Synthesis and Evaluation of Novel Acyclic Nucleoside Phosphonates as Inhibitors of Plasmodium falciparum and Human 6-Oxopurine Phosphoribosyltransferases

Article abstract of DOI:10.1002/cmdc.201500322

Acyclic nucleoside phosphonates (ANPs) are a promising class of antimalarial therapeutic drug leads that exhibit a wide variety of K_i values for Plasmodium falciparum (Pf) and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases [HG(X)PRTs]. A novel series of ANPs, analogues of previously reported 2-(phosphonoethoxy)ethyl (PEE) and (R,S)-3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) derivatives, were designed and synthesized to evaluate their ability to act as inhibitors of these enzymes and to extend our ongoing antimalarial structure-activity relationship studies. In this series, (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)propanoic acid (CPME), or (S)-2-(phosphonoethoxy)propanoic acid (CPEE) are the acyclic moieties. Of this group, (S)-3-hydroxy-2-(phosphonoethoxy)propylguanine (HPEPG) exhibits the highest potency for PfHGXPRT, with a K_i value of 0.1 μM and a K_i value for human HGPRT of 0.6 μM. The crystal structures of HPEPG and HPEPHx (where Hx=hypoxanthine) in complex with human HGPRT were obtained, showing specific interactions with active site residues. Prodrugs for the HPEP and CPEE analogues were synthesized and tested for in vitro antimalarial activity. The lowest IC₅₀ value (22 μM) in a chloroquine-resistant strain was observed for the bis-amidate prodrug of HPEPG. Combating malaria: An efficient inhibition of plasmodial 6-oxopurine phosphoribosyltransferase, a key enzyme of the parasitic purine nucleotide salvage pathway, is a promising way to combat malaria. Novel acyclic nucleoside phosphonates were designed as potent inhibitors of phosphoribosyltransferases, and the mode of their binding in the enzyme active site was studied in detail.

Full text of DOI:10.1002/cmdc.201500322

DOI: 10.1002/cmdc.201500322
Full Papers
Very Important Paper
Synthesis and Evaluation of Novel Acyclic Nucleoside
Phosphonates as Inhibitors of Plasmodium falciparum and
Human 6-Oxopurine Phosphoribosyltransferases
[a]
[a]
[b]
[a]
ˇ
´
ˇ
Martin M. Kaiser, Dana Hockovµ, Tzu-Hsuan Wang, Martin Dracínsky, Lenka Postovµ-
[a]
[a]
[c]
[c]
ˇ
ˇ
Slavetínskµ, Eliska Prochµzkovµ, Michael D. Edstein, Marina Chavchich,
Dianne T. Keough,[b] Luke W. Guddat,*[b] and Zlatko Janeba*[a]
Acyclic nucleoside phosphonates (ANPs) are a promising class
of antimalarial therapeutic drug leads that exhibit a wide varie-
ty of Ki values for Plasmodium falciparum (Pf) and human hypo-
acid (CPEE) are the acyclic moieties. Of this group, (S)-3-hy-
droxy-2-(phosphonoethoxy)propylguanine (HPEPG) exhibits
the highest potency for PfHGXPRT, with a Ki value of 0.1 mm
and a Ki value for human HGPRT of 0.6 mm. The crystal struc-
tures of HPEPG and HPEPHx (where Hx=hypoxanthine) in
complex with human HGPRT were obtained, showing specific
interactions with active site residues. Prodrugs for the HPEP
and CPEE analogues were synthesized and tested for in vitro
antimalarial activity. The lowest IC50 value (22 mm) in a chloro-
quine-resistant strain was observed for the bis-amidate pro-
drug of HPEPG.
xanthine-guanine-(xanthine)
phosphoribosyltransferases
[HG(X)PRTs]. A novel series of ANPs, analogues of previously re-
ported 2-(phosphonoethoxy)ethyl (PEE) and (R,S)-3-hydroxy-2-
(phosphonomethoxy)propyl (HPMP) derivatives, were designed
and synthesized to evaluate their ability to act as inhibitors of
these enzymes and to extend our ongoing antimalarial struc-
ture–activity relationship studies. In this series, (S)-3-hydroxy-2-
(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)-
propanoic acid (CPME), or (S)-2-(phosphonoethoxy)propanoic
Introduction
Malaria remains one of the most serious infectious diseases in
the world today with forty percent of the world’s population
living in areas of risk resulting in more than 200 million infect-
ed individuals and 500000 fatalities each year.[1] Plasmodium
falciparum (Pf) and Plasmodium vivax (Pv) are the most wide-
spread species that cause malaria in humans.[2,3] While Pf is re-
puted to be the most lethal, Pv is responsible for serious illness
with relapsing infections. This is due to the fact that the Pv
parasite can lie dormant in the liver of the host.[3] The current
standard treatment for uncomplicated Pf and Pv malaria are ar-
temisinin-based combination therapies and chloroquine plus
primaquine, respectively.[4] However, antimalarial drug resist-
ance has developed and spread for these antimalarial medica-
tions, particularly in Southeast Asia, where both Plasmodium
species are prevalent.[5–7] Therefore, alternative drug candidates
with novel mechanisms of action are urgently needed.
A promising approach for the development of new antima-
larials is based on the inhibition of Pf hypoxanthine-guanine-
xanthine phosphoribosyltransferase (HGXPRT).[8,9] HG(X)PRT is
a key enzyme of the purine salvage pathway where it catalyzes
the formation of the N-glycosidic bond between the N9 atom
of the 6-oxopurine base and the C1 atom of 5-phospho-a-d-ri-
bosyl-1-pyrophosphate (PRib-PP) (Figure 1).[10]
A significant difference between the metabolic pathways of
Plasmodium and its human host cell is the ability to synthesize
the purine nucleoside monophosphates essential for the pro-
duction of DNA/RNA. Mammalian cells are able to produce
these metabolites either by de novo synthesis or by salvage of
the preformed purine bases. In contrast, Pf and Pv parasites do
not possess the de novo pathway, relying on salvage of the 6-
ˇ
´
[a] Dr. M. M. Kaiser, Dr. D. Hockovµ, Dr. M. Dracínsky,
ˇ
ˇ
Dr. L. Postovµ-Slavetínskµ, E. Prochµzkovµ, Dr. Z. Janeba
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic v.v.i.
Flemingovo nµm. 2, 16610 Prague 6 (Czech Republic)
E-mail: janeba@uochb.cas.cz
[b] T.-H. Wang, Dr. D. T. Keough, Dr. L. W. Guddat
School of Chemistry and Molecular Biosciences
The University of Queensland, Brisbane, Queensland, 4068 (Australia)
E-mail: luke.guddat@uq.edu.au
[c] Dr. M. D. Edstein, Dr. M. Chavchich
Department of Drug Evaluation, Australian Army Malaria Institute
Enoggera, Brisbane, Queensland 4051 (Australia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500322.
Figure 1. Reaction catalyzed by HG(X)PRT. The naturally occurring bases are
hypoxanthine (R=H), guanine (R=NH2) and xanthine (R=OH).
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oxopurines transported from their host cell. Thus, the activity
of HG(X)PRT is crucial in the replication and survival of these
parasites making this enzyme a promising target for the
design of novel antiparasitic drugs.[3]
Acyclic nucleoside phosphonates (ANPs)[11] containing either
a pyrimidine or 6-amino purine base constitute a distinguished
class of successful therapeutic antiviral agents inhibiting either
RNA reverse transcriptases or DNA polymerases.[12] Three of
them have been approved worldwide for clinical use (adefovir,
cidofovir and tenofovir).[12] In this type of nucleotide analogues,
pounds may have enhanced affinity. Herein, we report the syn-
thesis and inhibitory properties of ANPs containing (S)-2-(phos-
phonomethoxy)propanoic acid (or 2’-carboxy-PME, CPME, 17,
18),[16] (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP, 25–27)
or (S)-2-(phosphonoethoxy)propanoic acid (or 2’-carboxy-PEE,
CPEE, 32, 33, 36, 37) as the acyclic moieties together with the
synthesis of the corresponding prodrugs of HPEP and CPEE an-
alogues and their antimalarial activities.
the heterocyclic base and phosphonate group are linked by Results and Discussion
various acyclic chains that mimic the sugar moiety. There are
Chemistry
several advantages that the ANPs possess with regard to their
potential as therapeutic drugs. These include:[12] 1) resistance
toward degradation by enzymes in vivo because of the stable
carbon-phosphorus bond; 2) attractive conformational flexibili-
ty; and 3) low cost of synthesis making them suitable thera-
peutics, especially for third world countries. Several ANPs con-
taining hypoxanthine or guanine as the nucleobase (e.g., 2-
(phosphonoethoxy)ethyl (PEE) hypoxanthine, PEE guanine and
(R,S)-3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) guanine,
Figure 2, Table S1) have proven to be good inhibitors of
PfHGXPRT and PvHGPRT.[13] These compounds were the first
(S)- and (R)-tritylglycidol 1a and 1b (Scheme 1) were chosen as
the starting materials for the synthesis of the target ANP
series. Intermediate 4 (Scheme 1), prepared through 2a and 3,
was oxidized using TEMPO/NaClO2/NaClO under mild reaction
conditions to give 5 (in a 34% overall yield from 1a),[16,17]
which, under treatment with diazomethane, quantitatively pro-
duced the methyl ester 6 (Scheme 1). The key intermediate 7
was then obtained in a 95% yield after removal of the benzyl
group from compound 6.
Modification of the previously described oxa-Michael addi-
tion of the secondary alcohol to diethyl vinylphosphonate
(DEVP)[18] was used for introducing the ethylphosphonate
moiety. Treatment of alcohols 2a and 2b with DEVP and a cata-
lytic amount of KOH in dioxane,[19] to give compounds 8a and
8b (Scheme 1), followed by the removal of the trityl groups by
holding at reflux in acetic acid, gave derivatives 9a and 9b, re-
spectively, in 37–39% overall yields (from compounds 2). Com-
pound 9a was further oxidized using TEMPO/NaClO2/NaClO
system to give carboxyl derivative 10 (83%) which was then
quantitatively converted with diazomethane to its methyl ester
11 (Scheme 1). Finally, the removal of the benzyl group from
Figure 2. Structures of two types of acyclic nucleoside phosphonates (ANPs)
shown to be potent inhibitors of human HGPRT and PfHGXPRT.[13]
HG(X)PRT inhibitors found to se-
lectively discriminate between
the human and parasitic en-
zymes. Subsequently,
a broad
spectrum of ANPs as inhibitors
of human, Escherichia coli (Ec),
Mycobacterium tuberculosis (Mt),
Pf and Pv HG(X)PRTs has been in-
vestigated.[14,15]
The ANPs in Figure 2 are sub-
micromolar
inhibitors
of
PfHGXPRT.[13] Therefore, a compu-
tational study based on the crys-
tal structures of human HGPRT
in complex with PEEHx, PEEG or
(S)-HPMPG was undertaken. The
results suggested that if a hy-
droxymethyl, carboxy or me-
thoxycarbonyl group could be
attached to the acyclic linker at
the second carbon atom from
Scheme 1. Synthesis of key intermediates 7, 9b and 12. Reagents and conditions: a) BnOH, NaH, DMF, 1008C, 2 h;
b) 1. NaH, DMF, 08C, 15 min, 2. BrCH2P(O)(OiPr)2, DMF, RT, 48 h; c) 80% AcOH, reflux, 2 h; d) TEMPO, NaClO2,
NaClO, phosphate buffer, MeCN, 408C, 48 h; e) CH2N2, EtOAc, RT, 0.5 h; f) H2/Pd/C, MeOH, RT, 72 h; g) KOH,
the purine ring, these com- CH2 =CHP(O)(OEt)2, dioxane, RT, 70 h.
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To obtain desired free phos-
phonic acids 17, 18, 25, 26, 32,
33, 36 and 37, the ester func-
tions on the phosphonate
moiety were removed under
standard reaction conditions
using Me3SiBr/MeCN followed by
hydrolysis (Schemes 2–4).[22] The
guanine derivative 26 was then
converted by treatment with iso-
Scheme 2. Preparation of (S)-CPMEHx (17) and (S)-CPMEG (18). Reagents and conditions: a) Mitsunobu conditions:
1. 6-chloro or 2-amino-6-chloropurine, compound 7, PPh3, dioxane, 2. DIAD, RT, 24 h; b) DABCO, K2CO3, dioxane/
H2O (4:1), 908C, 3–5 h; c) Me3SiBr, MeCN, RT, overnight.
compound 11 afforded the desired derivative 12 in a 93%
yield.
amylnitrite in AcOH to xanthine analogue 27 to give a 35%
yield (Scheme 3).
Mitsunobu conditions[20] were employed for introduction of
the acyclic moieties, namely alcohols 7, 9b and 12, to the N9-
position of 6-chloropurine and 2-amino-6-chloropurine in satis-
factory yields (40–80%; Schemes 2–4). For 2-amino-6-chloro-
purine derivatives 14, 20 and 29, the crude Mitsunobu reaction
mixtures were heated in water/dioxane to decompose triphe-
nylphosphoranylidene intermediates which were formed due
to presence of free amino groups in the C2 position.[21] The re-
sulting 6-chloropurine and 2-amino-6-chloropurine derivatives
can be, in general, transformed into the corresponding 6-oxo-
purine analogues using nucleophilic aromatic substitution in
either basic or acidic conditions. When the basic procedure
(DABCO/K2CO3) was applied, the methyl ester group in com-
pounds 13, 14, 28 and 29 was simultaneously cleaved to pro-
duce carboxylic acids 15 (59%), 16 (56%), 30 (57%) and 31
(55%), respectively (Scheme 2
The current SAR study of the (S)-HPEP analogues was also
extended for derivatives containing a bromo or oxo group at
the C8 position of guanine. Two methods were employed for
bromination of guanine containing ANPs: bromination of com-
pound 24 with a bromine/CCl4/DMF mixture to yield 85% of 8-
bromoguanine derivative 38 (Scheme 5); bromination of O-
benzyl derivative 22 using NaBrO3/Na2S2O4 to afford brominat-
ed product 40 in a 67% yield (the anticipated simultaneous
bromination and benzyl moiety removal[23] was not achieved).
Compound 40 was subsequently heated in a AcONa/AcOH
mixture to afford 8-oxoguanine derivative 41 (80% yield),[24]
which was then converted by hydrogenolysis to compound 42
in an 82% yield (Scheme 5). Finally, desired ANPs 39 (77%) and
43 (77%) were obtained in good yields after removal of phos-
phonate ethyl ester moieties (Scheme 5).
and 4). Treatment of compounds
28 and 29 (Scheme 4) under
acidic conditions (75% aqueous
trifluoroacetic acid at room tem-
perature) led preferentially to
the formation of methyl esters
34 (84%) and 35 (81%), respec-
tively. Analogous acidic hydroly-
sis (75% aqueous trifluoroacetic
acid) at elevated temperature
Scheme 3. Preparation of (S)-HPEPHx (25), (S)-HPEPG (26) and (S)-HPEPX (27). Reagents and conditions: a) Mitsuno-
bu conditions: 1. 6-chloro or 2-amino-6-chloropurine, PPh3, dioxane, 2. DIAD, RT, 24 h; b) DABCO, K2CO3, dioxane/
H2O (4:1), 908C, 3 h; c) H2/Pd/C, AcOH, RT, 72 h; d) Me3SiBr, MeCN, RT, overnight; e) isoamylnitrite, 80% AcOH, RT,
overnight.
(508C) and prolonged time
(overnight) also leads to a cleav-
age of the ester moiety and
treatment of compound 29 by
this method gave the carboxylic
acid 31 in
a
78% yield
(Scheme 4).
In the HPEP series, 6-chloro-
purine derivatives 19 and 20
(Scheme 3) were converted into
6-oxopurine analogues 21 and
22, respectively, by the basic
procedures (DABCO/K2CO3) de-
scribed above. Subsequently,
compounds
23
and
24
(Scheme 3) were prepared by
hydrogenolysis of the protecting
O-benzyl groups in derivatives
21 and 22, respectively.
Scheme 4. Preparation of (S)-CPEEHx (32), (S)-CPEEG (33) and their methyl esters 36 and 37. Reagents and condi-
tions: a) Mitsunobu conditions: 1. 6-chloro or 2-amino-6-chloropurine, PPh3, dioxane, 2. DIAD, RT, 24 h; b) DABCO,
K2CO3, dioxane/H2O (4:1), 908C, 3–5 h; c) Me3SiBr, MeCN, RT, overnight; d) 75% CF3COOH, RT, 24 h.
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The Ki value for HPEPG (26) is
similar to that of PEEG for both
enzymes (0.6 mm vs 1 mm for
human HGPRT and 0.1 mm (both
compounds)
for
PfHGXPRT,
Tables 1 and S1). Thus, the addi-
tion of the CH2OH group to the
acyclic linker does not signifi-
cantly affect the Ki value.
In contrast, when the base is
hypoxanthine, the Ki value for
human HGPRT decreases for the
HPEP compound relative to
PEEHx (from 3 to 0.5 mm). How-
Scheme 5. Preparation of 8-substituted (S)-HPEPG analogues 39 and 43. Reagents and conditions: a) Br2/CCl4, DMF,
RT, 3 h; b) Me3SiBr, MeCN, RT, overnight; c) NaBrO3, Na2S2O4, EtOAc/H2O (5:4), RT, 1 h; d) AcONa, AcOH, 1308C, 4 h;
e) H2/Pd/C, AcOH, RT, 24 h.
The above described 6-chloropurine intermediates 19
Table 1. Comparison of the Ki values of (S)-3-hydroxy-(2-phosphonoe-
thoxy)propyl (HPEP) ANPs for human HGPRT and PfHGXPRT.
(Scheme 3) and 28 (Scheme 4) were also used for the prepara-
tion of the corresponding adenine containing ANPs bearing
CH2OH, COOH and CONH2 moieties at C2 side chain position,
that is, compounds 46, 48 and 50, respectively (Scheme S1,
Supporting Information). These compounds have been de-
signed, in an analogy to previously reported adenine based
ANPs,[25] as potential inhibitors of Bordetella pertussis adenylate
cyclase toxin (ACT, CyA), but no activity was observed in the
ACT inhibitory assay[25] with these compounds (the assay de-
tails in Supporting Information).
Attachment to Compd Base
C2 in the linker
Ki [mm]
human
>30[a]
Pf
ÀCH2OH
ÀCH2OH
ÀCH2OH
ÀCH2OH
ÀCH2OH
43
27
39
26
25
8-oxoguanine
xanthine
8-bromoguanine
guanine
>30[a]
>30[b]
>30[c]
>30[b]
13Æ2
0.6Æ0.1
0.5Æ0.2
0.1Æ0.05
2Æ0.8
hypoxanthine
[a] Assay [inhibitor]: 31 mm. [b] Assay [inhibitor]: 49 mm. [c] Assay [inhibi-
tor]: 33 mm.
Several bis-amidate prodrugs of the HPEP and CPEE ana-
logues, namely compounds 51–56 (Scheme S2, Supporting In-
formation), were then prepared by the previously reported
methodology[26] (see Supporting Information for details) to test
their effectiveness as antimalarial compounds in cell based
assays.
ever, for PfHGXPRT, the reverse occurs and the Ki value increas-
es from 0.3 mm to 2 mm (Tables 1 and S1). This results in the Ki
value for HPEPHx for human HGPRT becoming similar to
HPEPG, suggesting that the contribution of the exocylic amino
group of the purine base may not be as important in human
HGPRT as that of the ANP moiety itself. For PfHGXPRT, HPEPG
has the highest affinity from this group of ANPs and has selec-
tivity in favor of this enzyme over human HGPRT of 6.
Inhibition studies of novel 6-oxopurine acyclic nucleoside
phosphonates
The synthesized ANPs analogues containing (S)-3-hydroxy-2-
(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)-
propanoic acid (CPME) or (S)-2-(phosphonoethoxy)propanoic
acid (CPEE) moieties were tested as potential inhibitors of
human HGPRT and PfHGXPRT.
(S)-2-(Phosphonoethoxy)propanoic acid (CPEE) derivatives
and their methyl esters
(S)-3-Hydroxy-2-(phosphonoethoxy)propyl (HPEP) derivatives
The Ki values for human HGPRT and PfHGXPRT when CPEE (or
its corresponding methyl ester) is the acyclic moiety are given
in Table 2. For human HGPRT, CPEEHx (32) and its methyl ester
36 show a significant decrease in affinity compared with the
corresponding HPEP analogue 25 (Ki values increase from
0.5 mm to 6 mm). However, when the base is guanine, the Ki
value is virtually unchanged with values of 0.6, 0.8 and 0.3 mm
for the three ANPs (Tables 1 and 2). Once again, the reverse is
true for PfHGXPRT, with the Ki values for CPEEHx and its ester
being similar to HPEPHx (1–2 mm cf. 1 mm; Tables 1 and 2).
When guanine is the base, the Ki value for CPEEG (33) com-
pared with HPEPG (26) increases from 0.1 mm to 0.5 mm and
the Ki value for its methyl ester 37 increases to 5 mm with a re-
sultant decrease in affinity of ~50-fold.
Table 1 compares the Ki values for human HGPRT and
PfHGXPRT when HPEP is the acyclic linker and is attached to
five different 6-oxopurine bases. In this series of ANPs, if the
purine base has a high KM for the enzymes,[10] or does not bind
at all, then the ANP containing that particular purine base ex-
hibits high Ki values. This data, therefore, emphasizes the im-
portant contribution of the purine base to the degree of inhib-
ition for these compounds. For example, xanthine is not a sub-
strate for human HGPRT and its KM for PfHGXPRT is 189 mm.[10]
Therefore, this ANP has an extremely high Ki value for the en-
zymes and, at the concentration of inhibitor added in the
assay, no inhibition could be detected. Thus, >30 mm is likely
to be a low estimate of its true Ki value.
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Table 2. Comparison of the Ki values for the (S)-2-carboxy-(phosphonoe-
thoxy)ethyl (CPEE) derivatives for human HGPRT and PfHGXPRT.
Table 3. In vitro antimalarial activity of ANP prodrugs against the chloro-
quine-sensitive (D6) and the chloroquine-resistant (W2) P. falciparum lines.
Attachment to
C2 in the linker
Compd
Base
Ki [mm]
human
ANP
moiety
Base
Compound Parent
IC50 [mm]
Pf
D6
W2
ÀCOOH
ÀCOOH
ÀCOOMe
ÀCOOMe
33
32
37
36
guanine
hypoxanthine
guanine
0.8Æ0.2
6.3Æ1.5
0.3Æ0.1
6Æ2
0.5Æ0.2
2Æ1
HPEP
HPEP
HPEP
HPEP
CPEE
CPEE
–
hypoxanthine
guanine
hypoxanthine
guanine
hypoxanthine
guanine
–
51
52
53
54
55
25
26
25
26
32
33
–
103
70
695
76
22
523
261Æ25
60Æ11
51Æ5
5Æ1
hypoxanthine
1Æ0.9
274Æ37
60Æ12
54Æ8
56
chloroquine
0.012Æ0.004 0.169Æ0.075
This data shows the importance of the character of the
purine base and the chemical structure of the acyclic moiety in
positioning the inhibitor in the active site of these two en-
zymes to obtain the highest affinity and selectivity. In some
cases, these two moieties work in synergy but, in other cases,
the contribution of one of them is more influential. Thus, it
can be hypothesized that it is the chemical structure of the
acyclic moiety that is responsible for positioning both the
purine base and the phosphonate moiety in the active site of
either of these two enzymes to obtain optimal binding.
Compounds 51, 52, 55 and 56 (Scheme S2) are bisphosphor-
amidate prodrugs with two ethyl l-phenylalanyl moieties at-
tached to the parent compound. Compounds 53 and 54 are
monophosphoramidates where the remaining phosphonate
oxygen is replaced by NH2 group (Scheme S2). The second
strategy was not successful as it resulted in a dramatic increase
in the IC50 value (7-fold when the base is hypoxanthine and
12-fold when the base is guanine). One suggestion for this in-
crease is that the amino group replacing the phosphonate
oxygen is not easily hydrolyzed in vivo and the resulting ANP
could be a weak inhibitor of the Pf enzyme. In all cases, the
prodrugs containing guanine as the base have lower IC50
values than when the purine base is hypoxanthine. This is con-
sistent with the fact that the Ki values for the parent com-
pound are lower for guanine than for the ANPs with hypoxan-
thine as the nucleobase.
(S)-2-(Phosphonomethoxy)propanoic acid (CPME) derivatives
Compounds 17 and 18 are very weak inhibitors with Ki values
of much greater than 30 mm as no inhibition was observed at
the concentration of these compounds in the assay (32.4 and
46.7 mm for compounds 17 and 18, respectively). It has been
shown that when the acyclic linker connecting the phosphorus
and N9 atoms is only four atoms long, the affinity for such
ANPs in the active site decreases significantly.[27] This has been
attributed to the fact that the phosphonate group cannot
reach into the 5’-phosphate binding pocket to form maximal
interactions with the amino acid side chains or the backbone
atoms of those residues that surround this group (residues
D137-T141 in human HGPRT and D148-T152 in PfHGXPRT).
However, (S)-3-hydroxy-2-(phosphonomethoxy)propyl guanine
[(S)-HPMPG] does bind to both the human and Pf enzymes,
albeit weakly, with Ki values of 177 and 28 mm, respectively.[13]
The crystal structure of (S)-HPMPG in complex with human
HGPRT suggests that one of the reasons for this affinity is a hy-
drogen bond between the hydroxy group and the NZ atom of
K68 which occurs in one of the two subunits. However, it may
be that, for the CPMEG compound, the conformation of the
acyclic moiety has changed the orientation of the ANP in the
active site and that the side chain of this residue is not in a po-
sition to form this hydrogen bond. Also, for (S)-HPMPG the
phosphonate group does indeed reach into the 5’-phosphate
binding pocket due to its location in the active site.[13a] This
may not be the case for inhibitors containing CPME as the acy-
clic moiety.
Crystal structures of compounds 25 and 26 in complex with
human HGPRT
These two structures contain a tetramer in the asymmetric
unit. The overall fold of each subunit is similar to that of PEEG
(PDB code: 3GGJ) and PEEHx (PDB code: 3GGC).[13a] In these
latter two structures, the asymmetric unit is a dimer though
the normal tetrameric molecule for human HGPRT is observed
when adjoining asymmetric units are considered. Thus, the
quaternary structure is the same in all four structures. Data col-
lection and refinement statistics for the two complexes are
given in Table S2 (Supporting Information). The electron densi-
ty for both inhibitors bound in the active site is shown in
Figure 3, unequivocally demonstrating their location together
with those of a magnesium ion and water molecules in the
active site.
The movement of flexible loops and the specific interactions
surrounding the purine base and phosphonate group when
HPEPG (compound 26) and HPEPHx (compound 25) bind in
the active site of human HGPRT (Figure 4). The chemical
nature of the five atoms connecting the N9 of the purine ring
to the phosphorus atom in the phosphonate group is the
same in the HPEP and PEE compounds, that is, -(CH2)2O(CH2)2-.
The design of this linker places the purine base under the aro-
matic ring of F186, forming a P-stacking arrangement, and the
phosphonate group in the 5’-phosphate binding pocket
(Figure 4). The figure also demonstrates the movement of two
IC50 values of the prodrugs of novel ANPs for Pf in cell culture
Table 3 shows the activity of six prodrugs of the HPEP and
CPEE compounds against Pf in cell culture (synthesis and struc-
tures are given in Supporting Information).
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tances between these two carbonyl oxygen atoms and the
exocyclic nitrogen atom in the purine ring (3.2 À3.9 Š to the
carbonyl oxygen of V187 and 3–3.3 Š to the carbonyl oxygen
of D193) are altered. In the presence of compound 26, the
loop containing these two residues moves by ~2.4 Š com-
pared with when HPEPHx binds (Figure 5). This is very different
to that found when PEEG and PEEHx bind as there is no
change in the structure of the loop between residues 187–193.
This is attributed to the fact that the location in the active site
of these two inhibitors is virtually identical except for that ad-
dition of the exocyclic amino group. The movement observed
when compound 25 binds appears to cause a cascade effect
on the structure of the enzyme in this region (Figure 5). Thus,
though the integrity of the b-sheets and a-helices is main-
tained, there is a shift so that the areas in this region are no
longer completely superimposable (Figure 5).
Figure 3. The crystal structure of the active site of human HGPRT in complex
with a) HPEPHx (25) and b) HPEPG (26). The FoÀFc omit electron density is
shown as green wire. Human HGPRT is presented as a Connolly surface. Hy-
drogen bonds to the inhibitors are shown as black dashed lines. Coordina-
tion to the Mg2+ ions is shown as purple dashed lines. Water molecules
within hydrogen bonding distance or that coordinate to the Mg2+ ions are
depicted as pink spheres.
This structural change also affects the association between
subunits in the two structures of human HGPRT in complex
with compounds 25 and 26. Thus, in the complex with com-
pound 26, the shift of the flexible loop (residues 187-193) re-
sults in the formation of two hydrogen bonds between subu-
nits (A with C and B with D). In this structure, the hydroxy
group of the side chain Y71 (subunit B) forms a hydrogen
bond with the carbonyl oxygen of Y71 in the adjoining subunit
(subunit D). The hydroxy group of this amino acid residue
(Y71; subunit D) can then form a hydrogen bond with the NZ
atom of K82 in subunit B. These two hydrogen bonds assist in
stabilizing the interface between these two subunits. These
two hydrogen bonds cannot form in the complex with com-
pound 25 because the side chain of Y71 is pointing in the op-
posite direction (Figure 4). The difference in the movement of
the flexible loop between residues 67–71 and, in particular
K68, is discussed later.
Figure 4. Stereoview of the superimposition of the active site of human
HGPRT in complex with HPEPHx (25) (cyan carbon atoms) and HPEPG (26)
(green carbon atoms).
flexible loops that occurs when these inhibitors bind and, in
particular, the loops that comprise the amino acid residues be-
tween F186–196E and L67–71Y.
How these structural changes influence the Ki value cannot
be determined but they do demonstrate the difficulties in pre-
dicting precisely how these compounds could bind and how
binding affects the positioning of the mobile loops in the
active site.
The mobile loops between residues 187–193 and 67–71
When the transition state analogue, (1S)-1-(9-deazaguanin-9-
yl)-1,4-dideoxy-1,4-imino-d-ribitol
(ImmGP),[28] binds to human
HGPRT, the nitrogen atom of the
exocyclic amino group attached
to the C2 atom in the purine
ring forms two hydrogen bonds:
to the carbonyl oxygen atoms of
V187 (2.6–2.8 Š) and D193 (2.7 Š
but 3.1 Š in one subunit). These
residues are located on the flexi-
ble loop (187–193). When PEEG
binds, there is also a hydrogen
bond to the carbonyl of V187
(3.1 Š) but the carbonyl of D193
is too far away for such bonds to
5-phosphate
guanine
form (4 Š in one subunit and Figure 5. Superimposition of subunit A of the human HGPRT–compound 25 complex (brown) with subunit A of
the human HGPRT–compound 26 complex (pink, red, green, blue). a) Overall view showing the relative move-
3.5 Š in the second). However,
ments of the subunits. b) The origin of the movement is the presence of the amino group in C2 position of com-
when compound 26 binds in the
pound 26 when the closest loop moves by 2.7 Š. This transmits a movement of the helix in subunit C by 4.1 Š
active site, the location of this
loop is altered so that the dis- binding pocket does not relocate.
and the upper helix in subunit A by 2.7 Š. However, the lower helix in subunit A, bordered by the 5’-phosphate
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K165
pound 25. This water molecule is not observed in the complex
with compound 26.
In both the HPEP structures, the invariant lysine residue (com-
pletely conserved across all the 6-oxopurine PRTases) forms
a hydrogen bond with the 6-oxo group (Figure 4). This confers
specificity for compounds containing an oxygen, halogen or
thiol at this position of the purine ring in contrast with an
amino group, which abolishes binding.
A water molecule is also found in this location when ANPs,
containing a second phosphonate group, are bound in the
active site.[14d–f] In the majority of instances, this water molecule
is coordinated to the second magnesium and acts as a bridge
to the phosphonate oxygen. However, on some occasions, it
has been found to form a hydrogen bond with the side chain
of D193. A water molecule has also been found in hydrogen
bond distance to the N3 atom when ImmGP binds together
with pyrophosphate.[28] Here, it is coordinated to the magnesi-
um ion coordinated to the phosphoryl oxygen atoms of pyro-
phosphate. The complex with compound 25 is the first in-
stance that the presence of a water molecule linking the N3
atom to the D193 side chain is observed when a second diva-
lent ion, together with pyrophosphate or its mimic, are not
found in the active site.
The 5’-phosphate binding pocket
The phosphonate group of the two structures is locked into
position by hydrogen bonds to the D137-T141 amino acid resi-
dues that surround the 5’-phosphate binding pocket (the
amides of D137, T138, G139 and T141 together with bonds to
the side chains of T138 and T141) (Figure 4).[13a]
Two water molecules can be observed in the binding pocket
of the phosphonate group in both structures. The first forms
a bridge between one of the phosphonate group oxygen
atoms and the OD1 atom of D137 residue (complex with 26,
subunits A and C; and complex with 25, subunits A, C and D).
These bonds assist in anchoring the phosphonate group in po-
sition and also help to restrict the movement of the side chain
of D137. This is another of the flexible regions of the enzyme
and demonstrates how different network of interactions con-
tribute to the affinity of the inhibitor. There is a second water
molecule located at the “bottom” of this pocket (Figure 4). This
molecule forms a hydrogen bond with the amide of M142
(complex with 26, subunit B; and complex with 25, subunits A,
C and D). In the other subunits, no other hydrogen bond is
formed except in subunit B (complex with 25) where there is
an interaction with the carbonyl oxygen atoms of I135 and
E133. This is also another flexible part of the enzyme. This ar-
rangement of water molecules is the same as in the structure
of PEEG and PEEHx in complex with human HGPRT.[13a] In the
transition state structure, the water molecule which forms a hy-
drogen bond with the side chain of D137 is absent. This is due
to the fact that this region of the enzyme has moved so that
the OD1 atom of D137 forms a hydrogen bond with the N7
atom of the purine ring. This is an important bond as it has
been proposed that it is necessary to promote catalysis. Thus,
the variable network of hydrogen bonds in this area of the
active site seems to be another factor that can influence the
affinity of an inhibitor for these enzymes. The electron density
for all the water molecules is unequivocal and, thus, this
strongly suggests that these molecules play a crucial role in
the binding of the inhibitors.
The large mobile loop (residues 100–120)
One difference between the structures of PEEG, PEEHx and
compounds 25 and 26, is found in the large mobile loop. In
the former two structures,[13a] this loop is visible though some
residues are missing. In the PEEG structure, the loop is com-
plete in chain A but residues D107 and T110 are not visible in
chain B. For PEEHx, residues N106 and D107 are missing chain
A and G117 in chain B. The structure of this loop is mainly
random coil and it adopts a position that appears to be inter-
mediate between that found in the ImmGP complex with
human HGPRT[28] and when this enzyme is in complex with the
nucleoside monophosphate, GMP. However, unlike the com-
plex with ImmGP, there are no interactions between the amino
acids in this loop and atoms in either PEEG or PEEHx. This sug-
gests that, for these ANPs, the positioning of the loop may not
affect their affinity. In comparison, there is no electron density
for residues 100–120 in the structures of human HGPRT in
complex with compounds 25 and 26 (Table S2) indicating that,
when these two ANPs bind, the loop is mobile and not fixed in
any one position. It is deduced, however, that like the PEEG
and PEEHx complexes, this loop does not play a direct role in
the binding of either of the two new ANPs. However, it cannot
be negated that the positioning of this loop may influence the
movement of other flexible loops that surround the active site
of the enzyme(s). The influence of the CH2OH group and its in-
teractions with magnesium and water ions and the resultant
location of the carbonyl, amide and side chain of K68 in this
complex.
Interactions with Mg2+ and water molecules
The effect of a water molecule found within hydrogen
bonding distance to the N3 atom of the purine ring
There is one magnesium ion present in the structure of human
HGPRT in complex with compounds 25 and 26. This ion
adopts regular near octahedral geometry and is coordinated to
the side chains of the two acidic amino acid residues, E133
and D134, and four waters. These two amino acids are com-
pletely conserved in all the 6-oxopurine PRTases known to
date suggesting that their role is of high importance in the
The positioning of a water molecule around the N3 atom of
the purine ring differs between the structures in complex with
compounds 25 and 26. As shown in Figure 3, there is a bridg-
ing water molecule between the N3 atom of the purine ring
and the carbonyl oxygen of D193 in the complex with com-
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binding of substrates/products of the catalytic reaction
(Figure 3). Crystal structures of both the human and Pf en-
zymes suggest that a magnesium ion can enter this region of
the active site by two ways: 1) coordinated to the hydroxy
groups of the ribose ring of PRib-PP; or 2) entering independ-
ently to assist in preparing the structure of the active site to
accept substrates or inhibitors.[29] For compounds 25 and 26,
only the latter can apply. It has been purported that when
magnesium is found in this area of the active site, the side
chain of K68 (K77 in Pf) rotates by 1808 where it can then set
up communication with the adjacent subunit of the tetrameric
structure (the carbonyl of V96 in human HGPRT and the car-
bonyl of E108 in PfHGXPRT).
the acyclic linker as it is only 3–3.3 Š away. This interaction is
not as definitive in the structure with compound 26. In subu-
nits A and C, it is ~3.8 Š away from the oxygen in the linker
though, in subunit D, it forms a strong interaction (2.5 Š).
There are no magnesium ions in the PEE structures and the
bonding pattern in the HPEPHx with magnesium could be part
of the reason that the Ki for compound 25 is lower for human
HGPRT compared with PEEHx (cf. 0.5 with 3.6 um). It is specu-
lated that the role of the divalent metal ion could be different
in PfHGXPRT accounting, in some part, for the differences in af-
finity for these ANPs.
Communication between subunits: the loop containing K68
(residues 67–72)
A second magnesium ion has been found in the active site
of other structures of the human and Pf structures in complex
with inhibitors but this only occurs in two instances: 1) when it
is coordinated to PPi either as it is entering or leaving the
active site;[28,30] and 2) in the case of some ANP inhibitors
When compound 26 binds in the active site, the side chain of
K68 does not form any interactions with amino acid residues
either within its own subunit or with those in an adjacent sub-
unit. This is because the side chain in this complex is flexible
(i.e., there is no electron density) and is therefore modeled in
as far as the Cb atom. This is not the case for compound 25
where there is strong density for this side chain and it forms
hydrogen bonds not only within its own subunit (the OE1
atom of E196) but also with the carbonyl oxygen of V96, thus
assisting in stabilizing the two dimers which constitute the bio-
logically active tetramer. The structure of this mobile loop is
thus different in these two structures (Figure 4 and 5). The con-
tribution to the dimeric interface for compound 25 is similar to
that found in the complex of PfHGXPRT with ImmHP and
when
a second phosphonate group (mimicking PPi) is
bound.[14d–f] Therefore, a second divalent metal ion is not
found in the complex with 25 or 26 as there are no phospho-
ryl oxygen atoms present.
The magnesium ion that is coordinated to the side chains of
E133 and D134 does have a role in binding of compounds 25
and 26 though this is slightly different in both complexes
(Figure 3). This is either by direct coordination to the inhibitor
or via bridging water molecules to the inhibitor. Two water
molecules are found in the vicinity of the hydroxy group of
the inhibitor and the magnesium ion coordinated to the side
chains of E133 and D134. For the complex with compound 25,
one of these water molecules forms a hydrogen bond with the
carbonyl of G69 (subunit A) or a hydrogen bond with D134
(subunit C) and in two subunits (B and D) with the carbonyl of
K68. This network helps to keep the flexible loop that contains
K68 close to the active site (167–172). K68 is located at the top
of this loop that connects a b-sheet (residues 61–65) to an a-
helix (residues 70–86). In contrast, such a bonding pattern
does not exist in the complex with compound 26 and this
water molecule does not partake in any other interactions at
the active site. In the complex with compound 25, the ar-
rangement is slightly different due to the fact that the hydroxy
group of the inhibitor is in a slightly different position. In this
case, it still forms a hydrogen bond with a water molecule co-
ordinated to magnesium (subunits A and B) but, in subunits C
and D, it has moved closer to magnesium (~3 Š distant). As
a result, there is insufficient room to accommodate a water
molecule in this location. One of the biggest differences be-
tween the two structures is the positioning of the flexible loop
containing K68. In the complex with compound 25, it has
moved further away from the active site and too far for the
oxygen of the carbonyl group to form a hydrogen bond to the
water molecule which is coordinated to magnesium and also
forms a hydrogen bond to the hydroxy group of the inhibitor.
The second water molecule performs an interesting function
though it appears to be absent in one of the subunits in com-
plex 26 (subunit B) and in complex 25 (subunit D). In the latter
structure, this second water interacts with the oxygen atom in
PPi·Mg2+ [30]
The side chain of K77 in PfHGXPRT forms a hydro-
.
gen bond to the carbonyl oxygen of E108 (V96 in human
HGPRT). The side chain of E108 is then available to form a hy-
drogen bond with the side chain of R80 which is found in the
same adjacent subunit as K77.
Thus, two of the contributing factors in stabilizing the di-
meric interface differ in the structures with compounds 25 and
26. For compound 25, one of these is the mobile loop be-
tween 67–71 while, for compound 26, it appears to be the
loop containing residues 187-194.
The CPEE compounds and their methyl esters
For the CPEEHx compound (32) and its methyl ester 36, there
is a 10-fold increase in the Ki value compared with HPEPHx
(25). From the crystal structure of HPEPHx, it could be specu-
lated that this could be due to the fact that the network of hy-
drogen bonds that holds the hydroxy group in place are no
longer able to form. The Ki values when guanine is the base
are similar for all three ANPs. Thus, the positioning of the hy-
droxy group may not be as influential for these ANPs. It is un-
known if the binding of magnesium ions effects the affinity of
this group of ANPs for PfHGXPRT as the only available struc-
tures have pyrophosphate bound as well as
ligand.[28,30]
a second
Thus, a number of factors come into play when these ANPs
bind. One of the most critical appears to be the movement of
the flexible loops surrounding the active site. At this stage, it
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Bruker Avance II500 (1H at 500 MHz and 13C at 126 MHz) spectrom-
eters in CDCl3, DMSO or D2O (NaOD additive) and referenced to
TMS, 13C chloroform signal (d 77.0) or dioxane used as internal
standard (d=3.75 and d=67.19). The numbering system for as-
signment of NMR signals is outlined in Figure S1 (Supporting Infor-
cannot be determined if these occur in synchrony or consecu-
tively or what is the trigger(s), that prompts these movements.
What is known from the crystal structures is that these move-
ments do occur. Further structures of the enzymes and, in par-
ticular, that of PfHGXPRT should assist in explaining this inter-
esting phenomenon.
1
mation). H and 13C NMR spectra of the corresponding (S)-isomers,
as well as racemates, are identical and are given for one isomer
only. Mass spectra were measured on a ZABEQ (VG Analytical)
spectrometer using FAB (ionization by Xe, accelerating voltage
8 kV, glycerol matrix) or by ESI technique. Optical rotations were
measured on an AUTOPOL IV polarimeter (Rudolph research ana-
lytical) at 208C; [a]D values are given [10À1 degcm2 gÀ1] and con-
centrations c are [g/100 mL]. Microwave experiments were carried
out in 10 mL or 80 mL vial in CEM Discover (Explorer) microwave
apparatus operated at a frequency of 2.45 GHz with continuous ir-
radiation power from 0 to 300 W. Chemicals were purchased from
Sigma–Aldrich (Prague, Czech Republic). Diethyl vinylphosphonate
was purchased from Epsilon Chemie (France). Solvents were dried
by standard procedures and stored over molecular sieves (4 Š). l-
Phenylalanine ethyl ester hydrochloride was dried in vacuo at 308C
and 0.1 mbar for 1 day. (S)- and (R)-tritylglycidole (1a and 1b, re-
spectively) were purchased from DAISO Co. Ltd., Japan. Chromato-
graphic system H1: ethyl acetate/acetone/ethanol/water=15:3:4:3.
Conclusions
A novel series of acyclic nucleoside phosphonates (ANPs) con-
taining either (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP)
or (S)-2-(phosphonomethoxy)propanoic acid (CPME) or (S)-2-
(phosphonoethoxy)propanoic acid (CPEE) moieties have been
synthesized. For the HPEP compounds, the nature of the
purine base has a strong influence on the Ki values as only
ANPs containing bases that are good substrates for either
enzyme, that is, guanine and hypoxanthine, exhibit low Ki
values. The chemical nature of the acyclic linker also contrib-
utes to the affinity of these inhibitors as shown for the three
different acyclic phosphonate moieties, HPEP, CPEE and its
methyl ester. None of the CPME derivatives exhibited inhibition
of either enzyme at the concentrations used in the assay (Ki >
30 mm) indicating again the importance of the nature and
length of the acylic phosphonate moiety in conferring affinity.
Crystal structures of HPEPG and HPEPHx demonstrate the mo-
bility of a number of loops surrounding the active site. The
flexibility of these loops which contain amino acid residues di-
rectly involved in the binding of the ANPs is a factor which has
to be considered in effective selective and potent drug design.
Thus, to accomplish inhibition with sub-nanomolar Ki values,
one goal should be to “lock” these flexible loops firmly in
place to achieve more interactions with the inhibitors. The im-
portance of water molecules at key locations in the active site
cannot be overlooked. The ability of these inhibitors to maxi-
mize subunit interactions at the interface of each pair of
dimers may also contribute to stabilizing the structure of en-
zyme(s) and could be another factor leading to tight binding
inhibitors. Finally, phosphoramidate prodrugs of HPEPG,
HPEPHx, CPEEG and CPPEHx were synthesized and tested for
their ability to arrest the growth of Pf lines in cell culture. This
data shows that the chemical nature of the prodrug has a sig-
nificant effect on the IC50 values and highlights the importance
of further prodrug design in improving their biological activity.
Procedures
Purification of the phosphonic acids (procedure A): Crude phos-
phonic acid was dissolved in water and purified by preparative
HPLC. Product was eluted by linear gradient from water to 50%
methanol in water. UV absorbing fractions containing product
were collected and evaporated in vacuo. Compounds were crystal-
lized from the water/ethanol mixture.
Hydrolysis of the phosphonate diester to corresponding phos-
phonic acids (procedure B): The appropriate diester (0.5 mmol)
was dissolved in acetonitrile (10 mL) and bromotrimethylsilane
(1 mL) was added dropwise. The mixture was stirred overnight and
evaporated to dryness. Residue was co-distilled with acetonitrile
(3”20 mL) and water (2”10 mL). Crude product was crystallized
from the ethanol/water mixture or further purified by procedure A.
Basic hydrolysis of the 6-chloropurine derivatives to corre-
sponding 6-oxopurine phosphonates (procedure C): The 6-chlor-
opurine derivative (1 mmol) was dissolved in mixture of dioxane
and water (5:1) (120 mL), DABCO (0.8 mmol) and potassium car-
bonate (1 mmol) were added. The reaction mixture was heated at
908C for 3–5 h. After cooling down, volatiles were evaporated and
a crude residue was purified by silica gel chromatography to give
the corresponding products.
Acidic hydrolysis of the 6-chloropurine derivatives to corre-
sponding 6-oxopurine phosphonates (procedure D): The 6-chlor-
opurine derivative (1 mmol) was dissolved in trifluoroacetic acid
(75%, 15 mL) and the mixture was stirred overnight. Volatile mate-
rials were evaporated in vacuo, the residue co-distilled with water
(3 times) and neutralized with ammonia (1:10 with water) to pH 6–
7. Volatiles were evaporated and a crude residue was purified by
silica gel chromatography (gradient from 1–12% methanol in
chloroform) to give the corresponding products.
Experimental Section
General
Unless otherwise stated, solvents were evaporated at 408C/2 kPa,
and compounds were dried at 2 kPa over P2O5. TLC was performed
on TLC aluminum sheets (silica gel 60 F254, Merck), chromatograph-
ic systems are described in the text. Column chromatography was
performed on silica gel 230–400 mesh, 60 Š (Merck). Reverse phase
HPLC separation were performed on a Waters Delta 600 instrument
with a Waters 486 Tunable Absorbance Detector using column
Phenomenex Gemini C18 (10 mm, 250”21.2 mm, flow 10 mLminÀ1
preparative column). 1H and 13C NMR spectra were measured on
a Bruker Avance II600 (1H at 600 MHz and 13C at 151 MHz) and/or
Synthesis of the 6-chloropurine derivatives from phosphonate
alcohols under Mitsunobu conditions (procedure E): A solution
of DIAD (2.95 mL, 15 mmol) in dioxane (50 mL) was added drop-
wise under argon atmosphere to a mixture of alcohol (7, 9b, 12)
(10 mmol), 6-chloropurine (2.0 g, 13 mmol), and Ph3P (3.41 g,
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13 mmol) in dioxane (150 mL) and the reaction mixture was stirred
at RT for 24 h. When the reaction was finished, the solvent was re-
moved in vacuo and the residue was purified by silica gel chroma-
tography (gradient from 20–100% ethyl acetate in iso-hexanes and
0-6% methanol in ethyl acetate).
J
gem =13.7 Hz, J4bÀP =9.2 Hz, 1H, H-4b), 3.82 (m, 2H, H-3), 1.31–
1.34 ppm (m, 12H, CH3-iPr); 13C NMR (MeOD): d=172.73 (C1),
139.35 (C1’), 129.32 (C3’), 128.86 (C2’), 128.70 (C4’), 81.55 (d,
J
CÀOÀCÀP =12.7 Hz, C2), 74.36 (C5), 73.33–73.46 (m, CH-iPr), 71.52
(C3), 65.40 (d, JCÀP =167.5 Hz, C4), 24.23–24.38 ppm (m, CH3-iPr);
MS-ESI+ m/z (%): 375 (10) [M+H]+, 397 (100) [M+Na]+) 419 (40)
[M+2Na]+; HRMS-ESI+: m/z calcd for C17H27O7NaP [M+Na]+
397.1387, found 397.1388; Anal. calcd for C17H27O7P: C 54.54, H
7.27, P 8.27, found: C 54.37, H 7.32, P 8.09; [a]2D0 =À14.2 (c=
0.435 g/100 mL, CHCl3).
Synthesis of the 2-amino-6-chloropurine derivatives from phos-
phonate alcohols under the Mitsunobu conditions (procedure F):
The procedure is identical with the procedure E, but 2-amino-6-
chloropurine was used instead of 6-chloropurine. When the reac-
tion was finished, water was added (50 mL) into reaction mixture
and stirred at 808C for 6 h (to remove triphenylphosphine ad-
ducts). The mixture was evaporated and the residue was purified
by silica gel chromatography (gradient from 20–100% ethyl ace-
tate in iso-hexanes and 0–12% methanol in ethyl acetate).
Methyl (S)-3-(benzyloxy)-2-((diisopropoxyphosphoryl)methoxy)-
propanoate (6): Compound 5 (11.2 g, 30 mmol) was dissolved in
ethyl acetate (150 mL) and solution of diazomethane (1m CH2N2 in
dry Et2O) was added dropwise until the solution wasn’t yellowish.
The reaction was stirred at RT for 0.5 h. Excess of diazomethane
was decomposed by adding of several drops of ethanol. The solu-
tion was concentrated in vacuo and a crude product was purified
by silica gel chromatography (gradient from 40–60% ethyl acetate
in iso-hexanes) to give (after evaporation) the title compound as
Hydrogenolysis of benzyl group (procedure G): The correspond-
ing derivative (2 mmol) was dissolved in glacial acetic acid
(120 mL) and a flask was purged with argon and evacuated
(3 times). After that catalytic amount of 10% Palladium on carbon
under argon atmosphere was added. Then the flask was evacuated
and purged with hydrogen (3 times) and the mixture was vigorous-
ly stirred at RT until the reaction was complete (~3 days). The reac-
tion mixture was filtered through short silica gel column and the
filter pad was washed with acetic acid (50 mL) and methanol
(50 mL). The filtrate was evaporated and a crude product was puri-
fied by silica gel chromatography (gradient from 2–12% methanol
in chloroform) to afford the corresponding products.
1
a colorless oil (11.4 g, yield 98%). H NMR (CDCl3): d=7.26–7.36 (m,
5H, H-2’, 3’, 4’), 4.72–7.79 (m, 2H, CH-iPr), 4.61 (d, Jgem =12.1 Hz,
1H, H-5a), 4.55 (d, Jgem =12.1 Hz, 1H, H-5b), 4.38 (t, J2À3 =4.3 Hz,
1H, H-2), 4.06 (dd, Jgem =13.9 Hz, J4aÀP =8.7 Hz, 1H, H-4a), 3.76–3.80
(m, 3H, H-3, 4b), 3.76 (s, 3H, O-CH3), 1.31–1.34 ppm (m, 12H, CH3-
iPr); 13C NMR (CDCl3): d=170.19 (C1), 137.72 (C1’), 128.31 (C3’),
127.70 (C2’), 127.66 (C4’), 80.00 (d, JCÀOÀCÀP =9.6 Hz, C2), 73.40 (C5),
71.41 (d, JCÀOÀP =6.5 Hz, CH-iPr), 71.20 (d, JCÀOÀP =6.5 Hz, CH-iPr),
70.21 (C3), 64.93 (d, JCÀP =166.4 Hz, C4), 52.08 (O-CH3), 23.89–
24.10 ppm (m, CH3-iPr); MS-ESI+ m/z (%): 389 (100) [M+H]+, 411
(95) [M+Na]+; HRMS-ESI+: m/z calcd for C18H30O7P [M+H]+
389.1535, found 389.1536; [a]2D0 =À20.3 (c=0.370 g/100 mL,
CHCl3).
Synthesis and compound characterization
(S)- and (R)-1-(Benzyloxy)-3-trityloxypropan-2-ol (2a and 2b):
The compounds 2a and 2b were prepared according to a previous-
ly described procedure[31] in 95 and 97% yields, respectively.
Methyl (S)-2-((diisopropoxyphosphoryl)methoxy)-3-hydroxypro-
panoate (7): Compound 6 (9.7 g, 25 mmol) from the previous reac-
tion step was dissolved in methanol (250 mL) and a flask was
purged with argon and evacuated (3-times). After that catalytic
amount of 10% palladium on carbon under argon atmosphere
was added. Then the flask was evacuated and purged with hydro-
gen (3-times) and the mixture was vigorously stirred at RT until the
reaction was complete (~3 days). The reaction mixture was filtered
through short silica gel column and the filter pad was washed with
methanol (100 mL). The filtrate was evaporated and a crude prod-
uct was purified by silica gel chromatography (gradient from 60-
80% ethyl acetate in iso-hexanes) to afford (after evaporation) the
Diisopropyl (S)-(((1-(benzyloxy)-3-(trityloxy)propan-2-yl)oxy)me-
thyl)phosphonate (3): Compound 3 was prepared according to
a previously described procedure[31] and was used in the next reac-
tion step without further purification.
Diisopropyl
(R)-(((1-benzyloxy)-3-hydroxypropan-2-yl)oxy)me-
thyl)phosphonate (4): Compound 4 was prepared according to
a previously described procedure[31] in a 40% yield (from 2a).
(S)-3-(Benzyloxy)-2-((diisopropoxyphosphoryl)methoxy)propano-
ic acid (5): To a solution of compound 4 (14.4 g, 40 mmol), TEMPO
(625 mg, 4 mmol), sodium chlorite (80%) (9.0 g, 80 mmol), acetoni-
trile (200 mL) and 0.67m sodium phosphate buffer (160 mL,
pH 6.7) was added dropwise a solution of dilute sodium hypochlor-
ite (40 mL of household bleach SAVO (40.72 mgmLÀ1) with 40 mL
of water). The reaction mixture was heated at 408C for 48 h. After
cooling the reaction to RT, water (300 mL) was added and the pH
was adjusted to 8.0 by addition 48 mL of 2m NaOH. Then the reac-
tion mixture was cooled to 08C (ice bath) and solution of sodium
sulfite (12 g Na2SO3 in 200 mL H2O) was added. After stirring at RT
for 0.5 h, the solution was acidified with 2m HCl to pH 3–4 and ex-
tracted with ethyl acetate (3”300 mL). The combined organic
phases were washed with water (2”200 mL), brine (2”200 mL),
dried over MgSO4 overnight, and then concentrated to give (after
evaporation) the crude product as a yellowish oil. This was subject-
ed to silica gel chromatography (gradient from 0–10% methanol in
ethyl acetate) to afford (after evaporation) compound 5 as a color-
less oil which solidified (13.2 g, yield 88%). 1H NMR (MeOD): d=
7.25–7.35 (m, 5H, H-2’, 3’, 4’), 4.70–4.79 (m, 2H, CH-iPr), 4.60 (d,
1
title compound as a colorless oil (7.1 g, yield 95%). H NMR (CDCl3):
d=4.72–4.82 (m, 2H, CH-iPr), 4.23 (dd, J2À3b =6.4 Hz, J2À3a =3.3 Hz,
1H, H-2), 4.11 (dd, Jgem =14.0 Hz, J4aÀP =8.2 Hz, 1H, H-4a), 3.93 (dd,
J
J
gem =12.2 Hz, J3aÀ2 =3.3 Hz, 1H, H-3a), 3.85 (dd, Jgem =12.2 Hz,
3bÀ2 =6.4 Hz, 1H, H-3b), 3.77 (s, 3H, O-CH3), 3.77 (dd, Jgem
=
14.0 Hz, J4bÀP =7.7 Hz, 1H, H-4b), 3.22 (bs, 1H, OH), 1.31–1.37 ppm
(m, 12H, CH3-iPr); 13C NMR (CDCl3): d=170.12 (C1), 81.54 (d,
J
J
CÀOÀCÀP =8.7 Hz, C2), 71.69 (d, JCÀOÀP =6.7 Hz, CH-iPr), 71.42 (d,
CÀOÀP =6.7 Hz, CH-iPr), 64.97 (d, JCÀP =167.8 Hz, C4), 62.84 (C3),
52.08 (O-CH3), 23.85–24.05 ppm (m, CH3-iPr); MS-ESI+ m/z (%): 299
(5) [M+H]+, 321 (100) [M+Na]+; HRMS-ESI+: m/z calcd for
C11H24O7P [M+H]+ 299.1254, found 299.1255; [a]2D0 =À23.3 (c=
0.510 g/100 mL, CHCl3).
Diethyl (S)-(2-((1-benzyloxy)-3-(trityloxy)propan-2-yl)oxy)ethyl)-
phosphonate (8a): A mixture of compound 2a (17 g, 40 mmol)
and catalytic amount of KOH (560 mg, 10 mmol) in dry dioxane
(200 mL) was stirred at RT for 15 min. Diethyl vinylphosphonate
(8 mL, 52 mmol) was added dropwise and the reaction was vigo-
J
gem =12.0 Hz, 1H, H-5a), 4.55 (d, Jgem =12.0 Hz, 1H, H-5b), 4.30 (m,
1H, H-2), 4.11 (dd, Jgem =13.7 Hz, J4aÀP =8.8 Hz, 1H, H-4a), 3.84 (dd,
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Full Papers
rously stirred at RT for 70 h. The mixture was poured into water
and extracted with ethyl acetate (3”300 mL). The combined or-
ganic phases were washed with brine (2”200 mL), dried over
MgSO4 and concentrated in vacuo. The crude mixture was subject-
ed to silica gel chromatography (gradient from 20–70% ethyl ace-
tate in iso-hexanes with 0.1% Et3N) to give (after evaporation) the
crude product as a yellowish oil (13 g, yield 55% of the crude
product, used in the next step without further purification). MS-
ESI+ m/z (%): 611 (100) [M+Na]+; HRMS-ESI+: m/z calcd for
C35H41O6NaP [M+Na]+ 611.2533, found 611.2530.
Methyl
(S)-2-(2-(diethoxyphosphoryl)ethoxy)-3-hydroxypropa-
noate (12): Compound 11 (3.56 g, 9.5 mmol) was dissolved in the
mixture of methanol and acetic acid (3:1, 120 mL) and treated anal-
ogously compound
methanol in chloroform) to give the title compound as a colorless
oil (2.5 g, yield 93%). H NMR (CDCl3): d=4.04–4.19 (m, 6H, H-2, 4a,
P-O-CH2-CH3), 3.90 (dd, Jgem =12.0 Hz, J1aÀ2 =3.2 Hz, 1H, H-1a),
3.69–3.80 (m, 5H, H-1b, 4b, O-CH3), 2.05–2.24 (m, 2H, H-5), 1.34 (t,
6 (chromatography: gradient from 0–5%
1
J
CH3ÀCH2 =7.1 Hz, 3H, P-O-CH2-CH3), 1.33 ppm (t, JCH3ÀCH2 =7.1 Hz,
3H, P-O-CH2-CH3); 13C NMR (CDCl3): d=170.63 (C3), 80.49 (C2),
64.79 (d, JCÀCÀP =5.9 Hz, C4), 62.76 (C1), 62.09 (d, JCÀOÀP =6.5 Hz, P-
O-CH2-CH3), 61.73 (d, JCÀOÀP =6.5 Hz, P-O-CH2-CH3), 52.08 (O-CH3),
26.73 (d, JCÀP =142.1 Hz, C5), 16.35 ppm (d, JCÀCÀOÀP =6.2 Hz, P-O-
CH2-CH3); MS-ESI+ m/z (%): 285 (5) [M+H]+, 307 (100) [M+Na]+;
HRMS-ESI+: m/z calcd for C10H21O7NaP [M+Na]+ 307.0917, found
307.0916; Anal. calcd for C10H21O7P H2O: C 39.74, H 7.67, P 10.25,
found: C 39.91, H 7.69, P 9.98; [a]2D0 =À41.7 (c=0.281 g/100 mL,
CHCl3).
Diethyl (R)-(2-((1-(benzyloxy)-3-hydroxypropan-2-yl)oxy)ethyl)-
phosphonate (9a): Compound 8a (13 g of the crude product) was
dissolved in 80% acetic acid (150 mL) and heated to reflux for 2 h.
After cooling down, the reaction mixture was diluted with 100 mL
of water/dioxane (1:1) and white crystals of trityl alcohol were fil-
tered off. The solution was extracted with ethyl acetate (3”
150 mL), the combined organics were washed with water (2”
50 mL), brine (2”50 mL), dried over MgSO4 and concentrated in
vacuo to give a crude oil. This was subjected to silica gel chroma-
tography (gradient from 40–100% ethyl acetate in iso-hexanes and
0–5% methanol in ethyl acetate) to afford (after evaporation) the
title compound as a colorless oil (5.1 g, 37% overall yield from 2a).
1H NMR (CDCl3): d=7.27–7.35 (m, 5H, H-2’, 3’, 4’), 4.53 (s, 2H, H-6),
4.05–4.16 (m, 4H, P-O-CH2-CH3), 3.98 (m, 1H, H-4a), 3.81 (m, 1H, H-
4b), 3.72 (dd, Jgem =11.7 Hz, J3aÀ2 =2.8 Hz, H-3a), 3.62 (m, 1H, H-2),
Diethyl (R)-(2-((1-benzyloxy)-3-(trityloxy)propan-2-yl)oxy)ethyl)-
phosphonate (8b): Compound 2b (42.5 g, 100 mmol) was placed
into 2 reaction flasks and treated analogously compound 2a to
give the title compound as a yellowish oil (33 g, yield 56% of the
crude product, used in the next step without further purification).
MS spectrum is identical to that of compound 8a.
Diethyl (S)-(2-((1-(benzyloxy)-3-hydroxypropan-2-yl)oxy)ethyl)-
phosphonate (9b): Compound 8b (33 g) was treated analogously
compound 8a to give the title compound as a colorless oil (13.5 g,
3.49–3.59 (m, 3H, H-1, 3b), 2.02–2.16 (m, 2H, H-5), 1.32 (t, JCH3ÀCH2
=
7.1 Hz, 3H, P-O-CH2-CH3), 1.31 ppm (t, JCH3ÀCH2 =7.1 Hz, 3H, P-O-
CH2-CH3); 13C NMR (CDCl3): d=137.98 (C1’), 128.34 (C3’), 127.62
(C4’), 127.54 (C2’), 80.02 (C2), 73.40 (C6), 70.19 (C1), 63.93 (d,
39% overall yield from 2b). H NMR, 13C NMR and MS spectra are
1
identical to those of compound 9a; [a]2D0 =À7.4 (c=0.298 g/
J
CÀCÀP =5.9 Hz, C4), 62.17 (C3), 61.87 (d, JCÀOÀP =6.4 Hz, P-O-CH2-
100 mL, CHCl3).
CH3), 61.57 (d, CÀOÀP =6.5 Hz, P-O-CH2-CH3), 27.08 (d, JCÀP
J
=
141.5 Hz, C5), 16.32 ppm (d, JCÀCÀOÀP =6.2 Hz, P-O-CH2-CH3); MS-
ESI+ m/z (%): 347 (20) [M+H]+, 369 (100) [M+Na]+; HRMS-ESI+:
m/z calcd for C16H28O6P [M+H]+ 347.1618, found 347.1617; Anal.
calcd for C16H28O6P H2O: C 52.74, H 8.02, P 8.50, found: C 52.95, H
7.98, P 8.40; [a]2D0 = +8.3 (c=0.289 g/100 mL, CHCl3).
Methyl
3-(6-chloropurin-9-yl)-2-((diisopropoxyphosphoryl)me-
thoxy)propanoate (13): From alcohol 7 (1.19 g, 4 mmol) and 6-
chloropurine (804 mg, 5.2 mmol), applied procedure E, obtained
the title compound as a yellowish oil (835 mg, yield 48%). H NMR
(CDCl3): d=8.75 (s, 1H, H-2), 8.28 (s, 1H, H-8), 4.75 (dd, Jgem
1
=
14.2 Hz, J1’aÀ2’ =3.3 Hz, 1H, H-1’a), 4.62–4.72 (m, 2H, CH-iPr), 4.61
(S)-3-(Benzyloxy)-2-(2-(diethoxyphosphoryl)ethoxy)propanoic
acid (10): Compound 9a (4.2 g, 12 mmol) was treated analogously
compound 4 (chromatography: gradient from 0-3% methanol in
chloroform) to give the title compound as a colorless oil (3.6 g,
(dd, Jgem =14.2 Hz, J1’bÀ2’ =6.8 Hz, 1H, H-1’b), 4.57 (dd, J2’À1’b
6.8 Hz, J2’À1’a =3.3 Hz, 1H, H-2’), 4.06 (dd, Jgem =14.0 Hz, J4’aÀP
=
=
8.8 Hz, 1H, H-4’a), 3.77 (s, 3H, O-CH3), 3.67 (dd, Jgem =14.0 Hz,
J
4’bÀP =7.9 Hz, 1H, H-4’b), 1.31 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.30
1
yield 83%). H NMR (CDCl3): d=7.27–7.36 (m, 5H, H-2’, 3’, 4’), 4.57
(d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.26 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-
iPr), 1.23 ppm (d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr); 13C NMR (CDCl3): d=
168.81 (COOH), 151.97 (C2), 151.82 (C4), 151.06 (C6), 146.34 (C8),
(s, 2H, H-6), 4.08–4.18 (m, 5H, H-2, P-O-CH2-CH3), 3.99 (m, 1H, H-
4a), 3.91 (dd, Jgem =10.5 Hz, J1aÀ2 =2.7 Hz, 1H, H-1a), 3.85 (m, 1H,
H-4b), 3.78 (dd, Jgem =10.5 Hz, J1bÀ2 =6.6 Hz, 1H, H-1b), 2.25 (m,
1H, H-5a), 2.10 (m, 1H, H-5b), 1.33 (t, JCH3ÀCH2 =7.0 Hz, 3H, P-O-CH2-
CH3), 1.29 ppm (t, JCH3ÀCH2 =7.0 Hz, 3H, P-O-CH2-CH3); 13C NMR
(CDCl3): d=171.39 (C3), 137.64 (C1’), 128.30 (C3’), 127.65 (C4’),
131.26 (C5), 77.50 (d, JCÀOÀCÀP =9.5 Hz, C2’), 71.46 (d, JCÀOÀP
=
6.8 Hz, CH-iPr), 65.20 (d, JCÀP =166.7 Hz, C4’), 52.73 (O-CH3), 45.31
(C1’), 23.87–24.00 ppm (m, CH3-iPr); MS-ESI+ m/z (%): 435 (40) [M+
H]+, 457 (100) [M+Na]+; HRMS-ESI+: m/z calcd for C16H25O6N4ClP
[M+H]+ 435.1195, found 435.1194; FTIR (CHCl3): n˜ =3126, 3002,
127.55 (C2’), 79.29 (C2), 73.45 (C6), 70.90 (C1), 65.42 (d, JCÀCÀP
6.4 Hz, C4), 62.50 (d, CÀOÀP =6.3 Hz, P-O-CH2-CH3), 62.00 (d,
CÀOÀP =6.7 Hz, P-O-CH2-CH3), 26.51 (d, CÀP =141.7 Hz, C5),
=
J
1754, 1594, 1567, 1499, 1438, 1339, 1233, 1145, 998 cmÀ1; [a]D20
À7.8 (c=0.275 g/100 mL, DMSO).
=
J
J
16.23 ppm (d, JCÀCÀOÀP =5.9 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%):
361 (45) [M+H]+, 383 (100) [M+Na]+; HRMS-ESI+: m/z calcd for
C16H26O7P [M+H]+] 361.1411, found 361.1409; Anal. calcd for
C16H26O7P 0.3H2O: C 52.54, H 7.06, P 8.01, found: C 52.68, H 7.08, P
7.83; [a]2D0 =À23.4 (c=0.295 g/100 mL, CHCl3).
Methyl
3-(2-amino-6-chloropurin-9-yl)-2-((diisopropoxyphos-
phoryl)methoxy) propanoate (14): Compound 7 (1.19 g, 4 mmol)
and 2-amino-6-chloropurine (882 mg, 5.2 mmol), applied procedure
F to give the title compound as a yellowish oil which solidified
1
(722 mg, yield 40%). H NMR ([D6]DMSO): d=7.99 (s, 1H, H-8), 6.93
Methyl (S)-3-(benzyloxy)-2-(2-(diethoxyphosphoryl)ethoxy)pro-
panoate (11): Compound 10 (3.6 g, 10 mmol) was treated analo-
gously compound 5 to give the title compound as a colorless oil
(3.7 g, yield 99%). MS-ESI+ m/z (%): 375 (15) [M+H]+, 397 (100)
[M+Na]+; HRMS-ESI+: m/z calcd for C17H27O7NaP [M+Na]+
397.1387, found 397.1386.
(bs, 2H, NH2), 4.56 (dd, J2’À1’b =7.9 Hz, J2’À1’a =3.6 Hz, 1H, H-2’),
4.40–4.52 (m, 3H, H-1’a, CH-iPr), 4.33 (dd, Jgem =14.7 Hz, J1’bÀ2’
=
7.9 Hz, 1H, H-1’b), 3.91 (dd, Jgem =13.8 Hz, J4’aÀP =8.7 Hz, 1H, H-4’a),
3.76 (dd, Jgem =13.8 Hz, J4’bÀP =9.3 Hz, 1H, H-4’b), 3.69 (s, 3H, O-
CH3), 1.18 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.15 (d, JCH3ÀCH =6.2 Hz,
3H, CH3-iPr), 1.14 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.09 ppm (d,
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Full Papers
J
CH3ÀCH =6.2 Hz, 3H, CH3-iPr); 13C NMR ([D6]DMSO): d=169.42 (C3’),
160.04 (C2), 154.32 (C4), 149.49 (C6), 143.77 (C8), 123.15 (C5), 77.56
(d, JCÀOÀCÀP =12.4 Hz, C2’), 70.57 (m, CH-iPr), 64.21 (d, JCÀP
Na]+, 376 (15) [M+2Na]+; HRMS-ESIÀ: m/z calcd for C9H11O7N5P
[MÀH]+ 332.0401, found 332.0404; FTIR (KBr): n˜ =1693, 1607, 1540
1481, 1409, 1376, 1176, 1108, 1069, 914 cmÀ1; [a]D20 =À20.2 (c=
0.316 g/100 mL, H2O).
=
164.2 Hz, C4’), 52.52 (O-CH3), 44.29 (C1’), 23.66–23.91 ppm (m, CH3-
iPr); MS-ESI+ m/z (%): 450 (40) [M+H]+, 472 (100) [M+Na]+;
HRMS-ESI+: m/z calcd for C16H26O6N5ClP [M+H]+ 450.1304, found
450.1308; FTIR (CHCl3): n˜ =3532, 3424, 3205, 2987, 2958, 2939,
1753, 1612, 1568, 1438, 1388, 1234, 1180, 1104 cmÀ1; [a]D20 =À10.5
(c=0.357 g/100 mL, DMSO).
Diethyl
(S)-(2-((1-(benzyloxy)-3-(6-chloropurin-9-yl)propan-2-
yl)oxy)ethyl)phosphonate (19): From alcohol 9b (3.46 g, 10 mmol)
and 6-chloropurine (2.0 g, 13 mmol), applied procedure E, obtained
1
the title compound as a yellowish oil (3.85 g, yield 80%). H NMR
([D6]DMSO): d=8.76 (s, 1H, H-2), 8.66 (s, 1H, H-8), 7.26–7.35 (m,
5H, H-2’’, 3’’, 4’’), 4.38–4.52 (m, 4H, H-1’, 4’), 3.99 (m, 1H, H-2’),
3.87–3.93 (m, 4H, P-O-CH2-CH3), 3.68 (m, 1H, H-5’a), 3.53 (m, 1H,
H-5’b), 3.50 (d, J3’À2’ =5.0 Hz, 2H, H-3’), 1.89 (dt, J6’À5’ =7.4 Hz,
(S)-2-((Diisopropoxyphosphoryl)methoxy)-3-(hypoxanthin-9-yl)-
propanoic acid (15): Treatment of compound 13 (435 mg, 1 mmol)
by procedure C (chromatography: linear gradient of H1 in ethyl
acetate) gave the title compound as a yellowish solid (237 mg,
yield 59%). H NMR ([D6]DMSO): d=8.46 (bs, 1H, NH), 8.04 (s, 1H,
H-2), 7.99 (s, 1H, H-8), 4.50 (dd, Jgem =14.4 Hz, J1’aÀ2’ =3.1 Hz, 1H, H-
J
6’ÀP =18.3 Hz, 2H, H-6’), 1.15 ppm (t, JCH3ÀCH2 =7.0 Hz, 6H, P-O-CH2-
1
CH3); 13C NMR ([D6]DMSO): d=152.42 (C4), 151.68 (C2), 149.12 (C6),
148.33 (C8), 138.22 (C1’’), 130.78 (C5), 128.42 (C3’’), 127.72 (C2’’),
127.71 (C4’’), 76.12 (C2’), 72.63 (C4’), 69.46 (C3’), 63.99 (C5’), 61.15
1’a), 4.36–4.50 (m, 2H, CH-iPr), 4.21 (ddm, Jgem =14.4 Hz, J1’bÀ2’
=
9.2 Hz, 1H, H-1’b), 4.11 (bdd, Jgem =14.4 Hz, J4’aÀP =8.1 Hz, 1H, H-
4’a), 3.97 (dm, J2’À1’b =9.2 Hz, 1H, H-2’), 3.57 (dd, Jgem =13.7 Hz,
(d, JCÀOÀP =6.1 Hz, P-O-CH2-CH3), 44.96 (C1’), 26.30 (d, JCÀP =
136.9 Hz, C6’), 16.37 ppm (d, JCÀCÀOÀP =5.9 Hz, P-O-CH2-CH3); MS-
ESI+ m/z (%): 483 (100) [M+H]+, 505 (65) [M+Na]+; HRMS-ESI+:
m/z calcd for C21H29O5N4ClP [M+H]+ 483.1559, found 483.1558;
[a]2D0 =À11.7 (c=0.403 g/100 mL, DMSO).
J
4’bÀP =8.8 Hz, 1H, H-4’b), 1.16 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.11
(d, JCH3ÀCH =6.2 Hz, 3H, CH3-iPr), 1.10 (d, JCH3ÀCH =6.2 Hz, 3H, CH3-
iPr), 1.07 ppm (d,
CH3ÀCH =6.2 Hz, 3H, CH3-iPr); 13C NMR
J
([D6]DMSO): d=170.77 (C3’), 157.06 (C6), 148.73 (C4), 145.44 (C2),
140.72 (C8), 123.77 (C5), 81.15 (d, JCÀOÀCÀP =12.5 Hz, C2’), 70.26 (d,
Diethyl (S)-(2-((1-(2-amino-6-chloropurin-9-yl)-3-(benzyloxy)pro-
pan-2-yl)oxy)ethyl) phosphonate (20): Compound 9b (1.73 g,
5 mmol) and 2-amino-6-chloropurine (1.1 g, 6.5 mmol), applied
procedure F to give the title compound as a yellowish oil (1.4 g,
J
J
CÀOÀP =6.3 Hz, CH-iPr), 70.16 (d, JCÀOÀP =6.3 Hz, CH-iPr), 63.23 (d,
CÀP =163.6 Hz, C4’), 46.18 (C1’), 23.65–23.94 ppm (m, CH3-iPr); MS-
ESIÀ m/z (%): 401 (100) [MÀH]+; HRMS-ESIÀ: m/z calcd for
C15H22O7N4P [MÀH]+ 401.1232, found 401.1231; FTIR (KBr): n˜ =
3423, 2981, 2936, 1699, 1611, 1550, 1465, 1386, 1376, 1243, 1126,
993 cmÀ1; [a]2D0 =À14.9 (c=0.356 g/100 mL, MeOH).
1
yield 55%). H NMR ([D6]DMSO): d=8.07 (s, 1H, H-8), 7.27–7.36 (m,
5H, H-2’’, 3’’, 4’’), 6.91 (bs, 2H, NH2), 4.49 (s, 2H, H-4’), 4.22 (dd,
J
J
gem =14.5 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.12 (dd, Jgem =14.5 Hz,
1’bÀ2’ =7.5 Hz, 1H, H-1’b), 3.87–3.94 (m, 5H, H-2’, P-O-CH2-CH3),
(S)-3-(Guanin-9-yl)-2-((diisopropoxyphosphoryl)methoxy) propa-
noic acid (16):[16] Treatment of compound 14 (450 mg, 1 mmol) by
procedure C (chromatography: linear gradient of H1 in ethyl ace-
tate) gave compound 16 as a yellowish solid (234 mg, yield 56%).
1H NMR, 13C NMR and MS spectra are identical with the original
spectroscopic data.[16]
3.66 (m, 1H, H-5’a), 3.45–3.52 (m, 3H, H-3’, 5’b), 1.84–1.91 (m, 2H,
H-6’), 1.20 (t, JCH3ÀCH2 =7.1 Hz, 3H, P-O-CH2-CH3), 1.16 ppm (t,
J
CH3ÀCH2 =7.1 Hz, 3H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=159.99
(C2), 154.53 (C4), 149.48 (C6), 144.01 (C8), 138.24 (C1’’), 128.47
(C3’’), 127.77 (C2’’), 127.59 (C4’’), 123.28 (C5), 76.18 (C2’), 72.67
(C4’), 69.66 (C3’), 64.00 (C5’), 61.19 (d, JCÀOÀP =6.1 Hz, P-O-CH2-CH3),
44.36 (C1’), 26.37 (d, JCÀP =136.2 Hz, C6’), 16.39 ppm (d, JCÀCÀOÀP
=
(S)-3-(Hypoxanthin-9-yl)-2-(phosphonomethoxy)propanoic acid
(17): Treatment of compound 15 (201 mg, 0.5 mmol) by procedure
B followed by procedure A gave the title compound as a white
solid (110 mg, 69%). 1H NMR (D2O): d=8.17 (s, 2H, H-2, 8), 4.60
5.9 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%): 498 (75) [M+H]+, 520 (100)
[M+Na]+; HRMS-ESI+: m/z calcd for C21H29O5N5ClNaP [M+Na]+
520.1487, found 520.1487; Anal. calcd for C21H29O5N5ClP: C 50.66, H
5.87, N 14.07, P 6.22, Cl 7.12, found: C 50.48, H 5.99, N 14.10, P
5.97, Cl 7.03; [a]2D0 =À19.0 (c=0.415 g/100 mL, DMSO).
(dd,
J
gem =14.8 Hz,
J
1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.54 (dd, Jgem
=
=
14.8 Hz, J1’bÀ2’ =5.6 Hz, 1H, H-1’b), 4.21 (dd, J2’À1’b =5.6 Hz, J2’À1’a
4.1 Hz, 1H, H-2’), 3.72 (dd, Jgem =12.9 Hz, J4’aÀP =8.8 Hz, 1H, H-4’a),
3.48 ppm (dd, Jgem =12.9 Hz, J4’bÀP =9.9 Hz, 1H, H-4’b); 13C NMR
(D2O): d=177.04 (C3’), 159.31 (C6), 149.78 (C4), 146.15 (C2), 143.70,
Diethyl
(S)-(2-((1-benzyloxy)-3-(hypoxanthin-9-yl)propan-2-yl)-
oxy)ethyl)phosphonate (21): Treatment of compound 19 (966 mg,
2 mmol) by procedure C (chromatography: gradient from 1-5%
methanol in chloroform) gave the title compound as a yellowish
oil (761 mg, yield 82%). H NMR ([D6]DMSO): d=12.29 (s, 1H, NH),
8.04 (s, 2H, H-2, 8), 7.27–7.36 (m, 5H, H-2’’, 3’’, 4’’), 4.49 (s, 2H, H-
4’), 4.32 (dd, Jgem =14.3 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.21 (dd,
(C8), 123.47 (C5), 81.63 (d, JCÀOÀCÀP =12.4 Hz, C2’), 67.06 (d, JCÀP
=
155.2 Hz, C4’), 46.43 ppm (C1’);MS-ESIÀ m/z (%): 317 (100) [MÀH]+,
339 (25) [M+Na]+, 355 (15) [M+2Na]+; HRMS-ESIÀ: m/z calcd for
C9H10O7N4P [MÀH]+ 317.0293, found 317.0293; FTIR (KBr): n˜ =1700,
1
1586, 1552, 1480, 1414, 1349, 1289, 1152, 1059, 968, 906, 788 cmÀ1
[a]2D0 =À14.9 (c=0.470 g/100 mL, H2O).
;
Jgem =14.3 Hz, J1’bÀ2’ =7.3 Hz, 1H, H-1’b), 3.88–3.95 (m, 5H, H-2’, P-
O-CH2-CH3), 3.65 (m, 1H, H-5’a), 3.43–3.56 (m, 3H, H-3’, 5’b), 1.88
(dt, J6’ÀP =18.3 Hz, 2H, H-6’), 1.16 ppm (t, JCH3ÀCH2 =7.1 Hz, 6H, P-O-
CH2-CH3); 13C NMR ([D6]DMSO): d=156.88 (C6), 148.78 (C4), 145.68
(C2), 141.15 (C8), 138.30 (C1’’), 128.46 (C3’’), 127.75 (C2’’), 127.72
(C4’’), 123.86 (C5), 76.61 (C2’), 72.63 (C4’), 69.62 (C3’), 64.07 (C5’),
61.18 (d, JCÀOÀP =6.1 Hz, P-O-CH2-CH3), 61.17 (d, JCÀOÀP =6.1 Hz, P-
O-CH2-CH3), 44.54 (C1’), 26.35 (d, JCÀP =136.5 Hz, C6’), 16.39 ppm
(d, JCÀCÀOÀP =5.9 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%): 465 (30) [M+
H]+, 487 (100) [M+Na]+; HRMS-ESI+: m/z calcd for C21H29O6N4NaP
[M+Na]+ 487.1717, found 487.1716; Anal. calcd for C21H29O6N4P: C
54.31, H 6.29, N 12.06, P 6.67, found: C 54.11, H 6.35, N 11.89, P
6.51; [a]2D0 =À10.3 (c=0.455 g/100 mL, DMSO).
(S)-3-(Guanin-9-yl)-2-(phosphonomethoxy)propanoic acid (18):
Treatment of compound 16 (209 mg, 0.5 mmol) by procedure B fol-
lowed by procedure A gave the title compound as a white solid
1
(112 mg, yield 67%). H NMR (D2O): d=7.84 (s, 1H, H-8), 4.40 (dd,
J
J
gem =14.7 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.32 (dd, Jgem =14.7 Hz,
1’bÀ2’ =6.1 Hz, 1H, H-1’b), 4.17 (dd, J2’À1’b =6.1 Hz, J2’À1’a =4.1 Hz, 1H,
H-2’), 3.73 (dd, Jgem =13.0 Hz, J4’aÀP =8.7 Hz, 1H, H-4’a), 3.51 ppm
(dd, Jgem =13.0 Hz, J4’bÀP =9.8 Hz, 1H, H-4’b); 13C NMR (D2O): d=
177.21 (C3’), 159.57 (C6), 154.24 (C2), 152.37 (C4), 141.38 (C8), 116.08
(C5), 81.73 (d, JCÀOÀCÀP =12.5 Hz, C2’), 66.85 (d, JCÀP =156.1 Hz, C4’),
45.82 ppm (C1’); MS-ESIÀ m/z (%): 332 (100) [MÀH]+, 354 (60) [M+
ChemMedChem 2015, 10, 1707 – 1723
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Full Papers
Diethyl (S)-(2-((guanin-9-yl)-3-(benzyloxy)propan-2-yl)oxy)ethyl)-
phosphonate (22): Treatment of compound 20 (996 mg, 2 mmol)
by procedure C (chromatography: gradient from 1-7% methanol in
chloroform) gave the title compound as a yellowish solid (631 mg,
yield 66%). H NMR ([D6]DMSO): d=10.57 (s, 1H, NH), 7.63 (s, 1H,
H-8), 7.27–7.37 (m, 5H, H-2’’, 3’’, 4’’), 6.44 (s, 2H, NH2), 4.49 (s, 2H,
H-4’), 3,97–4.12 (m, 2H, H-1’), 3.84–3.95 (m, 5H, H-2’, P-O-CH2-CH3),
2H, H-3’b, 4’b), 1.60–1.76 ppm (m, 2H, H-5’); 13C NMR (D2O): d=
158.46 (C6), 149.02 (C4), 145.79 (C2), 142.75 (C8), 122.92 (C5), 78.08
(C2’), 65.90 (C4’), 60.52 (C3’), 44.68 (C1’), 28.86 ppm (d, JCÀP
=
129.9 Hz, C5’); MS-ESI+ m/z (%): 319 (100) [M+H]+, 341 (80) [M+
Na]+; HRMS-ESI+: m/z calcd for C10H16O6N4P [M+H]+ 319.0802,
found 319.0802; FTIR (KBr): n˜ =3050, 1689, 1592, 1552, 1521, 1414,
1343, 1211, 1097, 1055, 1041, 909, 789 cmÀ1; [a]D20 =À5.3 (c=
0.338 g/100 mL, H2O).
1
3.64 (m, 1H, H-5’a), 3.38–3.52 (m, 3H, H-3’, 5’b), 1.88 (dt, J6’À5’
=
7.5 Hz, J6’ÀP =18.3 Hz, 2H, H-6’), 1.17 ppm (t, JCH3ÀCH2 =7.0 Hz, 6H,
P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=157.03 (C6), 153.72 (C2),
151.56 (C4), 138.30 (C1’’), 138.27 (C8), 128.49 (C3’’), 127.80 (C2’’),
127.74 (C4’’), 116.49 (C5), 76.56 (C2’), 72.66 (C4’), 69.83 (C3’), 64.05
(S)-(2-((Guanin-9-yl)-3-hydroxypropan-2-yl)oxy)ethyl) phosphonic
acid (26): Treatment of compound 24 (195 mg, 0.5 mmol) by pro-
cedure B followed by procedure A gave the title compound as
a white solid (127 mg, yield 76%). H NMR (D2O): d=7.74 (s, 1H, H-
8), 4.19 (dd, Jgem =14.8 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.04 (dd,
1
(C5’), 61.21 (d, JCÀOÀP =6.1 Hz, P-O-CH2-CH3), 61.20 (d, JCÀOÀP
=
6.1 Hz, P-O-CH2-CH3), 44.09 (C1’), 26.36 (d, JCÀP =136.2 Hz, C6’),
16.40 ppm (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%):
480 (55) [M+H]+, 502 (100) [M+Na]+; HRMS-ESI+: m/z calcd for
C21H31O6N5P [M+H]+ 480.2007, found 480.2006; Anal. calcd for
C21H31O6N5P: C 52.61, H 6.31, N 14.61, P 6.46, found: C 52.37, H
6.32, N 14.47, P 6.37; [a]2D0 =À10.5 (c=0.342 g/100 mL, DMSO).
Diethyl (S)-(2-((1-hydroxy-3-(hypoxanthin-9-yl)propan-2-yl)oxy)-
ethyl)phosphonate (23): Compound 21 (720 mg, 1.55 mmol) was
treated with procedure G to give the title compound as a yellowish
oil which solidified (465 mg, yield 80%). 1H NMR ([D6]DMSO): d=
8.04 (m, 2H, H-2, 8), 4.90 (bs, 1H, OH), 4.30 (dd, Jgem =14.3 Hz,
J
gem =14.8 Hz, J1’bÀ2’ =7.1 Hz, 1H, H-1’b), 3.75 (m, 1H, H-2), 3.52–
3.69 (m, 3H, H-3’a, 4’), 3.45 (dd, Jgem =12.4 Hz, J3’bÀ2’ =5.5 Hz, 1H,
H-3’b), 1.62–1.77 ppm (m, 2H, H-5’); 13C NMR (D2O): d=158.54 (C6),
153.46 (C2), 151.47 (C4), 140.24 (C8), 115.16 (C5), 65.61 (d, JCÀCÀP
=
1.8 Hz, C4’), 60.22 (C3’), 43.46 (C1’), 28.61 ppm (d, JCÀP =130.0 Hz,
C5’); MS-ESI+ m/z (%): 334 (100) [M+H]+, 356 (20) [M+Na]+;
HRMS-ESI+: m/z calcd for C10H17O6N5P [M+H]+ 334.0911, found
334.0910; FTIR (KBr): n˜ =3379, 3320, 3222, 1688, 1655, 1611, 1580,
1539, 1480, 1411, 1097, 1049, 909, 781 cmÀ1; [a]D20 =À15.4 (c=
0.240 g/100 mL, H2O).
(S)-(2-((1-(Xanthin-9-yl)-3-hydroxypropan-2-yl)oxy)ethyl)phos-
phonic acid (27): Compound 26 (250 mg, 0.75 mmol) was dis-
solved in 80% acetic acid (50 mL) and iso-amyl nitrite (1.5 mL) was
added. The reaction mixture was stirred at RT overnight. Volatiles
were evaporated and a residue was co-distilled with water (3”
10 mL) and toluene (3”20 mL). The crude product was purified by
procedure A to give the title compound as a white solid (87 mg,
yield 35%). 1H NMR (D2O+NaOD): d=7.73 (s, 1H, H-8), 4.23 (dd,
J
1’aÀ2’ =3.9 Hz, 1H, H-1’a), 4.14 (dd, Jgem =14.4 Hz, J1’bÀ2’ =7.6 Hz,
1H, H-1’b), 3.88–3.96 (m, 4H, P-O-CH2-CH3), 3.58–3.71 (m, 2H, H-2’,
4’a), 3.39–3.51 (m, 3H, H-3’, 4’b), 1.85–1.92 (m, 2H, H-5’), 1.18 ppm
(t, JCH3ÀCH2 =7.1 Hz, 6H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=
156.90 (C6), 148.77 (C4), 145.68 (C2), 141.19 (C8), 123.87 (C5), 78.64
(C2’), 63.86 (C4’), 61.22 (m, P-O-CH2-CH3), 60.91 (C3’), 44.48 (C1’),
26.34 (d, JCÀP =136.7 Hz, C5’), 16.41 ppm (d, JCÀCÀOÀP =6.0 Hz, P-O-
CH2-CH3); MS-ESI+ m/z (%): 375 (100) [M+H]+, 397 (50) [M+Na]+;
HRMS-ESI+: m/z calcd for C14H24O6N4P [M+H]+ 375.1428, found
375.1427; FTIR (KBr): n˜ =3052, 1696, 1593, 1551, 1414, 1341, 1221,
1118, 1100, 1054, 1042, 1028, 961, 789 cmÀ1; Anal. calcd for
C14H24O6N4P: C 44.92, H 6.19, N 14.97, P 8.27, found: C 45.13, H
6.33, N 14.75, P 8.13; [a]2D0 =À12.5 (c=0.208 g/100 mL, MeOH).
J
J
J
gem =14.8 Hz, J1’bÀ2’ =4.3 Hz, 1H, H-1’b), 4.10 (dd, Jgem =14.8 Hz,
1’aÀ2’ =6.9 Hz, 1H, H-1’a), 3.85 (ddt, J2’À1’a =6.9 Hz, J2’À3’a =5.2 Hz,
2’À1’b =J2’À3’b =4.4 Hz, 1H, H-2’), 3.78 (m, 1H, H-4’b), 3.69 (m, 1H, H-
4’a), 3.59 (dd, Jgem =12.4 Hz, J3’bÀ2’ =4.5 Hz, 1H, H-3’b), 3.52 (dd,
gem =12.4 Hz, J3’aÀ2’ =5.3 Hz, 1H, H-3’a), 1.68–1.81 ppm (m, 2H, H-
J
5’); 13C NMR (D2O+NaOD): d=161.65 (C6), 160.15 (C2), 154.03 (C4),
140.63 (C8), 115.07 (C5), 78.40 (C2’), 67.49 (C4’), 60.99 (C3’), 43.98
(C1’), 30.46 ppm (d, JCÀP =126.2 Hz, C5’); MS-ESIÀ m/z (%): 333 (100)
[MÀH]+, 355 (75) [M+Na]+; HRMS-ESIÀ: m/z calcd for C10H14O7N4P
[MÀH]+ 333.0606, found 333.0607; FTIR (KBr): n˜ =3500, 3193, 3124,
2294, 1720, 1695, 1612, 1580, 1393, 1149, 1100, 1051, 1010,
747 cmÀ1; [a]D20 =À4.7 (c=0.363 g/100 mL, H2O).
Diethyl
(S)-(2-((guanin-9-yl)-3-hydroxypropan-2-yl)oxy)ethyl)-
phosphonate (24): Compound 22 (580 mg, 1.21 mmol) was treat-
ed with procedure G to give the title compound as a white solid
(367 mg, yield 78%). H NMR ([D6]DMSO): d=7.62 (s, 1H, H-8), 4.10
(dd, Jgem =14.3 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 3.91–3.99 (m, 5H, H-
1’b, P-O-CH2-CH3), 3.61–3.68 (m, 2H, H-2’, 4’a), 3.53 (m, 1H, H-4’b),
1
3.38–3.41 (m, 2H, H-3’), 1.86–1.95 (m, 2H, H-5’), 1.21 (t, JCH3ÀCH2
=
Methyl
(S)-3-(6-chloropurin-9-yl)-2-(2-(diethoxyphosphoryl)-
7.1 Hz, 3H, P-O-CH2-CH3); 1.20 ppm (t, JCH3ÀCH2 =7.1 Hz, 3H, P-O-
CH2-CH3); 13C NMR ([D6]DMSO): d=156.82 (C6), 153.58 (C2), 151.38
(C4), 138.10 (C8), 116.40 (C5), 78.45 (C2’), 63.64 (C4’), 61.07 (m, P-O-
CH2-CH3), 60.87 (C3’), 43.70 (C1’), 26.36 (d, JCÀP =136.7 Hz, C5’),
16.20 ppm (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%):
390 (100) [M+H]+, 412 (15) [M+Na]+; HRMS-ESI+: m/z calcd for
C14H25O6N5P [M+H]+ 390.1537, found 390.1536; FTIR (KBr): n˜ =
3423, 3123, 2982, 1691, 1657, 1609, 1583, 1540, 1481, 1412, 1229,
1167, 1117, 1051, 1027, 961, 782 cmÀ1; Anal. calcd for C14H25O6N5P:
C 43.19, H 6.21, N 17.99, P 7.96, found: C 42.95, H 6.25, N 17.91, P
7.83; [a]2D0 =À22.6 (c=0.261 g/100 mL, MeOH).
ethoxy)propanoate (28): From alcohol 12 (1.71 g, 6 mmol) and 6-
chloropurine (1.21 g, 7.8 mmol), applied procedure E, obtained the
title compound as
a
yellow oil (1.1 g, yield 44%). 1H NMR
([D6]DMSO): d=8.75 (s, 1H, H-2), 8.32 (s, 1H, H-8), 4.72 (dd, Jgem
14.5 Hz, J1’aÀ2’ =3.9 Hz, 1H, H-1’a), 4.58 (dd, Jgem =14.6 Hz, J1’bÀ2’
=
=
6.7 Hz, 1H, H-1’b), 4.31 (dd, J2’À1’a =3.8 Hz, J2’À1’b =6.8 Hz, 1H, H-2’),
4.03–4.14 (m, 4H, P-O-CH2-CH3), 3.96 (m, 1H, H-4’a), 3.75 (s, 3H, O-
CH3), 3.62 (m, 1H, H-4’b), 2.00–2.14 (m, 2H, H-5’), 1.31 ppm (t,
J
CH3ÀCH2 =7.1 Hz, 6H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=169.32
(C3’), 151.98 (C2), 151.71 (C4), 151.02 (C6), 146.53 (C8), 131.13 (C5),
76.55 (C2’), 65.43 (C4’), 61.87 (m, P-O-CH2-CH3), 52.67 (O-CH3), 45.43
(C1’), 26.87 (d, JCÀP =140.8 Hz, C5’), 16.37 ppm (d, JCÀCÀOÀP =6.0 Hz,
P-O-CH2-CH3); MS-ESI+ m/z (%): 421 (35) [M+H]+, 443 (100) [M+
Na]+; HRMS-ESI+: m/z calcd for C15H23O6N4ClP [M+H]+ 421.1038,
found 421.1037; Anal. calcd for C15H22ClN4O6P: C 42.82, H 5.27, Cl
8.43, N 13.31, P 7.36, found: C 42.65, H 5.37, Cl 8.25, N 13.03, P
7.15; [a]2D0 =À5.3 (c=0.316 g/100 mL, DMSO).
(S)-(2-((1-Hydroxy-3-(hypoxanthin-9-yl)propan-2-yl)oxy)ethyl)-
phosphonic acid (25): Treatment of compound 23 (187 mg,
0.5 mmol) by procedure B followed by procedure A gave the title
1
compound as a white solid (116 mg, yield 73%). H NMR (D2O): d=
8.12 (s, 1H, H-2), 8.10 (s, 1H, H-8), 4.40 (dd, Jgem =14.7 Hz, J1’aÀ2’
=
3.8 Hz, 1H, H-1’a), 4.26 (dd, Jgem =14.8 Hz, J1’bÀ2’ =7.7 Hz, 1H, H-
1’b), 3.81 (m, 1H, H-2’), 3.63–3.70 (m, 2H, H-3’a, 4’a), 3.47–3.54 (m,
ChemMedChem 2015, 10, 1707 – 1723
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Methyl
(S)-3-(2-amino-6-chloropurin-9-yl)-2-(2-(diethoxyphos-
d=8.02 (s, 1H, H-2), 8.01 (s, 1H, H-8), 4.40 (dd, Jgem =14.4 Hz,
phoryl)ethoxy)propanoate (29): Compound 12 (1.99 g, 7 mmol)
and 2-amino-6-chloropurine (1.54 g, 9.1 mmol), applied procedure
F to give the title compound as a yellowish oil (1.16 g, yield 38%).
1H NMR ([D6]DMSO): d=8.05 (s, 1H, H-8), 6.93 (s, 2H, NH2), 4.46
(dd, J2’À1’a =4.1 Hz, J2’À1’b =7.1 Hz, 1H, H-2’), 4.41 (dd, Jgem =14.5 Hz,
J1’aÀ2’ =3.2 Hz, 1H, H-1’a), 4.19 (dd, Jgem =14.4 Hz, J1’bÀ2’ =8.4 Hz,
1H, H-1’b), 3.83 (dd, J2’À1’a =3.2 Hz, J2’À1’b =8.4 Hz, 1H, H-2’), 3.56
(m, 1H, H-4’a), 3.31 (m, 1H, H-4’b), 1.45–1.58 ppm (m, 2H, H-5’);
13C NMR (D2O): d=175.67 (C3’), 158.67 (C6), 150.00 (C4), 146.78
(C2), 143.17 (C8), 124.16 (C5), 80.58 (C2’), 67.39 (C4’), 47.30 (C1’),
31.2 and 30.2 ppm (C5’); MS-ESIÀ m/z (%): 331 (100) [MÀH]+, 353
(30) [M+Na]+; HRMS-ESIÀ: m/z calcd for C10H12O7N4P [MÀH]+
331.0449, found 331.0450; FTIR (KBr): n˜ =3058, 1692, 1614, 1590,
1551, 1520, 1415, 1349, 1291, 1112, 1063, 900 cmÀ1; [a]D20 =À5.0
(c=0.301 g/100 mL, H2O).
J
1’aÀ2’ =4.0 Hz, 1H, H-1’a), 4.31 (dd, Jgem =14.5 Hz, J1’bÀ2’ =7.1 Hz,
1H, H-1’b), 3.90–3.99 (m, 4H, P-O-CH2-CH3), 3.72 (m, 1H, H-4’a),
3.66 (s, 3H, O-CH3), 3.54 (m, 1H, H-4’b), 1.91–2.05 (m, 2H, H-5’),
1.19 ppm (t,
([D6]DMSO): d=170.01 (C3’), 160.02 (C2), 154.35 (C4), 149.48 (C6),
J
CH3ÀCH2 =7.1 Hz, 6H, P-O-CH2-CH3); 13C NMR
144.00 (C8), 123.08 (C5), 76.03 (C2’), 64.50 (C4’), 61.29 (d, JCÀOÀP
6.1 Hz, P-O-CH2-CH3), 52.40 (O-CH3), 44.46 (C1’), 26.11 (d, JCÀP
137.3 Hz, C5’), 16.41 ppm (d, JCÀCÀOÀP =5.7 Hz, P-O-CH2-CH3); MS-
ESI+ m/z (%): 436 (40) [M+H]+, 458 (100) [M+Na]+; HRMS-ESI+:
m/z calcd for C15H24O6N5ClP [M+H]+ 436.1147, found 436.1146;
[a]2D0 =À7.4 (c=0.398 g/100 mL, DMSO).
=
=
(S)-3-(Guanin-9-yl)-2-(2-phosphonoethoxy)propanoic acid (33):
Treatment of compound 31 (202 mg, 0.5 mmol) by procedure B
followed by procedure A gave the title compound as a yellowish
1
solid (127 mg, yield 73%). H NMR (D2O): d=7.69 (s, 1H, H-8), 4.32
(dd,
J
gem =14.7 Hz,
J
1’aÀ2’ =3.9 Hz, 1H, H-1’a), 4.16 (dd, Jgem
=
=
14.7 Hz, J1’bÀ2’ =7.1 Hz, 1H, H-1’b), 4.03 (dd, J2’À1’a =3.9 Hz, J2’À1’b
(S)-2-(2-(Diethoxyphosphoryl)ethoxy)-3-(hypoxanthin-9-yl)propa-
noic acid (30): Treatment of compound 28 (421 mg, 1 mmol) by
procedure C (chromatography: linear gradient of H1 in ethyl ace-
tate) gave the title compound as a yellowish solid (221 mg, yield
7.0 Hz, 1H, H-2’), 3.65 (m, 1H, H-4’a), 3.50 (m, 1H, H-4’b), 1.68–
1.81 ppm (m, 2H, H-5’); 13C NMR (D2O): d=176.79 (C3’), 158.87
(C6), 153.54 (C2), 151.72 (C4), 140.44 (C8), 115.47 (C5), 78.98 (C2’),
65.86 (C4’), 43.25 (C1’), 28.54 ppm (d, JCÀP =129.5 Hz, C5’); MS-ESIÀ
m/z (%): 346 (100) [MÀH]+, 368 (35) [M+Na]+; HRMS-ESIÀ: m/z
calcd for C10H13O7N5P [MÀH]+ 346.0558, found 346.0556; FTIR
(KBr): n˜ =3388, 3142, 1696, 1601, 1540, 1478, 1410, 1299, 1107,
1063, 906, 781 cmÀ1; [a]D20 =À23.7 (c=0.207 g/100 mL, H2O).
1
57%). H NMR ([D6]DMSO): d=12.48 (bs, 1H, NH), 8.022 and 8.017
(2 s, 2”1H, H-2, 8), 4.47 (dd, Jgem =14.2 Hz, J1’aÀ2’ =3.5 Hz, 1H, H-
1’a), 4.21 (dd, Jgem =14.2 Hz, J1’bÀ2’ =8.5 Hz, 1H, H-1’b), 3.88–3.97
(m, 4H, P-O-CH2-CH3), 3.86 (dd, J2’À1’a =3.5 Hz, J2’À1’b =8.4 Hz, 1H, H-
2’), 3.80 (m, 1H, H-4’a), 3.39 (m, 1H, H-4’b), 1.86–1.97 (m, 2H, H-5’),
Methyl (S)-2-(2-(diethoxyphosphoryl)ethoxy)-3-(hypoxanthin-9-
yl)propanoate (34): Treatment of compound 28 (421 mg, 1 mmol)
by procedure D gave the title compound as a colorless oil which
1.17 ppm (t,
([D6]DMSO): d=172.00 (C3’), 157.01 (C6), 148.73 (C4), 145.53 (C2),
141.06 (C8), 123.70 (C5), 79.92 (C2’), 63.17 (C4’), 61.23 (d, JCÀOÀP
J
CH3CH2 =7.1 Hz, 6H, P-O-CH2-CH3); 13C NMR
=
1
solidified (338 mg, yield 84%). H NMR ([D6]DMSO): d=8.04 (s, 1H,
6.2 Hz, P-O-CH2-CH3), 61.22 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 46.07
(C1’), 26.19 (d, JCÀP =136.8 Hz, C5’), 16.39 ppm (d, JCÀCÀOÀP =5.8 Hz,
P-O-CH2-CH3); MS-ESIÀ m/z (%): 387 (100) [MÀH]+; HRMS-ESIÀ: m/z
calcd for C14H20O7N4P [MÀH]+ 387.1075, found 387.1074; FTIR
(KBr): n˜ =2982, 1698, 1608, 1549, 1522, 1390, 1350, 1229, 1124,
1052, 1028, 963 cmÀ1; [a]D20 =À14.0 (c=0.377 g/100 mL, MeOH).
H-2), 8.01 (s, 1H, H-8), 4.38–4.52 (m, 3H, H-1’, 2’), 3.90–3.98 (m, 4H,
P-O-CH2-CH3), 3.72 (m, 1H, H-4’a), 3.64 (s, 3H, O-CH3), 3.53 (m, 1H,
H-4’b), 1.91–2.05 (m, 2H, H-5’), 1.19 ppm (t, JCH3ÀCH2 =7.1 Hz, 6H, P-
O-CH2-CH3); 13C NMR ([D6]DMSO): d=169.95 (C3’), 156.83 (C6),
148.64 (C4), 145.81 (C2), 141.16 (C8), 123.72 (C5), 76.38 (C2’), 64.52
(C4’), 61.29 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 52.35 (O-CH3), 44.78
(C1’), 26.08 (d, JCÀP =139.8 Hz, C5’), 16.41 ppm (d, JCÀCÀOÀP =5.8 Hz,
P-O-CH2-CH3); MS-ESI+ m/z (%): 403 (30) [M+H]+, 425 (100) [M+
Na]+; HRMS-ESI+: m/z calcd for C15H24O7N4P [M+H]+ 403.1377,
found 403.1376; FTIR (KBr): n˜ =3056, 2980, 2955, 1752, 1695, 1587,
(S)-3-(Guanin-9-yl)-2-(2-(diethoxyphosphoryl)ethoxy)propanoic
acid (31): Treatment of compound 29 (436 mg, 1 mmol) by proce-
dure C (chromatography: linear gradient of H1 in ethyl acetate)
gave the title compound as a yellowish solid (222 mg, yield 55%).
An alternative procedure: The compound 29 (436 mg, 1 mmol)
was dissolved in 15 mL of 75% CF3COOH and the solution was
heated at 508C overnight. Volatiles were evaporated and a residue
was purified by silica gel chromatography (gradient from 10–50%
methanol in chloroform) to afford (after evaporation) the title com-
1550, 1441, 1415, 1391, 1220, 1121, 1090, 1045, 1029, 964 cmÀ1
[a]2D0 =À8.0 (c=0.263 g/100 mL, MeOH).
;
Methyl (S)-3-(guanin-9-yl)-2-(2-(diethoxyphosphoryl)ethoxy)pro-
panoate (35): Treatment of compound 29 (436 mg, 1 mmol) by
procedure D gave the title compound as a white solid (338 mg,
pound as
a
yellowish solid (314 mg, yield 78%). 1H NMR
1
yield 81%). H NMR ([D6]DMSO): d=10.69 (bs, 1H, NH), 7.60 (s, 1H,
([D6]DMSO): d=11.49 (bs, 1H, NH), 7.60 (s, 1H, H-8), 6.98 (bs, 2H,
NH2), 4.32 (dd, Jgem =14.1 Hz, J1’aÀ2’ =3.2 Hz, 1H, H-1’a), 3.87–3.99
(m, 5H, H-1’b, P-O-CH2-CH3), 3.75–3.84 (m, 2H, H-2’, 4’a), 3.29–3.42
H-8), 6.57 (bs, 2H, NH2), 4.40 (dd, J2’À1’a =4.1 Hz, J2’À1’b =7.3 Hz, 1H,
H-2’), 4.28 (dd, Jgem =14.4 Hz, J1’aÀ2’ =4.1 Hz, 1H, H-1’a), 4.18 (dd,
J
gem =14.4 Hz, J1’bÀ2’ =7.3 Hz, 1H, H-1’b), 3.89–4.00 (m, 4H, P-O-CH2-
(m, 1H, H-4’b), 1.88–1.96 (m, 2H, H-5’), 1.18 ppm (t, JCH3ÀCH2
=
CH3), 3.71 (m, 1H, H-4’a), 3.66 (s, 3H, O-CH3), 3.51 (m, 1H, H-4’b),
1.92–2.02 (m, 2H, H-5’), 1.19 ppm (t, JCH3ÀCH2 =7.1 Hz, 6H, P-O-CH2-
CH3); 13C NMR ([D6]DMSO): d=170.09 (C3’), 156.99 (C6), 153.90
(C2), 151.46 (C4), 138.26 (C8), 116.28 (C5), 76.43 (C2’), 64.50 (C4’),
61.32 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 52.31 (O-CH3), 44.18 (C1’),
26.13 (d, JCÀP =136.5 Hz, C5’), 16.40 (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-
CH3), 16.39 ppm (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z
(%): 418 (50) [M+H]+, 440 (100) [M+Na]+, 462 (45) [M+2Na]+;
HRMS-ESI+: m/z calcd for C15H25O7N5P [M+H]+ 418.1486, found
418.1485; FTIR (KBr): n˜ =3434, 3140, 2756, 1681, 1605, 1539, 1470,
7.1 Hz, 6H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=172.66 (C3’),
157.46 (C6), 154.18 (C2), 151.50 (C4), 138.21 (C8), 116.26 (C5), 80.02
(C2’), 63.26 (C4’), 61.24 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 45.57 (C1’),
26.74 (d, JCÀP =136.5 Hz, C5’), 16.41 ppm (d, JCÀCÀOÀP =5.8 Hz, P-O-
CH2-CH3); MS-ESI+ m/z (%): 404 (25) [M+H]+, 426 (100) [M+Na]+,
448 (85) [M+2Na]+; HRMS-ESI+: m/z calcd for C14H23O7N5P [M+
H]+ 404.1330, found 404.1329; FTIR (KBr): n˜ =3413, 3319, 3128,
2983, 2930, 2775, 1740, 1693, 1621, 1538, 1481, 1369, 1101 cmÀ1
;
[a]2D0 =À49.8 (c=0.283 g/100 mL, DMSO).
(S)-3-(6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(2-phosphonoethoxy)-
propanoic acid (32): Treatment of compound 30 (194 mg,
1459, 1414, 1358, 1301, 1212, 1143, 1026, 971, 801 cmÀ1; [a]D20
À13.5 (c=0.310 g/100 mL, MeOH).
=
0.5 mmol) by procedure B followed by procedure A gave the title
1
compound as a yellowish solid (125 mg, yield 75%). H NMR (D2O):
ChemMedChem 2015, 10, 1707 – 1723
www.chemmedchem.org
1720
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
(S)-(2-((1-Methoxy-1-oxo-3-(hypoxanthin-9-yl)propan-2-yl)oxy)-
ethyl)phosphonic acid (36): Treatment of compound 34 (201 mg,
0.5 mmol) by procedure B followed by procedure A gave the title
ESIÀ: m/z calcd for C10H14O6N5BrP [MÀH]+ 409.9871, found
409.9871; FTIR (KBr): n˜ =3561, 3463, 3328, 3162, 2311, 1689, 1636,
1599, 1410, 1236, 1117, 1043, 931, 776 cmÀ1; [a]D20 =À4.3 (c=
0.257 g/100 mL, H2O).
1
compound as a yellowish solid (123 mg, yield 71%). H NMR (D2O):
d=8.12 (s, 1H, H-2), 8.06 (s, 1H, H-8), 4.56–4.66 (m, 2H, H-1’), 4.50
(m, 1H, H-2’), 3.45–3.81 (m, 5H, H-4’, O-CH3), 1.72–1.99 ppm (m,
2H, H-5’); 13C NMR (D2O): d=172.23 (C3’), 158.54 (C6), 148.89 (C4),
145.83 (C2), 142.87 (C8), 123.03 (C5), 76.35 (C2’), 66.95 (C4’), 53.01
(O-CH3), 45.27 (C1’), 28.80 ppm (d, JCÀP =129.4 Hz, C5’); MS-ESI+ m/
z (%): 347 (100) [M+H]+, 369 (95) [M+Na]+, 391 (55) [M+2Na]+;
HRMS-ESI+: m/z calcd for C11H16O7N4P [M+H]+ 347.0751, found
347.0751; FTIR (KBr): n˜ =1742, 1677, 1587, 1551, 1434, 1402, 1348,
1123, 1045, 900 cmÀ1; [a]D20 =À5.2 (c=0.278 g/100 mL, H2O).
Diethyl (S)-(2-((1-(8-bromoguanin-9-yl)-3-(benzyloxy)propan-2-
yl)oxy)ethyl)phosphonate (40): Compound 22 (1.92 g, 4 mmol)
was dissolved in ethyl acetate (20 mL) and aqueous solution
(15 mL) of sodium bromate (1.81 g, 12 mmol) was added. To a vigo-
rously stirred two-phase system a solution of sodium hydrosulfite
(2.6 g, 12 mmol) in water (30 mL) was added dropwise. The mixture
was vigorously stirred at RT for 2 h. The reaction was diluted with
water and extracted with ethyl acetate (3”150 mL). The combined
organic layers were washed with brine (2”100 mL), dried over
MgSO4 and concentrated in vacuo. A residue was purified by silica
gel chromatography (gradient from 1-8% methanol in chloroform)
to give (after evaporation) the title compound as a yellow foam
(1.50 g, yield 67%). 1H NMR ([D6]DMSO): d=10.71 (s, 1H, NH-1),
7.27–7.37 (m, 5H, H-2’’, 3’’, 4’’), 6.59 (bs, 2H, NH2), 4.51 (s, 2H, H-4’),
3.99–4.07 (m, 2H, H-1’), 3.83–3.95 (m, 5H, H-2’, P-O-CH2-CH3), 3.61
(m, 1H, H-5’a), 3.55 (dd, Jgem =10.5 Hz, J3’aÀ2’ =4.2 Hz, 1H, H-3’a),
3.49 (dd, Jgem =10.5 Hz, J3’bÀ2’ =5.4 Hz, 1H, H-3’b), 3.38 (m, 1H, H-
5’b), 1.72–1.88 (m, 2H, H-6’), 1.16 ppm (t, JCH3ÀCH2 =7.1 Hz, 6H, P-O-
CH2-CH3); 13C NMR ([D6]DMSO): d=155.75 (C6), 154.02 (C2), 152.84
(C4), 138.27 (C1’’), 128.46 (C3’’), 127.75 (C2’’), 127.71 (C4’’), 121.61
(C8), 116.94 (C5), 76.19 (C2’), 72.67 (C4’), 70.22 (C3’), 64.35 (C5’),
61.23 (d, JCÀOÀP =6.1 Hz, P-O-CH2-CH3), 61.18 (d, JCÀOÀP =6.1 Hz, P-
O-CH2-CH3), 45.41 (C1’), 26.42 (d, JCÀP =135.8 Hz, C6’), 16.36 (d,
(S)-(2-((Guanin-9-yl)-1-methoxy-1-oxopropan-2-yl)oxy)ethyl)-
phosphonic acid (37): Treatment of compound 35 (209 mg,
0.5 mmol) by procedure B followed by procedure A gave the title
1
compound as a white solid (125 mg, yield 69%). H NMR (D2O): d=
7.77 (s, 1H, H-8), 4.48–4.53 (m, 2H, H-1’a, 2’), 4.42 (dd, Jgem
=
15.6 Hz, J1’bÀ2’ =6.0 Hz, 1H, H-1’b), 3.83 (m, 1H, H-4’a), 3.74 (s, 3H,
O-CH3), 3.73 (m, 1H, H-4’b), 1.85–1.95 ppm (m, 2H, H-5’); 13C NMR
(D2O): d=173.03 (C3’), 159.49 (C6), 154.31 (C2), 152.26 (C4), 141.18
(C8), 116.14 (C5), 76.98 (C2’), 67.44 (C4’), 53.62 (O-CH3), 45.20 (C1’),
29.41 ppm (d, JCÀP =129.9 Hz, C5’); MS-ESIÀ m/z (%): 360 (100)
[MÀH]+, 382 (60) [M+Na]+; HRMS-ESIÀ: m/z calcd for C11H15O7N5P
[MÀH]+ 360.0715, found 360.0714; FTIR (KBr): n˜ =3344, 3158, 2954,
1630, 1598, 1482, 1438, 1412, 1362, 1108, 1074, 900, 783 cmÀ1
[a]2D0 =À3.1 (c=0.285 g/100 mL, H2O).
;
J
CÀCÀOÀP =5.8 Hz, P-O-CH2-CH3), 16.35 ppm (d, JCÀCÀOÀP =5.8 Hz, P-
Diethyl
(S)-(2-((1-(8-bromoguanin-9-yl)-3-hydroxypropan-2-yl)-
O-CH2-CH3); MS-ESI+ m/z (%): 558 (100) [M+H]+, 560 (95) [M+
H]+, 580 (45) [M+Na]+, 582 (43) [M+Na]+; HRMS-ESI+: m/z calcd
for C21H30O6N5BrP [M+H]+ 558.1112, found 558.1114; [a]2D0 =À20.7
(c=0.304 g/100 mL, DMSO).
oxy)ethyl)phosphonate (38): Compound 24 (779 mg, 2 mmol) was
dissolved in DMF (10 mL) and a solution of bromine (0.15 mL,
3 mmol) in carbon tetrachloride (10 mL) was added. The reaction
mixture was stirred at RT for 3 h. Volatiles were evaporated and
a residue was co-distilled with toluene (3”20 mL). This was sub-
jected to silica gel chromatography (gradient from 5-12% metha-
nol in chloroform) to afford (after evaporation) the crude product.
Crystallization from ethyl acetate gave the title compound as a yel-
Diethyl (S)-(2-((1-(8-oxoguanin-9-yl)-3-(benzyloxy)propan-2-yl)-
oxy)ethyl)phosphonate (41): Compound 40 (860 mg, 1.54 mmol)
was dissolved in a solution of sodium acetate (1.26 g, 15.4 mmol)
in glacial acetic acid (100 mL). The reaction mixture was heated at
1308C for 4 h. Acetic acid was evaporated and a residue was co-
distilled with water (1”20 mL), toluene (3”20 mL) and purified by
silica gel chromatography (gradient from 2-8% methanol in chloro-
form) to afford (after evaporation) the title compound as a white
1
lowish solid (800 mg, yield 85%). H NMR ([D6]DMSO): d=10.71 (s,
1H, NH), 6.63 (bs, 2H, NH2), 3.84-4.05 (m, 6H, H-1’, P-O-CH2-CH3),
3.71 (m, 1H, H-2’), 3.57 (m, 1H, H-4’a), 3.47 (dd, Jgem =11.8 Hz,
J
3’aÀ2’ =4.5 Hz, 1H, H-3’a), 3.40 (dd, Jgem =11.8 Hz, J3’bÀ2’ =5.5 Hz, 1H,
H-3’b), 3.39 (m, 1H, H-4’b), 1.73–1.91 (m, 2H, H-5’), 1.18 ppm (t,
CH3ÀCH2 =7.0 Hz, 6H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=155.72
1
foam (608 mg, yield 80%). H NMR ([D6]DMSO): d=10.72 (bs, 1H,
J
NH-1), 10.56 (s, 1H, NH-7), 7.25–7.36 (m, 5H, H-2’’, 3’’, 4’’), 6.48 (bs,
2H, NH2), 4.47 (bs, 2H, H-4’), 3.86–3.98 (m, 5H, H-2’, P-O-CH2-CH3),
3.74 (dd, Jgem =14.0 Hz, J1’aÀ2’ =7.2 Hz, 1H, H-1’a), 3.67 (m, 1H, H-
5’a), 3.63 (dd, Jgem =14.0 Hz, J1’bÀ2’ =5.6 Hz, 1H, H-1’b), 3.58 (m, 1H,
H-5’b), 3.52 (dd, Jgem =10.6 Hz, J3’aÀ2’ =3.8 Hz, 1H, H-3’a), 3.44 (dd,
(C6), 154.02 (C2), 152.81 (C4), 121.74 (C8), 116.88 (C5), 78.26 (C2’),
64.18 (C4’), 61.45 (C3’), 61.30 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 61.22
(d,
JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 45.20 (C1’), 26.38 (d, JCÀP =
135.9 Hz, C5’), 16.39 (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3), 16.38 ppm
(d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%): 468 (10) [M+
H]+, 470 (9) [M+H]+, 490 (100) [M+Na]+, 492 (97) [M+Na]+;
HRMS-ESI+: m/z calcd for C14H23O6N5BrNaP [M+Na]+ 490.0462,
found 490.0461; FTIR (KBr): n˜ =3417, 3311, 3215, 3149, 2983, 2933,
J
gem =10.6 Hz, J3’bÀ2’ =5.9 Hz, 1H, H-3’b), 1.81–1.96 (m, 2H, H-6’),
1.18 (t, JCH3ÀCH2 =7.1 Hz, 3H, P-O-CH2-CH3), 1.17 ppm (t, JCH3ÀCH2
=
7.1 Hz, 3H, P-O-CH2-CH3); 13C NMR ([D6]DMSO): d=153.71 (C6),
152.57 (C8), 151.36 (C2), 148.35 (C4), 138.44 (C1’’), 128.42 (C3’’),
127.71 (C2’’), 127.64 (C4’’), 98.45 (C5), 75.67 (C2’), 72.58 (C4’), 70.81
(C3’), 63.90 (C5’), 61.22 (d, JCÀOÀP =6.2 Hz, P-O-CH2-CH3), 61.19 (d,
1690, 1633, 1599, 1523, 1465, 1365, 1221, 1117, 1029, 964 cmÀ1
[a]2D0 =À19.9 (c=0.371 g/100 mL, DMSO).
;
J
CÀOÀP =6.2 Hz, P-O-CH2-CH3), 40.64 (C1’), 26.52 (d, JCÀP =135.6 Hz,
(S)-(2-((1-(8-Bromoguanin-9-yl)-3-hydroxypropan-2-yl)oxy)ethyl)-
phosphonic acid (39): Treatment of compound 38 (234 mg,
C6’), 16.41 ppm (d, JCÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z
(%): 496 (15) [M+H]+, 518 (100) [M+Na]+, 540 (15) [M+2Na]+;
HRMS-ESI+: m/z calcd for C21H30O7N5NaP [M+Na]+ 518.1775,
found 518.1775; FTIR (KBr): n˜ =3423, 3318, 3025, 1716, 1682, 1596,
1455, 1387, 1365, 1219, 1099, 1027, 962, 736 cmÀ1; [a]D20 =À14.6
(c=0.274 g/100 mL, MeOH).
0.5 mmol) by procedure B followed by procedure A gave the title
1
compound as a yellowish solid (159 mg, yield 77%). H NMR (D2O):
d=4.13–4.20 (m, 2H, H-1’), 3.88 (m, 1H, H-2’), 3.72–3.80 (m, 2H, H-
3’a, 4’a), 3.60 (dd, Jgem =12.4 Hz, J3’bÀ2’ =5.3 Hz, 1H, H-3’b), 3.57 (m,
1H, H-4’b), 1.79–1.96 ppm (m, 2H, H-5’); 13C NMR (D2O): d=158.07
(C6), 154.30 (C2), 153.14 (C4), 125.48 (C8), 116.67 (C5), 78.52 (C2’),
65.90 (C4’), 61.38 (C3’), 45.37 (C1’), 28.61 ppm (d, JCÀP =132.6 Hz,
C5’); MS-ESIÀ m/z (%): 410 (100) [MÀH]+, 412 (97) [MÀH]+; HRMS-
Diethyl (S)-(2-((1-(8-oxoguanin-9-yl)-3-hydroxypropan-2-yl)oxy)-
ethyl)phosphonate (42): Compound 41 (533 mg, 1.1 mmol) was
treated with procedure G to give the title compound as a white
ChemMedChem 2015, 10, 1707 – 1723
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
solid (361 mg, yield 82%). 1H NMR ([D6]DMSO): d=10.67 (bs, 1H,
NH-1), 10.60 (s, 1H, NH-7), 6.49 (bs, 2H, NH2), 4.75 (t, JOHÀ3’ =6.0 Hz,
OH), 3.88–4.01 (m, 4H, P-O-CH2-CH3), 3.57–3.72 (m, 5H, H-1’, 2’, 4’),
3.42 (ddd, Jgem =12.0 Hz, J3’aÀOH =6.1 Hz, J3’aÀ2’ =3.9 Hz, 1H, H-3’a),
3.34 (dt, Jgem =12.0 Hz, J3’bÀOH =J1’bÀ2’ =5.9 Hz, 1H, H-3’b), 1.85–1.97
(m, 2H, H-5’), 1.20 ppm (t, JCH3ÀCH2 =7.1 Hz, 6H, P-O-CH2-CH3);
13C NMR ([D6]DMSO): d=153.63 (C6), 152.69 (C8), 151.21 (C2),
148.36 (C4), 98.41 (C5), 77.77 (C2’), 63.61 (C4’), 61.75 (C3’), 61.28 (d,
coordinates was with PHENIX1.8-1069[35] and model building was
with COOT 0.7.[36] The structural restraints files for the inhibitors
were generated by use of the PRODRG2 Dundee server. The
atomic coordinates and structure factors of human HGPRT in com-
plex with the two inhibitors have been deposited in the Protein
Data Bank. The PDB accession code for (S)-(2-((1-hydroxy-3-(hypo-
xanthin-9-yl)propan-2-yl)oxy)ethyl)phosphonic acid (compound 25)
is 5BRN and, for the guanine derivative 26, the code is 5BSK.
J
CÀOÀP =6.3 Hz, P-O-CH2-CH3), 61.22 (d, JCÀOÀP =6.3 Hz, P-O-CH2-
In vitro antimalarial activity of ANP prodrugs: P. falciparum D6
(Sierra-Leone) laboratory line, sensitive to most antimalarial drugs
and W2 (Indochina) line, resistant to chloroquine and pyrimetha-
mine, were maintained in RPMI-1640-LPLF complete medium, con-
taining 10% human plasma, at 4% hematocrit and 1% to 8% para-
sitemia.[37] Cultures were routinely synchronized using d-sorbitol.[38]
To evaluate the antimalarial activity of the ANPs, the [3H]-hypoxan-
thine growth inhibition assay[39] was used, where the uptake of
[3H]-hypoxanthine by malaria parasites is used as a surrogate
marker for parasite growth. For these assays, stock solutions of
ANPs were made to concentrations of 20–40 mm in DMSO or
water and subsequently diluted in hypoxanthine-free complete
media prior to assay. The assays (in 96-well plate format) were initi-
ated when the majority of parasites (>90%) at early trophozoite
(ring) stage. Parasite cultures (100 mL per well) at 0.5% initial para-
sitemia and 2% hematocrit in hypoxanthine-free RPMI1640-LPLF
medium were exposed to ten 2-fold serial dilutions of the ANPs
and chloroquine (reference drug) for 96 h, with [3H]-hypoxanthine
(0.2 mCiwellÀ1) added ~48 h after beginning of the experiment.
The [3H]-hypoxanthine incorporation data were analyzed and sig-
moidal growth inhibition curves were produced by non-linear re-
gression analysis of the [3H]-hypoxanthine incorporation data
versus log-transformed concentrations of the compounds using
GraphPad Prism V5.0 software (GraphPad Software Inc. USA), from
which the inhibitory concentration (IC50) that cause 50% of para-
site growth were determined. The IC50 values were based on at
least two independent experiments with mean ÆSD calculated.
CH3), 40.14 (C1’), 26.46 (d, JCÀP =135.9 Hz, C5’), 16.42 ppm (d,
J
CÀCÀOÀP =5.8 Hz, P-O-CH2-CH3); MS-ESI+ m/z (%): 406 (20) [M+H]+,
428 (100) [M+Na]+; HRMS-ESI+: m/z calcd for C14H25O7N5P [M+
H]+ 406.1486, found 406.1486; FTIR (KBr): n˜ =3169, 1712, 1685,
1593, 1539, 1460, 1382, 1360, 1248, 1226, 1100, 1050, 960 cmÀ1
[a]2D0 =À4.0 (c=0.276 g/100 mL, MeOH).
;
(S)-(2-((1-(8-oxoguanin-9-yl)-3-hydroxypropan-2-yl)oxy)ethyl)-
phosphonic acid (43): Treatment of compound 42 (203 mg,
0.5 mmol) by procedure B followed by procedure A gave the title
compound as a yellowish solid (135 mg, yield 77%). H NMR (D2O):
d=3.91 (dd, Jgem =14.6 Hz, J1’aÀ2’ =5.1 Hz, 1H, H-1’a), 3.87 (dd,
J
3.71 (dd, Jgem =12.3 Hz, J3’aÀ2’ =3.9 Hz, 1H, H-3’a), 3.55 (dd, Jgem
12.3 Hz, J3’bÀ2’ =6.0 Hz, 1H, H-3’b), 1.74–1.91 ppm (m, 2H, H-5’);
13C NMR (D2O): d=154.55 (C8), 154.21 (C6), 153.39 (C2), 150.09
(C4), 99.77 (C5), 78.23 (C2’), 66.44 (d, JCÀCÀP =1.6 Hz, C4’), 61.75
(C3’), 40.59 (C1’), 29.57 ppm (d, JCÀP =129.8 Hz, C5’); MS-ESI+ m/z
(%): 350 (100) [M+H]+, 372 (65) [M+Na]+; HRMS-ESI+: m/z calcd
for C10H17O7N5P [M+H]+ 350.0860, found 350.0860; FTIR (KBr): n˜ =
3444, 3366, 3205, 2646, 1684, 1652, 1607, 1538, 1461, 1222, 1126,
1047, 1014, 978, 964 cmÀ1; [a]D20 =À7.3 (c=0.399 g/100 mL, H2O).
1
gem =14.6 Hz, J1’bÀ2’ =6.0 Hz, 1H, H-1’b), 3.73–3.87 (m, 3H, H-2’, 4’),
=
Biological assays
Determination of Ki values: Human HGPRT was stored in 0.1m Tris-
HCl, 0.01m MgCl2, pH 7.4, 200 mm PRib-PP, 1 mm DTT, À808C.
PfHGXPRT was stored in 0.01m phosphate, 60 mm Hx, 200 mm PRib-
PP, pH 7.2, 1 mm DTT as previously described.[9] This difference is
because the Pf enzyme is completely inactive under the conditions
used to store the human enzyme. The human and Pf enzymes con-
tain a hexa-histidine tag at the N-terminal. For enzyme assays for
human HGPRT, the buffer was 0.1m Tris-HCl, 0.01m MgCl2, pH 7.4.
For the Pf enzyme, the assays were performed in 0.01m phosphate,
0.01m MgCl2. The Ki values were calculated by Hanes’ plots at
a fixed concentration of guanine (60 mm) and at variable concentra-
tions of PRib-PP (14–1000 mm) depending on the KM(app) in the pres-
ence of the inhibitor.
Abbreviations: ANP: acyclic nucleoside monophosphonate, PRib-PP:
5-phospho-a-d-ribosyl-1-pyrophosphate, PEE: 2-(phosphonoeth-
oxy)ethyl,
HPMP:
(S)-3-hydroxy-2-(phosphonomethoxy)propyl,
ImmGP: (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-d-ribitol
5-phosphate, HPEP: (S)-3-hydroxy-2-(phosphonoethoxy)propyl,
CPME: (S)-2-(phosphonomethoxy)propanoic acid, CPEE: (S)-2-(phos-
phonoethoxy)propanoic acid, HG(X)PRTs : hypoxanthine-guanine-
(xanthine) phosphoribosyltransferases.
Acknowledgements
Crystallization and structure determination: For crystallization ex-
periments, human HGPRT was concentrated and stored as previ-
ously described.[9] The hanging drop method was used where 1 mL
of enzyme in complex with the inhibitor was added to 1 mL of well
solution. The trays were then incubated at 188C. The reservoir so-
lution for compound 25 was 20% PEG3000, 0.2m calcium acetate,
0.1m Tris-HCl, pH 7.4. For compound 26, the reservoir solution was
the same except that the concentration of PEG3000 was decreased
to 15%. The crystals were placed in a cryostream (100 K). X-ray
data were collected remotely by BLU-ICE[32] using beamline MX1 at
the Australian synchrotron. Data were scaled and merged using
XDS.[33] The structure was solved by molecular replacement with
the program PHASER[34] within PHENIX 1.8-1069[35] with the protein
coordinates of the tetramer of human HGPRT in complex with ([(2-
[(guanine-9H-yl)methyl]propane-1,3-diyl)bis(oxy)bis(methylene))di-
phosphonic acid (PDB code: 4IJQ). Subsequent refinement of the
The initial crystallographic conditions were determined using the
Mosquito and Rockimager facilities at the University of Queens-
land Remote-Operation Crystallization facility (UQROCX). Prelimi-
nary X-ray data were measured at this facility. Final measure-
ments were made at beamline MX1, Australian Synchrotron,
Clayton, Victoria with the assistance of Tom Caradoc-Davies. The
views expressed here are those of the authors and not necessarily
those of the Australian Synchrotron. We thank the technical ex-
cellence of Kerryn Rowcliffe for in vitro antimalarial drug testing
and the Australian Red Cross Blood Service for the provision of
human blood and sera for in vitro cultivation of P. falciparum
lines. The opinions expressed are those of the authors and do
not necessarily reflect those of the Australian Defence Organi-
sation or any extant policy. This study is a part of research proj-
ChemMedChem 2015, 10, 1707 – 1723
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Full Papers
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Biochemistry, Academy of Sciences of the Czech Republic and
was supported by MV CR (VG20102015046), a grant from the
Czech Science Foundation (P207/11/0108), funds from the Na-
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Received: July 22, 2015
Published online on August 25, 2015
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim