Synthesis of {[5-(adenin-9-yl)-2-furyl]methoxy}methyl phosphonic acid and evaluations against human adenylate kinases

Article abstract of DOI:10.1016/j.bmcl.2014.07.036

AMP mimics constitute an important class of therapeutic derivatives to treat diseases where the pool of ATP is involved. A new phosphonate derivative of 9-(5-hydroxymethylfuran-2-yl)adenine was synthesized in a multi-step sequence from commercially available adenosine. Its ability to behave as a substrate of human adenylate kinases 1 and 2 was assessed. The phosphonate was shown to be a moderate but selective substrate of the mitochondrial human AK2, better than well-known antiviral acyclic phosphonates 9-(2-phosphonomethoxyethyl)adenine (PMEA, Adefovir) and (R)-9-(2-phosphonomethoxypropyl)adenine (PMPA, Tenofovir). Putative binding mode within adenylate kinase NMP site revealed by molecular docking in comparison to AMP native substrate allowed to illustrate this selective behavior.

Full text of DOI:10.1016/j.bmcl.2014.07.036

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4227–4230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of {[5-(adenin-9-yl)-2-furyl]methoxy}methyl phosphonic
acid and evaluations against human adenylate kinases
b
Malika Kaci a, Jean-Pierre Uttaro a, Valérie Lefort b, Christophe Mathé a, , Chahrazade El Amri ,
⇑
Christian Périgaud a
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM 1-UM 2, cc 1705, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5, France
b Enzymologie Moléculaire et Fonctionnelle, UMR 8256 UPMC-CNRS, ERL INSERM U1164, Université Pierre et Marie Curie, 7 Quai St-Bernard, 75252 Paris cedex 5, France
a r t i c l e i n f o
a b s t r a c t
Article history:
AMP mimics constitute an important class of therapeutic derivatives to treat diseases where the pool of
ATP is involved. A new phosphonate derivative of 9-(5-hydroxymethylfuran-2-yl)adenine was synthe-
sized in a multi-step sequence from commercially available adenosine. Its ability to behave as a substrate
of human adenylate kinases 1 and 2 was assessed. The phosphonate was shown to be a moderate but
selective substrate of the mitochondrial human AK2, better than well-known antiviral acyclic phospho-
nates 9-(2-phosphonomethoxyethyl)adenine (PMEA, Adefovir) and (R)-9-(2-phosphonomethoxypro-
pyl)adenine (PMPA, Tenofovir). Putative binding mode within adenylate kinase NMP site revealed by
molecular docking in comparison to AMP native substrate allowed to illustrate this selective behavior.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 27 May 2014
Revised 11 July 2014
Accepted 12 July 2014
Available online 17 July 2014
Keywords:
Adenosine
Analogue/AMP mimics
Phosphotransfer
Nucleoside
Phosphonate
Kinase
9-(5-Hydroxymethylfuran-2-yl)adenine derivative (5) (Fig. 1)
has been known since 1974 when Robins et al. described its forma-
tion as a side-product following treatment of the parent 10,20-dide-
hydro-20-deoxynucleoside adenosine under mild acidic conditions.1
Chemical modifications and hydrogenation of this derivative
provided di- and trideoxy nucleosides as a racemic mixture.2 After-
wards, improved procedures of the preparation have been reported
in the literature.3,4 However, data can be found neither on the bio-
logical evaluations of this mimic nucleoside of adenosine nor on
any of the phosphorylated forms. The discovery of new analogues
of adenosine can be of interest in the drug discovery process.
Adenosine mimics represent an important class of therapeutical
molecules, due to the importance of the regulation of ATP levels
in metabolic processes that are deregulated in various diseases,
including cancer,5 type 2 diabetes,6 and viral infections.7 Particu-
larly, AMP signaling also plays an important role in hypoxic
response, immune function and taste reception.8 These nucleotide
forms are usually obtained from adenosine via phosphorylation
steps by cellular kinases.9,10
Phosphorus-modified nucleoside analogues, bearing a phospho-
nate group, display interesting biological properties, mainly as
antiviral agents. As examples, Adefovir dipivoxil and Tenofovir
disoproxil fumarate, the prodrugs of Adefovir (PMEA) and Tenofo-
vir (PMPA), acyclic nucleoside phosphonate analogues have been
approved as anti-HBV, and anti-HIV/HBV agents, respectively.11
Indeed, phosphonates are structurally and electronically similar
to phosphates and have been successfully applied as phosphate
mimics.12 Additionally, the phosphonate group has the advantage
of being more stable than its phosphate counterpart owing to the
resistance of phosphorus–carbon bond to hydrolytic cleavage.13
Finally, the presence of a phosphonate group allows bypassing
the first phosphorylation step required for nucleoside activation.
In this work, we report on the synthesis of (8) designed as a
structurally related analog of adenosine 50-monophosphate in
order to test its ability to behave as substrate of human adenylate
kinases. Adenylate kinases (AK, EC 2.7.4.4.3) that catalyze the
nucleotide phosphoryl exchange reaction (2ADP ? ATP + AMP)
maintain the consistent concentration and fixed ratio of adenine
Abbreviations: ADP, adenosine diphosphate; AK, adenylate kinase; ANP, acyclic
nucleotide phosphonate; AMP, adenosine monophosphate; AMPK, AMP-activated
kinase; ATP, adenosine triphosphate; Ap4P, P1,P4-di(adenosine-50) tetraphosphate;
Ap5P, P1,P5-di(adenosine-50)pentaphosphate; GSK3b, glycogen synthase kinase 3
beta; HBV, hepatitis B virus; HIV, human immunodeficiency virus; NMP, nucleoside
monophosphate kinase; PMEA, 9-(2-phosphonomethoxyethyl)adenine; PMPA,
(R)-9-(2-phosphonomethoxypropyl)adenine; TEAB, triethylammonium hydrogen
carbonate buffer; THF, tetrahydrofuran; DMAP, dimethylamino pyridine; DBN, 1,5-
diazabicyclo[4.3.0]non-5-ene; TIPSCl2, 1,3-dichloro-1,1,3,3-tetraisopropyldisilox-
ane; TMSBr, trimethylsilyl bromide.
⇑
Corresponding author. Tel.: +33 (0)4 67 14 47 76; fax: +33 (0)4 67 04 20 29.
E-mail address: christophe.mathe@univ-montp2.fr (C. Mathé).
http://dx.doi.org/10.1016/j.bmcl.2014.07.036
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

4228
M. Kaci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4227–4230
NH2
NH2
N
N
N
N
N
N
OH
P
O
N
N
O
HO
RO
HO
O
O
8
5
NH2
NH2
N
N
N
N
N
N
N
N
OR
P
OR
P
RO
O
O
HO2C
O
O
(R= H) Adefovir (PMEA)
Adefovir dipivoxil
(R= pivaloyloxymethyl)
(R= H) Tenofovir (PMPA)
CO2H
Tenofovir disoproxil fumarate
(R= isopropoxycarbonyloxymethyl)
Figure 2. Geometry optimization of compound 8.
Figure 1.
gave 9-(furan-2-yl)adenine derivative (5). The N6-triphenylphos-
phoranylidene group of 5 was used as a transient protection of
the exocyclic amino function in order to avoid N-alkylation during
installation of the phosphonate.
Nucleoside analogue 4 was treated by LiOtBu and coupled with
diethyl p-toluene sulfonyloxymethyl phosphonate in dry THF at
0 °C yielding the phosphonate derivative 6.17 Phosphonate diester
protecting groups of 6 were removed with TMSBr in anhydrous
acetonitrile to give 7 as triethyl ammonium salt after TEAB treat-
ment. Finally, treatment of 7 with an acetic acid/EtOH solution
afforded the target phosphonate 8 as an acid form.
nucleotides. AKs function as a sensitive reporter of cellular energy
state by regulating small changes in the ADP/ATP balance into rel-
atively larger changes in AMP levels.8 Indeed, dysfunction of AKs is
associated with various diseases, including genetic reticular dys-
genesis disease implying mutations in the AK2 gene,14 Alzheimer
disease through the neuropathogenic role of AK1 in Ab-mediated
tau phosphorylation via AMPK and GSK3b.15 Furthermore, AK2
plays an essential role in neutrophil differentiation. A possible
causative link between AK2 deficiency and neutropenia in reticular
dysgenesis has also been recently established.16 Thus, the develop-
ment of modulators of AKs’ activity could be of a great interest to
treat these latter diseases.
As a starting material, commercially available adenosine was
converted into 30,50-O-TIPS-adenosine (1) (Scheme 1). Application
from 1 of the methodology described by Gimisis et al.4 provided
the silylated 10,20-unsaturated N6-triphenylphosphoranylidene
nucleoside 3. Thermal reaction and desilylation afforded the pro-
tected furanyl nucleoside 4.3 Cleavage of the N6 protecting group
Conformational analysis was performed using a geometry
optimization with MM + as implemented in the HyperChem 7.5
software.
A root-mean-square gradient termination cutoff of
0.1 kcal Åꢀ1 molꢀ1 was used for geometry optimization with the
Polak–Ribiere conjugate gradient algorithm. The calculations
provided a geometry optimized structure (Fig. 2). Within this
structure, both heterocyclic aromatic moieties are almost coplanar
with a syn orientation18 of adenine and an angle
v
(dihedral angle
0
0
O4 –C1 –N9–C4) with a value of 25.85°.
PPh3
N
N
N
PPh3
N
N
NH2
N
N
N
N
N
N
N
(i)
(iii)
(ii)
O
O
O
adenosine
N
O
Si
O
N
O
Si
iPr
iPr
iPr
iPr
iPr
iPr
Si
O
O
O
I
O
O
iPr
O
OH
Si
iPr iPr
Si
Si
iPr iPr
iPr
2
1
3
(iv)
PPh3
PPh3
N
N
N
PPh3
N
N
N
N
HNEt3
O
N
N
N
N
N
OEt
P
(vi)
O
P
O
O
(v)
HO
N
O
N
EtO
N
HO
O
O
O
7
4
6
(vii)
vii)
(
NH2
N
NH2
N
N
N
N
N
OH
P
O
O
HO
N
N
HO
O
O
8
5
Scheme 1. Reagents and conditions: (i) TIPSCl2, DMAP, pyridine, rt, 94%; (ii) PPh3, I2, imidazole, toluene, reflux, 88%; (iii) DBN, pyridine, rt, 85%; (iv) (1) xylene, reflux; (2)
NH4F, MeOH, reflux, 45% overall yield; (v) (1) LiOtBu, THF, 0 °C; (2) (EtO)2POCH2OTs, THF, 0 °C, 62% overall yield; (vi) (1) TMSBr, CH3CN, 0 °C; (2) TEAB (pH 7, 1 M), 28% (vii)
MeOH, AcOH, 92% for 5, 86% for 8.

M. Kaci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4227–4230
4229
Table 1
Catalytic properties of AK1 and AK2 with compounds 5 and 8 obtained at 37 °C
Compounds
AK1
AK2
kcat (sꢀ1
)
KM (mM)
kcat/KM (Mꢀ1 sꢀ1) ꢁ 106
kcat (sꢀ1
)
KM (mM)
kcat/KM (Mꢀ1 sꢀ1) ꢁ 106
AMP
dAMP
5
500
240
nsa
ns
0.22
0.08
0.175 0.05
1.5 0.3
ns
ns
2.85
0.16
ns
ns
75
80
110
ns
1.06
2.4
1.5
0.38 0.11
0.21 0.05
ns
0.21
0.5
ns
0.025
800
250
8
0.42 0.013
PMPA
PMEA
3
6
0.3
1
3
6
0.3
0.5
14
a
ns: non substrate. The assay mix contained 50 mM Tris–HCl pH 7.4, 5 mM MgCl2, 1 mM ATP, 0.2 mM NADH, 10 mM DTT, 1 mM phosphoenolpyruvate and auxiliary
enzymes: pyruvate kinase (4 Units) and lactate dehydrogenase (4 Units) MgCl2 concentration in the AK2 experiments was 2 mM as slight inhibition were observed above
3 mM. Experiments were performed at least three times. Pyruvate kinase (404 Units/mg at 37 °C) was purchased from Sigma and lactate dehydrogenase (1100 U/mg at 37 °C)
from Roche.
Figure 3. Predicted binding mode of compound 8 in comparison with AMP. Molecular models of docked into the NMP site of AK1 (PDB code 1Z83). (A) Overview of the
binding into the NMP pocked AMP (green) versus compound 8 (magenta). (B) Interacting NMP site residues with AMP. (C) Interacting NMP site residues with compound 8.
Atom colors follow CPK nomenclature. Putative hydrogen bonds are illustrated by black dotted lines. Helices
Figure was prepared with Molegro Viewer software.
a3 and 4 that close up on acceptor molecules are indicated.
Compounds 8, designed as a structurally related analog of
adenosine 50-monophosphate, as well as compound 5, were evalu-
ated against nucleoside monophosphate kinases (NMP kinases)
involved in nucleosides metabolism in order to assess their sub-
strate properties.
display a large repertoire of substrates including purine deriva-
tives.19 The recombinant enzymes were prepared as described20
and kinetic measurements were obtained using spectroscopic
Pyruvate kinase/lactate dehydrogenase coupled assay to allow
measuring ADP formation.21 The enzymatic parameters of com-
pounds 5 and 8 in comparison with native substrates are presented
in Table 1.
The compounds 5 and 8 were screened against human GMP
kinase, AK1, AK2 and virus vaccine TMP kinase that is known to

4230
M. Kaci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4227–4230
The results clearly underline that compound 8 is a selective
In conclusion, we have reported here the synthesis of a new
phosphonate derivative of 9-(furan-2-yl)adenine thus providing a
novel nucleotide scaffold. This latter was shown to be a relatively
efficient substrate selective to AK2 when compared to well-known
ANPs PMEA and PMPA. Structure-based molecular docking
revealed that the binding mode, although correct, presents some
slight differences particularly in the positioning of phosphate
group when compared to AMP, which explains its lower catalytic
efficiency. Structural variations on compound 8 are ongoing as well
as studies aiming at deciphering its potential anticancer/antiviral
activities.
substrate of AK2, no substrate properties were observed for the
other screened kinases. The KM of 0.41 mM is in the same magni-
tude as AMP (KM = 0.38 mM) and dAMP (KM = 0.21 mM) meaning
that the affinity of compound 8 for AK2 is as strong as native sub-
strates. However catalytic efficiency (kcat = 1.06 sꢀ1) is roughly a
hundred times lower than that of AK’s biological substrates. These
data contrast with those previously reported by our group with
acyclic phosphonates PMPA and PMEA.20 Indeed, we found that
these latter derivatives were poorer substrates of human adenylate
kinases than compound 8.
To achieve more insights into structural basis of the recognition
of compound 8 by adenylate kinases, molecular docking was
performed using Molegro Virtual Docker.22 Although the crystallo-
graphic structure of AK2 in complex with Ap4P bivalent ligand is
available (PDB code: 2C9Y), the latter did not provide information
on the NMP acceptor site. While AK2 is strictly located in the
mitochondrial intermembrane space, AK1 is a cytosolic enzyme
expressed in high energy demanding tissues like, brain, heart,
and skeletal muscles.23 Three functional domains are important
in the primary structure of nucleoside monophosphate kinases:
the nucleoside triphosphate binding glycine-rich region, the
nucleoside monophosphate binding site, and the lid domain that
closes over the substrate upon binding.10,23 All these domains are
extremely well conserved over adenylate kinase isoforms, we thus
use structure of AK1 complexed to Ap5A (PDB code: 1Z83) to per-
form a docking calculation.24
One of the best poses is presented in Figure 3. Globally, com-
pound 8 positioning is close to the one of AMP, even though it is
more extended because it is slightly bulkier. Base moiety is in
interaction with same residues including the establishment of a
hydrogen bond between Thr 39 hydroxyl group and N9 of com-
pound 8 as well as N1 and N6 nitrogen atoms. Compound 8 base
moiety deeply accommodates the hydrophobic pocket composed
of residues Leu 43, Leu 66, Val 67, Val 72.
The most distinguishable features are located in the sugar/phos-
phate region. Indeed, while AMP sugar-phosphate hydrogen bond
network is clustered to Arg residues (44, 138, 140, 149), this of
compound 8 is restricted to Arg 44 and 48. Interestingly, the sugar
mimetic group seems to share hydrophobic contacts with Leu 43.
Moreover, the oxygen atom of the ethoxy-phosphonate linkage
shares a specific H-bond implying nitrogen atom of Arg guanidine
group. Stabilizing interactions of compound 8 as well as its
extended orientation with AK NMP site is in accordance with its
ability to be phosphorylated by AK2. In the complex AK1-Ap5A,
the phosphate group is in close contact with Arg44 belonging to
Acknowledgments
One of us M.K. is particularly grateful to the Ministère de l’Edu-
cation Nationale, de l’Enseignement Supérieur et de la Recherche
(France) for a doctoral fellowship. C.E wishes to thank Pr Bertrand
Friguet for laboratory facilities.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.
036.
References and notes
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helix
a3 and Arg97 from the CORE domain that carries the ATP
binding loop (P-loop). Both Arg residues are well conserved over
NMP kinase family and play a crucial role for an efficient nucleo-
phile attack on
c
-P of ATP.20 This configuration is disrupted for
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compound 8 as seen above, agreeing its lower catalytic efficiency.
Adenylate kinase constitutes an archetype for the study of confor-
mational dynamics during catalytic cycle.25 The selective action on
AK2 could thus be due to differential conformational plasticity of
both enzymes. Indeed, the greatest difference between AK1 and
AK2 is located in the lid domain.23
The lid domain of AK2 is more extended than the one of AK1
and may allow a more effective closure of the active site essential
for a proper catalytic reaction.
22. Thomsen, R.; Christensen, M. H. J. Med. Chem. 2006, 49, 3315.
23. Van Rompay, A. R.; Johansson, M.; Karlsson, A. Eur. J. Biochem. 1999, 261, 509.
24. Heteroatoms (water molecules, ions) and Ap5A ligand were removed from the
crystal structure and addition of hydrogen atoms was performed. The scoring
function Moldock Score and the Moldock SE search algorithm were used, the
resolution of the grid for estimation of the computed binding affinity was 0.3 Å
with a radius of 15 Å around the catalytic triad residues. Fifteen runs were
launched and up to 50 (maximum) poses subsequently analyzed.
25. Jana, B.; Adkar, B. V.; Biswas, R.; Bagchi, B. J. Chem. Phys. 2010, 134, 035101.