinoma,
and HCT116, colorectal adenocarcinoma). Cells were treated with
each compound for 48 h at a single dose (30 μM). Cells treated with 1
(30 μM) or DMSO (equivalent amount to that contained in the 30 μM
dose) were used as a reference and negative control, respectively,
and the cell cycle profiles were determined by flow cytometry after
staining of nuclear DNA with propidium iodide (PI) as previously
2
2
|
RESULTS AND DISCUSSION
|
Chemistry
.1
Compounds 9–16, 1i, and 12i–16i were synthesized according to
Scheme 1. Dichloropyrimidines 2 and 3 were substituted with dif-
ferent alkylamines at position C4 of the pyrimidine ring, providing
intermediates 4–8. These chloropyrimidines were treated with the
appropriate alcohol and an excess of the suitable N,N‐dimethylamide
under strong basic conditions to simultaneously carry out a dis-
placement at C6 and an annulation reaction, affording purine analogs
9
–15. This tandem reaction was controlled by the type of N,N‐
dimethylamide used as a solvent/reagent. Thus, dimethylformamide
DMF) drove the synthesis through route A, generating C8
[
12]
(
described.
The percentages of cells within the sub‐G
1
region
1
SCHEME 1 Structure of compound 1 and synthesis of compounds 9–16, 1i, and 12i–16i. Reagents and conditions: (a) H
2
N‐R , NaHCO
3
,
THF, 55°C, 18 h, or n‐butanol, 190°C (MW), 2 h, 46–84%; (b) NaH, DMF, 90°C, 18 h or 150°C (MW), 4 h, 7–45%; and (c) DMB, BnOH, or
a
4
‐bromobenzyl alcohol, NaH, 1,4‐dioxane, 90°C, 18 h or 150°C (MW), 4 h, 5–19%. 16 was not isolated

LORENTE‐MACÍAS ET AL.
3 of 13
|
TABLE 1 Compound library
1
2
3
a
Scaffold
X
R
R
R
MW
cLogD
Compound
Purine analogs
3
58.45 5.17
1
2
2
4
5
2
3
3
4
68.32 2.73
9
82.35 3.26
06.49 6.13
64.28 7.66
83.34 2.71
59.43 4.74
73.46 5.02
21.50 5.98
10
12
13
11
14
15
b
16
Pyrimidine Schiff base analogs
3
60.46 5.21
1i
4
5
3
3
4
08.51 6.16
66.30 7.70
61.45 4.69
75.48 4.97
23.52 5.92
12i
13i
14i
15i
16i
a
[13]
clogD values were calculated using Chemicalize by ChemAxon Ltd. at pH 7.4.
b
Not isolated.
(
(
apoptotic cells) were measured and values from negative controls
dimehthyl sulfoxide [DMSO]) were subtracted to show the variation
in the apoptotic populations (Figure 1).
1
Extending the length and bulkiness of the substituent at R by
the introduction of a phenethyl group afforded compounds with
different cellular activities. When these analogs were not sub-
2
stituted in R (= H) and also contained alkyloxy groups at the C6
position (9 and 10), the purine analogs did not show any significant
activity. In contrast, when both positions were occupied by lipo-
philic substituents such as the phenyl or 4‐bromophenyl group (12
and 13, respectively), a significant increment in the apoptotic
populations in Jurkat cells was detected. Compound 12 showed a
6
4% increase in this cell line and a moderate activity over MDA‐
MB‐231 cells (23%). Meanwhile, compound 13 showed very se-
lective activity over Jurkat cells with a sub‐G cell increment of
FIGURE 1 Phenotypic screening of compounds 9–15, 1i, and
2i–16i by cell cycle analysis. Cells were incubated for 48 h with the
1
1
51% while being negligible against the rest of the cancer cell lines.
The structural similarities between these two compounds to 1
suggest that the presence of bulky aromatic groups at C6 and C8
positions drives the apoptotic activity of these compounds toward
Jurkat cells.
indicated compounds at 30 μM, then stained with propidium iodide and
cell cycles were analyzed by flow cytometry. The heatmap represents
1
changes in the percentage of cells within the sub‐G region after
subtracting values from negative controls (dimethyl sulfoxide) in each cell
line. Compound 1 was used as the reference

4
of 13
LORENTE‐MACÍAS ET AL.
|
Compounds 11, 14, and 15 contained an amino group at the C2
active Jurkat‐selective compounds 12, 13 (12i and 13i) or the parent
compound 1 (1i), probably because of the different spatial arrange-
ment and physicochemical properties of the substituents compared
with the purine analogs. On the basis of the data from this pheno-
typic screening, compounds 12 and 13 were selected as the most
active compounds and their activities were further analyzed.
A titration of the activity of the new compounds was performed
by culturing the cells with 30, 10, and 3 μM for 48 h, followed by cell
cycle analysis (Figure 2). Compound 12 showed a stronger capacity
to induce cell death, as observed with the lowest dose of 3 μM
compared to the other two compounds.
position. None of these compounds showed any significant effect in
the cell cycle of the lines tested. Interestingly, the C2 aminated
analog of 1, compound 15, was completely inactive. This suggests
that the presence of the amino group disrupts the biological inter-
action of 1 with its biological target.
Imines 1i and 12i–16i were tested next. These molecules were
the pyrimidine Schiff base analogs of the corresponding compounds
1
, 12–16. Among these, only the C2 aminated imine of 1, compound
5i, exhibited a notable activity, inducing a 41% increase in the sub‐
1
1
G population of melanoma G361 cells, a phenotypic activity profile
only seen with this derivative. Also, moderate activities were ob-
served for compound 16i on Jurkat cells. However, apart from these
two imine derivatives, none of the others showed significant activ-
ities, not even the corresponding Schiff base analogs of the highly
To confirm the proapoptotic nature of these two molecules,
we stained the treated cells with annexin‐V, which selectively
binds to the phosphatidyl serine translocated to the cell mem-
brane during the process of apoptosis, followed by flow cytometric
FIGURE 2 Titration of the proapoptotic activity of compounds 1, 12, and 13. Jurkat cells were treated for 48 h with the indicated doses and
percentages of cells within the sub‐G1 region measured by flow cytometry after staining with propidium iodide. DMSO, dimethyl sulfoxide

LORENTE‐MACÍAS ET AL.
5 of 13
|
FIGURE 3 Membrane phosphatidyl serine expression after treatment of Jurkat cells with compounds 12 and 13. Jurkat cells were treated
for 48 h with the indicated doses and the percentages of apoptotic cells were measured by flow cytometry after double staining with propidium
iodide (PI) and annexin‐V‐FLUOS. Untreated cells (dimethyl sulfoxide [DMSO], 30 µM) and cells treated with 1 were included as negative and
−
−
positive controls of apoptosis, respectively. Cell populations (gating regions): R1, viable cells (annexin‐V /PI ); R2, early apoptotic cells
+
−
+
+
−
+
(
annexin‐V /PI ); R3, late apoptotic cells (annexin‐V /PI ); and R4, necrotic cells (annexin‐V /PI )
[
12]
analysis as previously described.
For this study, Jurkat cells
FMK (carbobenzoxy‐valyl‐alanyl‐aspartyl‐(O‐methyl)‐fluoromethylk-
etone). Indicated compounds were then added for 24 h at the dose of
30 μM and subjected thereafter to cell cycle analysis (Figure 4).
Whereas untreated cells showed a significant population within the
sub‐G1 region, this effect was completely abrogated in Z‐VAD‐FMK‐
pretreated cells. This indicates that induction of cell death mediated
by the tested compounds is fully dependent on caspase activation, a
hallmark of apoptosis pathways.
were treated for 48 h with compounds 12 and 13 at 30, 10, and
μM. DMSO (30 μM) and 1 (30, 10, and 3 μM) were used as
3
negative and positive apoptotic controls, respectively (Figure 3).
As expected, untreated Jurkat cells presented a negligible apop-
totic population, with the majority of cells gated within the viable
1
cell region (R ). However, when cells were treated with com-
pounds 12 and 13 at the indicated concentrations, a transition
2
3
behavior from early (R ) to late apoptotic (R ) stages was ob-
served. In addition, this effect was displayed in a dose–dependent
manner, and these results are fully coherent with those shown in
Figure 2, as compound 12 induced a significantly higher apoptosis
Additionally, to further compare the activities of compounds 12
and 13 with the reference molecule 1, cell viability was measured on
Jurkat cells using PrestoBlue™ reagent and analyzed by spectro-
fluorometry. Half‐maximal inhibitory concentration (IC50) values
2
3
as revealed in the percentages of R and R at the lowest con-
centration of 3 μM in both assays. Finally, no necrotic populations
were detected for any of these compounds even at the highest
doses. Hence, the unspecific necrotic potential of these molecules
was discarded. The cellular activity displayed by compounds 12
and 13 was equivalent to that shown by the parent compound 1,
suggesting that the three compounds share the same mechanism
of action and that the introduction of the phenethyl group at N9 or
were calculated using a 9‐point half‐log dose–response study
(0.78–200 μM). Table 2 shows the IC50 values obtained and the
theoretically calculated cLogD values.
The two new analogs exhibited superior activities than the
parent compound 1 (15.07 μM), exhibiting a direct correlation be-
tween the antiproliferative activity and their lipophilicity nature.
Thus, compound 12 showed an IC50 of 9.99 μM, whereas the value
for its brominated analog (13) was 7.29 μM. Therefore, even though
compound 13 was found to be the most potent compound of the
series on inhibition of proliferation, leading to a twofold potency
increase over the parent compound 1, compound 12 was the most
active in inducing apoptosis, as demonstrated by cell cycle and
annexin‐V analysis. These effects were mostly restricted to Jurkat
4
‐bromophenyl groups at C8 and C6 positions does not affect the
biological behavior of this chemotype.
To further demonstrate that the observed effect was due to the
induction of apoptosis, we carried out assays where Jurkat cells were
pretreated for 1 h at 37°C with the pan‐caspase inhibitor Z‐VAD‐

6
of 13
LORENTE‐MACÍAS ET AL.
|
FIGURE 4 Caspase‐dependent Jurkat cell death induced by compounds 12 and 13. Cells were either pretreated (bottom panels) or not with
the pan‐caspase inhibitor Z‐VAD‐FMK for 1 h and thereafter treated with the indicated compounds (30 µM) for 24 h. DMSO, dimethyl sulfoxide
TABLE 2 Antiproliferative activities of compounds 1, 12, and 13
on Jurkat cells
which were particularly conspicuous for compounds 12 and 13.
These effects, however, were restricted to T cell leukemia Jurkat
a
b
Compound
1
IC50 (μM)
15.07
9.99
cLogD
5.17
cells, and were fully coherent with induction of apoptosis as de-
monstrated by annexin‐V and Z‐VAD‐FMK assays. Additionally, this
study has informed the design of subsequent analogs to further
1
1
2
3
6.13
[
14]
enhance the cellular activity of this chemotype.
Follow‐on studies
7.29
7.66
will aim to identify the target of the analogs to facilitate future
medicinal chemistry campaigns.
a
IC50 values determined by the PrestoBlue™ cell viability assay after
treating Jurkat cells with compounds 1 (reference), 12 and 13 for 5 days,
nine‐dose range (0.78–200 μM).
b
cLogD values were calculated using Chemicalize by ChemAxon Ltd. at
[
13]
pH 7.4.
4
4
|
EXPERIMENTAL
|
Chemistry
.1
cells, showing a marginally superior selectivity over other cancer cell
lines.
4.1.1
|
General
Unless otherwise noted, reagents and solvents were obtained from
1
3
|
CONCLUSION
commercial suppliers and were used without further purification. H
13
and C NMR (nuclear magnetic resonance) spectra (see the Sup-
porting Information) were obtained using Varian Innova Unity
(300 MHz), Bruker Nanobay Avance III HD (400 MHz) or Bruker
Avance NEO (400 or 500 MHz) spectrometers, and chemical shifts
are reported in parts per million (ppm, δ) downfield from tetra-
methylsilane. Coupling constants (J) are reported in Hz. Spin
In summary, we have prepared a library of 6‐alkoxy purine analogs
aiming to further explore the previously reported proapoptotic ac-
tivity of this chemotype over human cancer cells. Our screening
campaign revealed that treatment with several of our analogs re-
sulted in increments in the sub‐G1 cell population on the cell cycle,

LORENTE‐MACÍAS ET AL.
7 of 13
|
13
multiplicities are described as s (singlet), bs (broad singlet), d
bs, NH
2 2
), and 3.01 (2H, t, J = 7.0 Hz, CH ). C‐NMR (101 MHz,
(
doublet), t (triplet), q (quartet) and m (multiplet). High‐resolution
CDCl ): δ 155.09 (C), 149.60 (CH), 142.57 (C), 138.95 (C), 128.91 (2×
3
mass spectra (HRMS) were obtained using a Waters LCT Premier XE
spectrometer. Analytical thin‐layer chromatography (TLC) was per-
formed using TLC silica gel 60 F254 plates and visualized under a UV
lamp. Purifications were carried out by flash chromatography using
silica gel 220−440 mesh (Sigma‐Aldrich). All compounds used in the
2
CH), 128.80 (2× CH), 126.72 (CH), 122.04 (C), 42.78 (CH ), and
+
35.54 (CH
2
). HRMS (ES +ve), C12
H
14
N
4
Cl (M+H) : Calculated
249.0907. Obtained 249.0902.
1
13
biological experiments were over 95% pure by H/ C NMR. Stock
solutions (100 mM) were prepared in DMSO and stored at −20°C
until used.
4.1.3 General procedure for the synthesis of
|
compounds 6–8
The InChI codes of the investigated compounds, together with
some biological activity data, are provided in the Supporting
Information.
A microwave vial was charged with a 0.4 M solution of 4,6‐
dichloropyrimidine‐2,5‐diamine (3) (1 equiv.) in n‐butanol, NaHCO
3
(2.5 equiv.), and the appropriate amine (5 equiv.). Then, the mixture
was heated at 190°C for 2 h using microwave radiation. The mixture
was diluted with dichloromethane, filtered, the solvent was removed
under reduced pressure and the crude material was purified by silica
gel column chromatography.
4.1.2 General procedure for the synthesis of
|
compounds 4 and 5
To a stirred 0.48 M solution of 5‐amino‐4,6‐dichloropyrimidine (2)
(
3
1 equiv.) in dry THF, NaHCO (2 equiv.) was added. The mixture was
then heated to 55°C and at this temperature, a 1.6 M solution of the
appropriate amine (2 equiv.) in THF was added dropwise. The mix-
ture was heated at reflux for 18 h and then cooled to room tem-
perature. The mixture was diluted with dichloromethane, filtered, the
solvent was removed under reduced pressure and the crude material
was purified by silica gel column chromatography.
2,5‐Diamino‐6‐chloro‐4‐(isopropylamino)pyrimidine (6)
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 66%) to provide the title compound as an off‐
1
white solid (189.7 mg, 0.978 mmol, 58% yield). H‐NMR (400 MHz,
CDCl
.66 (2H, bs, NH
126 MHz, CDCl
3
): δ 5.39 (1H, s, CH), 4.79 (2H, bs, NH
2
), 4.28–4.07 (1H, m, CH),
13
2
2
), and 1.23 (6H, d, J = 6.5 Hz, 2× CH ). C‐NMR
3
(
3
): δ 158.45 (C), 157.37 (C), 146.61 (C), 111.73 (C),
+
4
2.92 (CH), and 22.96 (2× CH
3
). HRMS (ES +ve), C
7
H
13
N
5
Cl (M+H) :
Calculated 202.0859. Obtained 202.0849.
5
‐Amino‐4‐(tert‐butylamino)‐6‐chloropyrimidine (4)
The crude product was purified via silica gel column chromatography
(
ethyl acetate/hexane 25%) to provide the title compound as a white
1
solid (851.2 mg, 4.233 mmol, 46% yield). H‐NMR (400 MHz, CDCl
3
):
δ 8.05 (1H, s, CH), 4.82 (2H, bs, NH
2
), 1.92 (1H, bs, NH), and 1.46 (9H,
). C‐NMR (101 MHz, CDCl ): δ 158.11 (C), 152.49 (CH),
35.25 (C), 101.82 (C), 51.47 (C), and 29.86 (3× CH ). HRMS (ES
Cl (M+H) : Calculated 201.0907. Obtained 201.0893.
2,5‐Diamino‐4‐(tert‐butylamino)‐6‐chloropyrimidine (7)
13
s, 3× CH
3
3
The crude product was purified via silica gel column chromatography
1
+
3
(ethyl acetate/hexane 20–33%) to provide the title compound as a
+
1
ve), C
8
H
14
N
4
dark green solid (187 mg, 0.865 mmol, 47 yield%). H‐NMR
(400 MHz, CDCl
3
): δ 5.37 (1H, bs, NH), 4.67 (2H, bs, NH
2
), 2.73
13
(
2H, bs, NH ), and 1.41 (9H, s, 3× CH
2
3
). C‐NMR (126 MHz, CDCl ):
3
δ 158.91 (C), 157.27 (C), 147.53 (C), 111.80 (C), 51.98 (C), and 28.97
+
(
3× CH
3
). HRMS (ES +ve), C
8
H
15
N
5
Cl (M+H) : Calculated 216.1016.
Obtained 216.1009.
5
‐Amino‐6‐chloro‐4‐(phenethylamino)pyrimidine (5)
The crude product was purified via silica gel column chromatography
(
ethyl acetate/hexane 33%) to provide the title compound as a yel-
1
low solid (1.13 g, 4.533 mmol, 50% yield). H‐NMR (400 MHz,
CDCl
3
): δ 8.13 (1H, s, CH), 7.37 (2H, t, J = 7.2 Hz, 2× CH), 7.34–7.24
(
3H, m, 3× CH), 5.19 (1H, bs, NH), 3.88–3.78 (2H, m, CH
2
), 3.25 (2H,

8
of 13
LORENTE‐MACÍAS ET AL.
|
2
,5‐Diamino‐6‐chloro‐4‐(phenethylamino)pyrimidine (8)
6‐Ethoxy‐9‐phenethyl‐9H‐purine (9)
The crude product was purified via silica gel column chromatography
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 33%) to provide the title compound as a yellow
(ethyl acetate/hexane 33–100%) to provide the title compound as a
1
1
solid (406.8 mg, 1.54 mmol, 84% yield). H‐NMR (400 MHz, CDCl
.32 (2H, t, J = 7.4 Hz, 2× CH), 7.28–7.14 (3H, m, 3× CH), 5.42 (1H, bs,
NH), 4.65 (2H, bs, NH ), 3.66 (2H, q, J = 6.7 Hz, CH ), 2.90 (2H, t,
J = 6.7 Hz, CH ), and 2.65 (2H, bs, NH ). C‐NMR (101 MHz, CDCl ): δ
59.21 (C), 157.91 (C), 148.36 (C), 139.20 (C), 128.91 (2× CH), 128.76
2× CH), 126.63 (CH), 111.75 (C), 42.37 (CH ), and 35.76 (CH ). HRMS
Cl (M+H) : Calculated 264.1016. Obtained 264.1022.
3
): δ
yellow solid (50.4 mg, 0.187 mmol, 45% yield). H‐NMR (400 MHz,
7
CDCl
CH), 7.13–7.07 (2H, m, 2× CH), 4.72 (2H, q, J = 7.1 Hz, OCH
(2H, t, J = 7.0 Hz, CH ), 3.24 (2H, t, J = 7.0 Hz, CH ), and 1.57 (3H, t,
). C‐NMR (101 MHz, CDCl ): δ 160.95 (C), 152.16
(CH), 152.05 (C), 142.15 (CH), 137.34 (C), 128.95 (2× CH), 128.77
3
): δ 8.60 (1H, s, CH), 7.58 (1H, s, CH), 7.37–7.24 (3H, m, 3×
2
2
2
), 4.54
13
2
2
3
2
2
13
1
J = 7.1 Hz, CH
3
3
(
2
2
+
(ES +ve), C12
H
15
N
5
(2× CH), 127.17 (CH), 121.57 (C), 63.21 (CH
2
), 45.73 (CH
2
), 36.25
+
(
CH ), and 14.61 (CH ). HRMS (ES +ve), C15
2
3
H
17
N
4
O (M+H) : Cal-
culated 269.1402. Obtained 269.1402.
4.1.4 General procedure for the synthesis of
|
compounds 1i, 9, 10, 12, 12i, 13, and 13i
A suspension of NaH (60%) in mineral oil (10 equiv.) was dissolved in a
mixture cooled at 0°C constituted both from the appropriate alcohol (10
equiv.) and amide (10 equiv.). When N,N‐dimethylbenzamide was used as
the amide source, dioxane was employed as a solvent. The solution was
stirred at room temperature for 30 min and then at 90°C for the same
time. At 90°C, the corresponding diamino pyrimidine (4, 5) (1 equiv.)
dissolved in the appropriate amide (50 equiv.) was added dropwise and
the mixture was heated for 18 h. The reaction mixture was quenched to
9‐Phenethyl‐6‐propoxy‐9H‐purine (10)
The crude product was purified via silica gel column chromatography
pH 7 with saturated aqueous NH
and washed with saturated brine. The combined organic extracts were
dried over anhydrous Na SO , filtered, and the solvent was removed
4
Cl, extracted with dichloromethane,
(ethyl acetate/hexane 50–66%) to provide the title compound as a
1
brown solid (24.9 mg, 0.088 mmol, 19% yield). H‐NMR (400 MHz,
2
4
CDCl
CH), 7.08–7.01 (2H, m, 2× CH), 4.56 (2H, t, J = 7.0 Hz, OCH
(2H, t, J = 7.0 Hz, CH ), 3.18 (2H, t, J = 7.0 Hz, CH ), 2.00–1.86 (2H, m,
CH ), and 1.08 (3H, t, J = 7.5 Hz, CH ). C‐NMR (101 MHz, CDCl ): δ
61.18 (C), 152.23 (CH), 152.08 (C), 142.18 (CH), 137.36 (C), 129.02
2× CH), 128.84 (2× CH), 127.26 (CH), 121.51 (C), 69.01 (CH ), 45.83
). HRMS (ES +ve),
3
): δ 8.54 (1H, s, CH), 7.54 (1H, s, CH), 7.31–7.18 (3H, m, 3×
under reduced pressure. The residue was purified by silica gel column
chromatography.
2
), 4.48
2
2
13
2
3
3
1
(
(
2
CH
2
), 36.32 (CH
2
2 3
), 22.36 (CH ), and 10.57 (CH
+
C
16
H
19
N
4
O (M+H) : Calculated 283.1559. Obtained 283.1559.
5
‐(Benzylideneamino)‐6‐(benzyloxy)‐4‐(tert‐butylamino)‐
pyrimidine (1i)
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 10–33%) to provide the title compound as a yellow
1
solid (15.6 mg, 0.043 mmol, 6% yield). H‐NMR (300 MHz, CDCl
3
): δ 9.17
(1H, s, CH), 8.15 (1H, s, CH), 7.81–7.71 (2H, m, 2× CH), 7.49–7.33 (8H, m,
8
× CH), 6.28 (1H, bs, NH), 5.50 (2H, s, OCH
2
), and 1.54 (9H, s, 3× CH
3
).
6‐(Benzyloxy)‐9‐phenethyl‐8‐phenyl‐9H‐purine (12)
+
HRMS (ES +ve), C22
H N
25 4
O (M+H) : Calculated 361.2028. Obtained
The crude product was purified via silica gel column chromatography
361.2041.
(ethyl acetate/hexane 25%) to provide the title compound as a white
1
solid (7.6 mg, 0.019 mmol, 5% yield). H‐NMR (400 MHz, CDCl
3
): δ
8
.58 (1H, s, CH), 7.60–7.53 (2H, m, 2× CH), 7.52–7.41 (3H, m, 3× CH),
.39–7.24 (5H, m, 5× CH), 7.21–7.13 (3H, m, 3× CH), 6.96–6.87 (2H,
), 4.56 (2H, t, J = 7.4 Hz, CH ), and 3.10
). C‐NMR (126 MHz, CDCl ): δ 161.23 (C),
58.80 (C), 154.40 (CH), 139.32 (C), 137.42 (C), 136.96 (C), 131.17
CH), 129.89 (C), 129.12 (2× CH), 128.81 (2× CH), 128.78 (2× CH),
7
m, 2× CH), 5.71 (2H, s, OCH
2
2
13
(2H, t, J = 7.4 Hz, CH
2
3
1
(

LORENTE‐MACÍAS ET AL.
9 of 13
|
+
1
1
28.66 (2× CH), 128.34 (2× CH), 128.13 (CH), 127.85 (2× CH),
26.59 (CH), 111.10 (C), 68.25 (CH ), 42.36 (CH ), and 36.18 (CH ).
O (M+H) : Calculated 407.1872. Obtained
35.58 (CH
2 21 4 2
). HRMS (ES +ve), C26H N OBr (M+H) : Calculated
2
2
2
563.0082. Obtained 563.0092.
+
23 4
HRMS (ES +ve), C26H N
4
07.1848.
6‐(4‐Bromobenzyloxy)‐5‐[(4‐bromobenzylidene)amino]‐4‐
5
‐(Benzylideneamino)‐6‐(benzyloxy)‐4‐(phenethylamino)‐
(phenethylamino)pyrimidine (13i)
pyrimidine (12i)
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 25%) to provide the title compound as an
The crude product was purified via silica gel column chromatography
1
(
ethyl acetate/hexane 20%) to provide the title compound as a
orange solid (9.7 mg, 0.017 mmol, 4% yield). H‐NMR (400 MHz,
1
brown solid (30.1 mg, 0.074 mmol, 19% yield). H‐NMR (500 MHz,
CDCl
7.69–7.62 (1H, m, CH), 7.58–7.41 (5H, m, 5× CH), 7.38–7.27 (6H, m,
6× CH), 5.45 (2H, s, OCH ), 3.83 (2H, q, J = 6.5 Hz, CH ), and 2.96
): δ 159.66 (C),
3
): δ 9.11 (1H, s, CH), 9.06 (1H, s, CH), 8.17 (1H, s, CH),
CDCl
CH), 7.47–7.40 (7H, m, 7× CH), 7.40–7.35 (3H, m, 3× CH), 7.34–7.27
3H, m, 3× CH), 6.30 (1H, bs, NH), 5.51 (2H, s, OCH ), 3.83 (2H, q,
J = 6.5 Hz, CH ), and 2.97 (2H, t, J = 6.8 Hz, CH
126 MHz, CDCl ): δ 161.22 (CH), 158.79 (C), 154.40 (CH), 139.32
C), 137.42 (C), 136.95 (C), 131.17 (CH), 129.89 (C), 129.12 (2× CH),
3
): δ 9.17 (1H, s, CH), 8.19 (1H, s, CH), 7.68–7.61 (2H, m, 2×
2
2
13
(
2
(2H, t, J = 6.5 Hz, CH
2
). C‐NMR (126 MHz, CDCl
3
1
3
2
2
).
C‐NMR
158.66 (CH), 154.57 (CH), 139.21 (C), 137.24 (C), 136.21 (C), 135.90
(C), 132.09 (2× CH), 131.81 (2× CH), 129.90 (C), 129.61 (2× CH),
129.12 (2× CH), 128.84 (2× CH), 128.38 (2× CH), 126.66 (CH),
(
(
3
1
1
4
28.81 (2× CH), 128.77 (2× CH), 128.66 (2× CH), 128.34 (2× CH),
122.18 (C), 110.83 (C), 67.50 (CH
2
2 2
), 42.29 (CH ), and 36.09 (CH ).
+
28.12 (CH), 127.84 (2× CH), 126.58 (CH), 111.09 (C), 68.25 (CH ),
2
HRMS (ES +ve), C26
tained 565.0245.
H
23
N
4
OBr
2
(M+H) : Calculated 565.0239. Ob-
+
2 2 25 4
2.37 (CH ), and 36.18 (CH ). HRMS (ES +ve), C26H N O (M+H) :
Calculated 409.2028. Obtained 409.2039.
4.1.5 General procedure for the synthesis of
|
compounds 11, 14, 14i, 15, 15i, 16, and 16i
A microwave vial was charged with a suspension of NaH (60%) in
mineral oil (10 equiv.), N,N‐dimethylformamide (40 equiv.) and the
appropriate alcohol (10 equiv.). Then, the corresponding triamino-
pyrimidine (6–8) (1 equiv.) dissolved in DMF (80 equiv.) was added
dropwise and the mixture was heated at 150°C for 4 h using mi-
crowave radiation. When DMB was used as the amide source, di-
oxane was employed as a solvent. The reaction mixture was
6
‐(4‐Bromobenzyloxy)‐8‐(4‐bromophenyl)‐9‐phenethyl‐9H‐
purine (13)
The crude product was purified via silica gel column chromatography
quenched to pH 7 with saturated aqueous NH
dichloromethane, and washed with saturated brine. The combined
organic extracts were dried over anhydrous Na SO , filtered, and the
4
Cl, extracted with
2
4
(
ethyl acetate/hexane 25%) to provide the title compound as an
solvent was removed under reduced pressure. The residue was
purified by silica gel column chromatography.
1
orange solid (15.1 mg, 0.027 mmol, 7% yield). H‐NMR (500 MHz,
CDCl
3
): δ 8.56 (1H, s, CH), 7.58–7.54 (1H, m, CH), 7.53–7.41 (7H, m,
× CH), 7.31–7.27 (2H, m, 2× CH), 7.20–7.14 (2H, m, 2× CH),
.97–6.89 (1H, m, 1× CH), 5.02 (2H, s, OCH ), 4.56 (2H, t, J = 6.8 Hz,
× CH), and 3.10 (2H, t, J = 6.8 Hz, 2× CH). C‐NMR (126 MHz,
): δ 159.93 (C), 158.10 (C), 151.48 (CH), 137.12 (C), 136.01 (C),
35.37 (C), 133.68 (C), 131.72 (2× CH), 130.19 (C), 130.14 (2× CH),
29.48 (C), 129.20 (2× CH), 129.02 (2× CH), 128.66 (2× CH), 126.89
), 45.52 (CH ), and
7
6
2
2
1
3
CDCl
3
1
1
(
CH), 121.86 (C), 114.93 (2× CH), 69.28 (CH
2
2

10 of 13
LORENTE‐MACÍAS ET AL.
|
2
‐Amino‐6‐(benzyloxy)‐9‐isopropyl‐9H‐purine (11)
103.59 (C), 67.78 (CH
2
3
), 42.56 (CH), and 23.38 (2× CH ). HRMS (ES
+
The crude product was purified via silica gel column chromatography
+ve), C21
H
24
N
5
O (M+H) : Calculated 362.1981. Obtained 362.1978.
(
ethyl acetate/hexane 50–100%) to provide the title compound as a
1
red solid (8.5 mg, 0.03 mmol, 7% yield). H‐NMR (500 MHz, CDCl
3
): δ
7
5
1
1
.68 (1H, s, CH), 7.53–7.47 (2H, m, 2× CH), 7.37–7.27 (3H, m, 3× CH),
.56 (2H, s, OCH ), 4.84 (2H, bs, NH ), 4.73–4.63 (1H, m, CH), and
.54 (6H, d, J = 6.8 Hz, 2× CH
61.14 (C), 159.00 (C), 153.78 (C), 137.08 (CH), 136.71 (C), 128.49
2
2
1
3
3
3
). C‐NMR (126 MHz, CDCl ): δ
(
2× CH), 128.38 (2× CH), 128.06 (CH), 116.10, 68.10 (CH
CH), and 22.75 (2× CH ). HRMS (ES +ve), C15
2
), 46.66
+
(
3
H
18
N
5
O (M+H) :
Calculated 284.1511. Obtained 284.1510.
2‐Amino‐6‐(benzyloxy)‐9‐(tert‐butyl)‐8‐phenyl‐9H‐purine (15)
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 20%) to provide the title compound as a
1
brown solid (10.7 mg, 0.029 mmol, 7% yield). H‐NMR (400 MHz,
CDCl
bs, NH
61.04 (C), 157.69 (C), 150.44 (C), 136.76 (C), 135.52 (C), 130.20 (2×
CH), 129.22 (CH), 128.73 (2× CH), 128.42 (2× CH), 128.04 (CH),
3
): δ 7.58–7.25 (10H, m, 10× CH), 5.54 (2H, s, OCH
2
), 4.79 (2H,
13
2
), and 1.58 (9H, s, 3× CH
3
). C‐NMR (101 MHz, CDCl ): δ
3
1
1
27.88 (2× CH), 127.14 (C), 115.92 (C), 68.00 (CH
30.88 (3× CH ). HRMS (ES +ve), C22
374.1981. Obtained 374.1965.
2
), 60.10 (C), and
+
2
‐Amino‐6‐(benzyloxy)‐9‐isopropyl‐8‐phenyl‐9H‐purine (14)
3
H
24
N
5
O (M+H) : Calculated
The crude product was purified via silica gel column chromato-
graphy (ethyl acetate/hexane 33%) to provide the title compound
1
as
400 MHz, CDCl
m, 5× CH), 7.39–7.31 (3H, m, 3× CH), 5.58 (2H, s, OCH
a
brown solid (11 mg, 0.031 mmol, 6% yield). H‐NMR
(
3
): δ 7.64–7.56 (2H, m, 2× CH), 7.58–7.42 (5H,
), 4.84
2
2H, bs, NH ), 4.68–4.55 (1H, m, CH), and 1.64 (6H, d, J = 6.8 Hz,
2
(
1
3
2
1
1
1
× CH
3
3
). C‐NMR (126 MHz, CDCl ): δ 160.95 (C), 158.20 (C),
50.34 (C), 136.81 (C), 131.74 (C), 129.74 (CH), 129.61 (2× CH),
28.68 (2× CH), 128.61 (2× CH), 128.45 (2× CH), 128.05 (CH),
27.13 (C), 116.15 (C), 68.02 (CH
). HRMS (ES +ve), C21
Obtained 360.1820.
2
), 49.31 (CH), and 21.16 (2×
+
CH
3
H
22
N
5
O (M+H) : Calculated 360.1824.
2‐Amino‐5‐(benzylideneamino)‐6‐(benzyloxy)‐4‐(tert‐butylamino)‐
pyrimidine (15i)
The crude product was purified via silica gel column chromatography
(ethyl acetate/hexane 20%) to provide the title compound as a yellow
1
solid (7.3 mg, 0.019 mmol, 5% yield). H‐NMR (500 MHz, CDCl
1H, s, CH), 7.72–7.66 (2H, m, 2× CH), 7.48–7.30 (8H, m, 8× CH), 6.50
1H, bs, NH), 5.45 (2H, s, OCH ), 4.69 (2H, bs, NH ), and 1.53 (9H, s, 3×
). C‐NMR (126 MHz, CDCl ): δ 161.77 (C), 160.22 (C), 159.22 (C),
54.67 (CH), 138.60 (C), 137.49 (C), 129.79 (CH), 128.66 (2× CH),
28.54 (2× CH), 127.86 (CH), 127.77 (2× CH), 127.45 (2× CH), 103.94
C), 67.49 (CH ), 51.41 (C), and 29.47 (3× CH ). HRMS (ES +ve),
O (M+H) : Calculated 376.2137. Obtained 376.2152.
3
): δ 9.11
(
(
2
2
13
CH
3
3
1
1
(
2
3
+
2
‐Amino‐5‐(benzylideneamino)‐6‐(benzyloxy)‐4‐(isopropylamino)‐
22 26 5
C H N
pyrimidine (14i)
The crude product was purified via silica gel column chromatography
(
ethyl acetate/hexane 33%) to provide the title compound as a yel-
1
low solid (29.9 mg, 0.083 mmol, 16% yield). H‐NMR (400 MHz,
CDCl
3
): δ 9.10 (1H, s, CH), 7.73–7.65 (1H, m, CH), 7.47–7.27 (9H, m,
× CH), 6.22 (1H, bs, NH), 5.43 (2H, s, OCH ), 4.84 (2H, bs, NH ),
.32–4.19 (1H, m, CH), and 1.27 (6H, d, J = 6.5 Hz, 2× CH ). C‐NMR
): δ 160.51 (C), 155.24 (CH), 141.04 (C), 138.48 (C),
9
4
2
2
13
3
(
126 MHz, CDCl
3
1
1
37.29 (C), 134.60 (C), 129.94 (CH), 128.68 (2× CH), 128.58 (CH),
27.96 (CH), 127.82 (CH), 127.76 (CH), 127.55 (CH), 127.11 (2× CH),

LORENTE‐MACÍAS ET AL.
11 of 13
|
2
‐Amino‐6‐(benzyloxy)‐9‐phenethyl‐8‐phenyl‐9H‐purine (16)
2
1% L‐glutamine (Gibco) and incubated at 37°C under 5% CO . Cell
The crude product was purified via silica gel column chromatography
cultures were split at 60–80% confluence. Suspension cells were
centrifuged (1500 rpm, 5 min). Cell pellets were resuspended in
supplemented RPMI growth media, seeded at a dilution factor of
1:10 (T75–15 ml) and incubated until 60–80% confluence was
achieved. Adherent cells were washed with PBS (T75–10 ml) and
trypsinized (Trypsin: T75–1 ml, 5 min, 37°C). Trypsin was in-
activated with supplemented DMEM growth media (9 ml) and
centrifuged (1500 rpm, 5 min). Cell pellets were resuspended in
supplemented DMEM growth media, seeded at a dilution factor of
1:10, unless otherwise stated, in the same media (T75–15 ml) and
incubated until 60–80% confluence was achieved.
(
ethyl acetate/hexane 25%) to provide the title compound as an
1
orange solid (traces). H‐NMR (400 MHz, CDCl
3
): δ 7.51–7.47 (3H, m,
3
7
6
4
× CH), 7.41 (2H, d, J = 7.2 Hz, 2× CH), 7.38–7.32 (4H, m, 4× CH),
.31–7.27 (2H, m, 2× CH), 7.20–7.17 (2H, m, J = 5.1, 2.0 Hz, 2× CH),
.97–6.93 (2H, m, 2× CH), 5.58 (2H, s, OCH
2
), 4.70 (2H, bs, NH
2
),
).
.36 (2H, t, J = 7.6 Hz, CH ), and 3.04 (2H, t, J = 7.6 Hz, CH
2
2
13
3
C‐NMR (101 MHz, CDCl ): δ 160.68 (C), 158.73 (C), 156.00 (C),
1
1
1
6
37.88 (C), 136.94 (C), 130.41 (C), 129.57 (CH), 129.16 (2× CH),
28.87 (2× CH), 128.70 (2× CH), 128.66 (CH), 128.62 (2× CH),
28.54 (2× CH), 128.44 (2× CH), 127.68 (CH), 126.81 (C), 115.22 (C),
8.00 (CH
2 2 2
), 46.27 (CH ), and 45.23 (CH ). HRMS (ES +ve),
+
C
26
H
23
N
5
ONa (M+Na) : Calculated 444.1800. Obtained 444.1814.
4.2.2 Cell cycle analysis
|
For the analysis of cell cycle, 250,000 cells/well (Jurkat, K562),
00,000 cells/well (G361), and 150,000 cells/well (MDA‐MB‐231,
2
HCT116) in 24‐well plates or 50,000 cells/well (HeLa) in six‐well
plates were cultured in 500 µl (24‐well) or 1.5 ml (six‐well) of the
appropriate media and incubated with the chosen compounds at
3
0 µM for 48 h. Untreated cells were incubated with the maximum
DMSO concentration used. Cell cycles were analyzed by staining of
cells with PI staining. Thus, after treatment with the corresponding
compounds, the cells were collected and washed with 2 ml
phosphate‐buffered saline (PBS) at 4°C and fixed with 100 µl of PBS
and 900 µl of 70% ethanol on ice for 5 min. After washing with PBS,
cells were resuspended in 250 µl of PBS and another 250 µl of a DNA
2
‐Amino‐5‐(benzylideneamino)‐6‐(benzyloxy)‐4‐(phenethylamino)‐
pyrimidine (16i)
The crude product was purified via silica gel column chromatography
(
ethyl acetate/hexane 25%) to provide the title compound as a yel-
1
low solid (21.1 mg, 0.05 mmol, 14% yield). H‐NMR (400 MHz,
2 4 6 8 7
extraction buffer (0.2 M Na HPO , 0.1 M C H O , pH 7.8) and in-
CDCl
3
): δ 9.08 (1H, s, CH), 7.62–7.54 (2H, m, 2× CH), 7.45–7.25
12H, m, 12× CH), 7.21–7.13 (1H, m, CH), 6.47 (1H, bs, NH), 5.44
2H, s, OCH ), 5.20 (2H, bs, NH ), 3.75 (2H, q, J = 6.7 Hz, CH ), and
.92 (2H, t, J = 6.7 Hz, CH ): δ 160.62
C), 158.99 (C), 153.98 (CH), 140.13 (C), 139.73 (C), 138.73 (C),
37.56 (C), 129.65 (CH), 129.11 (2× CH), 128.75 (2× CH), 128.57
2× CH), 127.85 (2× CH), 127.70 (2× CH), 127.50 (2× CH), 127.18
cubated at 37°C for 10 min. The supernatant was removed and
200 µl of the staining solution (8 µl PI [1 mg/ml] and 2 µl RNAse
100 [µg/ml]) was added and the samples were incubated for 10 min
at 37°C in the dark. Fluorescence was measured in the FL2 detector
of the FACSCalibur cytometer (Becton Dickinson & Co.) and the
analysis of the sub‐G1 population (apoptotic cells) was done using
Cell Quest software (BD Biosciences).
(
(
2
2
2
1
3
2
2
). C‐NMR (101 MHz, CDCl
3
(
1
(
(
(
CH), 126.43 (CH), 103.47 (C), 67.58 (CH
CH ). HRMS (ES +ve), C26
2 2
), 45.85 (CH ), and 42.27
+
2
H
26
N
5
O (M+H) : Calculated 424.2137.
Obtained 424.2140.
4.2.3
|
Annexin‐V staining
For the analysis of phosphatidyl serine exposure, Jurkat cells
(250,000 cells/well) were cultured in 24‐well plates in 500 µl of
supplemented RPMI medium and incubated with the chosen com-
4.2
|
Pharmacological/biological assays
4.2.1
|
Cell subculture
2
pounds at the indicated doses for 48 h at 37°C and 5% CO . Eto-
poside was used as the positive control and untreated cells were
used as controls of live cells. After treatment, cells were collected
and washed with annexin V binding buffer (10 mM HEPES pH 7.4,
Jurkat (acute T cell leukemia) and K562 (chronic myelogenous
leukemia) cell lines were grown in T75 flasks in Roswell Park
Memorial Institute (RPMI) 1640 medium (BioWhittaker) with 10%
fetal bovine serum (Gibco), 1% L‐glutamine (Gibco), and incubated
2
140 mM NaCl, 2.5 mM CaCl ) and resuspended in 100 µl of this
buffer. Then, 2.5 µl of annexin‐V‐FLUOS solution (Roche) was added
to each sample, followed by incubation for 20 min in the dark at
room temperature. After a final wash, the pellet was resuspended in
annexin V binding buffer containing 10 µl of PI at 10 µg/ml. Stained
samples were immediately analyzed by flow cytometry using a
2
at 37°C under 5% CO . HeLa (cervical cancer), G361 (malignant
melanoma), MDA‐MB‐231 (breast cancer), and HCT116 (colon
cancer) cell lines were grown in T75 flasks in Dulbecco's Modified
Eagle Medium (DMEM) with 10% fetal bovine serum (Gibco) and

12 of 13
LORENTE‐MACÍAS ET AL.
|
FACSCalibur flow cytometer and analyzed using Cell Quest
software.
100 μl/well. The final compound concentration ranged from 0.05 to
200 μM. Untreated cells were incubated with the maximum DMSO
concentration used (0.2% v/v). After 5 days, PrestoBlue™ cell viabi-
lity reagent (10 µl) was added to each well and the plates were in-
cubated for 90–120 min. Fluorescence emission was detected using
an Envision® fluorescence plate reader (excitation 530/20 nm,
emission 590/35 nm).
4.2.4
|
Z‐VAD‐FMK treatment
For the study of the induction of caspase‐dependent cell death,
Jurkat cells (200,000 cells/well) were cultured in 24‐well plates in
All conditions were normalized to the untreated cells (100%) and
curves were fitted using a four‐parameter logistic fit with the mini-
mum value constrained to zero using GraphPad Prism software to
calculate half‐maximal inhibitory concentration (IC50) values. IC50
values are expressed as the mean of three independent experiments.
5
00 µl of supplemented RPMI medium and incubated with Z‐VAD‐
FMK (50 µM) for 1 h at 37°C and 5% CO . Thereafter, cells were
treated with the chosen compounds (30 µM) and incubated for 24 h
at 37°C and 5% CO . 6d and DMSO were used as positive and ne-
2
2
gative controls, respectively. Cell cycles were analyzed by staining of
cells with PI staining. Thus, after treatment with the corresponding
compounds, the cells were collected and washed with 2 ml of
phosphate‐buffered saline (PBS) at 4°C and fixed with 100 µL of PBS
and 900 µl of 70% ethanol on ice for 5 min. After washing with PBS,
cells were resuspended in 250 µl of PBS, and another 250 µl of a
4.3
|
cLogD determinations
Calculated distribution coefficients (clogD) were determined using
[
13]
Chemicalize by ChemAxon Ltd.
at the pH of 7.4.
DNA extraction buffer (0.2 M Na
incubated at 37°C for 10 min. The supernatant was removed and
00 µl of the staining solution (8 µl PI [1 mg/ml] and 2 µl RNAse 100
µg/ml]) was added and the samples were incubated for 10 min at
7°C in the dark. Fluorescence was measured in the FL2 detector of
2 4 6 8 7
HPO , 0.1 M C H O , pH 7.8) and
ACKNOWLEDGMENTS
2
Álvaro Lorente‐Macías acknowledges support from the Spanish
MECD (FPU 14/00818). This study was supported by grant RTC‐
2017‐6620 from the Spanish Ministry of Science and Innovation.
Funding for open access charge: Universidad de Granada/CBUA.
[
3
the FACSCalibur cytometer (Becton Dickinson & Co.) and the ana-
lysis of the sub‐G1 population (apoptotic cells) was done using Cell
Quest software (BD Biosciences).
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interest.
4.2.5
|
Dose–response cell viability assays
ORCID
Álvaro Lorente‐Macías
http://orcid.org/0000-0001-9510-714X
Hematopoietic cells (Jurkat and K562): Compounds, including DMSO,
were prepared as intermediate solutions at 2× in supplemented
RPMI medium. The compounds were then added in serial dilution in
María J. Pineda de las Infantas
http://orcid.org/0000-0002-
0617-6408
9
6‐well flat‐bottomed plates to give a final volume of 50 μl/well.
Then, 50 µl of a previously prepared supplemented RPMI medium at
000 cells per well (Jurkat, K562) was added to each well and the
plates were incubated for 5 days in an incubator at 37°C and 5%
CO . The final compound concentration ranged from 0.05 to 200 μM.
REFERENCES
[1] H. Rosemeyer, Chem. Biodivers. 2004, 1, 361. https://doi.org/10.
002/cbdv.200490033
2] M. E. Welsch, S. A. Snyder, B. R. Stockwell, Curr. Opin. Chem. Biol.
010, 14, 347. https://doi.org/10.1016/j.cbpa.2010.02.018
1
1
[
[
[
2
2
3] A. M. Pedley, S. J. Benkovic, Trends Biochem. Sci. 2017, 42, 141.
https://doi.org/10.1016/j.tibs.2016.09.009
Untreated cells were incubated with the maximum DMSO con-
centration used (0.2% v/v). After 5 days, PrestoBlue™ cell viability
reagent (10 µl) was added to each well and the plates were incubated
for 90–120 min. Fluorescence emission was detected using a Sy-
nergy™ HTX Multi‐Mode Microplate Reader (excitation 530/20 nm,
emission 590/35 nm).
4] C. Benson, J. White, J. de Bono, A. O'Donnell, F. Raynaud,
C. Cruickshank, H. McGrath, M. Walton, P. Workman, S. Kaye,
J. Cassidy, A. Gianella‐Borradori, I. Judson, C. Twelves, Br. J. Cancer
2
007, 96, 29. https://doi.org/10.1038/sj.bjc.6603509
[5] E. de Clercq, J. Med. Chem. 2019, 62, 7322. https://doi.org/10.1021/
acs.jmedchem.9b00175
[
6] S. Sharma, J. Singh, R. Ojha, H. Singh, M. Kaur, P. M. S. Bedi,
Nonhematopoietic (G361, MDA‐MB‐231, HCT116, HeLa): Cells
were seeded in 96‐well flat‐bottomed plates at 500 cells/well (G361
and HCT116 cells), 1000 cells/well (HeLa cells) and 2000 cells/well
K. Nepali, Eur. J. Med. Chem. 2016, 112, 298. https://doi.org/10.
1
016/j.ejmech.2016.02.018
[7] C. R. Coxon, E. Anscombe, S. J. Harnor, M. P. Martin, B. Carbain,
B. T. Golding, I. R. Hardcastle, L. K. Harlow, S. Korolchuk,
C. J. Matheson, D. R. Newell, M. E. M. Noble, M. Sivaprakasam,
S. J. Tudhope, D. M. Turner, L. Z. Wang, S. R. Wedge, C. Wong,
R. J. Griffin, J. A. Endicott, C. Cano, J. Med. Chem. 2017, 60, 1746.
https://doi.org/10.1021/acs.jmedchem.6b01254
(
MDA‐MB‐231 cells) in 50 μl of supplemented medium and incubated
for 24 h in an incubator at 37°C and 5% CO . After 24 h, the medium
2
was aspirated from each well and replaced with 100 μl of fresh
medium. Compounds, including DMSO, were prepared as inter-
mediate solutions at 2× in supplemented DMEM medium. Then,
compounds were added in serial dilution to give a final volume of
[8] N. S. Gray, S. Kwon, P. G. Schultz, Tetrahedron Lett. 1997, 38, 1161.
https://doi.org/10.1016/S0040-4039(97)00014-2

LORENTE‐MACÍAS ET AL.
13 of 13
|
[
9] Á. Lorente‐Macías, M. Benítez‐Quesada, I. J. Molina, A. Unciti‐Broceta,
J. J. Díaz‐Mochón, M. J. Pineda de las Infantas Villatoro, Magn. Reson.
Chem. 2018, 56, 852. https://doi.org/10.1002/mrc.4743
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[
[
10] P. G. Baraldi, A. U. Broceta, M. J. P. D. Las Infantas, J. J. D. Mochun,
A. Espinosa, R. Romagnoli, Tetrahedron 2002, 58, 7607. https://doi.
org/10.1016/S0040-4020(02)00867-0
11] M. J. Pineda De Las Infantas Y Villatoro, J. D. Unciti‐Broceta,
R. Contreras‐Montoya, J. A. Garcia‐Salcedo, M. A. G. Mezo,
A. Unciti‐Broceta, J. J. Diaz‐Mochon, Sci. Rep. 2015, 5, 2. https://doi.
org/10.1038/srep09139
How to cite this article: Á. Lorente‐Macías, I. Iañez, M. C.
Jiménez‐López, M. Benítez‐Quesada, S. Torres‐Rusillo, J. J.
Díaz‐Mochón, I. J. Molina, M. J. Pineda de las Infantas.
Synthesis and screening of 6‐alkoxy purine analogs as cell
type‐selective apoptotic inducers in Jurkat cells. Arch. Pharm.
[
12] M. J. Pineda de las Infantas, S. Torres‐Rusillo, J.D. Unciti‐Broceta,
P. Fernandez‐Rubio, M.A. Luque‐Gonzalez, M. Gallo, A. Unciti‐
Broceta, I. J. Molina, J. J. Diaz‐Mochon, Org. Biomol. Chem. 2015, 13,
2
021, e2100095. https://doi.org/10.1002/ardp.202100095
5
224. https://doi.org/10.1039/C5OB00230C
13] M. Swain, J. Chem. Inf. Model. 2012, 52, 613. https://doi.org/10.
021/ci300046g
14] Á. Lorente‐Macías, Doctoral Thesis, Universidad de Granada
Granada, Spain) 2019.
[
[
1
(