Semisynthesis, characterization and evaluation of new adenosine derivatives as antiproliferative agents

Article abstract of DOI:10.3390/molecules23051111

We describe the semisynthesis and biological effects of adenosine derivatives, which were anticipated to function as agonists for the A₃ receptor. Molecular docking was used to select candidate compounds. Fifteen nucleoside derivatives were obtained through nucleophilic substitutions of the N⁶-position of the nucleoside precursor 6-chloropurine riboside by amines of different origin. All compounds were purified by column chromatography and further characterized by spectroscopic and spectrometric techniques, showing moderate yield. These molecules were then evaluated for their antiproliferative activity in human gastric cancer cells expressing the A₃ receptor. We found that the compounds obtained have antiproliferative activity and that new structural modifications can enhance their biological activity. The ADME (Absorption, Distribution, Metabolism and Excretion) properties of the most active compounds were also evaluated theoretically.

Full text of DOI:10.3390/molecules23051111

molecules
Article
Semisynthesis, Characterization and Evaluation of
New Adenosine Derivatives as
Antiproliferative Agents
Francisco Valdés Zurita 1, Nelson Brown Vega 2,3 and Margarita Gutiérrez Cabrera 1,
*
1
Institute of Chemistry of Natural Resources, University of Talca, 3460000 Talca, Chile;
franciscovaldesz@hotmail.es
Medical School, University of Talca, 3460000 Talca, Chile; nbrown@utalca.cl
Programa de Investigación Asociativa en Cáncer Gástrico (PIA-CG), Universidad de Talca,
2
3
3460000 Talca, Chile
*
Correspondence: mgutierrez@utalca.cl; Tel.: +56-712-200-448
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 April 2018; Accepted: 5 May 2018; Published: 8 May 2018
Abstract: We describe the semisynthesis and biological effects of adenosine derivatives, which were
anticipated to function as agonists for the A3 receptor. Molecular docking was used to select candidate
compounds. Fifteen nucleoside derivatives were obtained through nucleophilic substitutions of
the N6-position of the nucleoside precursor 6-chloropurine riboside by amines of different origin.
All compounds were purified by column chromatography and further characterized by spectroscopic
and spectrometric techniques, showing moderate yield. These molecules were then evaluated for
their antiproliferative activity in human gastric cancer cells expressing the A3 receptor. We found that
the compounds obtained have antiproliferative activity and that new structural modifications can
enhance their biological activity. The ADME (Absorption, Distribution, Metabolism and Excretion)
properties of the most active compounds were also evaluated theoretically.
Keywords: nucleoside derivatives; adenosine; cancer; antiproliferative activity
1. Introduction
Nucleosides are endogenous compounds that play critical roles in various cellular processes [
1
].
].
This multifunctionality renders these molecules interesting for both design and drug discovery [
2
This group of compounds encompasses analogues of the naturally occurring DNA and RNA bases,
as well several derivatives of nucleosides and nucleotides of these bases.
Due to the ubiquitous nature and widespread functions of nucleoside derivatives (NDs), structural
modifications can be interesting as tools for the generation of agonists or antagonists. Several reports
have shown that a number of chemical modifications in nucleosides confer important improvements
in their bioactivity. For example, NDs have shown antiviral and antibacterial activities [
3–5], and some
of them have also proven effective as anticancer agents [ ]. Indeed, many nucleoside analogues have
6,7
already been approved by the Food and Drug Administration (FDA) for cancer treatment. For example,
the base analogue 6-mercaptopurine (Figure 1a) has been used to treat acute lymphoblastic leukemia,
as well as various autoimmune disorders [
8]. Likewise, azacytidine (Figure 1b), a pyrimidine analogue,
has been used to treat myelodysplastic syndromes [
9], and nelarabine (Figure 1c) was approved in
2005 for the treatment of T-cell lymphoblastic lymphoma/leukemia [10].
Among nucleosides, those containing purine bases are of great importance in medicinal chemistry.
This medical potential has led to the organic synthesis and production of new purine derivatives in
order to enhance their biological effects or improve yields when directly obtained from natural sources.
Molecules 2018, 23, 1111; doi:10.3390/molecules23051111
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1111
2 of 17
Not surprisingly, derivatives and analogues of purine bases have been shown to possess a wide range
of biological properties [5,11–13].
OCH3
NH2
N
S
N
N
N
N
N
N
N
H
O
HN
N
NH2
HO
HO
O
O
N
H
H
H
H
OH
H
H
H
H
OH
OH
OH
(a)
(b)
(b)
Figure 1. (a) 6-mercaptopurine, (b) azacitidine, and (c) nelarabine act as nucleic acid synthesis inhibitors
and have been approved as anticancer drugs by the Food and Drug Administration (FDA).
Adenosine, a natural nucleoside present in different organs and a known modulator of
physiological and pathophysiological processes, can bind to at least four subtypes of G-protein-coupled
receptors [14]. Chemical modifications of the nucleus of adenosine have produced compounds
with interesting biological applications. For example, N6-focused structural modifications of
adenosine-containing nucleosides have given rise to cytokinin nucleosides containing a hydrophilic
ribofuranose moiety and a purine heterocyclic nucleus [5
]. Previously, N6-substituted adenosines
had been reported as natural products isolated from a cell culture of anise [15] in the endosperm
liquid of fresh young coconut fruits [16] and as an abundant terpene nucleoside derived from
Mycobacterium tuberculosis [17]. Also, N6-substituted-adenosines have been further modified in order
to specifically target any one of the four receptors of adenosine (A1, A2A, A2B and A3) [18
antitumor and antiproliferative effects [20,21].
,19], revealing
The assessment of adenosine and its derivatives as candidates for adjuvant anti-cancer therapy [22
]
has had a considerable impact on the treatment of certain types of tumors [23]. In this context,
all subtypes of purinergic receptors (A1, A2A, A2B and A3) could be exploited as potential targets for
the development of new anti-tumor drugs [24]. In particular, the activation of signaling pathways
dependent on A3 receptor activation can lead to reduced proliferation or induction of apoptosis in
some tumor cells [24,25]. Nevertheless, the activation of the A3 receptor may also prove advantageous
to cancer cells during their adaptation to hypoxia, a condition commonly observed in rapidly growing
solid tumors [26].
Herein, we report the semisynthesis and chemical characterization of fifteen adenosine derivatives
with different substituents at the N6-position. These substitutions were predicted to confer agonist
activity on the A3 receptor. In addition, proliferative assays were used to assess the ability of these
derivatives to impair proliferation of A3 receptor-expressing human gastric epithelial adenocarcinoma
cells (AGS).
2. Results and Discussion
2.1. Molecular Docking of Adenosine Derivatives
The Gaussview 5.0 (Semichem Inc., Shawnee, KS, USA) and the Maestro suite programs
(Schrödinger, LLC, New York, NY, USA) were used for the in silico design of adenosine analogs
from the precursor 6-chloropurine-riboside. This procedure gave rise to a set of 284 molecules that
included compounds with modifications at the ribose ring, as well as compounds in which the N6
position of the purine ring was substituted by aliphatic and aromatic amines. Conformations of the

Molecules 2018, 23, 1111
3 of 17
most stable structures were obtained, which represent those with less repulsion. Following docking,
a total of 1572 molecular coupling conformers were obtained. Subsequently, a filter of selection
was applied based on the interactions and/or contacts between the compounds and the amino acid
residues of interest present in the active site of the A3 receptor. After application of the pose-filter
script, only 42 conformers were obtained, corresponding to fourteen compounds. These compounds
were then compared to the commercially available agonist (IB-MECA) and antagonist (MRS-1523).
The most favorable energy values for each ligand-A3 receptor binding were identified, selected,
and registered (Table 1). As an auxiliary discrimination method, free energies of interaction were
calculated through MMGB-SA (Molecular Mechanics–Generalized Born Surface Area) for each
compound, including an agonist and an antagonist as references.
Table 1. Binding energies and free energies (∆Gb) for the fourteen compounds that were post-filtered
according to their interactions with the active site of the A3 receptor.
Energies
Ligands
Binding Energy (Kcal/mol)
∆Gb (Kcal/mol)
1
2
3
4
5
6
7
8
9
10
11
12
13
−44.94
−41.69
−38.72
−40.73
−44.56
−43.68
−38.71
−37.88
−30.06
−38.95
−46.53
−35.32
−37.20
−37.41
−47.29
−23.46
−84.71
−79.75
−78.28
−77.74
−76.52
−76.07
−70.76
−69.60
−68.64
−68.29
−67.00
−66.36
−64.83
−57.93
−67.391
−77.363
14
IB-MECA
MRS-1523
Post-docking selected compounds were grouped and numbered in Figure 2. These compounds
showed structural diversity, with several modifications in the ribose region of the adenosine nucleus
(protection of the 20-OH and 30-OH groups; oxidation of the 50-OH group to a carboxylic acid) and
different aromatic substitutions at the N6-position.
1
2
3
4
Figure 2. Cont.

Molecules 2018, 23, 1111
4 of 17
5
6
7
8
9
10
11
12
13
14
IB-MECA
MRS-1523
Figure 2. Chemical structures of selected compounds derived from in silico analyses.
Molecular dockings were visualized through the PyMOL software, identifying interactions
of some ligands with the active site of the A3 receptor (Figure 3). Compound displayed the
1
highest binding energy compared to the other compounds, including the selective agonist IB-MECA
(N6-(3-Iodobenzyl) adenosine-50-N-methyluronamide). Figure 3A shows some of the interactions
established by compound 1 with important amino acid residues of the active site of the A3 receptor.
As a comparison, Figure 3B, C show the interactions displayed by the selective agonist and the selective
antagonist, B-MECA and MR-1523, respectively.
Similar results to those reported by Valdés et al. [27] were obtained. The fourteen compounds
presented interactions pi–pi stacking between the purine ring and Phe 168, and a ring-type interaction
with Asn 250. N6-substituted compounds also displayed three hydrogens bonds between Ser 271 and
the oxygens of 20-OH and 30-OH groups. However, some compounds presented hydrogen bonds
between the Gln 261 and the 50-COOH group. For example, the docking for compound
1 showed
hydrogen bonds, hydrophobic and mild polar interactions, as well as pi–pi stacking interactions.
The residue of Ser 271 displayed a hydrogen bond interaction with the oxygen of the OH-Ar
substitution group and two hydrogen bonds with Gln 261. The Phe 168 showed pi–pi interaction with
the purine ring.

Molecules 2018, 23, 1111
5 of 17
3D
2D
A
Compound 1
B
IB-MECA
C
MRS-1523
Figure 3. 3D and 2D interactions of the semi-synthesized compound
1
(
A
), the selective commercial
selective agonist, IB-MECA (N6-(3-Iodobenzyl) adenosine-50-N-methyluronamide) (
B), and the
commercial selective antagonist, MRS-1523 (C), with residues at the active site of the A3 receptor.
The interactions found for the selective agonist IB-MECA confirmed previous reports [27–29].
It is important to mention that most of the compounds that were able to form hydrogen bonds between
the 30-O-isopropylidene group and Ser 271 acted as agonist molecules [29]. In addition, when the

Molecules 2018, 23, 1111
6 of 17
compounds showed pi–pi stacking interactions with Phe 168 residue, these interactions were indicative
of increased agonist-receptor binding affinity [30].
Likewise, the interactions shown by the selective antagonist for the A3 receptor, known as
MRS-1523, depended on a single hydrogen bond between the thiocarbonyl group and the Ser 271
residue, allowing the compound to be recognized as an antagonist.
Therefore, the interactions displayed by compound 1 were energetically more favorable than those
shown by the selective agonist IB-MECA. When compared to the antagonist MRS-1523, the interactions
were weaker in proportion and less energetically favorable than compound 1.
Regarding the binding energies of the different compounds to the active site of the A3
receptor, these were difficult to assert, which led us to perform free energy calculations of
interactions between two systems (ligand-protein). This methodology is called MMGB-SA (molecular
mechanics-generalized surface area) and was used as an auxiliary discrimination method [31,32].
However, there were not significant differences between the energy values presented by the compounds
and the energies of the agonist (IB-MECA), which would be a positive indicator in the search for new
agonists with the same or better coupling shape and affinity for the A3 receptor.
2.2. Chemical Synthesis
Next, we synthesized a subset of adenosine derivatives with different patterns of substitution at
the N6 position of the aromatic ring. Each substitution was carried out using amines of different origin
according to the bioinformatic procedures previously described [27]. In total, six of the compounds
proposed by molecular docking were synthesized. In addition, we also synthesized compounds
that were not selected by molecular docking for obtaining greater structural diversity of adenosine
derivative compounds. Series
1
(Figure 4a–i) was obtained by nucleophilic substitutions with aromatic
(Figure 5a–f) was obtained
and aliphatic amines, with yields between 43% and 79%. The series
2
by a modification of the precursor at the diol (20-OH and 30-OH groups) of the ribose and by a total
0
oxidation of 5 -OH group, followed by a nucleophilic substitution with amines at the N6 position of the
precursor, with yields between 43% and 98%. All compounds were isolated by column chromatography.
The structures of all derivatives were confirmed by IR and NMR spectroscopy, showing signals that
were characteristic of aromatic protons near to hete−ro1atom and aliphatic atom, being identified in the
1H-NMR. The IR spectra showed signals of 3500 cm that were characteristic of hydroxyl groups and
signals between 1640 and 1560 cm−1 for primary amines, and 1500 cm−1 for secondary amines.
In the particular case of compounds 1f and 2d, the substitution was performed between the
chlorine atom of the nucleoside and the thiol group of the amine, this because of the higher reactivity
and nucleophilia of the thiol group compared to the amine group.
The structural diversity of both synthesized series is presented in Figures 4 and 5, where each
“R” represents a substituent that was coupled to the N6-position of the purine ring. It is important to
mention that series 1 and 2 preserve a common structural core, formed by a ribose (with or without
modifications) and a purine ring.

Molecules 2018, 23, 1111
7 of 17
1a
(10) *
1b
(6) *
1c
(5) *
1d
1h
1e
1f
1g
1i
Figure 4. Structures of adenosine derivatives that belong to the series 1. (* Compounds suggested by
molecular docking).
2a
(3) *
2b
(4) *
2c
Figure 5. Cont.

Molecules 2018, 23, 1111
8 of 17
2f
(1) *
2d
2e
Figure 5. Structures of adenosine derivatives that belong to series 2. (* Compounds suggested by
molecular docking).
2.3. Biological Activity of Adenosine Derivatives
All derivatives were tested for their ability to impair proliferation of A3-expressing AGS gastric
cancer cells. For each compound, final concentrations of 10, 100 and 1000 nM were tested for 72 h.
The percentage of inhibition of cell proliferation was determined and compared to the inhibitory effects
of the positive control IB-MECA. The results are shown in Figure 6.
Based on their antiproliferative activity, the most interesting compounds were 1e, 1h, 2b and
2f. These compounds displayed the highest degrees of inhibition with the lowest concentrations.
It is important to mention that the compound IB-MECA showed antiproliferative activity only at the
highest concentration (1000 nM) tested, which is in stark contrast with its efficacy shown in other
cell lines [33]. Overall, most of the structures obtained by semisynthesis showed improvements in
biological activity (i.e., antiproliferative activity), corroborating the hypothesis that modifications at
the N6-position influence the biological effects of adenosine derivatives.
Figure 6. The ability of adenosine derivatives to inhibit the proliferation AGS cells (gastric
adenocarcinoma) was assessed. Three concentrations (10 nM, 100 nM and 1000 nM) for each compound
were tested. Compounds of the series 1 (1a
antagonist (MRS-1523), the protected adenosine (A) modified 5 -OH group and protected on the ribose
ring (B) (see Figure 7), adenosine (Ado) and 6-chloropurine riboside were all tested. The values
–
1h), 2 (2a
–
2f),0the selective agonist (IB-MECA) and
represent the percentage of inhibition with respect to the negative control used (0.2% DMSO)
standard deviation (SD). Interestingly, most of the compounds tested display better percentages of
inhibition at concentrations of 10 and 100 nm when compared to the selective agonist (IB-MECA).
±

Molecules 2018, 23, 1111
9 of 17
Figure 7. General synthesis steps of series 2.
Taking the in silico and biological data together, two compounds showed a good correlation
between computational calculations (in silico) and results obtained by cell proliferation assays (in vitro).
Thus, docking analyses first revealed that compounds 2b and 2f had better binding energies to the
active site of the A3 receptor (
∆
Gb >
−
75 Kcal/mol). After testing these compounds in AGS cells,
they also showed better inhibitory effects at concentrations of 100 nM and 1000 nM [20,24].
Some selected molecular properties were predicted using Molinspiration [34]. Lipinski’s rule of
five is commonly used to predict oral bioavailability of potential lead or drug molecules. According to
this, a candidate molecule will likely be active orally, if: (i) the molecular weight is under 500; (ii) the
calculated octanol/water partition coefficient (Log P) is <5 L; (iii) it has fewer than five hydrogen
bond donors (OH and NH groups) and; (iv) it has less than ten hydrogen bond acceptors (notably
N and O) [34]. The molecular properties of the most active adenosine derivatives (1e, 1h, 2b and 2f)
were calculated using the Molinspiration cheminformatics software [34]. The parameters obtained are
presented in Table 2. Only one example of violation of the Lipinski’s rules [35] was observed in the
four compounds analyzed (1e, 1h, 2b, 2f), namely the number of hydrogen bond acceptors. The Log
P values (partition coefficients, an indicator of lipophilicity) of all compounds were found to be less
than 5, suggesting good permeability across cell membranes. Similarly, the molecular weight of the
selected derivatives was found to be less than 500. Thus, these molecules were predicted to be easily
transported, diffused and absorbed across biological membranes. On the other hand, the number
of hydrogen bond donors (NH and OH) in compound 1h, 2b, and 2f were also in accordance with
Lipinski’s rules (i.e., less than 5). Overall, the compound 2f is most likely to be orally active as it obeys
most of Lipinski’s rules.
The topological polar surface area (TPSA) is a useful parameter for predicting drug transport
properties. It is defined as the sum of surfaces of polar atoms (normally oxygens, nitrogens and
attached hydrogens) in a molecule. TPSA can be correlated with hydrogen bonding, being a good
indicator of bioavailability of drugs. Thus, this parameter has been shown to correlate very well with
human intestinal absorption, in vitro monolayer permeability, and blood–brain barrier penetration.
All TPSA values of the adenosine derivatives were observed in order of 150 Å, that is, below the limit
of 160 Å. From the results, we can conclude that the compounds 1e, 1h, 2b, 2f can be used as starting
points in the development of potential drugs.
Table 2. Theoretical ADME properties of the most active adenosine derivatives.
Compound
Log P
Molecular Weight
TPSA
n-ON Acceptors
n-OHNH Donors
Volume
1e
1h
2b
2f
1.09
1.70
2.11
2.85
358.36
403.39
426.43
447.83
151.58
144.02
140.87
140.87
10
11
11
11
6
4
3
3
302.34
342.14
358.53
353.32
Log P octanol/water partition coefficient; TPSA topological polar surface area; n-ON number of hydrogen acceptors;
n-OHNH number of hydrogen bond donors. The data was determined with molinspiration calculation software [34].

Molecules 2018, 23, 1111
10 of 17
3. Materials and Methods
3.1. Molecular Docking’s Study of Adenosine Analogues
All the ligand structures were constructed using Gaussview 5.0 (Semichem Inc., Shawnee, KS,
USA). The glide tool from Maestro suite (Glide, version 5.7, Schrödinger, LLC, New York, NY, USA) [36
]
was also used. These software were used to perform the molecular docking of the compounds designed,
applying a hierarchical series of filters that allow to find possible locations of the ligand in the region of
the active site of the human receptor A3. Information of conformational, positional, and orientational
space of the ligands coupled to the active site was obtained under a force field OPLS-SAA (All-Atom
Optimized Potentials for Liquid Simulations) [37].
3.2. Selection of the Best Conformers
After performing molecular docking, the pose-filter script was used to select the pool of
conformers that had some interaction with amino acid residues considered important by different
authors [28,37]. To select the best conformers for each ligand, criteria used by Valdes et al. were
applied [27].
3.3. Chemistry
Melting points were measured using a Büchi apparatus and are uncorrected. The purity of
compounds was checked through analytical TLC (thin layer chromatography) on silica gel plates
(Merck 60 F254, KGaA, Darmstadt, Germany). Compounds were purified by column chromatography
when necessary. Chemicals were bought from Aldrich (KGaA, Darmstadt, Germany) and used without
1
further purification. H and 13C-NMR spectra (400.1 MHz for proton and 100.6 MHz for carbon) were
recorded in an AM-400 spectrometer (Bruker, Rheinstetten, Germany), using DMSO-d6 as solvent.
Tetramethylsilane (TMS) was used as an internal standard. Chemical shifts (δ) and J values are reported
in ppm and Hz, respectively; relative to the solvent peak DMSO-d6 2.5 ppm for protons and 39.7 ppm
for carbon atoms. Signals are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; m, multiplet; br.s, broad singlet. IR spectra (KBr pellets, 500–4000 cm−1) were recorded on a
NEXUS 670 FT-IR spectrophotometer (Thermo Nicolet, Madison, WI, USA).
3.4. Synthesis
All adenosine derivatives were prepared by condensing 6-chloropurine riboside
(6-chloro-9-b-D-ribofuranosyl-9H-purine) with amines of different nature, including amines
and diamines with straight and branched chains, as well as aromatic amines by nucleophilic aromatic
substitution. This gave rise to two series of compounds:
Series 1: these derivatives were obtained by nucleophilic aromatic substitution (SNAr) in absolute
ethanol, using N,N-diisopropylethylamine (DIPEA) as Lewis acid (Figure 8).
The derived nucleosides were obtained through semisynthetic methods described previously
by Ottria et al. [12]. To a solution of 6-chloropurine riboside (0.35 mmol) in absolute EtOH or DMF,
DIPEA (1.05 mmol) and the appropriate amine (4.5 mmol) were added. The mixture was stirred at 80
◦
C for 8 h and then cooled down to room temperature. The solvent was removed by filtration or under
vacuum to leave a residue that was analyzed by TLC. The residue was washed with hexane, dried and
purified by SiO2 column chromatography (CH2Cl2-MeOH, 97:3). In some cases, the addition of dry
Et2O was used to precipitate DIPEACl (N,N-Diisopropylethylamine chloride), which was then filtered
off. The crude residue obtained after evaporation was purified by column chromatography.

Molecules 2018, 23, 1111
11 of 17
Figure 8. General scheme of synthesis of series 1.
2-Hydroxymethyl-5-[6-(2-hydroxy-5-methyl-phenylamino)-purin-9-yl]-tetrahydro-furan-3,4-diol (1a): was
prepared following the above describe procedure starting from 6-chloropurine riboside and
◦
2-amine-4-methylphenol. Yellow solid 50.9% yield; m.p. 237–238 C. 1H-NMR (DMSO-d6, 400.1 MHz)
δ
: 9.96 (s, 1H, NH); 8.58 (s, 1H, OH); 8.53 (s, 1H, CH-Ar purine); 8.44 (s, 1H, CH-Ar purine); 8.13 (d,
J = 4.00 Hz, 1H, CH-Ar); 6.82 (m, 1H, CH-Ar); 6.74 (m, 1H, CH-Ar); 5.96 (d, J = 6.00 Hz, 1H, CH-10);
5.48 (0d, J = 6.11 Hz, 1 H, 20-OH); 5.22 (m, 2H, CH2-50); 4.64 (q, J = 5.83 Hz, 1H, CH-20); 4.19 (m, 1H,
CH-3 );3.99 (q, J = 3.55 Hz, 1H, CH-4 ); 3.64 (m, 2H, CH2-5 ); 2.25 (s, 3H, CH3-Ar). 13C–NMR (DMSO-d6,
0
0
100.6 MHz)
21.1. IR (KBr)
δ
: 152.6, 152.3, 149.3, 145.6, 141.6, 128.3, 127.2, 124.6, 122.3, 120.9, 115.5, 88.4, 86.3, 74.1, 70.9,
λ
/cm−1 3358, 3117, 2926, 2857, 1636, 1476, 1216.Anal. Cal. C17H19N5O5: C = 54.64%,
H = 5.09%, N = 18.75%.
2-[6-(3-Chloro-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol (1b): was prepared
following the above describe procedure starting from 6-chloropurine riboside and chloroaniline.
◦
Yellow solid 50.1% yield; m.p. 190–193 C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ: 10.14 (s, 1H, NH);
8.59 (s, 1H, CH-Ar purine); 8.47 (s, 1 H, CH-Ar purine); 8.20 (s, 1H, CH-Ar); 7.90 (d, J = 8.07 Hz, 1H,
CH-Ar); 7.33 (t, J = 7.83 Hz, 1H, CH-Ar); 7.08 (d, J = 7.83 Hz, 1H, CH-Ar); 5.98 (d, J = 5.98 Hz, 1H,
0
0
0
0
CH-1 ); 5.48 (d, J = 5.38 Hz, 1H, 2 -OH); 5.23 (d, J = 6.85 Hz, 2H, 3 -OH, 5 -OH); 4.65 (d, J = 4.89 Hz, 1H,
0
0
0
0
CH-2 ); 4.18 (s, 1H, CH-3 ); 3.99 (s, 1H, CH-4 ); 3.64 (m, 2H, CH2-5 ). 13C–NMR (DMSO-d6, 100.6 MHz)
: 151.7, 151.8, 149.7, 141.2, 141.0, 132.8, 130.0, 122.1, 120.5, 119.9, 118.9, 87.8, 85.8, 73.6, 70.5, 61.5. IR
(KBr)
/cm−1 3441, 2923, 2860, 1641, 1482, 1219.Anal. Cal. C16H16ClN5O4: C = 50.82%, H = 4.25%,
δ
λ
Cl = 9.38%, N = 18.53%. The spectroscopic dates were concordance with literature reported [38,39].
2-[6-(3-Ethyl-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol (1c): was prepared
following the above describe procedure starting from 6-chloropurine riboside and 3-ethyl aniline.
◦
White solid 54.4% yield; m.p. 181–184 C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ: 9.81 (s, 1H, NH); 8.53 (s,
1H, CH-Ar purine); 8.40 (s, 1H, CH-Ar purine); 7.82 (s, 1H, CH-Ar); 7.20 (t, J = 8.07 Hz, 1H, CH-Ar);
0
6.89 (d, J = 7.46 Hz, 1H, CH-Ar); 7.08 (d, J = 7.83 Hz, 1H, CH-Ar); 5.97 (d, J = 5.87 Hz, 1H, CH-1 ); 5.48 (s,
0
0
0
0
0
1H, 2 -OH); 5.25 (m, 2H, 3 -OH, 5 -OH); 4.61 (s, 1H, CH-2 ); 4.18 (s, 1H, CH-3 ); 3.99 (d, J = 2.93 Hz, 1H,
CH-4 ); 3.65 (m, 2H, CH2-5 );2.60 (q, J= 7.54 Hz, 2H, CH2-1 ); 1.21 (t, J= 7.52 Hz, 3H, CH3-2”).13C–NMR
(DMSO-d6, 100.6 MHz) :152.7, 152.4, 149.8, 144.4, 141.1, 139.9, 128.7, 122.8, 120.8, 120.8, 118.9, 88.3,
86.3, 74.1, 71.0, 62.0, 28.8, 15.9. IR (KBr)
/cm−1 3331, 2970, 2921, 1647, 1592, 1545, 1378, 1218. Anal.
0
0
00
δ
λ
Cal. C18H21N5O4: C = 58.16%, H = 5.65%, N = 18.85%. The spectroscopic dates were concordance with
literature reported [40].
2-[6-(3-Amino-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol (1d): was prepared
following the above describe procedure starting from 6-chloropurine riboside and 1,3-phenylendiamine.
Solid amorphous gray, 71.8% yield; m.p. 198–200 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ: 9.54 (s, 1H,
NH); 8.49 (s, 1H, CH-Ar purine); 8.35 (s, 1H, CH-Ar purine); 7.21 (d, J = 1.83 Hz, 1H, CH-Ar); 6.96 (m,
2H, CH-Ar); 6.29 (dt, J = 2.00 Hz, J3 = 7.00 Hz, 1H, CH-Ar); 5.94 (d, J = 5.99 Hz, 1H, CH-10); 5.47 (d,
J = 6.11 Hz, 1H, 20-OH); 5.30 (m, 1H, 30-OH); 5.20 (d, J = 4.65 Hz, 1H, 50-OH); 5.00 (s, 2H, NH2); 4.63
(q, J = 5.91 Hz, 1H, CH-20); 4.17 (m, 1H, CH-30); 3.98 (q, J = 3.34 Hz, 1H, CH-40); 3.63 (m, 2H, CH2-50).

Molecules 2018, 23, 1111
12 of 17
13C–NMR (DMSO-d6, 100.6 MHz)
109.72, 107.30, 88.33, 86.32, 74.04, 71.02, 62.04. IR (KBr)
δ
: 153.55, 152.80, 149.60, 149.22, 140.92, 140.38, 129.12, 120.75, 109.79,
−1
λ/cm 3415, 3332, 3147, 2924, 2861, 1642, 1546,
1477, 1215. Anal. Cal. C16H18N6O4: C = 53.57%, H = 5.02%, N = 23.44%.
2-[6-(2-Amino-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol
(
1e):
was
prepared following the above describe procedure starting from 6-chloropurine riboside and
1,2-diphenylendiamine. Yellow solid 62.9% yield; m.p. 201–205 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ
: 9.10 (s, 1H, NH); 8.46 (s, 1H, CH-Ar purine); 8.20 (s, 1H, CH-Ar purine); 7.23 (dd, J = 7.83 Hz,
J = 0.98 Hz, 1H, CH-Ar); 6.96 (m, 1H, CH-Ar); 6.77 (dd, J = 7.95 Hz, J = 1.10 Hz, 1H, CH-Ar); 6.59 (m,
1H, CH-Ar); 5.92 (d, J = 6.11 Hz, 1H, CH-10); 5.46 (d, J = 5.87 Hz, 1H, 20-OH); 5.36 (m, 1H, 30-OH),
5.20 (d, J = 4.16 Hz, 1H, 50-OH); 4.63 (q, J = 5.54 Hz, 1H, CH-20); 4.17 (d, J = 3.18 Hz, 1H, CH-30); 3.98
(q, J = 3.42 Hz, 1H, CH-40); 3.69 (m, 1H, CH2-50); 3.56 (m, 1H, CH2-50); 3.35 (s, 2H, NH2). 13C–NMR
(DMSO-d6, 100.6 MHz)
88.3, 86.1, 73.3, 70.9, 61.6. IR (KBr)
δ
: 161.3, 159.5, 150.6, 150.2, 134.7, 130.8, 126.9, 124.5, 120.9, 116, 2, 110.6,
λ
/cm−1 3375, 3335, 3272, 2944, 2918, 1640, 1494, 1215. Anal. Cal.
C16H18N6O4: C = 53.58%, H = 5.02%, N = 23.44%.
2-[6-(5-Amino-[1,2,4]thiadiazol-2-ylsulfanyl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol
(
1f):
was
prepared following the above describe procedure starting from 6-chloropurine riboside and
◦
1
2-amine-5-mercaptothiadiazole. Solid amorphous white, 70.5% yield; m.p. 220–223 C. H–NMR
(DMSO-d6, 400.1 MHz) δ: 8.83 (s, 1H, CH-Ar purine); 8.75 (s, 1H, CH-Ar purine); 7.65 (s, 2H, NH2);
5.99 (d, J = 5.38 Hz, 1H, CH-10); 5.54 (d, J = 4.40 Hz, 1H, 20-OH); 5.24 (s, 1H, 30-OH); 5.07 (s, 1H,
50-OH); 4.59 (m, 1H, CH-20); 4.20 (m, 1H, CH-30), 3.97 (d, J = 3.42 Hz, 1H, CH-40); 3.70(m, 2H, CH2-50).
13C–NMR (DMSO-d6, 100.6 MHz)
70.6, 61.6. IR (KBr)
δ
: 173.6, 156.4, 152.2, 149.7, 144.9, 141.0, 131.1, 88.4, 86.2, 74.4,
λ
/cm−1 3511, 3389, 3112, 2955, 2929, 1644, 1566, 1219. Anal. Cal. C12H13N7O4S2:
C = 37.56%, H = 3.39%, N = 25.56%, S = 16.69%.
2-[6-(4-Amino-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol
(
1g):
was
prepared following the above describe procedure starting from 6-chloropurine riboside and
1,4-diphenylendiamine. Solid gray, 52.0% yield; m.p. 250–253 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ
: 9.46 (s, 1H, NH); 8.42 (s, 1H, CH-Ar purine); 8.25 (s, 1H, CH-Ar purine); 7.42 (d, J = 8.31 Hz, 2H,
CH-Ar); 6.50 (d, 2H, CH-Ar); 5.93 (d, J = 5.87 Hz, 1H, CH-10); 5.48 (s, 1H, 20-OH); 5.34 (s, 1H, 30-OH),
5.19 (s, 1H, 50-OH); 4.87 (s, 2H, NH2); 4.62 (s, 1H, CH-20); 4.16 (s, 1H, CH-30); 3.98 (s, 1H, CH-40); 3.69
(m, 1H, CH2-50); 3.53 (m, 1H, CH2-50). 13C–NMR (DMSO-d6, 100.6 MHz)
δ
: 152.9, 152.5, 149.2, 145.3,
−1
140.5, 128.6, 123.7, 114.1, 120.4, 88.4, 86.3, 71.1, 73.9, 62.1. IR (KBr)
λ
/cm 3404, 3338, 3224, 2918, 2867,
1651, 1512, 1477, 1218. Anal. Cal. C16H18N6O4: C = 53.58%, H = 5.02%, N = 23.44%.The spectroscopic
dates were concordance with literature reported [41].
2-[6-(2,4-Dimethoxy-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol
prepared following the above describe procedure starting from 6-chloropurine riboside and
2,4-dimethoxyaniline. Solid gray, 63.1% yield; m.p. 173–176 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
(
1h):
was
δ
:
8.57 (d, J = 1.22 Hz, 1H, NH); 8.42 (d, J = 2.20 Hz, 1H, CH-Ar purine); 8.27 (d, J = 2.20 Hz, 1H, CH-Ar
purine); 7.84 (dd, J = 8.68 Hz, J = 1.59 Hz, 1H, CH-Ar); 6.67 (d, J = 2.20 Hz, 1H, CH-Ar); 6.53 (m, 1H,
CH-Ar); 5.93 (dd, J = 5.99 Hz, J = 2.08 Hz, 1H, CH-10); 5.47 (d, J = 4.40 Hz, 1H, 20-OH); 5.33 (s, 1H,
30-OH); 5.19 (s, 1H, 50-OH); 4.64 (d, J = 3.67 Hz, 1H, CH-20); 4.16 (s, 1H, CH-30); 3.97 (s, 1H, CH-40);
3.82 (d, J = 2.20 Hz, 3H, -OCH3); 3.76 (d, J = 2.45 Hz, 3H, -OCH3); 3.61 (m, 2H, CH2-50). 13C–NMR
(DMSO-d6, 100.6 MHz) δ: 157.55, 153.26, 152.92, 152.58, 121.05, 149.37, 141.12, 124.95, 120.60, 104.74,
99.43, 88.46, 86.35, 74.02, 71.04, 62.05, 56.28, 55.82. IR (KBr) λ/cm−1 3392, 2941, 2832, 1626, 1540, 1426,
1286, 1212, 1046. Anal. Cal. C18H21N5O6: C = 53.55%, H = 5.21%, N = 17.35%.
2-[6-(5-Chloro-2-hydroxy-phenylamino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol
(1i):
was prepared following the above procedure, starting from 6-chloropurine riboside and
2-amine-4-chlorophenol. Brown solid, 79.2% yield; m.p. 214–218 ◦C. 1H–NMR (DMSO-d6,
400.1 MHz) δ: 10.82 (s, 1H, OH-Ar); 8.60 (s, 1H, CH-Ar purine); 8.50 (s, 1H, CH-Ar purine); 8.48 (s, 1H,

Molecules 2018, 23, 1111
13 of 17
CH-Ar); 7.04 (m, 1H, CH-Ar); 6.94 (d, J = 8.56 Hz, CH-Ar); 5.98 (d, J = 5.87 Hz, 1H, CH-10); 5.55 (d,
J = 5.62 Hz, 1H, 20-OH); 5.26 (d, J = 4.40 Hz, 2H, 30-OH, 50-OH); 4.63 (q, J = 5.14 Hz, 1H, CH-20); 4.15
(m, 1H, CH-30); 3.96 (d, J = 3.18 Hz, 1H, CH-40); 3.64 (m, 2H, CH2-50). 13C–NMR (DMSO-d6, 100.6
MHz)
IR (KBr)
δ
: 152.5, 151.9, 149.6, 146.5, 141.9, 128.7, 123.1, 122.8, 120.9, 120.2, 116.5, 88.3, 86.3, 74.2, 70.9, 61.9.
λ
/cm−1 3352, 3106, 2926, 1630, 1575, 1424, 1219, 652. Anal. Cal. C16H16ClN5O5: C = 48.76%,
H = 4.06%, Cl = 9.00%, N = 17.78%.
Series 2: These derivatives of adenosine were obtained by nucleophilic substitution of
10-deoxy-10-(6-chloro-9H-purin-9-yl)-20,30-O-isopropylidene-beta-D-ribofuranuronic acid, obtained
from successive modifications of the commercial precursor 6-chloropurine riboside: protection of
vicinal diols 20-OH and 30-OH [42] and, then, total oxidation of 50-OH group [43]. Figure 7 shows the
general procedure and reaction conditions.
200 mg (0.7 mmol) of the commercial precursor 6-chloropurine riboside and acetone (10 mL)
were mixed under agitation at room temperature for 30 min. This was followed by the slow
addition of p-toluensulfonic acid (5.57 mmol). The solution was kept under stirring conditions
at room temperature for 3 h. The progress of the reaction was monitored by TLC. Sodium
bicarbonate (1.5 g) was added and maintained under agitation. Once the reaction was finished,
the solid phase was removed and washed with ethyl acetate (
purified by column chromatography with mixtures of CH2Cl2-MeOH, obtaining the compound
[6-(6-Chloro-purin-9-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-methanol ( ); Yellow solid,
: 8.72 (s, 1H, CH-Ar purine); 8.31 (s, 1H,
×
2). The product was then
A
74.1% yield; m.p. 155–158 ◦C 1H–NMR (CDCl3, 400.1 MHz)
δ
CH-Ar purine); 6.01 (d, J = 8.0 Hz, 1H, CH-10); 5.16 (m, 1H, CH-20); 4.97 (d, J = 7.83 Hz, 1H, CH-30);
4.52 (d, J = 1.22 Hz, 1H, CH-40); 3.83 (m, 2H, CH2-50); 5.06 (m, 1 OH); 1.62 (s, 3H, ketal); 1.35 (s, 3H,
ketal). 13C–NMR (CDCl3, 100.6 MHz)
62.7, 27.1, 24.8. IR (KBr)
δ
: 151.6, 151.4, 148.8, 144.4, 132.5, 114.0, 93.4, 86.2, 83.2, 81.1,
/cm−1 3320, 2906, 2863, 959, 733. Anal. Cal. C13H15ClN4O4: C = 47.75%,
λ
H = 4.59%, Cl = 10.85%, N = 17.14%.
For oxidation of 5’–OH, 100 mg of
A
(0.31 mmol) were added to H2O/CH3CN (1:1 mixture)
and placed in an ultrasound bath for 30 min. The solvent was removed by vacuum, and
the residue obtained was stirred with diethyl ether (50 mL), filtered and then dried before
being purified by a SiO2 column chromatography with mixtures of CH2Cl2-MeOH, obtaining
6-(6-Chloro-purin-9-yl)-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic acid (
B), yellow solid,
80.5% yield; m.p. 209–211 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ
: 9.20 (s, 1H, CH-Arpurine); 8.73 (s,
1H, CH-Ar purine); 6.31 (s, 1H, CH-10); 5.28 (d, J = 5.62, 1H, CH-20); 5.20 (d, J = 5.62 Hz, 1H, CH-30);
4.54 (s, 1H, CH-40); 1.52 (s, 3 H, ketal); 1.31 (s, 3H, ketal). 13C–NMR (DMSO-d6, 100.6 MHz)
δ
: 173.1,
−1
151.9, 150.9, 149.1, 147.3, 131.4, 112.8, 91.7, 88.2, 84.9, 84.4, 27.2, 25.5. IR (KBr)
λ/cm 3409, 2990, 2937,
1592, 1336, 1206, 1086, 635. Anal. Cal. C13H13ClN4O5: C = 45.79%, H = 3.82%, Cl = 10.40%, N = 16.44%.
Compound
B was fused with amines by nucleophilic aromatic substitution giving the series 2
of compounds:
6-[6-(2-Hydroxy-phenylamino)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic acid
2a): was prepared following the above describe procedure starting from and 2-aminephenol gray
solid, 43.4% yield; m.p. 275–277 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
: 8.91 (s, 1H, CH-Ar purine);
(
B
δ
8.89 (s, 1H, OH-Ar); 8.42 (s, 1H, CH-Ar purine); 8.32 (d, J = 7.58 Hz, 1H, CH-Ar); 6.87 (m, 3H, CH-Ar);
6.25 (s, 1H, CH-10); 5.15 (m, 2H, CH-20, CH-30); 4.47 (s, 1H, CH-40); 1.51 (s, 3H, CH3-ketal); 1.30 (s,
3H,CH3-ketal). 13C–NMR (DMSO-d6, 100.6 MHz)
δ
: 173.1, 152.5, 152.0, 149.4, 142.0, 127.8, 127.8, 123.8,
121.0, 120.2, 118.9, 115.7, 112.9, 90.8, 87.8, 85.2, 84.5, 27.4, 25.6. IR (KBr)
/cm−1 3407, 2984, 2935, 1624,
1219, 1092. Anal. Cal. C19H19N5O6: C = 55.16%, H = 4.60%, N = 16.93%.
λ
6-[6-(2-Hydroxy-5-methyl-phenylamino)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic
acid (2b): was prepared following the above describe procedure, starting from
B and
2-amine-4-methylphenol. Black soli0d amorphous, 98.5% yield; m.p. 247–252 ◦C. 1H–NMR
(DMSO-d6, 400.1 MHz) δ:12.46 (s, 1H, 5 -COOH); 8.91 (s, 1H, OH-Ar); 8.50 (s, 1H, CH-Ar purine); 8.43

Molecules 2018, 23, 1111
14 of 17
(d, J = 4.00 Hz, 1H, CH-Ar); 8.42 (s, 1H, CH-Ar purine); 8.23 (d, J = 7.83 Hz, 1H, CH-Ar); 8.20 (s, 1H,
CH-Ar); 6.67 (d, J = 7.83 Hz, 1H, CH-Ar); 6.24 (s, 1H, CH-10); 5.14 (d, J = 12.72 Hz, 2H, CH-20, CH-30);
4.46 (s, 1H, CH-40); 2.22 (s, 3H, CH3-Ar); 1.52 (s, 3H, CH3-ketal); 1.30 (s, 3H, CH3-ketal). 13C–NMR
(DMSO-d6, 100.6 MHz)
δ
: 173.1, 152.6, 151.9, 149.3, 146−.01, 127.6, 127.3, 123.9, 121.1, 120.2, 115.4, 112.2,
90.9, 87.8, 85.2, 84.5, 65.4, 27.5, 25.6, 21.2. IR (KBr) λ/cm 3468, 2987, 2926, 1627, 1216, 1092. Anal. Cal.
C20H21N5O6: C = 56.15%, H = 4.91%, N = 16.38%.
6-[6-(4-Amino-phenylamino)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic acid (2c):
was prepared following the above describe procedure starting from and 1,4-phenylendiamine. Black
solid, 95.6% yield; m.p. 250–253 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
: 9.32 (s, 1H, NH); 8.83 (s, 1H,
B
δ
CH-Ar); 8.23 (s, 1H, CH-Ar purine); 7.40 (d, J = 8.56 Hz, 2H, CH-Ar); 6.55 (d, J = 8.56 Hz, 2H, CH-Ar)
6.19 (s, 1H, CH-10); 5.10 (m, 2H, CH-20, CH-30); 4.43 (s, 1H, CH-40); 3.64 (s, 3H, CH3-ketal); 3.37 (s, 3H,
CH3-ketal); 3.15 (s, 2H, NH2). 13C–NMR (DMSO-d6, 100.6 MHz)
140.8, 128.5, 123.4, 119.4, 113.9, 112.7, 90.4, 87.4, 84.9, 84.2, 27.3, 25.4. IR (KBr) λ/cm 3447, 3213, 3109,
δ
: 172.9, 152.6,−1152.4, 149.2, 145.1,
2987, 2932, 1624, 1514, 2095, 1213. Anal. Cal. C19H20N6O5: C = 55.30%, H = 4.85%, N = 20.37%.
6-[6-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic
acid (2d): was prepared following the above describe procedure starting from
B and
3-amine-1,2,4-triazol-5-tiol. Ocher solid, 48.2% yield; m.p. 142–145 ◦C. 1H–NMR (DMSO-d6,
400.1 MHz) δ
: 12.25 (s, 1H, -COOH); 8.78 (s, 1H, CH-Ar); 8.56 (s, 1H, CH-Ar); 6.38 (s, 1H, CH-10); 6.16
(m, 2H, NH2); 5.81 (s, 1H, NH); 5.46 (d, J = 5.62 Hz, 1H, CH-20); 5.37 (d, J = 5.38, 1H, CH-30); 4.62 (d,
J = 8.80, 1H, CH-40); 1.55 (s, 3H, CH3-ketal); 1.33 (s, 3H, CH3-ketal). 13C–NMR (DMSO-d6, 100.6 MHz)
δ
: 172.5, 166.8, 163.0, 152.8, 151.7, 149.1, 145.2, 130.7, 112.7, 90.5, 84.8, 84.2, 83.9, 26.8, 25.2. IR (KBr)
/cm−1 3566, 3389, 2984, 2936, 1643, 1574, 1095. Anal. Cal. C15H16N8O5S: C = 42.82%, H = 3.81%,
λ
N = 26.64%, S = 7.61%.
6-[6-(3-Amino-phenylamino)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic acid
(
2e): was prepared following the above describe procedure starting from
B
and 1,3-phenylendiamine.
:9.43 (s, 1H, NH); 8.39
Purple solid, 60.8% yield; m.p. 193–195 ◦C. 1H–NMR (DMSO-d6, 400.1 MHz)
δ
(m, 1H, CH-Ar purine); 8.78 (s, 1H, CH-Ar purine); 7.20 (s,01H, CH-Ar); 6.93 (m, 1H, CH-Ar); 6.64 (t,
0
0
J = 7.76, 1H, CH-Ar); 6.25 (m, 1H, CH-Ar); 5.83 (m, 1H, CH-1 ), 5.74 (m, 1H, CH-2 ); 5.21 (m, 1H, CH-3 );
4.50 (m, 1H, CH-40); 3.15 (s, 2H, NH2); 1.55 (s, 3H, CH3-ketal); 1.29 (s, 3H, CH3-ketal). 13C–NMR
(DMSO-d6, 100.6 MHz)
107.15, 103.56, 100.47, 90.64, 84.97, 84.58, 45.56, 27.38, 25.57. IR (KBr)
δ
: 152.55, 152.39, 149.55, 149.22, 140.55, 132.45, 129.08, 120.12, 112.89, 109.54,
/cm−1 3450, 3213, 3114, 2987,
λ
2932, 1636, 1219, 1097. Anal. Cal. C19H20N6O5: C = 55.30%, H = 4.85%, N = 20.37%.
6-[6-(5-Chloro-2-hydroxy-phenylamino)-purin-9-yl]-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxole-4-carboxylic
acid (2f): was prepared following the above describe procedure starting from
B and
2-amine-4-chlorophenol. Brown solid, 75.3% yield; m.p. 247–251 ◦C. 1H–NMR (DMSO-d6,
400.1 MHz) δ: 8.90 (s, 1H, OH-Ar); 8.56 (s, 1H, CH-Ar purine); 8.48 (s, 1H, CH-Ar purine); 6.95 (d,
J = 8.31 Hz, 2H, CH-Ar); 6.80 (d, J = 8.31 Hz, 1H, CH-Ar); 6.25 (s, 1H, CH-10); 5.18 (m, 1H, CH-20);
5.09 (d, J = 5.14 Hz, 1H, CH-30); 4.44 (s, 1H, CH-40); 1.51 (s, 3H, CH3-ketal); 1.30 (s, 3H, CH3-ketal).
13C–NMR (DMSO-d6, 100.6 MHz)
115.5, 114.9, 112.8, 90.88, 87.9, 85.1, 84.5, 27.4, 25.6. IR (KBr)
δ
: 172.9, 152.5, 151.5, 149.4, 142.4, 129.3, 122.4, 120.4, 118.9, 116.2,
/cm−1 3434, 2925, 2849, 1623, 1209, 1085,
λ
646. Anal. Cal. C19H18ClN5O6: C = 52.32%, H = 4.13%, Cl = 8.13%, N = 16.06%.
3.5. Biological Activity
3.5.1. Cancer Cell Line and Cell Culture Conditions
Gastric cancer cell lines (AGS) were obtained from the American Type Culture Collection (ATCC).
Cells were cultured in the recommended media (RPMI-1640). The culture medium was supplemented
with 10% FBS, 5
µg/mL of Plasmocin (prophylactic), 250 µg/mL of amphotericin-B and 25 µg/mL of
gentamicin. The cell line was maintained at a 37 ◦C, 5% CO2 and with a humidity of 95%.

Molecules 2018, 23, 1111
15 of 17
3.5.2. General Screening of Adenosine Derivatives
Gastric cancer AGS cells were exposed to different concentrations of semisynthetic compounds
(series 1 and series 2; 10 nM, 100 nM and 1000 nM) for 72 h. Each experiment was repeated twice.
This procedure allowed us to select those compounds with higher activity, which was expressed as a
percentage of inhibition of proliferation compared to the vehicle.
A commercial agonist (IB-MECA), a noselective adenosine agonist (Ado), the commercial
precursor for semisynthesis (6-chloropurine riboside), as well as a selective antagonist (MRS-1523)
were included as controls.
3.5.3. Theoretical Evaluation of ADME Properties
Preliminary data for an ADME profile analysis was estimated using the Molinspiration property
calculation program. Molecular properties such as partition coefficient (Log P), topological polar
surface area (TPSA), hydrogen bond donors and acceptors, rotatable bonds, number of atoms,
molecular weight, and violations of Lipinski’s rule of five, were calculated for each compound [35].
3.5.4. Statistical Analysis
For determining biological activity, each experiment was performed in duplicate. Statistical
analysis of data was carried out using the Student t-test. p < 0.05 was considered statistically significant.
4. Conclusions
We describe the synthesis and structural determination of new adenosine derivatives.
These compounds were obtained through nucleophilic aromatic substitution reactions (SNAr) by
coupling commercially available chloropurine riboside to different amines. Overall, the yields
were moderate to good. Biological activity assays revealed several potential compounds with
antiproliferative effects that do not violate Lipinski’s rules. New modifications at the N6-position
of adenosine can therefore be an important alternative in the search of more selective and potent
compounds with antiproliferative potential.
Author Contributions: F.V., N.B. and M.G. conceived and designed the experiments; F.V. performed the syntheses;
F.V. and M.G. contributed with the spectroscopy data; F.V. and N.B. contributed the biological activity assays; F.V.,
N.B. and M.G. analyzed the data and wrote the paper.
Acknowledgments: This research was supported by Programa de Investigación Asociativa en Cáncer Gástrico
(PIA-CG, RU2107), F.V. thanks Scholarship CONICYT No. 21130335, for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
Sanyal, N.K.; Roychoudhury, M.; Ojha, R.P. Biological activity of the nucleoside analogs: A theoretical study.
Int. J. Quantum Chem. 1981, 20, 159–166. [CrossRef]
Jordheim, L.P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucleoside and
nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12, 447. [CrossRef]
Pertusat, F.; Serpi, M.; McGuigan, C. Medicinal Chemistry of Nucleoside Phosphonate Prodrugs for Antiviral
Therapy. Antivir. Chem. Chemother. 2012, 22, 181–203. [CrossRef]
Vitali, L.A.; Petrelli, D.; Lambertucci, C.; Prenna, M.; Volpini, R.; Cristalli, G. In vitro antibacterial activity of
different adenosine analogues. J. Med. Microbiol. 2012, 61, 525–528. [CrossRef]
Oslovsky, V.E.; Drenichev, M.S.; Sun, L.; Kurochkin, N.N.; Kunetsky, V.E.; Mirabelli, C.; Neyts, J.; Leyssen, P.;
Mikhailov, S.N. Fluorination of naturally occurring N6-benzyladenosine remarkably increased its antiviral
activity and selectivity. Molecules 2017, 22, 1219. [CrossRef]
6.
Shelton, J.; Lu, X.; Hollenbaugh, J.A.; Cho, J.H.; Amblard, F.; Schinazi, R.F. Metabolism, biochemical actions,
and chemical synthesis of anticancer nucleosides, nucleotides, and base analogs. Chem. Rev. 2016, 116,
14379–14455. [CrossRef]

Molecules 2018, 23, 1111
16 of 17
7.
Damaraju, V.L.; Damaraju, S.; Young, J.D.; Baldwin, S.A.; Mackey, J.; Sawyer, M.B.; Cass, C.E. Nucleoside
anticancer drugs: The role of nucleoside transporters in resistance to cancer chemotherapy. Oncogene 2003
,
22, 7524. [CrossRef]
8.
9.
Korelitz, B.I. Expert opinion: Experience with 6-mercaptopurine in the treatment of inflammatory bowel
disease. World J. Gastroenterol. 2013, 19, 2979. [CrossRef]
Khan, C.; Pathe, N.; Fazal, S.; Lister, J.; Rossetti, J.M. Azacitidine in the management of patients with
myelodysplastic syndromes. Ther. Adv. Hematol. 2012, 3, 355–373. [CrossRef]
10. Cohen, M.H.; Johnson, J.R.; Justice, R.; Pazdur, R. FDA drug approval summary: Nelarabine (Arranon®) for
the treatment of T-cell lymphoblastic leukemia/lymphoma. Oncologist 2008, 13, 709–714. [CrossRef]
11. Hocek, M. Syntheses of Purines Bearing Carbon Substituents in Positions 2, 6 or 8 by Metal-or
Organometal-Mediated C−C Bond-Forming Reactions. Eur. J. Org. Chem. 2003, 2003, 245–254. [CrossRef]
12. Ottria, R.; Casati, S.; Baldoli, E.; Maier, J.A.; Ciuffreda, P. N6-Alkyladenosines: Synthesis and evaluation of
in vitro anticancer activity. Bioorg. Med. Chem. 2010, 18, 8396–8402. [CrossRef] [PubMed]
13. Ren, H.; An, H.; Hatala, P.J.; Stevens, W.C., Jr.; Tao, J.; He, B. Versatile synthesis and biological evaluation of
0
novel 3 -fluorinated purine nucleosides. Beilstein J. Org. Chem. 2015, 11, 2509. [CrossRef] [PubMed]
14. Cristalli, G.; Cacciari, B.; Dal Ben, D.; Lambertucci, C.; Moro, S.; Spalluto, G.; Volpini, R. Highlights on
the development of A2A adenosine receptor agonists and antagonists. Chem. Med. Chem. 2007, 2, 260–281.
[CrossRef] [PubMed]
15. Ernst, D.; Schäfer, W.; Oesterhelt, D. Isolation and identification of a new, naturally occurring cytokinin
(6-benzylaminopurineriboside) from an anise cell culture (Pimpinella Anisum L.). Planta 1983, 159, 222–225.
[CrossRef] [PubMed]
16. Ge, L.; Yong, J.W.H.; Goh, N.K.; Chia, L.S.; Tan, S.N.; Ong, E.S. Identification of kinetin and kinetin riboside
in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography–tandem mass
spectrometry, high performance liquid chromatography and capillary electrophoresis. J. Chromatogr. B 2005
829, 26–34. [CrossRef] [PubMed]
,
17. Young, D.C.; Layre, E.; Pan, S.-J.; Tapley, A.; Adamson, J.; Seshadri, C.; Wu, Z.; Buter, J.;
Minnaard, A.J.; Coscolla, M. In vivo biosynthesis of terpene nucleosides provides unique chemical markers
of Mycobacterium tuberculosis infection. Chem. Biol. 2015, 22, 516–526. [CrossRef] [PubMed]
18. Bridges, A.J.;
Bruns, R.F.;
Ortwine, D.F.;
Priebe, S.R.;
Szotek, D.L.;
Trivedi, B.K.
N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine and its uronamide derivatives.
Novel adenosine agonists with both high affinity and high selectivity for the adenosine A2 receptor.
J. Med. Chem. 1988, 31, 1282–1285. [CrossRef] [PubMed]
19. Volpini, R.; Costanzi, S.; Lambertucci, C.; Taffi, S.; Vittori, S.; Klotz, K.-N.; Cristalli, G. N6-Alkyl-2-alkynyl
derivatives of adenosine as potent and selective agonists at the human adenosine A3 receptor and a starting
point for searching A2B ligands. J. Med. Chem. 2002, 45, 3271–3279. [CrossRef] [PubMed]
20. Cohen, S.; Stemmer, S.; Zozulya, G.; Ochaion, A.; Patoka, R.; Barer, F.; Bar-Yehuda, S.; Rath-Wolfson, L.;
Jacobson, K.; Fishman, P. CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory
effects in the liver. J. Cell. Physiol. 2011, 226, 2438–2447. [CrossRef] [PubMed]
21. Chung, H.; Jung, J.-Y.; Cho, S.-D.; Hong, K.-A.; Kim, H.-J.; Shin, D.-H.; Kim, H.; Kim, H.O.; Shin, D.H.;
Lee, H.W. The antitumor effect of LJ-529, a novel agonist to A3 adenosine receptor, in both estrogen
receptor–positive and estrogen receptor–negative human breast cancers. Mol. Cancer Ther. 2006, 5, 685–692.
[CrossRef] [PubMed]
0
0
22. Actis, A.; Croci, M.; Bergoc, R. Efectos farmacológicos de un análogo de adenosina-3 ,5 -monofosfato cíclico
sobre útero de ratones: Consideraciones terapéuticas. Acta Bioquim. Clin. Latinoam. 2005, 39, 197–202.
23. Antonioli, L.; Blandizzi, C.; Pacher, P.; Haskó, G. Immunity, inflammation and cancer: A leading role for
adenosine. Nat. Rev. Cancer 2013, 13, 842. [CrossRef] [PubMed]
24. Fishman, P.; Bar-Yehuda, S.; Synowitz, M.; Powell, J.; Klotz, K.; Gessi, S.; Borea, P. Adenosine receptors and
cancer. Handb. Exp. Pharmacol. 2009, 193, 399–441. [CrossRef]
25. Gessi, S.; Merighi, S.; Sacchetto, V.; Simioni, C.; Borea, P.A. Adenosine receptors and cancer. Biochim. Biophys.
Acta Biomembr. 2011, 1808, 1400–1412. [CrossRef] [PubMed]
26. Gessi, S.; Varani, K.; Merighi, S.; Cattabriga, E.; Iannotta, V.; Leung, E.; Baraldi, P.G.; Borea, P.A. A3 adenosine
receptors in human neutrophils and promyelocytic HL60 cells: A pharmacological and biochemical study.
Mol. Pharmacol. 2002, 61, 415–424. [CrossRef] [PubMed]

Molecules 2018, 23, 1111
17 of 17
27. Valdés, F.; Luna, V.; Arévalo, B.; Brown, N.; Gutiérrez, M. Docking’s study of 6-substituted
adenosine-50-N-ethyluronamide analogs as selective agonists at A3 adenosine receptor and selection of
compounds for to be synthesized later. Der Pharma Chem. 2015, 61, 212–225.
28. Costanzi, S.; Lambertucci, C.; Vittori, S.; Volpini, R.; Cristalli, G. 2-and 8-alkynyladenosines: Conformational
studies and docking to human adenosine A3 receptor can explain their different biological behavior. J. Mol.
Graph. Model. 2003, 21, 253–262. [CrossRef]
29. Van der Wenden, E.; Künzel, J.; Mathôt, R.; Ijzeman, A.; Danhof, M.; Soudijn, W. FC20 ribose modified adenosine
analogues as potential partial agonists for the adenosine receptor. Eur. J. Pharm. Sci. 1994, 2, 105. [CrossRef]
30. Gao, Z.-G.; Kim, S.-K.; Biadatti, T.; Chen, W.; Lee, K.; Barak, D.; Kim, S.G.; Johnson, C.R.; Jacobson, K.A.
Structural determinants of A3 adenosine receptor activation: Nucleoside ligands at the agonist/antagonist
boundary. J. Med. Chem. 2002, 45, 4471–4484. [CrossRef] [PubMed]
31. Kollman, P.A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.
Calculating structures and free energies of complex molecules: Combining molecular mechanics and
continuum models. Acc. Chem. Res. 2000, 33, 889–897. [CrossRef] [PubMed]
32. Zhang, L.Y.; Gallicchio, E.; Friesner, R.A.; Levy, R.M. Solvent models for protein–ligand binding: Comparison
of implicit solvent Poisson and surface generalized Born models with explicit solvent simulations.
J. Comput. Chem. 2001, 22, 591–607. [CrossRef]
33. Fishman, P.; Bar-Yehuda, S.; Ohana, G.; Pathak, S.; Wasserman, L.; Barer, F.; Multani, A. Adenosine acts as
an inhibitor of lymphoma cell growth: A major role for the A3 adenosine receptor. Eur. J. Cancer 2000, 36,
1452–1458. [CrossRef]
34. Molinspiration Cheminformatics. Bratislava, Slovak Republic. Available online: http://www.molinspiration.
com/cgi-bin/properties (accessed on 11 December 2017).
35. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997
,
23, 3–25. [CrossRef]
36. Release, S. 1: Desmond Molecular Dynamics System, version 3.7. Epik Version 2.2; Glide, Version 5.7;
Impact, Version 5.7; LigPrep, Version 2.5; Phase, Version 3.3; Prime, Version 2.3; SiteMap, Version 2.5; Virtual
Screening Workflow. DE Shaw Research: New York, NY, USA, Maestro-Desmond Interoperability Tools.
Schrödinger: New York, NY, USA, 2014.
37. Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.;
Shelley, M.; Perry, J.K. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739–1749. [CrossRef] [PubMed]
38. Daly, J.W.; Padgett, W.; Thompson, R.D.; Kusachi, S.; Bugni, W.J.; Olsson, R.A. Structure-activity relationships
for N6-substituted adenosines at a brain A1-adenosine receptor with a comparison to an A2-adenosine
receptor regulating coronary blood flow. Biochem. Pharmacol. 1986, 35, 2467–2481. [CrossRef]
39. De Zwart, M.; De Groote, M.; Van der Klein, P.A.; Van Dun, S.; Bronsing, R.; Von Frijtag Drabbe Künzel, J.K.;
0
Ijzerman, A.P. Phenyl-substituted N6-phenyladenosines and N6-phenyl-5 -N-ethylcarboxamidoadenosines
with high activity at human adenosine A2B receptors. Drug Dev. Res. 2000, 49, 85–93. [CrossRef]
40. Kwatra, M.M.; Leung, E.; Hosey, M.M.; Green, R.D. N6-Phenyladenosines: Pronounced effect of phenyl
substituents on affinity for A2 adenosine receptors. J. Med. Chem. 1987, 30, 954–956. [CrossRef] [PubMed]
41. Palczewski, K.; Kahn, N.; Hargrave, P.A. Nucleoside inhibitors of rhodopsin kinase. Biochemistry 1990, 29,
6276–6282. [CrossRef] [PubMed]
42. Kim, H.O.; Ji, X.-D0.; Siddiqi, S.M.; Olah, M.E.; Stiles, G.L.; Jacobson, K.A. 2-Substitution of
N6-benzyladenosine-5 -uronamides enhances selectivity for A3 adenosine receptors. J. Med. Chem. 1994, 37,
3614–3621. [CrossRef] [PubMed]
0
43. Epp, J.B.; Widlanski, T.S. Facile preparation of nucleoside-5 -carboxylic acids. J. Org. Chem. 1999, 64, 293–295.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).