Synthesis of Substituted 5′-Aminoadenosine Derivatives and Evaluation of Their Inhibitory Potential toward CD73

Article abstract of DOI:10.1002/cmdc.201900348

Derivatives of 5′-aminoadenosine containing methyl carboxylate, methyl phosphonate, gem-bisphosphonate, bis(methylphosphonate), and α-carboxylmethylphosphonate or phosphonoacetate moieties were synthesized from key intermediate 5′-aminonucleoside. These nucleotide analogues were envisaged as 5′-mono- or diphosphate nucleoside mimics. All compounds were evaluated for CD73 inhibition in a cell-based assay (MDA-MB-231) and toward the purified recombinant protein. Most of them failed to reach significant inhibition of AMP hydrolysis by CD73 at 100 μm. Among the new compounds, the most interesting candidates, 5 (5′-deoxy-5′-N-phosphonomethyladenosine) and 7 (5′-deoxy-5′-N-(ethoxyphosphorylacetate)adenosine), inhibited recombinant CD73 by 36 and 46 % and cellular CD73 by 61 and 45 % at 100 μm, respectively. Molecular modeling partially explains this lack of activity, as the initially predicted docking scores had been encouraging, especially for compound 9.

Full text of DOI:10.1002/cmdc.201900348

Accepted Article
Title: Synthesis of substituted 5’-aminoadenosine derivatives and
evaluation of their inhibitory potential towards CD73
Authors: Rayane Ghoteimi, Tai Van Nguyen, Rahila Rahimova,
Felix Grosjean, Emeline Cros-Perrial, Jean-Pierre Uttaro,
Christophe Mathé, Laurent Chaloin, Lars Peter Jordheim, and
Suzanne Peyrottes
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ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
Synthesis of substituted 5’-aminoadenosine derivatives and
evaluation of their inhibitory potential towards CD73
R. Ghoteimi,$[a] T. Nguyen Van,$[a] R. Rahimova, F. Grosjean, E. Cros-Perrial, J.-P. Uttaro, C.
[b]
[a]
[c]
[a]
[
a]
[b]
[c]
[a]
Mathé, L. Chaloin, L.P. Jordheim, and S. Peyrottes*
$
These authors contributed equally to this work
[
a]
R. Ghoteimi, Dr T. Nguyen Van, F. Grosjean, Dr J.-P. Uttaro, Prof. C. Mathé, Dr S. Peyrottes, ORCID: 0000-0003-1705-0576
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Univ. Montpellier, ENSCM
Campus Triolet, cc1705, Place Eugène Bataillon, 34095 Montpellier, France
E-mail: suzanne.peyrottes@umontpellier.fr
[
[
b]
c]
Dr R. Rahimova, Dr L. Chaloin, ORCID: 0000-0002-5757-5804
Institut de Recherche en Infectiologie de Montpellier (IRIM), Univ. Montpellier, CNRS
34293 Montpellier, France
E. Cros-Perrial, Dr L.P. Jordheim, ORCID: 0000-0003-3948-7090
Univ. Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286
Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon
69008 Lyon, France
Supporting information for this article is given via a link at the end of the document
Abstract: Derivatives of 5’-amino-adenosine containing methyl
carboxylate, methyl phosphonate, gem-bisphosphonate,
development of immunotherapy for the treatment of cancer
patients have been achieved. Thus, modulation of the immune
system through a small-molecule approach offers several unique
advantages (lower cost, facilitated administration…) being
complementary to and potentially synergistic with immune-
oncology approaches using monoclonal antibodies.[3] This is
exemplified by the association of two mAbs, one targeting CD73
bis(methylphosphonate), and -carboxylmethylphosphonate or
phosphonoacetic moieties were synthesized from a key intermediate
5’-aminonucleoside. These nucleos(t)ide analogues were envisaged
as 5’-mono or diphosphate nucleoside mimics. All compounds were
evaluated for CD73 inhibition in a cell-based assay (MDA-MB-231)
and towards the purified recombinant protein. Most of them failed to
reach significant inhibition of AMP hydrolysis by CD73 at 100 µM.
Among the new compounds, the most interesting ones were
derivatives 5 and 7, inhibiting recombinant CD73 by 36 and 47% and
cellular CD73 by 61 and 45% at 100 µM, respectively. Molecular
modeling allowed to explain partially this lack of activity as predicted
docking score calculated beforehand were initially encouraging,
especially for compound 7.
[
4]
(MEDI-9447 ) and one neutralizing the immune check point PD-
1. However, approaches currently using small molecules to
inhibit CD73 activity are not yet suitable for clinical use. Indeed,
one of the first and well-known small molecule inhibitor (APCP)
has low bioavailability, poor metabolic stability and off-target
related effects presumably associated with chemical instability
and adenosine release.[5] To our knowledge, the most potent
inhibitors of CD73 were described by the team of Prof. Müller
and are structurally related to the APCP skeleton (Figure 1).[ In
addition, nucleoside analogues incorporating two carboxylate
groups have been patented.[7] However, no data are available
on the in vivo activity of these two series of compounds.
Therefore, it is still of great interest to design new small-
molecule CD73 inhibitors.
6]
Introduction
Ecto‐5’‐nucleotidase CD73 is
a
glycophosphatidyl-inositol‐
2
+
anchored di‐Zn
metallo‐phosphatase. It is an important
Study of APCP in the active site of CD73 (Figure 2) showed key
structural interactions with the terminal domains in the substrate
enzyme involved in the extracellular catabolism of nucleosides
and nucleotides working together with the membrane‐bound
CD39. The latter catalyzes the dephosphorylation of ATP into
AMP, whereas CD73 dephosphorylates extracellular AMP into
[8]
binding site. The adenine-base formed hydrophobic π-stacking
interaction between two phenylalanine residues (F417 and
F500), and the diol group of the ribose interacts mainly with
D506. The α-phosphonate group binds with R354 and N245
residues, whereas the β-phosphonate group binds to R395 of C-
terminal domain as well as to N85 and H243 of the N-terminal
adenosine and inorganic phosphate. Thus, CD73, as
a
modulator of adenosine production, has been identified as an
active player and a validated drug target in several diseases,
such as cancer, autoimmunity, allergy and ischemia–reperfusion
injury.[ Numerous studies have shown that the inhibition of
CD73 activity, both in mice models and in cell cultures, by a
substrate analogue mimicking ADP (APCP, 5’-methylene-
bisphosphonate adenosine, Figure 1), a monoclonal antibody or
siRNA, is associated with decreased tumor growth and
metastasis development, decreased tumor angiogenesis and
domain. In addition, α,β-phosphonate groups chelate the two
Zn2+ ions through the formation of monodentate and bidentate
1]
interactions. Based on these observations and as illustrated in
Figure 1, two types of nucleos(t)ide analogues were designed
using adenosine as scaffold. In particular, 5’-amino-adenosine
was selected as the nitrogen atom allows di-substitution or
formation of additional H-bond interaction. Thus, compounds 1,
increased
activity
of
immune-modulating
therapeutic
4
and 5 incorporate either a carboxylate or a phosphonate group
antibodies.[ In the last few years, exciting progress in the
2]
that may be viewed as equivalent of the phosphate of AMP (the
1
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ChemMedChem
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natural CD73 substrate). In order to mimic the interaction of the Results and Discussion
α,β-methylene diphosphate group of APCP with R354 and N245,
a carboxyl oxygen or a phosphoryl oxygen was present in
derivatives 2, 3, and 6-9, whereas the two oxygen atoms of the
Synthesis
The synthesis of adenosine analogues 1-8 began with 5’-amino-
phosphonate group of bis(methylphosphonate), -carboxyl-
methylphosphonate or phosphonoacetic moieties may mimic the
chelation of the Zn2+ ions by the -phosphonate of APCP.
We report herein the synthesis of novel series of N-substituted,
2
’,3’-O-isopropylidene-adenosine 10 (Schemes 1 & 2), which
can be easily accessed in three steps from commercially
available adenosine with overall yield about 71%.[9] Synthesis of
derivatives
nucleophilic displacement by using methyl bromoacetate
diethyl p-toluene sulfonyloxymethylphosphonate (obtained
1
to
5
(Scheme 1) was envisaged through
5’-amino-adenosine derivatives (compounds 1-9, Figure 1) as
[10]
or
potential AMP or ADP mimics and evaluate their potential as
CD73 inhibitors in vitro.
[11]
before ) in presence of a base (either NEt
coupling of diethylphosphonoacetic acid[
3
or DBU), or
using previously
12]
described or adapted conditions. Attempts to obtain di-
substituted derivatives, by increasing the number of equivalents
and/or reaction time, and temperature were unsuccessful.
Concerning the synthesis of compound 1, the 5’-deoxy-2,3-O-
(
isopropylidene)-5’-N-methyl-acetate-5’-aminoadenosine
was
contaminated with triethylammonium salts and directly engaged
in the next step leading to N-alkylated derivative in 45% yield
over the two steps. The latter was treated with an equimolar
amount of 1M NaOH to afford derivative 1. Intermediates 11 and
1
3 were isolated in good yields, and removal of the sugar-
protecting group in acidic conditions afforded derivatives 12 and
4 in 90 and 33% yields, respectively. Deprotection of the
1
diethylphosphonoester group for compounds 12 and 14 was
performed with TMSBr in DMF (Scheme 1). Partially deprotected
derivatives were eventually isolated such as compounds 3 and 4,
as well as the desired phosphonic acids 2 and 5, which were
obtained as sodium salts through ion-exchange on Dowex (Na+
form).
Synthesis of derivative 6 incorporating two phosphonomethyl
groups and derivatives 7 and 8 incorporating a carboxylate and
a phosphonate groups was also carried out starting from 5’-
amino-5’-deoxy-2’,3’-O-isopropylidene-adenosine 10 (Scheme
2).
The
double
Kabachnik-Fields
reaction
of
10,
paraformaldehyde, and diethyl phosphite was performed under
microwave activation and solvent-free conditions.[13] The
resulting derivative was directly treated in acidic conditions to
afford compound 15 in 31% yield over the two steps. Then,
removal of the phosphonic protecting groups was carried out as
previously described and the desired 5’-N-[bisphosphonomethyl]
adenosine 6 was obtained quantitatively.
Figure 1. Structures of ADP analogues as potent CD73 inhibitors and studied
compounds 1-9.
The -carboxyphosphonate derivative 16 was obtained by using
the Aza-Pudovick reaction, consisting in the addition of
diethylphosphite to the in situ generated imine from 10 and
ethylglyoxylate. This step was also performed under microwave-
A
B
R354
N245
R395
H38
H243
R354
F417
N245
D506
D36
H220
D36
D85
N390
H118
assisted and solvent-free conditions as described in the
D85
[14]
R395
H220
literature.
Removal of the various protecting groups was
H38
N117
F500
carried out successively under acidic conditions, affording
compound 17, and then in presence of TMSBr. During this last
F500
H118
N117
D506
H243
F417
step, the carboxy(monoethyl)phosphonate ester
7 was
isolated as major derivative after 31 hours, whereas formation of
the fully deprotected derivative 8 required 6 days of reaction. In
both case, -carboxyphosphonate derivatives 7 and 8 were
isolated as equimolar mixture of the two diastereoisomers.
Figure 2. APCP binding mode within the active site of CD73 (crystal structure
of the closed form solved by Knapp et al.[8]). A) and B) correspond to two
orientations. APCP is depicted as pink sticks while connecting residues are
shown in blue thin sticks and zinc ions as grey spheres.
2
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Scheme 1. Synthetic pathways for derivatives 1-5.
Scheme 2. Synthetic pathways for derivatives 6-8.
Preparation of (aminomethylene)bisphosphonate derivative 9
commercially available adenosine, according to previously
published procedure.[16] Then azidation in position 5’ was
performed and compound 19 was subjected to hydrogenation to
(
Scheme 3) was envisaged through a three-component
condensation involving 10, triethyl orthoformate and diethyl
phosphite.[15] Despite several attempts, only small amounts of
the desired compound were obtained and formation of the bis-
adduct corresponding to the reaction of the exocyclic amino
function of the purine ring was also observed. Thus, the use of a
fully protected precursor appeared necessary. Protection of the
afford
the
9-(5-amino-5-deoxy-2,3-O-isopropylidene--D-
ribofuranosyl)-6-(2,5-dimethylpyrrol-1-yl)purine 20. With this fully
protected intermediate in hand, the method elaborated by E.
Balint et al.[
15]
was successfully applied to a nucleosidic
substrate, affording the gem(bisphosphonate) derivative 21 in
34% yield. Final steps consisted in removal of the various
protecting groups as previously reported and lead to the target
compound 9.
amino group in position
6 of the 2’,3’-O-isopropylidene-
adenosine by the 2,5-dimethylpyrrole was selected as it can be
hydrolyzed in the same conditions as of the ribose protecting
group. Thus, compound 18 was obtained in 36% yield from
3
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Scheme 3. Synthesis of derivative 9.
In addition to compounds 1-9, we synthesized two analogues,
such as the -carboxyphosphonate derivative of adenosine as a
reference (Figure 3, compound 23), to study the impact of the
nitrogen atom in position 5’ (in comparison to compound 8), and
the analogue of derivative 6 incorporating Clofarabine (Figure 3,
compound 24) instead of adenosine to study the influence of the
nucleosidic residue. Indeed, literature data[7a, 17] tend to show
that the use of Clofarabine as nucleoside scaffold led to
increased potency for 5’-ectonucleotidase inhibition (cf.
Clofarabine PCP, Figure 1). Synthetic pathways of these two
derivatives are detailed in the SI as compound 23 has already
been reported in the literature[18] and compound 24 was obtained
by adapting procedure described in Scheme 2.
were able to inhibit CD73 activity by more than 90% at high
concentrations. MTT assays were performed with MDA-MB-231
cells and studied compounds to estimate cytotoxicity, and
showed marginal (76% survival for compound 3) or no such
effect (data not shown).
Figure 3. Structures of compounds 23 and 24.
Inhibitory activity of synthesized compounds
towards CD73
The enzymatic activity of CD73 was assessed in presence of all
adenosine-based derivatives to determine their potential
inhibitory effect on CD73. Inhibition levels promoted by the
compounds are shown in Table 1 at 10 or 100 µM with
recombinant protein, and at 100 µM in the cell based assay.
Compounds 1-4, 6 and 8 inhibited CD73 activity only weakly at
high concentration (between 10-44% of inhibition) in both
conditions. Compounds 5, 7, 9 and 24 exhibited a moderate
enzymatic inhibition with inhibition values ranging from 45 to
Figure 4. A) In vitro enzymatic activity measured in the presence of
representative compounds: APCP (black bars), derivative 5 (grey), 6 (white), 7
(
7
green) and 23 (blue). B) IC50 determination for weak compounds 5 (♦), 6 (),
(■), modest 23 (∆) compared to strong inhibitor, APCP (○).
For the most interesting derivative (23), a detailed inhibition
curve was obtained in order to determine its IC50 values and
compared to the ones of weak (5, 6 and 7) or strong (APCP)
inhibitors (Figure 4B). For all compounds, dose response curves
followed a regular sigmoidal shape even for derivatives 5-7. IC50
was determined at 35 ± 6 µM for compound 5, around 35 µM
and 92 µM for compounds 6 and 7, respectively (with important
incertitude for compounds 5-7 in this concentration range). In
contrast, derivative 23 gave a value of 19 ± 6 µM and APCP at
61% at 100 µM in at least one of the two assays. In contrast,
derivative 23 was found to promote a strong inhibition on both
recombinant (76 ± 3%) and cellular (82 ± 4%) CD73 at high
concentrations, and still significantly (35 ± 4%) at 10 µM (Figure
4
A). As expected, APCP, as well as our previously developed
[
17]
inhibitor Clofarabine PCP,
used here as positive controls,
1.4 ± 1.0 µM.
4
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Table 1. Summary of biochemical properties of compounds 1- 9 and 24, and reference compounds (APCP, 23 and Clofarabine PCP).
%
of inhibition @ 100
µM (% ± SEM)
toward purified
% of inhibition @ 10
µM (% ± SEM)
toward purified
% of inhibition @
100 µM (% ± SEM)
in cell based assay
Compound #
Structure
Docking
Score
[
a]
recombinant-CD73
recombinant-CD73
147.2
99.5 ± 0.5
81.2 ± 6.7
91 ± 3
APCP
1
106.4
108.8
114.3
110.7
118.4
132
39.4 ± 5
13.7 ± 3
12 ± 1
2
3
4
5
6
7
32.3 ± 2.8
19.9 ± 3
36 ± 5
11.9 ± 7.4
18.9 ± 3.5
10 ± 8.5
18.5 ± 2
13 ± 8
27 ± 9
44 ± 5
29 ± 9
61 ± 8
36 ± 6
[
c]
18 ± 9
n.d.a.
143.7
46 ± 9
36.2 ± 5
51.9 ± 2
20.2 ± 3
5.7 ± 4
45 ± 6
23 ± 8
31 ± 7
135.3
8
9
136.6
134.8
132.5
10.5 ± 3
23
76 ± 2.5
35 ± 4
82 ± 4
[b]
[b]
24
n.a.
n.a.
45 ± 9
142.8
98.5 ± 1.5
96 ± 2.5
92 ± 4
Clofarabine PCP
[
a] Docking score for AMP is 108.5. [b] n.a.: not active. [c] n.d.a: no data available. Data represents Mean ± SEM of 3 independent experiments.
between D506 and ribose hydroxyls but also with R354 and
Molecular modeling
N245 due to the missing α-phosphonate and between H118 or
R395 with the missing oxygen of β-phosphonate (Figure 5B).
This may be explained by the length of the amino-phosphonate
chain that is shorter for 5 compared to the one of APCP and
since the electrostatic component is the main driving force to
connect oxygen near the magnesium ion; this induces a
translation of the rest of the molecule. The difference in inhibition
between APCP and the potent CD73 inhibitor (23) may be
explained by a weakening of electrostatic interactions between
R354 and oxygen atoms (5’-oxygen, as seen by the larger
distance of 5.4 Å compared to 3 Å with APCP) and similarly with
R395 (distance increases from 2.9 to 3.3 Å). In addition to this, a
slight shift of the ribose moiety is observed inducing an increase
of the H-bond distance between 2’-oxygen and D506 (from 2.4
to 2.9 Å) (Figure 5C). A lower activity was observed for
compound 9 compared to 23 although these two molecules
differ only by one atom at 5’ position (nitrogen versus oxygen)
and presented similar DS values. Firstly, docking analysis failed
to identify important modifications between binding modes for
these two compounds as they almost superimposed (Figure 6A).
To understand the relatively weak and disappointing inhibition
observed with most of the 5’-amino-adenosine derivatives, a
molecular docking study was carried out by using the crystal
structure of CD73 (4H2I). Best-ranked docking poses (according
to their docking scores given by the GoldScore fitness function
which takes into account H-bonding energy, van der Waals
energy, metal interaction and ligand torsion strain) were
analyzed for defining the various interactions with CD73
residues arising upon binding of the compound (Figure 5).
Binding orientation differed significantly for derivatives 1-4 as
compared to that of APCP (Figure 5A). Indeed, two major
features distinguish their binding, first the positioning of the
nucleobase that is shifted and made weaker π-π interactions
with the two phenyl residues (F417 and F500) from CD73, and
consequently the displacement of the ribose weakening its
interaction with D506 (two H-bonds). Overall, the binding of such
compounds altered electrostatic interactions between R354 (and
R395) and 5’-oxygen and those from the α-phosphonate for
APCP, as it is replaced by a single phosphonate and an amino
group (preferentially H-bond donor than H-bond acceptor).
For compound 5, the binding is strongly different to that of APCP
as seen for compounds 1-4 and again interactions are lost
5
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during the docking, no changes was observed at the vicinity of
the substituted atom. This loss of activity may also be due to a
change in the protonation state of H118 during the enzymatic
reaction (achieved at pH 6.5) leading to a decrease in binding
A
affinity of
Comparison of binding poses of compounds 6 and 24 (Figure
B), and compound 24 and Clofarabine PCP (Figure 6C),
highlighted the impact of both the nature of the nucleoside
adenosine or Clofarabine) and the bis(methylphosphonate)
9 (because of the supplementary hydrogen).
6
(
chain on the global positioning of these derivatives. These
modifications have a strong impact on the orientation of the
ribose moiety and consequently on the nucleobase positioning
for compounds 6 and 24.
Whereas, for methylene bisphosphonate derivatives (APCP and
Clofarabine PCP) modification of the nucleoside moiety did not
change the final orientation of the nucleobase, as previously
described[17]
. In Figure 6E, overlay of binding poses of
2.4
compounds 7 and 9 show the differences of interactions
between the gem-bisphosphonate (7) and -carboxyl-
2.4
methylphosphonate (9) with
a slight move of the ribose
weakening the interaction with D506 and R354. More notably,
this binding pose suggested the loss of a major interaction
between N245 and the third phosphate oxygen.
Discussion
We designed novel AMP or ADP analogues incorporating methyl
carboxylate, methyl phosphonate, bis(methylphosphonate),
gem-bisphosphonate, -carboxylmethylphosphonate or
-
phosphonoacetic moieties as potential CD73 inhibitors. The
replacement of the oxygen atom in the 5’-position of
endogenous adenosine could allow obtaining di-substituted
derivatives and may lead to additional interaction(s) in the
catalytic site of the protein. First of all, the docking scores (DS)
of the targeted compounds (1-9 and 24) were evaluated and
compared to the ones of AMP (the natural substrate), APCP,
Clofarabine PCP and compound 23 as reference compounds
C
(
(
Table 1). Most of the derivatives showed a DS higher than AMP
108.5), except the methyl carboxylate derivative (1). Based on
these data, bis(methylphosphonate) (6), gem-bisphosphonate
7), and α-carboxylmethylphosphonate (8 and 9) analogues
(
attracted our attention as they showed DS values in the same
range as APCP (147.2) and Clofarabine PCP (142.8).
Compound 7, including the gem-bisphosphonate moiety,
seemed to be the most promising one with a DS value of 143.7.
Thus, this novel series of 5’-aminoadenoside analogues was
successfully obtained in moderate to good yields and we next
investigated their ability to interfere with CD73. In all cases,
replacement of the methylene bisphosphonate group of APCP
lead to a decreased ability to inhibit CD73. Whereas substitution
of the oxygen in 5’-position by a nitrogen atom did not seem to
affect the ligand potency (DS values of 136.6 for compound 9
and 134.8 for compound 23), it significantly affects the ability to
inhibit the protein. When, the adenosine moiety was replaced by
Clofarabine (compounds 6 and 24), it slightly increased the
potential of the inhibitors but this effect was not able to
counterbalance the replacement of the nature of the hetero-atom
in position 5’. Despite our effort in designing original nucleotide
Figure 5. Binding prediction by docking for various compounds and
comparison of interactions mediated with CD73 amino acids. A) Docking
poses for weak inhibitors such as 2 (light green) and 3 (orange) and showing a
displacement (black arrow) compared to that of APCP (depicted as yellow
sticks). B) Large difference observed in binding orientation of compounds 5
(cyan) and APCP (yellow). C) Comparison between APCP (yellow) and 23
(pink sticks) highlighting changes of interaction distances between 5’-oxygen
and R354/R395, and for ribose hydroxyl groups and D506.
However, a thorough analysis showed that subtle changes
occurred such as the orientation of the nucleobase between
both phenyl residues (possibly lowering π-π interactions) and an
increase of the H-bond formed by the 2’-hydroxyl group and
D506 (2.9 Å for 23 and 3.2 Å for 9). Most importantly, this single
atom substitution may reflect either
a weakening of the
interaction with R395 (stronger with an oxygen than a nitrogen
because of the electronegativity) or a change / break in the H-
bond network formed between water molecules, amino acids
and compound. Although five water molecules were included
2
analogues, the replacement of the P-CH -P-O5’ pattern, and
related interaction within the catalytic site of CD73, appears not
suitable following the here proposed modifications.
6
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A
B
C
D
E
N245
Figure 6. A) Deciphering the differential activity of compound 9 (blue) and 23 (pink sticks) by analyzing their binding mode. B) Comparison of binding mode of 6
salmon sticks) versus 24 (dark green). C) Binding modes of 24 (dark green) and Clofarabine PCP (brown). D) Overlay of binding poses of derivatives 6 (salmon)
and 8 (white). E) Binding comparison of between compounds 7 (green) and 9 (blue sticks).
(
According to the important contribution of the negatively charged
groups over the final orientation of the inhibitor, it would be
interesting to increase the distance in between the carboxylate
and the phosphonate groups, and flexibility in this region may be
beneficial for zinc ions chelating. In addition, as the location of
carboxylate and/or phosphonate groups seems to govern the
correct positioning of the nucleoside moiety, increasing this
distance may lead to stronger interactions (reduction of the
bonding length with D506 and hydroxyl groups and improving
the -stacking between the nucleobase and aromatic residues).
assay (45-61% inhibition at 100 µM), as well as derivatives 7
and 9 with the purified recombinant protein (46-52% inhibition at
100 µM). We assume that these differences may arise from the
use of the soluble form of the protein, whereas in the cell-based
assay the protein is anchored to the membrane. In these two
cases, our derivatives may access and/or accommodate in a
different fashion the catalytic site of the protein. In addition,
molecular modeling showed that the modifications envisaged
deeply impact the binding modes of the new derivatives in
comparison to APCP and/or Clofarabine APCP.
Considering the literature data related to the design of
nucleos(t)ide analogues as CD73 inhibitors, these results will
certainly contribute to the establishment of a SAR study and
further development of novel derivatives.
Conclusions
Novel series of mono- or di-substituted 5’-aminonucleotide
analogues
including
carboxylated
or
phosphonylated
substituents, or both, were successfully obtained. All Experimental Section
synthesized compounds were evaluated for their ability to inhibit
1H NMR, 13C NMR and 31P spectra were recorded with proton decoupling
at ambient temperature on the following spectrometers: Bruker Avance III
5’-ectonucleotidase CD73. Among them, phosphonic acids 5
and 24 showed marginal inhibition of CD73 in the cell based
7
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(
(
600, 500 or 400 MHz). Chemical shifts () are quoted in parts per million
ppm) referenced to the residual solvent peak chloroform (CDCl ) at 7.26
) at 2.50 ppm and
OD) at 3.41 ppm and 49.00 ppm, and
O) at 4.63 ppm relative to tetramethylsilane (TMS).
For 31P NMR spectra, chemical shifts are reported relative to external
phosphoric acid (H PO ). COSY experiments were performed in order to
triethylammonium salts). This last was directly used in the next step
+
3
without further purification. R
m/z 379.17 (M+H) . HRMS Q-TOF MS E : Calculated for C16
f
(CH
2
Cl
2
/MeOH 9/1) 0.41. Q-TOF MS E :
+
+
ppm and 77.16 ppm, dimethyl sulfoxide (DMSO-d
9.52 ppm, methanol (CD
deuterium oxide (D
6
H N O
23 6 5
3
3
([M+H]+ 379.1730; found: 379.1728. This derivative (200 mg, 0.44 mmol)
was treated following general procedure A. Purification was achieved by
flash chromatography on silica gel (EtOAc/MeOH, 100/0 to 50/50) to give
the deprotected intermediate (as a white solid, 246 mg, 45% for two
2
3
4
confirm proton assignments as well as 2D 1H-13C heteronuclear COSY
for the attribution of 13C signals. Coupling constants, J, are given in Hertz.
Mass spectra were recorded on a Micromass Q-Tof mass spectrometer
using electrospray ionization. The high-resolution mass spectra (HRMS)
were obtained with a Waters Synapt G2S spectrometer equipped with
positive electrospray source ionization (ESI), using Leu-enkephalin as an
internal standard. The capillary voltage was set to 1.2 kV and the
sampling cone voltage was set to 30 V. Thin layer chromatography was
performed on pre-coated aluminum sheets of Silica Gel 60 F254 (Merck,
Art. 5554), visualization of products was accomplished by UV
absorbance followed by spraying with Hanes molybdate reagent. Column
chromatography was carried out on Silica Gel 60 (Merck, Art. 9385). All
moisture sensitive reactions were carried out in anhydrous conditions
under argon atmosphere using oven-dried glassware. Solvents were
steps) which was engaged in the saponification step. R
(EtOAc/MeOH
f
+
+
-
1/4) 0.40. Q-TOF MS E : m/z 339.14 (M+H) . Q-TOF MS E : m/z 337.10
(M-H)-. HRMS Q-TOF MS E : Calculated for
+
N O
19 6 5
[M+H]+
13
C H
339.1417; found: 339.1418. UV (EtOH abs) λmax = 259 nm (εmax = 9710).
Thus, methyl ester derivative (194 mg, 0.58 mmol) was dissolved in
NaOH 1M solution and stirred at room temperature for 2 h. The volatiles
were removed under reduced pressure and the crude was purified by
flash chromatography on silica gel (EtOAc/MeOH, 1/2 to 0/100) to afford
compound 1 as a white solid (118 mg, 60%). R
1H NMR (600 MHz, D
O): δ 8.31 (s, 1H, H-2), 8.20 (s, 1H, H-8), 6.08 (d, J
= 5.5 Hz, 1H, H-1’), H-2’ (signal in D O peak), 4.39 – 4.35 (m, 2H, H-3’,
H-4’), 3.45 – 3.38 (m, 2H, CH
-CO), 3.17 (d, J = 5.6 Hz, 2H, H-5’). 13C
NMR (151 MHz, D O): δ 176.5 (C=O), 155.5 (C-6), 152.8 (C-2), 148.7
(C-4), 140.3 (C-8), 118.9 (C-5), 88.1 (C-1’), 82.2 (C-4’), 73.5 (C-2’), 71.5
f
(EtOAc/MeOH 1/9) 0.05.
2
2
2
2
+
+
dried and distilled prior to use and solids were dried over P
reduced pressure at room temperature.
O
2 5
under
(C-3’), 51.3 (CH
HRMS Q-TOF MS E : Calculated for C12
found: 325.1261.
2
-CO), 49.9 (C-5’). Q-TOF MS E : m/z 347.11 (M+H) .
+
17 6 5
H N O
[M-Na+2H]+ 325.1260;
General Procedure (A) for removal of acid labile sugar protecting
groups. The protected derivative was dissolved in TFA-H O (ratio 1/1, 30
2
5’-Deoxy-5’-N-[(diethyl-phosphono)acetyl]-2’,3’-O-isopropylidene-
adenosine (11)[12] Diethyl phosphonoacetic acid (321 µL, 2 eq.) was
slowly added, at room temperature and under an argon atmosphere, to a
solution of compound 10 (306 mg, 1 mmol), DCC (495 mg, 2.4 eq.) and
4-DMAP (12 mg, 0.1 eq.) in anhydrous DMF (12 ml). After 20 h, the
reaction mixture was filtered and the filtrate was evaporated under
reduced pressure. The resulting oil was then co-evaporated 3 times with
mL/mmol) at room temperature and stirred until TLC indicated completion
of the reaction. Then, the solvents were evaporated and the crude was
co-evaporated with EtOH under reduced pressure. The crude material
was purified by flash chromatography on silica gel to afford the desired
product.
EtOH (10 mL) before dissolution with CH
washed with H O (twice), dried (MgSO ), filtered and concentrated to
dryness. The resulting oil was purified by silica gel column
chromatography (CH Cl /MeOH, 9/1) to give compound 11 as a yellow
foam (233 mg, 72%). R (CH Cl
/MeOH 9/1) 0.33. 1H NMR (300 MHz,
CDCl ): δ 8.39 (brs, 1H, NH), 8.37 (s, 1H, H-2), 7.93 (s, 1H, H-8), 6.00
brs, 2H, NH ), 5.87 (d, J = 4.5 Hz, 1H, H-1’), 5.37 (dd, J = 6.2, 4.5 Hz,
H, H-2’), 4.86 (dd, J = 6.3, 2.3 Hz, 1H, H-3’), 4.46 (dd, J = 5.9, 3.3 Hz,
H, H-4’), 4.20 – 4.01 (m, 5H, O-CH and H-5’), 3.38 – 3.33 (m, 1H, H-5’),
-P), 1.60 (s, 3H, CH ), 1.35 (s, 3H,
-CH ): δ
). 13C NMR (75 MHz, CDCl
65.4 (d, J = 4.9 Hz, C=O), 155.8 (C-6), 152.8 (C-2), 148.7 (C-4), 140.3
C-8), 119.8 (C-5), 114.9 (C(CH ), 91.1 (C-1’), 83.8 (C-4’), 82.9 (C-2’),
1.5 (C-3’), 63.1 (d, J = 11.2 Hz, O-CH -CH ), 41.2 (C-5’), 35.2 (d, J =
34.1 Hz, CH -P), 27.0 (C(CH ), 25.0 (C(CH
-CH
). 31P NMR (121 MHz, CDCl
2 2
Cl . The organic layer was
General Procedure (B) for diethyl phosphonate removal. The protected
phosphonate (1 eq.) was dissolved in anhydrous DMF (20 mL/mmol) and
trimethylsilyl bromide (15 eq.) was added dropwise at 0°C. The reaction
mixture was stirred at room temperature until completion of the reaction
2
4
2
2
was indicated by TLC (iPrOH/NH
reaction was stopped by adding triethylammonium bicarbonate buffer
TEAB 1 M, pH 7) and concentrated to dryness under high vacuum.
Column chromatography of the crude materials on reverse phase
4 2
OH/H O, 7/2/1, v/v/v). Then, the
f
2
2
3
(
1
1
2
(
2
(
gradient: H
2
O to MeOH 100%) gave the expected phosphonic acid (as
+
2.97 (dd, J = 21.4, 4.7 Hz, 2H, CH
CH ), 1.33 – 1.27 (m, 6H, O-CH
2
3
triethylammonium salt), which was passed through a Dowex Na ion
exchange column, the desired fractions were collected and freeze dried
leading to the title compound as sodium salt.
3
2
3
3
1
(
8
1
3 2
)
2
3
5’-Amino-5’-deoxy-2’,3’-O-(isopropylidene)adenosine
(10)
was
2
3
)
2
3
)
2
), 15.98 (d, J = 6.1 Hz,
obtained from commercially available adenosine in 3 steps with 71%
overall yield according to previously published procedures. [9]
+
O-CH
2
3
3
): δ 22.1. Q-TOF MS E : m/z
+
+
1
13
485.19 (M+H) . HRMS Q-TOF MS E : Calculated for C19
29 6 7
H N O P
Characterizations ( H, C and MS) were in agreement with the literature.
+
1
[M+H] 485.1914; found: 485.1923.
R
f
2
(CH Cl
2
/MeOH 9/1) 0.04. H NMR (300 MHz, CDCl
3
): δ 8.31 (s, 1H, H-
2
5
4
1
), 7.91 (s, 1H, H-8), 6.11 (s, 2H, NH
.46 (dd, J = 6.5, 3.0 Hz, 1H, H-2’), 5.01 (dd, J = 6.5, 3.5 Hz, 1H, H-3’),
.24 (dd, J = 9.3, 4.5 Hz, 1H, H-4’), 2.99 (qd, J = 13.4, 5.2 Hz, 2H, H-5’),
2
), 6.02 (d, J = 3.0 Hz, 1H, H-1’),
5
’-Deoxy-5’-N-[(diethyl-phosphono)acetyl]adenosine (12)[12] The title
compound was obtained as a white solid (350 mg, 90%) from compound
11 (424 mg, 0.876 mmol) using general procedure A. Purification was
). 13C NMR (75
.72 (s, 2H, CH -NH ), 1.60 (s, 3H, CH ), 1.37 (s, 3H, CH
2 2 3 3
achieved by flash chromatography on silica gel (CH
CH Cl
/MeOH 9/1) 0.08. 1H NMR (300 MHz, DMSO-d6): δ 8.39 (s, 1H,
H-2), 8.19 (s, 1H, H-8), 7.40 (brs, 2H, NH ), 5.86 (d, J = 6.3 Hz, 1H, H-1’),
5.43 (brs, 1H, OH), 5.28 (brs, 1H, OH), 4.68 (t, J = 5.7 Hz 1H, H-2’), 4.07
2 2 f
Cl /MeOH, 9/1. R
MHz, CDCl
3
): δ 155.9 (C-6), 153.3 (C-2), 149.5 (C-4), 140.0 (C-8), 120.5
(
2
2
(
C-5), 114.7 (C(CH ), 90.8 (C-1’), 87.7 (C-4’), 83.8 (C-2’), 82.0 (C-3’),
3 2
)
+
+
2
44.0 (C-5’), 27.4 (CH
3 3
), 25.5 (CH ). Q-TOF MS E : m/z 307.15 (M+H) .
+
+
HRMS Q-TOF MS E : Calculated for C13
H N O
19 6 3
[M+H] 307.1519; found
–
3.93 (m, 6H, H-3’, H-4’ and O-CH
2
-CH
3
), H-5’ (signal in H
2
O peak.),
307.1521.
2.91 (d, J = 21.4 Hz, 2H, CH -P), 1.21 (t, J = 7.1 Hz, 6H, CH
2
3
). 13C NMR
(
4
3
CH
75 MHz, DMSO-d6): δ 164.3 (C=O), 156.0 (C-6), 152.3 (C-2), 149.3 (C-
5
’-N-acetate-5’-deoxyadenosine (1) To a suspension of compound 10
), 140.4 (C-8), 119.4 (C-5), 87.6 (C-1’), 83.5 (C-4’), 72.7 (C-2’), 71.1 (C-
’), 61.7 (d, J = 6.1 Hz, O-CH -CH ), 41.3 (C-5’), 34.6 (d, J = 132.3 Hz,
-P), 16.2 (d, J = 5.8 Hz, CH
(535 mg, 1.61 mmol) in anhydrous MeOH (32 mL) and under argon
2
3
atmosphere was added freshly distilled triethylamine (566 µL, 2.5 eq.).
Methyl bromoacetate (312 µL, 2.05 eq.) was then dropwise added over a
period of 2 h and the reaction mixture was refluxed overnight before
concentration under reduced pressure. Purification by flash
chromatography on silica gel (CH
alkylated intermediate as white solid (705 mg containing 20% of
31
2
3
). P NMR (121 MHz, DMSO-d6): δ 22.8.
+
+
Q-TOF MS E : m/z 445.16 (M+H) .
5
’-Deoxy-5’-N-(phosphono-acetyl)adenosine (disodium salts) (2)[12]
The title compound was obtained as a white solid (17 mg, 7%) from
2 2
Cl /MeOH, 19/1 to 9/1) afforded the
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
+
+
compound 12 (287 mg, 0.646 mmol) using general procedure B.
Purification was achieved by flash chromatography on reverse phase
m/z 417.17 (M+H) . HRMS Q-TOF MS E : Calculated for C15
H
26
N
6
O
6
P
+
[M+H] 417.1651; found: 417.1651.
2
O/MeOH, 100/0 to 50/50). R
NMR (400 MHz, D O): δ 8.31 (s, 1H, H-2), 8.19 (s, 1H, H-8), 6.01 (d, J =
.6 Hz, 1H, H-1’), H-2’ signal in D O peak, 4.35 (t, J = 4.8 Hz, 1H, H-2’),
.28 (q, J = 4.8 Hz, 1H, H-4’), 3.65 and 3.56 (ABX, J = 14.5, 5.3, 4.8 Hz,
H, H-5’), 2.62 (d, J = 19.2 Hz, 2H, CH O): δ
-P). 13C NMR (101 MHz, D
72.4 (d, J = 4.4 Hz, C=O), 155.4 (C-6), 152.7 (C-2), 148.7 (C-4), 140.2
f 2 4
(iPrOH/H O/NH H
OH, 6/3/1) 0.39. 1
(
H
2
5
’-Deoxy-5’-N-[(ethyl-phosphono)methyl]adenosine
(monosodium
5
4
2
1
2
salt) (4) The title compound was obtained as a white solid (40 mg, 29%)
from compound 14 (140 mg, 0.336 mmol) using general procedure B.
Purification was achieved by flash chromatography on reverse phase
2
2
O, 7/2/1) 0.43. 1
(
H
2
O/MeOH, 100/0 to 50/50). R
f
(iPrOH/NH
4
OH/H
2
H
(
(
C-8), 118.8 (C-5), 87.4 (C-1’), 83.2 (C-4’), 73.2 (C-2’), 70.9 (C-3’), 40.9
NMR (400 MHz, D
2
O): δ 8.30 (s, 1H, H-2), 8.24 (s, 1H, H-8), 6.07 (d, J =
.6 Hz, 1H, H-1’), 4.85 – 4.82 (m, 1H, H-2’), 4.39 – 4.35 (m, 2H, H-3’, H-
’), 3.89 – 3.84 (m, 2H, O-CH -CH ), 3.26 – 3.24 (m, 2H, H-5’), 3.05 –
). 13C NMR (101 MHz,
.93 (m, 2H, CH -P), 1.13 (t, J = 7.1 Hz, 3H, CH
O): δ 155.6 (C-6), 152.9 (C-2), 148.7 (C-4), 140.4 (C-8), 119.0 (C-5),
8.2 (C-1’), 81.9 (C-4’), 73.2 (C-2’), 71.6 (C-3’), 60.9 (d, J = 5.8 Hz, O-
31
C-5’), 38.4 (d, J = 115.1 Hz, CH
2
-P). P NMR (162 MHz, D
2
O): δ 12.45.
5
4
2
+
+
+
Q-TOF MS E : m/z 389.10 (M+H) . HRMS Q-TOF MS E : for Calculated
C
2
2
3
+
12
H
18
N
6
O
7
P [M+H] 389.0975; found: 389.0975. UV (EtOH abs) λmax =
2
3
61 nm (εmax = 12300).
D
2
8
5
’-Deoxy-5’-N-(ethyl-phosphono-acetyl)adenosine
(monosodium
CH
2
-CH
3
), 51.3 (d, J = 10.2 Hz, C-5’), 44.0 (d, J = 142.9 Hz, CH
2
-P), 15.8
). 31P NMR (162 MHz, D
O): δ 17.7. Q-TOF MS E :
+
salt) (3) Compound 3 was obtained as a white solid (60 mg, 20%) from
compound 12 (350 mg, 0.790 mmol) using general procedure B.
Purification was achieved by flash chromatography on reverse phase
(d, J = 5.9 Hz, CH
m/z 411.12 (M+H) . Q-TOF MS E : m/z 387.12 (M-Na) . HRMS Q-TOF
MS E : Calculated for
411.1161. UV (EtOH abs) λmax = 259 nm (εmax = 15800).
3
2
+
-
+
+
13 21 6 6
C H N O
NaP [M+H]+ 411.1158; found:
(
H
2
O/MeOH, 100/0 to 50/50). R
NMR (400 MHz, D O): δ 8.31 (s, 1H, H-2), 8.25 (s, 1H, H-8), 6.01 (d, J =
.6 Hz, 1H, H-1’), 4.84 – 4.81 (m, 1H, H-2’), 4.35 – 4.26 (m, 2H, H-3’ and
H-4’), 3.92 – 3.83 (m, 2H, O-CH -CH ), 3.70 and 3.51 (ABX, J = 15.5, 5.2,
.7 Hz, 2H, H-5’), 2.72 (d, J = 20.3 Hz, 2H, CH -P), 1.15 (t, J = 7.1 Hz,
H, CH O): δ 170.3 (d, J = 5.7 Hz, C=O), 155.6
).13C NMR (101 MHz, D
f
(iPrOH/H
2
O/NH
4
OH, 6/3/1) 0.59. 1
H
2
5
5’-Deoxy-5’-N-phosphonomethyladenosine (disodium salt) (5) The
2
3
title compound was obtained as a white solid (29 mg, 17% over the 2
steps) from compound 13 (182 mg, 0.4 mmol) using general procedure
B. The residue was purified on reverse phase (H O/MeOH: 100/0) to give
2
3
3
2
3
2
(
8
4
C-6), 152.9 (C-2), 148.8 (C-4), 140.5 (C-8), 119.0 (C-5), 87.7 (C-1’),
the intermediate deprotected phosphonate (37 mg). This last was
subsequently treated following procedure A. Purification on reverse
phase (H O/MeOH: 100/0) followed by an ion exchange on DOWEX
2
3.3 (C-4’), 73.1 (C-2’), 70.9 (C-3’), 61.3 (d, J = 5.9 Hz, O-CH
2
-CH
). 31
O): δ 16.0. Q-TOF MS E : m/z 439.11 (M+H) . Q-TOF
3
),
P
0.9 (C-5’), 36.4 (d, J = 122.0 Hz, CH -P), 15.83 (d, J = 6.2 Hz, CH
2
3
+
+
NMR (162 MHz, D
MS E : m/z 415.11 (M-Na) . HRMS Q-TOF MS E : Calculated for
2
+
1
5
1
4
5
0WX2 (Na form). H NMR (500 MHz, D
H, H-2), 6.07 (d, J = 5.4 Hz, 1H, H-1’), H-2’ signal in D
.45 (m, 1H, H-4’), 4.42 (t, J = 4.8 Hz, 1H, H-3’), 3.60 – 3.52 (m, 2H, H-
P). 13C NMR (126 MHz, D
’), 3.00 (d, J = 11.6 Hz, 2H, CH O): δ 155.5
2
O): δ 8.27 (s, 1H, H-8), 8.23 (s,
-
-
+
2
O peak, 4.48 –
C
H N O
14 21 6 7
NaP [M+H]+ 439.1107; found: 439.1108. UV (EtOH abs)
λmax = 259 nm (εmax = 13900).
2
2
(
C-6), 152.9 (C-2), 148.6 (C-4), 140.5 (C-8), 119.0 (C-5), 88.6 (C-1’),
80.0 (C-4’), 73.2 (C-2’), 71.4 (C-3’), 50.8 (C-5’), 46.6 (d, J = 128.9 Hz,
CH O): δ 7.34. Q-TOF MS E+: m/z 405.07
-P). 31P NMR (162 MHz, D
5’-Deoxy-5’-N-[(diethyl-phosphono)methyl]-2’,3’-O-isopropylidene-
adenosine (13) A solution of compound 10 (408 mg, 1.2 mmol) in
2
2
anhydrous THF (4 mL) was added, at room temperature, to a mixture of
(M+H) +. HRMS Q-TOF MS E+: calculated for C11
H
16
N
6
O
6
PNa
2
[M+H] +
(diethoxyphosphoryl)methyl 4-methylbenzenesulfonate (322 mg, 1.0
405.0664 found; 405.0663.
mmol) and DBU (152 µl, 1.0 mmol) under an argon atmosphere. The
reaction mixture was stirred at 50°C for 4 days before concentration
under reduced pressure. The crude was purified by flash
5
’-Deoxy-5’-N-[(tetraethyl-bisphosphono)methyl]adenosine
(15)
Compound 10 (302.4 mg, 0.98 mmol), paraformaldehyde (62.9 mg, 2
eq.) and diethylphosphite (0.26 mL, 2 eq.) were sealed in a microwave
reactor. Under stirring, microwave irradiations were applied for 1h at
chromatography on silica gel (CH
solid (232 mg, 51%). R (CH Cl
/MeOH 9/1) 0.40. 1H NMR (400 MHz,
CDCl ): δ 8.35 (s, 1H, H-2), 7.92 (s, 1H, H-8), 6.00 (d, J = 3.0 Hz, 1H, H-
’), 5.77 (s, 2H, NH ), 5.42 (dd, J = 6.3, 3.1 Hz, 1H, H-2’), 5.02 (dd, J =
.3, 3.3 Hz, 1H, H-3’), 4.35 – 4.33 (m, 1H, H-4’), 4.16 – 4.07 (m, 4H, O-
-CH ), 3.07 – 2.93 (m, 4H, H-5’ + CH -P), 1.60 (s, 3H, CH ), 1.37 (s,
H, CH ), 1.35 – 1.27 (m, 6H, O-CH -CH ):
). 13C NMR (101 MHz, CDCl
δ 155.7 (C-6), 153.4 (C-2), 149.6 (C-4), 140.0 (C-8), 120.5 (C-5), 114.7
C(CH ), 90.9 (C-1’), 85.6 (C-4’), 83.6 (C-2’), 82.3 (C-3’), 62.2 (d, J =
.6 Hz, O-CH -CH ), 52.5 (d, J = 13.0 Hz, C-5’), 45.3 (d, J = 154.5 Hz,
-P), 27.4 (CH
2 2
Cl /MeOH, 9/1) to give 13 as a white
f
2
2
3
100°C with a power of 850 Watts. The crude mixture was purified on
1
2
silica gel flash chromatography (CH Cl /MeOH: 90/10) to afford protected
intermediate (300 mg, 0.5 mmol). This latter was treated following
procedure A. The residue was co-evaporated with EtOH and gave, after
2
2
6
CH
2
3
2
3
3
3
2
3
3
purification on reverse phase (H
drying, the desired compound 15 as a white solid (172.3 mg, 31%). R
O, 7/1/2) 0.81. 1H NMR (500 MHz, CDCl
iPrOH/NH OH/H ): δ 8.25 (s,
H, H-2), 8.11 (s, 1H, H-8), 6.27 (br s, 2H, NH ), 6.01 (d, J = 3.9 Hz, 1H,
H-1’), 4.65 – 4.63 (m, 1H, H-2’), 4.55 – 4.53 (m, 1H, H-3’), 4.21 – 4.18 (m,
H, H-4’), 4.12 – 4.08 (m, 8H, O-CH -CH ), 3.26 – 3.11 (m, 6H, CH -P +
).13C NMR (126 MHz, CDCl
H-5’), 1.30 – 1.27 (m, 12H, O-CH -CH ): δ
55.6 (C-6), 152.9 (C-2), 149.6 (C-4), 139.6 (C-8) 119.8 (C-5), 89.2 (C-
’), 83.3 (C-4’), 74.5 (C-2’), 71.2 (C-3’), 62.5 (O-CH -CH ), 57.8 (C-5’),
-P), 51.1 (d, J = 7.5 Hz, CH -P), 16.6 (O-CH
).31P NMR (202 MHz, CDCl
): δ 24.6. Q-TOF MS E : m/z 567.21
2
O/MeOH: 100/0 to 0/100) and freeze-
f
(
3 2
)
(
1
4
2
3
6
CH
2
3
2
). 31
P
2
3
), 25.5 (CH
3
), 16.6 (d, J = 5.7 Hz, O-CH
2
-CH
3
+
+
NMR (162 MHz, CDCl
HRMS Q-TOF MS E : Calculated for C18
found: 457.1965.
3
): δ 25.7. Q-TOF MS E : m/z 457.20 (M+H) .
1
2
3
2
+
30 6 6
H N O
P [M+H]+ 457.1964;
2
3
3
1
1
2
3
5
’-Deoxy-5’-N-[(diethyl-phosphono)methyl]adenosine (14) The title
compound was obtained as a white solid (60 mg, 33%) from compound
3 (200 mg, 0.438 mmol) using general procedure A. Purification was
52.4 (d, J = 7.5 Hz, CH
2
2
2
-
+
CH
3
3
+
+
37 6 9 2
H N O P
[M+H]+
1
(M+H) . HRMS Q-TOF MS E : calculated for C20
567.2097; found 567.2103.
achieved by flash chromatography on reverse phase (H
2
O/MeOH, 100/0
1
to 50/50). R
f
2
(CH Cl
2
/MeOH 9/1) 0.06. H NMR (300 MHz, D
2
O): δ 8.27 (s,
1
1
CH
1
H, H-2), 8.25 (s, 1H, H-8), 6.07 (d, J = 5.4 Hz, 1H, H-1’), 4.86 – 4.82 (m,
H, H-2’), 4.47 – 4.41 (m, 2H, H-3’and H-4‘), 4.19 – 4.06 (m, 4H, O-CH
), 3.55 (d, J = 13.5 Hz, 2H, H-5’), 3.49 (d, J = 5.8 Hz, 2H, CH -P),
.23 (t, J = 6.9 Hz, 3H, CH ), 1.19 (t, J = 6.8 Hz, 3H, CH
). 13C NMR (75
MHz, D O): δ 155.3 (C-6), 152.3 (C-2), 148.4 (C-4), 141.0 (C-8), 119.3
C-5), 89.2 (C-1’), 80.1 (C-4’), 73.0 (C-2’), 71.6 (C-3’), 64.6 (d, J = 6.5 Hz,
5
’-Deoxy-5’-N-[(bisphosphono)methyl]adenosine (tetra sodium salt)
2
-
(
6) The title compound was obtained as white solid (158 mg,
a
3
2
quantitative yield) from compound 15 (165 mg, 0.3 mmol) using general
procedure B. After stirred at room temperature for 24 h, trimethylsilyl
bromide (200 µL, 5 eq.) was added and the mixture stirred at room
temperature for
chromatography with H
3
3
2
(
5
h. Purification by reverse-phase column
O followed by ion exchange on DOWEX 50WX2
O-CH
2
-CH
3
), 50.3 (d, J = 9.6 Hz, C-5’), 41.2 (d, J = 153.1 Hz, CH
). 31P NMR (121 MHz, D
2
-P),
2
+
15.5 – 15.3 (m, CH
3
2
O): δ 20.6. Q-TOF MS E :
Na+ form) provided compound 6 as a white solid (158 mg, quantitative
yield). 1H NMR (500 MHz, D
O): δ 8.38 (s, 1H, H-2), 8.30 (s, 1H, H-8),
(
2
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
6
1
3
2
2
.14 (d, J = 5.3 Hz, 1H, H-1’), H-2’ signal in D
H, H-4’), 4.43 – 4.41 (m, 1H, H-3’), 4.06 – 3.84 (m, 2H, H-5’), 3.61 –
.47 (m, 4H, CH O): δ 155.4 (C-6), 152.6 (C-
-P). 13C NMR (126 MHz, D
), 148.7 (C-4), 140.7 (C-8), 119.2 (C-5), 88.7 (C-1’), 78.7 (C-4’), 73.1 (C-
2
O peak, 4.67 – 4.63 (m,
5’-Deoxy-5’-N-[(ethoxyphosphorylacetate)]adenosine
(disodium
salt) (7) The title compound was obtained as a white solid (71.6 mg,
78%) from compound 17 (94.5 mg, 0.19 mmol) using general procedure
B. Purification by reverse-phase column chromatography with
2
2
’), 71.6 (C-3’), 58.1 (C-5’), 52.6 (CH
2
P), 51.6 (CH
2
P). 31P NMR (121
H
2
O/MeOH (100/0 to 0/100) afford the desired intermediate as
triethylammonium salt. This last was treated with NaOH solution (1M in
O, 300 µL, 1.5 eq.) and the mixture was stirred at room temperature
overnight. The mixture was neutralized with a saturated aqueous solution
of NH Cl. After freeze-drying, purification by reverse phase column
chromatography with H
a
-
-
MHz, D
E : calculated for C12
2
O): δ 7.9. Q-TOF MS E : m/z 475.05 (M-H) . HRMS Q-TOF MS
-
-
H
18
N
6
O
9
NaP
2
[M-H] 475.0508; found 475.0511.
H
2
5
’-Deoxy-5’-N-[(diethoxyphosphorylethylacetate)]-2’,3’-O-isopropyl-
idene-adenosine (16) To a stirred solution of 10 (419.4 mg, 1.37 mmol)
in freshly distilled dichloromethane (7 mL) was added MgSO (300 mg, 5
4
2
O/MeOH (100/0 to 0/100) and ion exchange on
DOWEX 50WX2 (Na+ form) afforded the desired mixture of
diastereomers 7. R
4
1
f
(iPrOH/NH
4
OH/H
2
O, 7/1/2) 0.62. H NMR (500 MHz,
eq.) and ethylglyoxylate (50% solution (wt.) in toluene, 272 µL, 1.2 eq.)
under an argon atmosphere. After stirring at room temperature for 3 h,
the mixture was filtered and concentrated in vacuum to afford the
intermediate imine. It was transferred in a microwave reactor and diethyl
phosphite (700 µL, 4 eq.) was added, then the mixture was irradiated by
microwave for 6 h at 100°C with a power of 850 Watts. Purification by
D
2
(
2
O): δ 8.33 – 8.29 (m, 4H, H-2 and H-8, dia1 + dia2), 6.11 – 6.09 (m,
H, H-1’, dia1 + dia2), H-2’ dia1 + dia2 signal in D O peak, 4.55 – 4.48
m, 4H, H-4’ and H-3’, dia1 + dia2), 4.01-3.86 (m, 6H, CHP and O-CH
CH , dia1 + dia2), 3.75 – 3.60 (m, 4H, H-5’, dia1 + dia2), 1.17 – 1.09 (m,
H, CH O): δ 168.9 (CO, dia1 +
, dia1 + dia2). 13C NMR (126 MHz, D
2
2
-
3
6
3
2
dia2), 155.2 (C-6, dia1 + dia2), 152.4 (C-2, dia1 or dia2), 152.3 (C-2, dia1
or dia2), 148.5 (C-4, dia2), 148.4 (C-4, dia1), 141.2 (C-8, dia1 + dia2),
119.4 (C-5, dia1 + dia2), 89.4 (C-1’, dia1 + dia2), 79.9 (C-4’, dia2), 79.2
2 2
flash chromatography on silica gel (CH Cl /MeOH: 90/10) afforded 1/1
mixture of diastereomers 16 as a white solid (375 mg, 53%).
R
f
1
2
(CH Cl
2
/MeOH, 9/1) 0.48. H NMR (500 MHz, CDCl
3
): δ 8.39 (s, 1H, H-2,
(
3
(
C-4’, dia1), 73.3 (C-2’, dia1), 73.0 (C-2’, dia2), 71.5 (C-3’, dia1), 71.4 (C-
’, dia2), 62.6 – 59.9 (m, CHP and O-CH -CH , dia1 + dia2), 49.3 – 48.8
m, C-5’, dia1 + dia2), 15.9 – 15.8 (m, CH
, dia1 + dia2). 31P NMR (202
MHz, D O): δ 7.9 (dia1 or dia2), 7.8 (dia1 or dia2). Q-TOF MS E : m/z
77.09 (M+H) . HRMS Q-TOF MS E : calculated for C14
dia1 or dia2), 8.36 (s, 1H, H-2, dia1 or dia2), 8.06 (s, 1H, H-8, dia1 or
dia2), 7.95 (s, 1H, H-8, dia1 or dia2), 6.06 (d, J = 2.9 Hz, 1H, H-1’, dia1),
2
3
3
6
(
.03 (d, J = 3.2 Hz, 1H, H-1’, dia2), 5.74 (br s, 4H, NH
dd, J = 6.5, 3.0 Hz, 1H, H-2’, dia1), 5.39 (dd, J = 6.4, 3.2 Hz, 1H, H-2’,
dia2), 5.07 (dd, J = 6.4, 3.2 Hz, 1H, H-3’, dia2), 5.02 (dd, J = 6.5, 3.4 Hz,
2
, dia1 + dia2), 5.42
+
2
+
+
4
20 6 8 2
H N O PNa
+
[M+H] 477.0876; found 477.0876.
1
1
1
2
1
H, H-3’, dia1), 4.37 – 4.32 (m, 2H, H-4’, dia1 + dia2), 4.24 – 4.11 (m,
2H, O-CH -CH , dia1 + dia2), 3.77 (s, 1H, CHP, dia1 or dia2), 3.72 (s,
2
3
H, CHP, dia1 or dia2), 3.10 (dd, J = 4.0, 12.7 Hz, 1H, H-5’, dia2), 2.98-
.86 (m, 2H, H-5’, dia1 + dia2), 2.79 (dd, J = 3.8, 12.7 Hz, 1H, H-5’ dia2),
5’-Deoxy-5’-N-[(phosphorylacetate)]adenosine (tri sodium salt) (8)
The title compound was obtained as a white solid (78.9 mg, 77%) from
compound 17 (219.2 mg, 0.45 mmol) using general procedure B.
Purification was carried out by reverse phase column chromatography
with H
triethylammonium salt. This last was then dissolved in a NaOH solution
(1M in H O, 2.1 mL, 10 eq.) and stirred at room temperature for a week
.61 (s, 6H, CH
3
dia1 + dia2), 1.38 (s, 6H, CH
-CH , dia1 + dia2), 1.24 – 1.21 (m, 6H, O-CH
dia2). 13C NMR (126 MHz, CDCl
): δ 169.2 (CO, dia1 or dia2), 169.1
3
, dia1 + dia2), 1.33 – 1.29
(m, 12H, O-CH
2
3
2
-CH , dia1
3
+
3
2
O/MeOH (100/0 to 0/100) to afford desired compound as a
(CO, dia1 or dia2), 155.6 (C-6, dia1 + dia2), 153.5 (C-2, dia1 or dia2),
1
1
1
53.3(C-2, dia1 or dia2), 149.6 (C-4, dia1 + dia2), 140.1 (C-8, dia1),
39.9 (C-8, dia2), 120.5 (C-5, dia1 or dia2), 120.4 (C-5, dia1 or dia2),
14.8 (C(CH ) , dia1), 114.7 (C(CH ) , dia2), 91.0 (C-1’, dia2), 90.4 (C-1’,
3 2 3 2
2
the solution was adjusted to pH = 5 with an ion exchange resin DOWEX
50WX2 (H+ form). Freeze drying, followed by reverse phase column
dia1), 86.3 (C-4’, dia1), 85.9 (C-4’, dia2), 83.9 (C-2’, dia1), 83.7 (C-2’,
dia2), 82.3 (C-3’, dia1 + dia2), 63.6 (O-CH -CH , dia1 + dia2), 63.5 –
-CH , dia1 + dia2), 61.4 –
1.3 (CHP, dia1 or dia2), 60.2 (CHP, dia1 or dia2), 50.8 – 50.6 (C-5’,
, dia1 + dia2), 27.4 (CH , dia1 + dia2), 16.6 –
, dia1 + dia2), 14.2 (C-O-CH -CH
, dia1 + dia2). 31P
): δ 17.6 (dia1 or dia2), 17.5 (dia1 or dia2). Q-TOF
2
chromatography with H O/MeOH (0 to 100) and ion exchange on
DOWEX 50WX2 (Na+ form) provided the desired mixture of
2
3
1
6
6
3.4 (O-CH
2
-CH
3
, dia1 + dia2), 61.9 (O-CH
2
3
diastereomers 8. R
f
(iPrOH/NH
4
OH/H
2
O, 5/1/4) 0.22. H NMR (500 MHz,
2
D O): δ 8.28 (d, 2H, H-8, dia1 + dia2), 8.22 (d, 2H, H-2, dia1 + dia2), 6.09
dia1 + dia2), 27.5 (CH
3
3
– 6.06 (m, 2H, H-1’, dia1 + dia2), H-2’ dia1 + dia2 signal in D O peak,
2
16.5 (P-O-CH -CH
2
3
2
3
4.53 – 4.48 (m, 2H, H-4’, dia1 + dia2), 4.46 – 4.44 (m, 1H, H-3’, dia1),
4.41 – 4.39 (m, 1H, H-3’, dia2), 3.76 (t, J = 14.0 Hz, 2H, CHP, dia1 +
NMR (202 MHz, CDCl
3
+
+
+
dia2), 3.69 – 3.52 (m, 4H, H-5’, dia 1 + dia 2). 13C NMR (126 MHz, D
MS E : m/z 529.22 (M+H) . HRMS Q-TOF MS E : calculated for
2
O):
δ 171.7 – 171.6 (m, CO, dia1 + dia2), 155.4 (C-6, dia1 + dia2), 153.0 (C-
, dia1), 152.8 (C-2, dia2), 148.5 (C-4, dia1 + dia2), 140.5 (C-8, dia1 or
dia2), 140.4 (C-8, dia1 or dia2), 119.0 (C-5, dia1 + dia2), 88.8 (C-1’, dia1),
+
C
21
H
34
N
6
O
8
P [M+H] 529.2176; found 529.2177.
2
5’-Deoxy-5’-N-[(diethoxyphosphorylethylacetate)]adenosine (17) The
8
7
8.6 (C-1’, dia2), 79.8 (C-4’, dia1), 79.5 (C-4’, dia2), 73.4 (C-2’, dia2),
3.2 (C-2’, dia1), 71.4 (C-3’, dia2), 71.2 (C-3’, dia1), 64.2 – 63.1 (m, CHP,
title compound was obtained as a white solid (330.2 mg, 98%) from
compound 16 (365.2 mg, 0.69 mmol) using general procedure A.
Purification was carried by flash chromatography on silica gel
31
dia1 + dia2), 50.1 – 49.9 (m, C-5’, dia1 + dia2). P NMR (202 MHz,
D
TOF MS E : calculated for
+
+
//MeOH, 9/1) 0.32. 1H NMR (300 MHz,
OD): δ 8.36 – 8.26 (m, 4H, H-2 & H-8, dia1 + dia2), 5.98 (d, J = 5.5
2
O): δ 5.8 (dia1 + dia2). Q-TOF MS E : m/z 405.09 (M+H) . HRMS Q-
(
CH
CD
Hz, 1H, H-1’, dia1), 5.94 (d, J = 5.9 Hz, 1H, H-1’, dia2), H-2’ dia2 signal in
O peak), 4.80 – 4.77 (m, 1H, H-2’, dia1), 4.38 (dd, J = 5.3, 3.5 Hz, 1H,
H-3’, dia2), 4.26 – 4.10 (m, 15H, O-CH -CH , H-3’ dia1 and H-4’ dia1 +
2
Cl
2 f 2 2
/MeOH, 9/1)). R (CH Cl
+
12 18 6 8
C H N O P
[M+H]+ 405.0924; found
3
405.0921.
H
2
2
3
9-(2,3-O-isopropylidene--D-ribofuranosyl)-6-(2,5-dimethylpyrrol-1-
yl)purine (18)[
16]
Compound 18 was obtained from commercially
dia2), 3.15 (dd, J = 12.8, 3.1 Hz, 1H, H-5’ dia2), 3.03 – 3.00 (m, 2H, H-5’,
dia1 + dia2), 2.83 (dd, J = 12.8, 4.9 Hz, 1H, H-5’, dia2), 1.34 – 1.20 (m,
available adenosine with 36% yield according previously published
procedure.[16] Characterization ( H) was in agreement with the literature.
2 3 3
-CH OD): δ 170.4
, dia1 + dia2). 13C NMR (126 MHz, CD
1
18H, O-CH
f 3
R ): δ
(Petroleum ether / EtOAc, 7/3) 0.23. 1H NMR (400 MHz, CDCl
(CO, dia1 + dia2), 156.6 (C-6, dia1 or dia2), 156.4 (C-6, dia1 or dia2),
1
1
9
7
6
5
52.8 (C-2, dia1), 152.7 (C-2, dia2), 150.6 (C-4, dia1), 150.5 (C-4, dia2),
42.5 (C-8, dia2), 142.1 (C-8, dia1), 120.8 (C-5, dia2), 120.7 (C-5, dia1),
0.8 (C-1’, dia2), 90.4 (C-1’, dia1), 86.3 (C-4’, dia2), 85.9 (C-4’, dia1),
5.0 (C-2’, dia1), 74.6 (C-2’, dia2), 73.0 (C-3’, dia1), 72.9 (C-3’, dia2),
8.92 (s, 1H, H-2), 8.17 (s, 1H, H-8), 6.00 (s, 2H, CH), 5.98 (d, J = 5.0 Hz,
1H, H-1’), 5.32 – 5.29 (m, 1H, H-2’), 5.16 (dd, J = 5.9, 1.2 Hz, 1H, H-3’),
4.59 (s, 1H, H-4’), 4.02 and 3.85 (AB, J = 12.8, 1.8 Hz, 2H, H-5’), 2.21 (s,
3 3 3
6H, CH ), 1.67 (s, 3H, CH ), 1.40 (s, 3H, CH ).
5.3 – 64.8 (m, O-CH
2
-CH
3
, dia1 + dia2), 63.0 (CHP, dia1 + dia2), 51.9 –
-CH and P(O)-CH
1.4 (C-5’, dia1 + dia2), 16.8 – 14.4 (m, C(O)CH
2
3
2
-
9
-(5-azido-5-deoxy-2,3-O-isopropylidene--D-ribofuranosyl)-6-(2,5-
dimethylpyrrol-1-yl)purine (19) To a stirred solution of compound 18
300 mg, 0.78 mmol) in anhydrous 1.4-dioxane (3 mL) was added
diphenyl phosphoryl azide (336 µL, eq.) and 1,8-
CH
3
, dia1 + dia2). 31P NMR (202 MHz, CD
3
OD): δ 18.4 (dia1 or dia2),
+
+
18.3 (dia1 or dia2). Q-TOF MS E : m/z 489.19 (M+H) . HRMS Q-TOF
(
+
+
30 6 8
MS E : calculated for C18H N O P [M+H] 489.1863; found 489.1866.
2
1
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
diazabicyclo[5.4.0]undec-7-ene (350 µL,
3
eq.) under an argon
5), 90.8 (C-1’), 86.4 (C-4’), 74.6 (C-2’), 72.9 (C-3’), 64.9 – 64.5 (m, O-
2 3 3
CH -CH ), 52.6 – 52.5 (m, CHP and C-5’), 16.8 – 16.7 (m, CH
). 31P
atmosphere. The mixture was stirred overnight at room temperature
before the addition of sodium azide (253 mg, 5 eq.) and a catalytic
amount of crown ether (15-crown-5, 1.7 mg, 0.01 eq.). This mixture was
heated at reflux for 4 h, before filtration and concentration to dryness
under reduced pressure. Purification by column chromatography with
Petroleum ether/EtOAc (80/20) afforded compound 19 as a white solid
+
NMR (202 MHz, CD
3
OD): δ 20.3 – 19.2 (m, 2P). Q-TOF MS E : m/z
+
+
553.19 (M+H) . HRMS Q-TOF MS E : calculated for C19
H
35
N
6
O P
9 2
+
[M+H] 553.1941; found, 553.1938.
5’-Deoxy-5’-N-(methylene bisphosphonate)adenosine (Tetra sodium
(
319 mg, quantitative yield). R
NMR (600 MHz, CDCl ): δ 8.95 (s, 1H, H-2), 8.24 (s, 1H, H-8), 6.23 (d, J
2.5 Hz, 1H, H-1’), 5.99 (s, 2H, 2xCH), 5.48 – 5,47 (m, 1H, H-2’), 5.08 –
.07 (m, 1H, H-3’), 4.44 – 4.42 (m, 1H, H-4’), 3.69 – 3.62 (m, 2H, H-5’),
.20 (s, 6H, CH pyrrole), 1.65 (s, 3H, CH ), 1.41 (s, 3H, CH
). 13C NMR
(Petroleum ether/EtOAc, 7/3) 0.73. 1
H
f
salts) (9) The title compound was obtained as a white solid (79 mg,
quantitative yield) from compound 22 (82 mg, 0.15 mmol) using general
procedure B (but with 20 eq. trimethylsilyl bromide). Purification was
3
=
5
2
achieved by reverse-phase column chromatography with H
ion exchange on DOWEX 50WX2 (Na+ form). R
(iPrOH/NH
2
O followed by
3
3
3
OH/H O,
f
4
2
(151 MHz, CDCl3): δ 152.9 (C-4), 152.7 (C-2), 150.8 (Cq), 143.9 (C-8),
1
6
2
1
1
2
/1/3) 0.36. H NMR (400 MHz, D
2
O): δ 8.38 (s, 1H, H-8), 8.36 (s, 1H, H-
), 6.13 (d, J = 5.7 Hz, 1H, H-1’), H-2’ signal in D O peak, 4.62 – 4.58 (m,
H, H-4’), 4.52 – 4.50 (m, 1H, H-3’), 3.99 – 3.84 (m, 2H, H-5’), 3.62 (t, J =
7.7 Hz, 1H, CHP). 13C NMR (151 MHz, D O): δ 154.2 (C-6), 151.6 (C-
), 147.2 (C-4), 139.22 (C-8), 117.7 (C-5), 87.3 (C-1’), 78.4 (C-4’), 72.1
130.0 (C-5), 129.4 (C-6), 115.4 (Cq), 109.3 (CH), 90.7 (C1’), 85.2 (C-4’),
2
8
4.1 (C-2’), 81.8 (C-3’), 52.4 (C-5’), 27.4 (CH
3
), 25.5 (CH
3
), 13.7 (CH
3
).
+
+
+
Q-TOF MS E : m/z 411.19 (M+H) . HRMS Q-TOF MS E : calculated for
C
2
+
H N O
19 23 8 3
[M+H] 411.1893; found 411.1891.
3
1
(
C-2’), 69.9 (C-3’), 53.4 (CHP), 48.3 (C-5’). P NMR (162 MHz, D
2
O): δ
-
-
9-(5-amino-5-deoxy-2,3-O-isopropylidene--D-ribofuranosyl)-6-(2,5-
7.28 (P), 7.07 (P). Q-TOF MS E : m/z 439.05 (M-H) . HRMS Q-TOF MS
-
-
dimethylpyrrol-1-yl)purine (20) Compound 19 (587.5 mg, 1.4 mmol)
17 6 9 2
E : calculated for C11H N O P [M-H] 439.0532 found; 439.0536.
was dissolved in absolute ethanol (7 mL) then Palladium on carbon 10%
(44 mg, 0.03 eq.) was added and the heterogeneous mixture was stirred
5’-O-[(phosphorylacetate)]adenosine (trisodium salt) (23) This
overnight under a hydrogen atmosphere. After filtration through a pad of
Celite, the residual mixture was concentrated in vacuum. Purification by
compound was obtained according to the procedure describe by
[18]
1
Debarge et al. Characterizations ( H and MS) were in agreement with
column chromatography with CH
0 as a white solid (539 mg, 98%). R
600 MHz, CDCl
Hz, 1H, H-1’), 5.98 (s, 2H, 2xCH), 5.48 – 5.46 (m, 1H, H-2’), 5.07 – 5.06
m, 1H, H-3’), 4.33 – 4.31 (m, 1H, H-4’), 3.13 – 3.02 (m, 2H, H-5’), 2.20 (s,
H, CH ), 1.65 (s, 3H, CH ), 1.40 (s, 3H, CH
). 13C NMR (151 MHz,
CDCl ): δ 153.0 (C-4), 152.6 (C-2), 150.7 (Cq), 144.2 (C-8), 129.9 (C-5),
29.5 (C-6), 115.2 (C), 109.3 (CH), 90.9 (C-1’), 87.1 (C-4’), 83.8 (C-2’),
2
Cl
2
/MeOH (90/10) afforded compound
1
the literature. H NMR (400 MHz, D
2
O): δ 8.62 (s, 1H, H-8, dia1 or dia2),
1
2
f
2
(CH Cl
2
/MeOH, 9/1) 0.58. H NMR
8.60 (s, H-8, 1H, dia1 or dia2), 8.18 (s, 2H, H-2, dia1 + dia2), 6.07 (s, 1H,
(
3
): δ 8.93 (s, 1H, H-2), 8.28 (s, 1H, H-8), 6.15 (d, J = 3.3
H-1’, dia1 or dia2), 6.06 (s, 1H, H-1’, dia1 or dia2), H-2’ signal in D
2
O
peak, 4.53 – 4.45 (m, 2H, H-4’, dia1 or dia2), 4.34 – 4.30 (m, 2H, H-3’,
dia1 or dia2), 3.99 – 3.94 (m, 2H, CHP, dia1 or dia2), 3.90 – 3.61 (m, 4H,
(
6
3
3
3
H-5’, dia1 + dia2). HRMS Q-TOF MS E+ calculated for C12
17 5 9
H N O P
3
[M+H] 406.0764 found; 406.0763.
+
1
8
1.8 (C-3’), 43.7 (C-5’), 27.4 (CH
3
), 25.5 (CH
3
), 13.7 (CH
3
). Q-TOF MS
2’-Arabino-fluoro-5’-N-[(bisphosphono)methyl]-2-chloro-2’,5’-
+
+
+
E : m/z 385.20 (M+H) . HRMS Q-TOF MS E : calculated for C19
H N O
25 6 3
dideoxyadenosine (24) The title compound was synthesized from
+
[M+H] 385.1988; found 385.1986.
commercially available Clofarabine according to procedure detailed in the
SI. R
f
(iPrOH/NH
4 2 2
OH/H O): δ 8.53
O, 7/1/2) 0.04. 1H NMR (600 MHz, D
9-(5-deoxy-2,3-O-isopropylidene--D-ribofuranosyl-5-N[(tetraethyl
(s, 1H, H-8), 6.46 (dd, J = 16.1, 4.1 Hz, 1H, H-1'), 5.36– 5.26 (m, 1H, H-
(
(
methylene bisphosphonate))]-6-(2,5-dimethylpyrrol-1-yl)- purine
21) Compound 20 (260mg, 0.68 mmol), triethyl orthoformiate (135 µL,
2
'), 4.72 – 4.66 (m, 1H, H-3'), 4.41– 4.38 (m, 1H, H-4'), 3.67– 3.51 (m, 2H,
).13C NMR (151 MHz, D
O) δ 156.4
C-2), 153.8 (C-6), 149.9 (C-4), 142.0 (C-8), 117.1 (C-5), 94.5 (d, J =
H-5'), 3.05 (d, J = 10.8 Hz, 4H, N-CH
(
2
2
1
.2 eq.) and diethylphosphite (306 µL, 3.5 eq.) were sealed in a
microwave reactor under stirring. Microwave irradiations were applied for
h at 125°C with a power of 850 Watts. The resulting brown oil was
purified by column chromatography CH Cl /MeOH (95/5) to afford the
aminomethylene) bisphosphonate 21 as a white solid (157 mg, 34%). R
191.3 Hz, C-2'), 82.2 (d, J = 17.1 Hz, C-1'), 80.1 (C-4'), 75.6 (d, J = 25.4
2
C-N). 31P NMR (400 MHz,
Hz, C-3'), 57.4 (C-5'), 55.4 (d, J = 137.1 Hz, H
MeOD) δ 13.2. F NMR (400 MHz, MeOD) δ -198.4. Q-TOF MS E : m/z
2
2
2
19
-
(
(
f
-
-
489.03 (M-H) . HRMS Q-TOF MS E . Calculated for C12
17 6 8 2
H N O FP Cl [M-
1
2
CH Cl
2
/MeOH, 9/1) 0.72. H NMR (500 MHz, CDCl
3
): δ 9.01 (s, 1H, H-2),
-
H] 489.0256; found: 489.0256
8
(
4
.33 (s, 1H, H-8), 6.12 (d, J = 3.8 Hz, 1H, H-1’), 5.98 (s, 2H, 2xCH), 5.43
dd, J = 6.5, 3.8 Hz, 1H, H-2’), 5.13 (dd, J = 6.5, 3.2 Hz, 1H, H-3’), 4.40 –
.38 (m, 1H, H-4’), 4.23 – 4.15 (m, 8H, O-CH ), 3.41 – 3.32 (m, 2H, H-5’
and CHP), 3.11 – 3.08 (m, 1H, H-5’), 2.20 (s, 6H, CH ), 1.64 (s, 3H, CH ),
.39 (s, 3H, CH ), 1.32 – 1.27 (m, 12H, O-CH -CH
). 13C NMR (101 MHz,
CDCl ): δ 153.2 (C-4), 152.9 (C-2), 150.7 (Cq), 144.0 (C-8), 130.0 (C-5),
29.4 (C-6), 115.1 (Cq), 109.2 (CH), 90.8 (C-1’), 85.7 (C-4’), 83.5 (C-2’),
Molecular modelling and docking. All compounds were modelled using
the VegaZZ molecular modelling suite and subjected to energy
minimization of the potential energy by 500 steps of steepest-descent
gradient followed by 5, 000 steps of conjugate gradient algorithm (Tripos
force field and Gasteiger-Marsili partial charges) until convergence with a
gradient tolerance of 0.001 kcal/mol. Å).[19] Molecular docking was
achieved with Gold v5.6 (Genetic Optimization for Ligand Docking) from
the Cambridge Crystallographic Data Centre (CCDC, Software Limited)
on the crystal structure of CD73 (PDB 4H2I) solved at 2 Å of resolution in
a closed conformation (crystal form III from Knapp K. et al.[8]). Target
binding site was defined by a spherical area around the CZ atom of
Arg395 (as this residue is located in the center of the substrate binding
site and in contact with phosphonate oxygen’s of APCP) with a radius 10
2
3
3
1
3
2
3
3
1
8
2
1.9 (C-3’), 63.3 – 63.0 (m, O-CH
2
), 56.3 – 53.4 (m, CHP), 51.5 (C-5’),
7.5 (CH ), 25.6 (CH ), 16.6 (O-CH
3
3
2
-CH
3
), 13.7 (CH
3
). 31P NMR (202
+
+
MHz, CDCl
HRMS Q-TOF MS E : calculated for C28
found 671.2720.
3
): δ 19.5 – 19.0 (m, 2P). Q-TOF MS E : m/z 671.27 (M+H) .
+
45 6 9 2
H N O P
[M+H]+ 671.2723;
5
(
’-Deoxy-5’-N-[(tetraethyl(methylene
22) The title compound was obtained as a white solid (89 mg, 74%) from
compound 21 (146.2 mg, 0.2 mmol) using general procedure A (with a
ratio of TFA/H O, 9/1). Purification was achieved by column
chromatography with CH Cl /MeOH (90/10). R (CH Cl /MeOH, 9/1) 0.22.
1H NMR (500 MHz, CD
OD): δ 8.32 (s, 1H, H-2), 8.29 (s, 1H, H-8), 5.94
5.93 (m, 1H, H-1’), H-2’ signal in H O peak, 4.39 – 4.38 (m, 1H, H-3’),
.24 – 4.14 (m, 9H, O-CH -CH and H-4’), 3.38 – 3.34 (m, 1H, H-5’), 3.06
3.03 (m, 1H, H-5’), 1.33 – 1.27 (m, 12H, CH
). 13C NMR (126 MHz,
OD): δ 157.0 (C-6), 153.6 (C-2), 150.6 (C-4), 142.2 (C-8), 120.9 (C-
bisphosphonate))]adenosine
Å
and scrutiny of cavity. Water molecules from the crystal were
preserved and allowed for translation / rotation freedoms within a 2 Å
window. The search of best docking poses was performed by executing
2
2
2
f
2
2
50 runs of genetic algorithms (search-based optimization technique
3
based on Genetics and natural selection) and the ranking of docking
solutions was computed by the GoldScore fitness function by using the
complete linkage clustering method from the RMSD matrix of generated
solutions.[20] The GoldScore scoring function was selected as it has been
–
4
–
2
2
3
3
CD
3
optimized for the prediction of ligand binding positions and takes into
1
1
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ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
account factors such as H-bonding energy, van der Waals energy, metal
interaction and ligand torsion strain (thus, the score reflects the
theoretical affinity and is expressed as arbitrary units in GOLD). The
highest-ranked poses were selected for structural analysis and
interaction measurements with CD73 amino acids using the PyMol
Molecular Graphics System (v1.8, Schrödinger, LLC).
Keywords: 5’-ectonucleotidase • enzyme inhibitors •
nucleos(t)ide analogue • immunotherapy • cancer
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(
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(
2
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inhibition activity was calculated as 1- (ODAMP+inhibitor – ODinhibitor) / (ODAMP
–
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PhD fellowship. RR was supported by a fellowship from the
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Conflict of Interest
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The authors declare no conflict of interest
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ChemMedChem
10.1002/cmdc.201900348
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ChemMedChem
10.1002/cmdc.201900348
FULL PAPER
Entry for the Table of Contents
Novel nucleos(t)ide analogues based on a 5’-aminoadenosine
scaffold were synthesized and their ability to inhibit 5’-
ectonucleotidase CD73 was assessed.
1
4
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