Synthesis of 2,6-diamino-substituted purine derivatives and evaluation of cell cycle arrest in breast and colorectal cancer cells

Article abstract of DOI:10.3390/molecules23081996

Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1-3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1-3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.

Full text of DOI:10.3390/molecules23081996

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molecules
Article
Synthesis of 2,6-Diamino-Substituted Purine
Derivatives and Evaluation of Cell Cycle Arrest
in Breast and Colorectal Cancer Cells
Bartolomeo Bosco 1, Andrea Defant 2, Andrea Messina 1, Tania Incitti 1 ID , Denise Sighel 1,2
,
Angela Bozza 1, Yari Ciribilli 1, Alberto Inga 1 ID , Simona Casarosa 1,* and Ines Mancini 2,
*
1
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123 Trento, Italy;
bartolomeo.bosco@unitn.it (B.B.); andrea.messina@unitn.it (A.M.); tania.incitti@gmail.com (T.I.);
denise.sighel@unitn.it (D.S.); angela.bozza@aulss8.veneto.it (A.B.); yari.ciribilli@unitn.it (Y.C.);
alberto.inga@unitn.it (A.I.)
Laboratory of Bioorganic Chemistry, Department of Physics, University of Trento, Via Sommarive 14,
38123 Trento, Italy; andrea.defant@yahoo.it
2
*
Correspondence: simona.casarosa@unitn.it (S.C.); ines.mancini@unitn.it (I.M.); Tel.: +39-461-281-548 (I.M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 July 2018; Accepted: 8 August 2018; Published: 10 August 2018
Abstract: Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora
kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe
three reversine-related molecules, designed by docking calculation, that present structural
modifications in the diamino units at positions 2 and 6. We investigated the conformations of
the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-
N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas
phase, water and chloroform, the last solvent considered to give insights into the detection of
broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the
HN(7) form, but the most stable conformations changed in different solvents. Molecules 1–3 were
evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being
less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy.
Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell
lines used, similar concentrations of 1–3 were effective only in cells where p53 was deleted or
down-regulated. Therefore, our findings support a potential selective role of these structurally
simplified, reversine-related molecules in p53-defective cancer cells.
Keywords: reversine; microwave-assisted synthesis; molecular docking; cell cycle arrest;
endoreduplication; p53
1. Introduction
Pluripotent cells play a relevant role in regenerative medicine, due to their capability of
differentiating into many cell types. Pluripotency can be achieved in vitro, by over-expressing
specific genes or by chemical induction, using a combination of synthetic molecules [
Among them the 2,6-diamino-substituted purine reversine (N6-cyclohexyl-N2-(4-morpholinophenyl)-
7H-purine-2,6-diamine), firstly synthesized by Schultz and coworkers [ ], is a promising molecule.
In fact, it induces de-differentiation of C2C12 murine myoblasts and allows their subsequent
differentiation in adipocytes, osteoblasts [ ], neural progenitor-like cells [ ] and cardiomyocytes [ ],
1].
2
3
4
5
under specific conditions. Besides showing dedifferentiation properties, reversine is a kinase inhibitor
able to interfere with the growth of several solid tumors, by specifically inhibiting the G2/M
checkpoint and cytokinesis. Reversine is an inhibitor of Aurora kinases, Cyclin Dependent Kinases
Molecules 2018, 23, 1996; doi:10.3390/molecules23081996
www.mdpi.com/journal/molecules

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Molecules 2018, 23, 1996
2 of 15
(CDK) and Monopolar Spindle 1 (Mps1), proteins which have a crucial role in mitotic regulation.
Aurora-A kinase is a Ser/Thr kinase that affects centrosomes maturation, mitotic entry, centrosome
separation, spindle formation and cytokinesis [
during mitosis, including chromosome migration to the spindle equator, chromosome segregation
and cytokinesis [ ]. Mps1 is involved in the regulation of spindle checkpoint in part by acting at the
level of CDK1-cyclinB complex [ ], therefore contributing to the proper attachment of chromosomes
6]. Aurora-B kinase can modulate various tasks
7
8
to the spindle microtubules before chromosome separation. Interestingly, this protein has emerged
as a potential target for cancer therapy and reversine is known to inhibit Mps1 more efficiently
than Aurora-B kinase, based on experimental and computational approaches [9]. As inhibitor of
Aurora-A and Aurora-B kinases and Mps1, reversine causes an incorrect mitotic spindle assembly
and chromosome separation, cell cycle arrest in G2/M phase, endoreduplication and eventually cell
death due to a phenomenon called “mitotic catastrophe” observed in many cancer cell lines [10–16].
Moreover, reversine treatment of human follicular thyroid cancer cells leads to the activation of
autophagy due to its capability to down-regulate mTOR [17]. Very recently, reversine has been
reported to significantly inhibit the growth of urothelial carcinoma cell lines through activation of
AMP activated protein kinase pathway, leading to cell death [18].
The transcription factor p53 is described as the guardian of the genome because it is implicated in
several important cell functions, such as DNA repair, cell cycle regulation and apoptosis, preventing
tumor formation. In normal conditions the p53 signaling pathway is in standby mode, but in response
to cellular stresses p53 is activated and through its sequence-specific DNA binding activity it mediates
the transcription of a large number of target genes. p53 is the most commonly mutated gene in many
types of human cancers, losing in this way its tumor suppressor properties. Loss of heterozygosity
(LOH) is also a frequent event resulting in complete lack of wild type p53 expression in cancer cells.
In breast cancer numerous p53 coding mutations were identified, even if the frequency of p53 mutation
in this cancer varies among subtypes and overall is approximately at 20% [19]. p53 has been observed
to play an important role in modulation of reversine effect. It has been demonstrated that silencing of
p53, and the consequent loss of transcriptional activation of its target p21, whose checkpoint function
can prevent endoreduplication, renders fibroblasts more sensitive to reversine [10].
A structure activity relationship (SAR) study on a series of reversine-like molecules showing
different substitutions on both C-6 purine and C-2 piperazine moieties was carried out with the aim to
optimize biological activity, selectivity and oral bioavailability [20]. We report here three 2,6-diamino
substituted purine derivatives, selected by docking calculation focused on Aurora-B kinase and Mps1
inhibition, which present the insertion of a new N-heterocycle unit in the C-2 substitution and a
N-methyl group in C-6 unit, enhancing the structural diversity of reversine analogues. In particular,
we focused on DFT calculations applied to the first structural investigation of the prototropic tautomers
of one of these molecules. The effect of molecules 1–3 has been also compared to reversine on breast and
colorectal cancer cell lines. We observed that they promoted aberrant mitosis and endoreduplication
leading to cell death, possibly via mitotic catastrophe. Moreover, the compounds had a stronger effect
when p53 function was ablated.
2. Results and Discussion
2.1. Selection and Synthesis of Compounds 1–3
The design of the molecules 1–3 (Figure 1) was performed via structure selection based on docking
calculations, considering both energy values and interactions of the complexes between the molecules
and the two known target proteins of reversine, Aurora-B kinase and Mps1. The results by Autodock
Vina indicated that compound
1, which preserves the reversine skeleton, and the structurally simplified
molecules 2 and 3 gave complexes with very similar energy values to those of reversine (Table S1).

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Figure 1. Molecular structures of reversine and synthetic compounds 1–3.
Products
were derived by nucleophilic substitution of cyclohexylamine or methylcyclohexylamine with
2,6-dichloropurine (Scheme 1). The production of proceeded with higher yields and in a cleaner way
compared to . In fact the methyl-N6 substituent as in prevented the formation of the by-product,
resulting by the substitution of a second unit of 2,6-dichloropurine at the N6 position, as observed in
the synthesis of product . The synthesis of was challenging due to the poor nucleophilicity of the
aryl amine
2 [21] and 3 were obtained by reacting aniline with 4 and 5, respectively, the latter
5
4
5
4
1
6
. Different reaction conditions were compared (Buchwald-Hartwing amination, butanol
in the presence of trimethylsilyl chloride or triethylamine; N-methyl-2-pyrrolidone (NMP), 150 ◦C,
p-toluenesulfonic acid monohydrate in DMSO, trifluoroacetic acid (TFA) in EtOH) under conventional
heating or microwave (MW) irradiation, as well as different leaving groups (chlorine as in
5 or a
fluorine analogue obtained by 6-chloro-2-fluoro-9H-purine). The MW irradiation of and in ethanol
5
6
in the presence of TFA proved to be the best procedure, although still giving a poor yield in line with
data for reactions using similar nucleophiles [21].
Scheme 1. Synthesis of molecules
1
–
3
. Reagents and conditions: (
a
) ethanol, TEA, reflux 75 ◦C, 14 h;
) H2, Pd/C, 2 h; ( ) ethanol,
(
b
) N-methyl-2-pyrrolidone, reflux 150 ◦C, 14 h; (
c) DCM, TEA, 12 h; (
d
e
◦
TFA, Microwave 120 C, 2.30 h.
The obtained products were characterized by spectrometric measurements including ESI-MS/MS
and high resolution experiments by EI-MS technique, and by extensive NMR analysis (see Materials
and Methods and Supplementary Material). Prior to their biological evaluation, compound
1
was
purified by preparative RP-18 HPLC and the purity of
(Materials and Methods).
2 and 3 was evaluated using silica gel HPLC

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2.2. Study on the Tautomeric Forms of Product 3
1
Both H- and 13C-NMR spectra of products
1
and
3
recorded in CDCl3 solution at room
temperature showed very broad H3C-N and HC-N signals (Figures S1 and S3), a behavior also
observed for the precursor . The broad nature of these signals would normally suggest the presence
5
of tautomers and/or restricted conformations. This is in line with the report of coexisting tautomers of
purine bases, with the prototropic HN(7) and HN(9) as the preferred species, where the intramolecular
proton-transfer is accompanied by migration of
π electrons. In general their relative populations could
be affected by structural factors such as aromaticity, intramolecular interactions, substituent effects
able to modify the electronic distribution within the molecule, repulsions of lone electron pairs and
also by external influences such as solvent and temperature [22].
In order to gather information on the effective structures of the molecule and to provide details
on the interactions involved with biological targets, we decided to further investigate the tautomeric
forms of 3. Thanls to MarvinSketch software analysis the two most probable tautomers were predicted
to be the prototropic HN(7) and HN(9) forms. Firstly, a series of conformations for each tautomer were
evaluated by Density Functional Theory (DFT) calculation [22] at a B3LYP/6-31G(d,p) level of theory
in the gas-phase, obtaining the corresponding energy values and indications on the energy-minimized
structures for the two prototropic forms. In both cases the most stable conformers displayed an
anti-position of the phenyl-N(2) and cyclohexyl-N(6) groups (Table 1, entries 1 and 8), with the HN(9)
structure as the favored one (E =
−
1027.75607832 a.u.). On the contrary, the structure associated to the
highest energy value is the syn-HN(7) tautomer (Table 1, entry 7). These predictions are in agreement
with HN(9) tautomer being more favored than HN(7), as reported for neutral purine in the gas phase
and in non-polar solvents [23], due to the possibility of a stabilizing intramolecular interaction between
the hydrogen at N(9) position and the lone electron pair on the N(1) atom. Besides, the energy
minimized structure reported in entry 1 displayed a length of 1.36139 Å for the C(6)-N bond, a value
which is really intermediate between 1.47357 Å calculated for a single C-N distance and 1.26715 Å for
a double C=N distance, indicating a restricted rotation around this bond.
Afterwards, the conformations of the two tautomers were also evaluated in chloroform since it is
the solvent used in NMR analysis. The most stable structure (Table 1, entry 1; E =
−
1027.76438045 a.u.)
presented the same conformation as obtained from calculation in the gas phase. Moreover, all the
conformations considered for the HN(9) tautomer were much more stable than the corresponding ones
for the HN(7) isomer. Notably, the latter result is in line with both the detections of a single tautomer
by NMR spectra and with the very similar energy values for the anti- and syn-HN(9) tautomer obtained
by rotation around the N-C(6) bond (entries 1 and 3, respectively). These data support their coexistence
in solution, responsible for the very broad signals attributable to N-CH3 group (δH 3.39 and δC 30.1
ppm), to the cyclohexylmethine carbon (δC 54.8 ppm) and to C(6) (δC 152.3 ppm).
The evaluation of the predominant structure of a ligand molecule present in solution is important
in theoretical drug design. It is known that a more biologically relevant water-phase environment
can significantly change the relative stability of various tautomers if compared with the gas phase
condition [23]. DFT calculations were thus performed in water for the HN(9) and HN(7) forms of
3
by using the Polarized Continuum Model (PCM). The data reported in Table 1 suggest a similar
behavior for the analyzed species both in gas phase and in chloroform solution: the HN(9) tautomer
resulted more stable than the HN(7), with the most stable conformation showing a syn-position of the
phenyl-N(2) and cyclohexyl-N(6) groups (E =
displaying the anti-position (Table 1, entries 3 and 4, respectively).
−
1027.76745565 a.u.), very close in energy to the one