A novel type of acyclic nucleoside phosphonates derived from 2-(phosphonomethoxy)propanoic acid

Article abstract of DOI:10.1016/j.tet.2012.03.066

A convenient and efficient synthesis of a novel class of acyclic nucleoside phosphonates derived from 2-(phosphonomethoxy)propanoic acid has been developed. The key step of the synthesis is the optimized oxidation of the 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) analogues to the corresponding 2′-carboxy-PME (CPME) derivatives using the TEMPO/NaClO₂/NaClO oxidizing system. Although (S)-3-(adenin-9-yl)-2-(phosphonomethoxy)propanoic acid ((S)-CPMEA) has been designed as a compound with potential anti-HIV activity, none of the newly prepared CPME analogues exhibited any antiviral activity.

Full text of DOI:10.1016/j.tet.2012.03.066

Tetrahedron 68 (2012) 4003e4012
Contents lists available at SciVerse ScienceDirect
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A novel type of acyclic nucleoside phosphonates derived from
2-(phosphonomethoxy)propanoic acid
*
ꢀ
ꢁ
Martin Maxmilian Kaiser, Petr Jansa, Martin Dracínsky, Zlatko Janeba
ꢁ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient and efficient synthesis of a novel class of acyclic nucleoside phosphonates derived from 2-
(phosphonomethoxy)propanoic acid has been developed. The key step of the synthesis is the optimized
oxidation of the 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) analogues to the corresponding 20-
carboxy-PME (CPME) derivatives using the TEMPO/NaClO2/NaClO oxidizing system. Although (S)-3-(ad-
enin-9-yl)-2-(phosphonomethoxy)propanoic acid ((S)-CPMEA) has been designed as a compound with
potential anti-HIV activity, none of the newly prepared CPME analogues exhibited any antiviral activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 10 October 2011
Received in revised form 27 February 2012
Accepted 19 March 2012
Available online 26 March 2012
Keywords:
Acyclic nucleoside phosphonates
CPMEA
HPMPA
PMEA
Oxidation
TEMPO
Microwave
Antiviral
1. Introduction
prototype of ANPs derived from (S)-DHPA (5), possesses broad-
spectrum activity against DNA viruses (including herpes-, adeno-,
Acyclic nucleoside phosphonates (ANPs) constitute a distin-
guished class of antiviral agents.1 They also exhibit important cy-
tostatic,2 antiparasitic3 and immunomodulatory activities.4 The
main advantages of ANPs are their resistance towards degradation
by enzymes in vivo, and the ability to by-pass the first phosphor-
ylation step necessary for the activation of the bioactive nucleo-
sides.5 Three ANPs have been approved worldwide for clinical use:5
HPMPC (cidofovir, VistideÒ) for the treatment of CMV retinitis in
AIDS patients,5,6 PMEA (1, Fig. 1) (as its oral prodrug adefovir
dipivoxil, HepseraÒ) for the treatment of chronic HBV infections,5
and PMPA (2, Fig. 1) (as its oral prodrug tenofovir disoproxil fu-
marate, VireadÒ) for the treatment of HIV (itself or in combination
with other antivirals) and chronic HBV infections.5 Many other
ANPs were found to exhibit important antiviral properties, e.g.,
derivatives of 2,6-diaminopurine (HPMPDAP, PMEDAP, PMPDAP)1f
and ‘open ring’ derivatives of 2,4-diaminopyrimidine (HPMPOeD-
APy, PMEOeDAPy, PMPOeDAPy).1f,7 Like nucleotides,8 due to their
poor bioavailability, all ANPs have to be administered in the form of
their prodrugs (or by injection in the case of cidofovir).9
pox- and iridoviruses), as well as against retroviruses. While only
the (S)-HPMP enantiomer 3 is active in the adenine series, its 30-O-
phosphonomethyl regioisomer (iso-HPMPA)10 exhibits no antiviral
activity.1a The approved adenine analogues PMEA (1) and PMPA (2)
are primarily active against hepadna- and retroviruses, while PMEA
(1) also exhibits notable activity against herpesviruses.5 Finally,
FPMPA (4), which proved to be less antivirally effective, but also less
toxic than PMEA (1), was thoroughly studied for its antiretroviral
activity but was not pursued further to the clinical use.11
A number of other adenine derivatives proved to have important
antiviral activities.1f Acyclic nucleoside analogues like (S)-DHPA (5,
Fig. 1), marketed in Czechoslovakia as DuviraÒ gel,12 or structurally
related
D
-eritadenine13 and 3-(adenin-9-yl)-2-hydroxypropanoic
acid (6, AHPA)14 constitute another class of broad-spectrum anti-
virals. These acyclic analogues of adenosine are targeted at S-ade-
nosyl-L
-homocysteine hydrolase,15 and thus at the capping process
(methylation of 50-end-guanine of viral mRNA).16
Herein we describe the synthesis and biological properties of
the novel type of acyclic nucleoside phosphonates structurally re-
lated to all above-mentioned biologically important compounds.
The novel type of ANPs is derived from 2-(phosphonomethoxy)
propanoic acid, with the adenine derivative (S)-20-carboxy-PMEA
(7a, (S)-CPMEA, Fig. 1) being the most promising candidate with
potential biological properties.
Analogues containing adenine play a special role among ANPs
for their potent antiviral properties. HPMPA (3, Fig. 1),1a the
* Corresponding author. Tel.: þ420 220183143; fax: þ420 220183560; e-mail
addresses: janeba@uochb.cas.cz, zjaneba@yahoo.com (Z. Janeba).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.03.066

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M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
Fig. 1. Examples of biologically important acyclic nucleoside and nucleotide analogues 1e6 and structure of the target compound 7a.
2. Results and discussion
the properly protected HPMPA followed by the removal of the
protecting groups and (b) alkylation of adenine (or 6-chloropurine)
with a suitable alkylating agent containing the protected carboxylic
function, followed by deprotection (and ammonolysis in the case of
6-chloropurine).
The X-ray structure of HIV-1 RTeDNA complex after in-
corporation of tenofovir diphosphate is an ideal starting point for
further rational and targeted drug design (Fig. 2a).17 In the complex,
two amino acid residues (Arg 72 and Gln 151) in the binding site of
the HIV-1 RT are oriented towards the C-20 methyl group of teno-
fovir diphosphate (Fig. 2a). Apparent incapability of the C-20 methyl
group to substantially interact with the two polar amino acids of
the HIV-1 RT led us to reason that replacement of the C-20 methyl
with a polar group able to interact with Arg 72 and/or Gln 151
would increase the binding affinity of such an inhibitor to HIV-1 RT.
Our docking studies indicated that replacement of the C-20 methyl
group of tenofovir diphosphate (Fig. 2a) with a carboxyl group
could, theoretically, lead to formation of up-to four new hydrogen
bonds with arginine 72 and glutamine 151 (Fig. 2b).
The initial docking studies indicated that (S)-CPMEA (7a, Fig. 1),
an analogue of PMEA (1) bearing a carboxyl group at the C-20 po-
sition of the aliphatic chain, potentially is a good candidate for
strong binding affinity in the enzyme pocket of HIV-1 RT. Moreover,
(S)-CPMEA (7a) can be considered to be a direct structural modi-
fication of PMPA (2) or HPMPA (3) with oxidized 20-methyl or 20-
hydroxymethyl functions, respectively, or can also be looked upon
as an O-phosphonomethyl derivative of AHPA (6, Fig. 1). Thus,
compound 7a, as well as the other CPME analogues, was expected
to display a wide range of biological properties.
The oxidation of the protected HPMPA derivative seems to be
the more relevant and straightforward method for the synthesis of
the compound 7a, while the ‘synthon’ approach seems to be too
laborious and less effective. Thus, diisopropyl (S)-9-[3-hydroxy-2-
(phosphonomethoxy)propyl]adenine (8a, Scheme 1) was chosen as
the suitable starting material. This can be prepared in sufficient
amounts according to the previously described procedures.18
Nowadays, there are a considerable number of oxidizing agents,
which can be used under relatively mild reaction conditions and
which tolerate number of functional groups.19 Our goal was to
develop a simple, mild and clean oxidation of the HPMPA derivative
8a, which would tolerate the presence of the unprotected amino
group at the C-6 position of the purine moiety.
The conversion of the 50-hydroxyl group of 20,30-protected pu-
rine nucleosides to the corresponding 50-uronic acids is often ac-
complished by oxidation with an excess of KMnO4 in aqueous KOH
solution under conditions of high dilution.20 An attempted oxida-
tion of compound 8a under analogous reaction conditions only led
to complex reaction mixtures (TLC).
A simple and efficient oxidative conversion of primary alcohols
to the corresponding methyl esters using I2/K2CO3 in MeOH has
been reported by Mori and Togo.21 Nevertheless, an attempt to
prepare the methyl ester of derivative 9a by the direct oxidative
Generally, two strategies could be utilized for the synthesis of
the target analogue, (S)-CPMEA (7a, Fig. 1): (a) direct oxidation of
Fig. 2. (a) Immediate binding site of the complex of HIV-1 RTeDNA and tenofovir diphosphate. (b) Docking of the complex of HIV-1 RT-DNA and (S)-CPMEA (7a) showing possible
hydrogen bonding (dotted green lines) between the carboxyl group of (S)-CPMEA diphosphate and Arg 72 and Gln 151.

M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
4005
Scheme 1. Synthesis of (S)-CPMEA (7a) and (R)-CPMEA (7b) through oxidation of the HPMPA derivatives 8a and 8b.
esterification of compound 8a under the reported reaction condi-
tions was not successful (Table 1).
offers a clean reaction and easy work-up based on the isolation of
the product by flash chromatography on silica gel followed by
crystallization. This efficient procedure offered compound 9a in
very high purity.
Finally, the desired product (S)-CPMEA (7a) was obtained in
a 76% yield by ester cleavage of compound 9a under the standard
conditions (Me3SiBr in MeCN).28
Analogously, oxidation of enantiomeric diisopropyl (R)-HPMPA
(8b) using the TEMPO/NaClO2/NaClO system afforded the de-
rivative 9b in a 70% yield. Subsequent deprotection of compound 9b
gave the free phosphonic acid 7b in a 50% yield.
Table 1
Oxidation of diisopropyl HPMPA (8a) to diisopropyl (S)-CPMEA (9a)
Oxidative agent; solvent
Desired product
Isolated yield
KMnO4/KOH; H2O
9a
Complex mixture
I2/K2CO3; MeOH
Methyl ester of 9a
Complex mixture
RuO2/NaIO4; MeCN, CH2Cl2, H2O
TEMPO/BAIB; MeCN, H2O
TEMPO/NaClO2/NaClO; MeCN, H2O
9a
9a
9a
63%
71%
87%
Unfortunately, none of the oxidizing systems listed in Table 1
afforded (S)-CPMEA (7a) by direct oxidation of free HPMPA (3,
Fig. 1), confirming the importance of the diester protection of the
phosphonate group during the procedure, and synthetic trans-
formations in general.
Eventually, we decided to verify the more general applicability of
our optimized oxidation protocol, and it was utilized for the prep-
aration of CPME derivatives bearing other nucleobases (Scheme 2,
Table 2). Thus, the expected diisopropyl CPME analogues 9 were
obtained by the oxidation of the corresponding HPMP derivatives 8
with TEMPO/NaClO2/NaClO in high yields (76e87%).
The functional groups of the unprotected nucleobases are usu-
ally very well tolerated during the oxidation process. The only
problem was observed with compounds bearing 2-aminopurine
bases containing a guanidine motif (guanine and 2,6-diamino
purine). Treatment of the HPMPG derivative 8g18b and HPMPDAP
derivative 8h18b with TEMPO/NaClO2/NaClO gave complex and
colourful reaction mixtures and no desired products were isolated
(entries 7 and 8, respectively, Table 2).
To overcome the undesirable reactivity of the 2-aminopurine
bases, we decided to protect the amino groups of compounds 8g
and 8h with the benzoyl group. Thus, the N2-benzoylguanine de-
rivative 8i18b was prepared in 49% yield from compound 8g in
pyridine by sequential treatment with Me3SiCl, BzCl, water and
aqueous ammonia (Scheme 3). Subsequent oxidation of the benzoyl
derivative 8i with TEMPO/NaClO2/NaClO afforded the carboxy an-
alogue 9i in 78% yield (Table 2, entry 9). Direct deprotection of the
phosphonate moiety of compound 9i using Me3SiBr in acetoni-
trile28 afforded the N2-benzoylguanine derivative 7i in 52% yield
(Table 2, entry 9, Scheme 3). In order to obtain CPMEG (7g), the
benzoyl group of derivative 9i was removed first, using MeONa
solution, followed by the removal of the isopropyl groups from the
intermediate 9g (Scheme 3).
Ruthenium tetroxide (RuO4), another widely used oxidizing
agent,19 is ideal when a very vigorous oxidizing agent is needed but
mild reaction conditions must be maintained. Since RuO4 can de-
compose explosively, it is mostly prepared in situ by oxidation of
RuCl3 or RuO2. A mild oxidation of nucleosides to the corresponding
50-carboxylic acids by use of RuCl3/NaIO4 and RuCl3/K2S2O8/KOH
systems has been reported.22 Since RuCl3 was found to be a very
hygroscopic reagent, not easy to handle, RuO2 was our oxidizing
agent of choice.
Oxidation of diisopropyl HPMPA (8a) with RuO2/NaIO4 afforded
the derivative 9a in a satisfactory yield (63%, Table 1). The oxidation
was carried out in a mixture of acetonitrile, chloroform, and water
(1:1:2) according to the described procedure used for the prepa-
ration of adenosine 50-uronamides,23 with an addition of concen-
trated HCl to pH 2.5. Despite the good isolated yield, purification
was tedious and column chromatography had to be repeated sev-
eral times to obtain pure (S)-CPMEA derivative 9a, probably owing
to the fact that phosphonates strongly bind metal ions. Thus,
a cleaner and less laborious procedure was still desirable.
Commercially available 2,2,6,6-tetramethyl-1-piperidinyloxyl
(TEMPO) is a reagent of choice for oxidation of various organic
substrates.24 TEMPO belongs to the class of stable organic nitroxyl
radicals commonly used for the oxidation of primary and secondary
alcohols.19,24
Mild and efficient oxidation of alcohols to ketones and aldehydes
using catalytic amounts of TEMPO and stoichiometric amounts of
[bis(acetoxy)iodo]benzene (BAIB) has been developed.25 This
method differs from most other TEMPO-mediated oxidations in that
it generates acetic acid and iodobenzene as by-products and avoids
inorganic contaminants.25 This oxidizing system was successfully
employed for the oxidation of 20,30-protected nucleosides to give
the corresponding 50-carboxylic acids.26 In our case, oxidation of
HPMPA derivative 8a with the TEMPO/BAIB oxidizing system
afforded the expected product 9a in a 71% yield (Table 1).
An even better yield (87%) of the (S)-CPMEA derivative 9a was
achieved when BAIB was replaced with NaClO2/NaClO (sodium
Analogously, the HPMPDAP analogue 8h was protected as its
dibenzoyl derivative 8j (in 47% yield), which was then efficiently
oxidized using the TEMPO/NaClO2/NaClO system to compound 9j in
80% yield (Table 2, entry 10, Scheme 4). Finally, CPMEDAP (7h) was
obtained in 38% yield from compound 9j by simultaneous hydro-
lysis of both benzoyl groups and diisopropyl esters using 1 M
hypochlorite is
a readily available and inexpensive oxidant,
household bleach). This oxidizing system (TEMPO/NaClO2/NaClO)27

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M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
Scheme 2. Synthesis of CPME compounds 7 starting from the HPMP analogues 8.
Table 2
Oxidation of the HPMP analogues 8 with TEMPO/NaClO2/NaClO to give 9, followed by diester removal to yield 7
Entry
Nucleobase B
Starting compound
Product (yield)a of oxidation
Product (yield)a of the
phosphonate deprotection
1
2
8a (S)-
8b (R)-
9a (87%)
9b (76%)
7a (68%),b (46%)c
7b (50%)b
3
4
8c (S)-
9c (83%)
9d (82%)
7c (62%),b (39%)c
8d (S)-
7dd (64%),b (44%)c
5
6
8e (S)-
8f (R)-
9e (81%)
9f (80%)
7e (42%)c
7f (48%)c
7
8
8g (S)-
Complex mixture (no 9g isolated)e
7g (61%),b (37%)c
8h (S)-
Complex mixture (no 9h isolated)
7h (38% from 9j)c,f
7i (52%)b
9
8i (S)-
9i (78%)

M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
4007
Table 2 (continued )
Entry
Nucleobase B
Starting compound
Product (yield)a of oxidation
Product (yield)a of the
phosphonate deprotection
10
8j (S)-
9j (80%)
N.A.g
a
Isolated yield.
Method A.
Method B.
b
c
d
Compound 7d contains thymine as a base.
Compound 9g prepared from 9i (Scheme 3).
Compound 7h prepared from 9j (Scheme 4).
N.A. not applicable.
e
f
g
Scheme 3. Synthesis of CPMEG (7g) using TEMPO/NaClO2/NaClO as a key step.
Scheme 4. Synthesis of CPMEDAP (7h) using TEMPO/NaClO2/NaClO as a key step.

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M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
aqueous HCl under microwave irradiation (Scheme 4), a general
method for the convenient deprotection of phosphonate diesters
recently developed in our laboratory.29
on a Q-Tof micro (Waters) and HRMS were taken on an LTQ Orbitrap
XL (Thermo Fisher Scientific) spectrometer. 1H and 13C NMR spectra
were recorded on a Bruker Avance 400 (1H at 400 MHz and 13C
100.6 MHz), Bruker Avance 500 (1H at 500 MHz and 13C 125.7 MHz)
and Bruker Avance 600 (1H at 600.1 MHz and 13C 150.9 MHz) in
All of the prepared diisopropyl CPME phosphonates 9 were
deprotected to the final free phosphonic acids 7 (Table 2, Scheme 2).
As already mentioned above, two general methods for the removal
of alkyl esters from the phosphonate group can be employed: (a)
commonly used deprotection with Me3SiBr in acetonitrile followed
by hydrolysis (method A),28 (b) recently described microwave-
assisted hydrolysis using aqueous HCl (method B).29 Method A
gave better yields (50e68%) of the free phosphonic acids compared
to the method B (37e46%). Diisopropyl phosphonate 9d, with the
methoxy group in position C-4 of the pyrimidine ring, afforded
under the deprotection conditions (by both methods A and B) the
corresponding thymine derivative 7d (Table 2, entry 4).
All newly prepared CPME analogues 7 were tested for their
antiviral properties. Although completely nontoxic in all assays,
none of the compounds (including the most promising (S)-CPMEA
(7a)) exhibited any promising activity against the HIV-1 virus or
any other viruses tested (HSV-1 (KOS), HSV-1 (KOS TKꢀ), HSV-2 (G),
RSV, Vaccinia virus, Vesicular stomatitis virus, Parainfluenza-3 vi-
rus, Reovirus-1, Sindbis virus, Coxsackie B4 virus, Punta Toro virus,
Feline Corona virus and Feline Herpes virus). One of the obvious
reasons for the lack of the biological properties of the CPME de-
rivatives 7 may be their poor bioavailability caused by their high
polarity (CPME derivatives are even more polar that the corre-
sponding PME, PMP and HPMP analogues). Since the majority of
the therapeutically successful ANPs have to be administered in the
form of their prodrugs, the preparation of various prodrugs of the
lead (S)-CPMEA (7a) is currently in progress and will be reported
elsewhere.
DMSO-d6 (referenced to the solvent signal
respectively), or in D2O (referenced to dioxane as an internal
standard
¼ppm). Complete assignment is based on heteronuclear
correlation experiments HSQC and H,C-HMBC. Chemical shifts (
d¼2.50 and 39.70 ppm,
d
d
)
are in parts per million and coupling constants (J) in hertz. UV
spectra were taken on a Beckman DU-65 spectrophotometer in
methanol or water solution. IR spectra were obtained on a FT IR
Bruker Equinox IFS 55 spectrometer in KBr pellets.
The microwave-assisted reactions were carried out in CEM
Discover (Explorer) microwave apparatus, 24-position system for
10-mL vessels sealed with a Teflon septum. It was operated at
a frequency of 2.45 GHz with continuous irradiation power from
0 to 300 W. The solutions were steadily stirred during the reaction.
The temperature was measured with an IR sensor on the outer of
the process vessel. The vials were cooled to ambient temperature
with gas jet cooling system. The pressure was measured with an
inboard CEM Explorer pressure control system (0e21 bar).
ꢁ
Household bleach SAVO purchased from BOCHEMIE a.s. (Lidicka
326, 735 95 Bohumín, Czech Republic) and the concentration of
sodium hypochlorite was determined by iodometric titration
(40.7 mg/mL). Starting HPMP analogues were prepared according
to the published procedures.18
4.2. Computational details
The docking studies (Fig. 2) were performed with the Argus-
Lab30 program with the use of the ‘ArgusDock’ algorithm. An ex-
haustive search was performed by enabling the ‘High precision’
option in the Docking precision menu, ‘Dock’ was chosen as the
3. Conclusion
ꢂ
A novel acyclic nucleoside analogue, (S)-3-(adenin-9-yl)-2-
(phosphonomethoxy)propanoic acid (7a, (S)-CPMEA), was
designed as an inhibitor of HIV-1 RT and its synthesis was de-
veloped and optimized. The key step of the (S)-CPMEA (7a) syn-
thesis consists of oxidation of the (S)-HPMPA derivative 8a with
TEMPO/NaClO2/NaClO to give compound 9a in a high yield (87%).
Subsequently, a whole series of CPME derivatives 9 was prepared in
high yields from the corresponding HPMP analogues 8 by the op-
timized oxidative methodology using TEMPO. The oxidation pro-
cess tolerates the functional groups present on the common
nucleobases (A, C, T, U), with the exception of compounds con-
taining the amino group in position C-2 of the purine ring (i.e., 8g
and 8h). Compounds 8g and 8h had to be oxidized as their benzoyl
derivatives 8i and 8j, respectively. Finally, two methods were
employed for the deprotection of the phosphonate diesters 9: (a)
treatment with Me3SiBr in acetonitrile followed by hydrolysis; (b)
microwave-assisted hydrolysis with aqueous HCl. Unfortunately,
none of the newly synthesized CPME compounds (including the
most promising (S)-CPMEA) showed any interesting activity against
the viruses tested.
calculation type, and ‘flexible’ for the ligand. Spacing of 0.2 A be-
tween the grid points was used. The atomic co-ordinates for the X-
ray structures of the HIV-1 RTeDNA complexes after incorporation
of the anti-HIV drug tenofovir were downloaded from the Protein
Data Bank (PDB code: 1T05).17 The final inhibitoreprotein complex
was visualized using ArgusLab.30
4.3. Synthesis of the N-benzoyl derivatives 8i and 8j
Starting HPMP derivative 8g or 8h (1.5 mmol) was dissolved in
dry pyridine (20 mL) in a round-bottomed flask with a drying tube,
and the reaction mixture was cooled in an ice bath. Me3SiBr
(7.5 mmol) was added to the reaction mixture and after 30 min
benzoyl chloride (7.5 mmol) was added. The reaction flask was
removed from the ice bath and the mixture was stirred for 2 h at
room temperature. Then the reaction mixture was cooled in ice
bath and cold water (4 mL) was added, followed after 15 min of
stirring by concentrated aqueous ammonia (4 mL). The mixture
was stirred at room temperature for another 30 min and solvents
were removed in vacuo to give crude oil. The chromatography on
silica gel column (0e3% methanol in chloroform) afforded the
product 8i or 8j as thick yellowish oil.
4. Experimental section
4.1. General
4.3.1. Diisopropyl
(S)-N2-benzoyl-9-[3-hydroxy-2-(phosphonome-
thoxy)propyl]guanine (8i).18b Yield 49%, yellowish solid (recryst
EtOAc/hexane), mp 171e173 ꢁC (mp 173e174 ꢁC);18b MS-ESIþ m/z
(%) 508 (15, MþHþ), 530 (100, MþNaþ); HRMS-ESIþ: m/z calcd for
C22H31O7N5P (MþHþ) 508.1956, found 508.1954. 1H NMR (DMSO-
d6) spectrum is identical with the original spectroscopic data.18b
Analytical TLC was performed on silica gel pre-coated alumin-
ium plates with fluorescent indicator (Merck 5554, 60 F254). Spots
were visualized with UV light (254 nm) or by spraying with 4-
nitrobenzylpyridine (2% solution in ethanol) followed by short
heating to 600 ꢁC and spraying with concentrated ammonia (26%
solution in water). Column chromatography was carried out on
4.3.2. Diisopropyl (S)-N2,N6-dibenzoyl-9-[3-hydroxy-2-(phosphono-
silica gel (Sigma S-0507, 40e63
mm). Mass spectra were measured
methoxy)propyl]-2,6-diaminopurine (8j). Yield 47%, thick yellowish

M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
4009
oil; 1H NMR (DMSO-d6)
d
: 11.16 (s, 1H, NH), 11.01 (s, 1H, NH), 8.31 (s,
J¼6.2 Hz, 3H, CH3ipr); 13C NMR (DMSO-d6)
d
: 170.61 (C-30), 155.93
1H, H-8), 8.06 (m, 2H, H-200), 7.99 (m, 2H, H-200), 7.50e7.66 (m, 6H,
(C-6), 152.37 (C-2), 149.75 (C-4), 141.47 (C-8), 118.55 (C-5), 77.99 (d,
H-300, 400), 5.04 (t, JOHeCH2¼5.7 Hz,1H, OH), 4.44e4.560(m, 3H, CHipr,
J2 -P¼12.7 Hz, C-20), 70.58 (d, JCeOeP¼6.5 Hz, CHipr), 70.53 (d,
0
H-10a), 4.35 (dd, 1H, Jgem¼14.7 Hz, J1 be2 ¼6.4 Hz, H-1 b), 3.89e3.94
(m, 3H, H-20, CH2P), 3.50 (m, 1H, H-30), 3.43 (m, 1H, H-30), 1.20 (d,
JCH3eCH¼6.2 Hz, 3H, CH3ipr), 1.17 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr),
1.16 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr), 1.13 (d, JCH3eCH¼6.2 Hz, 3H,
JCeOeP¼6.5 Hz, CHipr), 64.08 (d, JCeP¼163.9 Hz, CH2P), 44.36 (C-10),
23.92 (d, JCeCeOeP¼4.0 Hz, CH3ipr), 23.88 (d, JCeCeOeP¼3.9 Hz,
CH3ipr), 23.78 (d, JCeCeOeP¼4.5 Hz, CH3ipr), 23.67 (d,
JCeCeOeP¼4.5 Hz, CH3ipr); MS-ESIþ m/z (%) 402 (60, MþHþ), 825
(100, 2MþNaþ); HRMS-ESIþ: m/z calcd for C15H25O6N5P (MþHþ)
0
0
CH3ipr); 13C NMR (DMSO-d6)
d: 165.86 (CON), 165.74 (CON), 153.90
(C-4), 152.30 (C-2), 150.68 (C-6), 145.28 (C-8), 134.46 (C-100), 133.56
(C-100), 132.64 (C-400), 132.20 (C-400), 128.70 (C-200, 300), 128.63 (C-200,
402.1537, found 402.1536; FTIR (KBr, cmꢀ1
) n: 3396, 3181, 2981,
1688, 1651, 1599, 1233, 996. [
a]
20 ꢀ26.2 (c 0.302, MeOH).
D
300), 128.59 (C-200, 300), 128.28 (C-200, 300), 123.08 (C-5), 79.91 (d, J2 -
0
¼11.5 Hz, C-20), 70.48 (d, JCeOeP¼6.3 Hz, CHipr), 63.57 (d,
4.6.2. (R)-3-(6-Amino-9H-purin-9-yl)-2-[(diisopropoxyphosphono)
P
JCeP¼165.1 Hz, CH2P), 59.97 (C-30), 43.36 (C-10), 23.78e24.00 (m,
CH3ipr); MS-ESIþ m/z (%) 611 (5, MþHþ), 633 (100, MþNaþ), 655
(12, Mþ2Naþ); HRMS-ESIþ: m/z calcd for C29H36O7N6P (MþHþ)
611.2378, found 611.2376.
methoxy]propanoic acid (9b). Yellowish crystals, dec >150 ꢁC, yield
76%; 1H NMR (DMSO-d6)
d: 8.15 (s, 1H, H-2), 8.03 (s, 1H, H-8), 7.28
(br s, 2H, NH2), 4.37e4.55 (m, 5H, H-10, H-20, CHipr), 3.95 (dd,
Jgem¼13.7 Hz, JHeCeP¼8.7 Hz, 1H, CH2Pb), 3.73 (dd, Jgem¼13.7 Hz,
JHeCeP¼9.4 Hz, 1H, CH2Pa), 1.18 (d, J¼6.2 Hz, 3H, CH3ipr), 1.14 (d,
J¼6.2 Hz, 3H, CH3ipr), 1.13 (d, J¼6.2 Hz, 3H, CH3ipr), 1.07 (d,
4.4. Oxidation of diisopropyl (S)-HPMPA (8a) with ruthenium
tetroxide
J¼6.2 Hz, 3H, CH3ipr); 13C NMR (DMSO-d6)
d
: 170.61 (C-30), 155.93
(C-6), 152.37 (C-2), 149.75 (C-4), 141.47 (C-8), 118.55 (C-5), 77.99 (d,
J2 -P¼12.7 Hz, C-20), 70.58 (d, JCeOeP¼6.5 Hz, CHipr), 70.53 (d,
0
Compound 8a (1.0 mmol, 387 mg), RuO2 (0.15 mmol, 20 mg)
and NaIO4 (5.0 mmol, 1.1 g) were vigorously stirred in a mixture of
CH3CN (15 mL), CHCl3 (15 mL) and H2O (30 mL). Concentrated HCl
(eight drops) was added to wpH 2.5. The heterogenous mixture
was vigorously stirred at room temperature for 72 h. The solid was
removed by filtration of the reaction mixture through a silica gel
layer and washed with methanol. The solvents were evaporated
and the residue was purified by column chromatography on silica
gel (CHCl3/MeOH/AcOH, 88:6:6). The purification was repeated two
more times to remove colourful by-products and inorganic salts.
The desired product 9a (253 mg, 63%) was obtained as a yellowish
oil, which upon standing in EtOAc/MeOH mixture crystallized.
JCeOeP¼6.5 Hz, CHipr), 64.08 (d, JCeP¼163.9 Hz, CH2P), 44.36 (C-10),
23.92 (d, JCeCeOeP¼4.0 Hz, CH3ipr), 23.88 (d, JCeCeOeP¼3.9 Hz,
CH3ipr), 23.78 (d, JCeCeOeP¼4.5 Hz, CH3ipr), 23.67 (d,
JCeCeOeP¼4.5 Hz, CH3ipr); MS-ESIþ m/z (%) 402 (60, MþHþ), 825
(100, 2MþNaþ); HRMS-ESIþ: m/z calcd for C15H25O6N5P (MþHþ)
402.1537, found 402.1536; FTIR (KBr, cmꢀ1
) n: 3396, 3181, 2981,
1688, 1651, 1599, 1233, 996. [
a]
20 þ28.0 (c 0.613, MeOH).
D
4.6.3. (S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl)-2-[(diisopropox-
yphosphono)methoxy]propanoic acid (9c). Yellowish crystals, dec
>150 ꢁC, yield 83%; 1H NMR (DMSO-d6)
d
: 7.43 (d, J6e5¼7.3 Hz, 1H,
H-6), 7.34 (br s,1H, NH2), 7.19 (br s,1H, NH2), 5.66 (d, J5e6¼7.3 Hz,1H,
H-5), 4.49e4.58 (m, 2H, CHipr), 4.26 (dd, JCHeCH2¼8.7 and 3.7 Hz,1H,
1-CH2-CH), 4.15 (dd, Jgem¼13.8 Hz, JCH2eCH¼3.7 Hz,1H,1-CH2a), 3.91
(dd, Jgem¼13.8 Hz, JHeCeP¼8.6 Hz, 1H, CH2Pa), 3.64e3.71 (m, 2H,
4.5. Oxidation of diisopropyl (S)-HPMPA (8a) with TEMPO/
BAIB
Compound 8a (1.0 mmol, 387 mg), TEMPO (0.2 mmol, 32 mg)
and BAIB (2.2 mmol, 644 mg) in a mixture of CH3CN (2 mL) and H2O
(2 mL) were stirred at room temperature for 24 h and the solvents
were evaporated. Flash chromatography on silica gel (CHCl3/MeOH,
4:1) afforded the product 9a (285 mg, 71%) as a yellowish oil, which
upon standing in EtOAc/MeOH mixture crystallized.
CH2Pb, 1-CH2b), 1.17e1.23 (m, 12H, CH3ipr); 13C NMR (DMSO-d6)
d:
171.04 (COOH), 165.84 (C-4), 155.22 (C-2), 147.29 (C-6), 93.24 (C-5),
77.77 (d, JCeOeCeP¼12.8 Hz, 1-CH2eCH), 70.50 (d, JCeOeP¼6.1 Hz,
CHipr), 64.15 (d, JCeP¼163.9 Hz, CH2P), 50.39 (NeCH2), 23.77e23.98
(m, CH3ipr); MS-ESIþ m/z (%) 378 (20, MþHþ), 400 (85, MþNaþ), 422
(100, Mþ2Naþ); HRMS-ESIꢀ: m/z calcd for C14H23O7N3P (MꢀHþ)
376.1279, found 376.1280; FTIR (KBr, cmꢀ1
) n: 3357, 3183, 2979, 2901,
4.6. Oxidation of diisopropyl HPMP derivatives 8 with
TEMPO/NaClO2/NaClO. General procedure
1723, 1652, 1376, 1238, 1002. [
a]
20 ꢀ75.7 (c 0.264, MeOH).
D
4.6.4. (S)-2-[(Diisopropoxyphosphono)methoxy]-3-(4-methoxy-5-
methyl-2-oxopyrimidin-1(2H)-yl)propanoic acid (9d). Yellowish
crystals, mp 82e90 ꢁC, dec at 130 ꢁC, yield 82%; 1H NMR (DMSO-d6)
The corresponding HPMP derivative 8 (1.0 mmol), TEMPO
(0.3 mmol, 47 mg) and NaClO2 (2.0 mmol, 226 mg) in a mixture of
CH3CN (10 mL) and 0.67 M aqueous sodium phosphate buffer
(10 mL, wpH 6.7) were vigorously stirred at room temperature. An
aqueous solution of sodium hypochlorite (40.7 mg/ml), prepared by
dilution of household bleach SAVO (1.0 mL) with H2O (1.0 mL) was
added. The mixture was stirred at room temperature until the re-
action was complete (w24 h) and the solvents were evaporated.
Flash chromatography on silica gel column (CHCl3/MeOH, 4:1)
afforded the desired CPME products 9 in good yields (76e87%) as
yellowish oils, which upon standing in EtOAc/MeOH mixture gave
yellowish crystals.
d
: 7.63 (q, J6-CH3¼1.0 Hz, 1H, H-6), 4.43e4.54 (m, 2H, CHipr), 4.27
0
0
0
(dd, Jgem¼13.7 Hz, J1 ae2 ¼3.3 Hz, 1H, H-1 a), 4.03 (dd, Jgem¼13.8 Hz,
0
0
0
JHeCeP¼7.8 Hz, 1H, CH2Pa), 3.96 (dd, J2 e1 ¼9.8 and 3.3 Hz, 1H, H-20 ),
0
0
3.82 (s, 3H, OCH3), 3.62 (dd, Jgem¼13.7 Hz, J1 be2 ¼9.7 Hz, 1H, H-1 b),
3.54 (dd, JHeCeP¼9.1 Hz, Jgem¼13.8 Hz, 1H, CH2Pb), 1.84 (d, JCH3-
¼1.0 Hz, 3H, 5-CH3), 1.20 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr), 1.17 (d,
6
JCH3eCH¼6.2 Hz, 3H, CH3ipr), 1.15 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr),
1.12 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr); 13C NMR (DMSO-d6)
d: 171.38
(C-30), 169.99 (C-4), 155.45 (C-2), 147.59 (C-6), 101.43 (C-5), 79.65 (d,
J2 -P¼12.5 Hz, C-20), 70.16 (m, CHipr), 63.45 (d, JCeP¼163.6 Hz,
0
CH2P), 53.95 (OCH3), 51.64 (C-10), 23.66e23.90 (m, CH3ipr), 11.81
(5-CH3); MS-ESIþ m/z (%) 407 (5, MþHþ), 429 (15, MþNaþ), 451
(100, Mþ2Naþ); HRMS-ESIþ: m/z calcd for C16H27O8N2NaP (MþHþ)
4.6.1. (S)-3-(6-Amino-9H-purin-9-yl)-2-[(diisopropoxyphosphono)
methoxy]propanoic acid (9a). Yellowish crystals, dec >150 ꢁC, yield
87%; 1H NMR (DMSO-d6)
d: 8.15 (s, 1H, H-2), 8.03 (s, 1H, H-8), 7.28
429.1397, found 429.1397; FTIR (KBr, cmꢀ1
) n: 3440, 2981, 2935,
(br s, 2H, NH2), 4.37e4.55 (m, 5H, H-10, H-20, CHipr), 3.95 (dd,
Jgem¼13.7 Hz, JHeCeP¼8.7 Hz, 1H, CH2Pb), 3.73 (dd, Jgem¼13.7 Hz,
JHeCeP¼9.4 Hz, 1H, CH2Pa), 1.18 (d, J¼6.2 Hz, 3H, CH3ipr), 1.14 (d,
J¼6.2 Hz, 3H, CH3ipr), 1.13 (d, J¼6.2 Hz, 3H, CH3ipr), 1.07 (d,
1670, 1541, 1480, 1374, 1244, 1106, 994. [
a
]
20 ꢀ84.4 (c 0.399, MeOH).
D
4.6.5. (S)-2-[(Diisopropoxyphosphono)methoxy]-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoic acid (9e). Yellowish crystals,

4010
M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
dec >150 ꢁC, yield 81%; 1H NMR (DMSO-d6)
d: 11.23 (br s, 1H, H-3),
cmꢀ1
997. [
) n: 3423, 3163, 1686, 1630, 1579, 1485, 1410, 1242, 1178, 1105,
7.49 (d, J6e5¼7.9 Hz, 1H, H-6), 5.48 (dd, J5e6¼7.9 Hz, J5e3¼2.1 Hz, 1H,
H-5), 4.49e4.59 (m, 2H, CHipr), 4.14 (dd, Jgem¼14.0 Hz,
JCH2eCH¼3.5 Hz, 1H, 1-CH2a), 4.08 (dd, JCHeCH2¼9.1 and 3.5 Hz, 1H, 1-
CH2eCH), 3.97 (dd, Jgem¼13.9 Hz, JHeCeP¼8.3 Hz,1H, CH2Pa), 3.69(dd,
Jgem¼14.0 Hz, JCH2eCH¼9.2 Hz, 1H, 1-CH2b), 3.65 (dd, Jgem¼13.9 Hz,
JHeCeP¼8.5 Hz, 1H, CH2Pb), 1.17e1.22 (m, 12H, CH3ipr); 13C NMR
a
]
20 ꢀ35.6 (c 0.407, MeOH).
D
4.8. Deprotection of diisopropyl esters 9 to free phosphonic
acids 7
(DMSO-d6)
d: 171.22 (COOH), 164.01 (C-4), 151.06 (C-2), 146.76 (C-6),
4.8.1. Method A.28General procedure. Diisopropyl esters
9
100.42 (C-5), 78.92 (d, JCeOeCeP¼11.9 Hz, 1-CH2eCH), 70.39 (m,
CHipr), 63.72 (d, JCeP¼163.7 Hz, CH2P), 49.63 (1-CH2), 23.79e23.99
(m, CH3ipr); MS-ESIþ m/z (%) 379 (5, MþHþ), 401 (60, MþNaþ), 423
(100, Mþ2Naþ); HRMS-ESIꢀ: m/z calcd for C14H22O8N2P (MꢀHþ):
(0.5 mmol) in CH3CN (10 mL) were treated with Me3SiBr (2 mL) at
room temperature overnight. Volatiles were evaporated, and the
reaction mixture codistilled with dry acetonitrile (3ꢂ10 mL) and
water (1ꢂ10 mL). Crystallization from MeOH gave the products 7
(50e68%) as white or yellowish crystals.
377.1119, found: 377.1119; FTIR (KBr, cmꢀ1
) n: 3417, 2981, 1691, 1630,
1464, 1388, 1245, 1105, 1001. [
a
]
20 ꢀ53.0 (c 0.322, MeOH).
D
4.8.2. Method B.29General procedure. A solution of diisopropyl es-
ters 9 (0.5 mmol) in 0.5 M aqueous HCl (2 mL) was heated in a mi-
crowave at 140 ꢁC for 30 min. The volatiles were evaporated, the
residue was dissolved in H2O (0.5e1.0 mL) and the product was
isolated by HPLC (gradient: 2% aqueous MeOH to 80% aqueous
MeOH). Crystallization from a water/ethanol mixture afforded the
products 7 (37e46%) as white or yellowish crystals.
4.6.6. (R)-2-[(Diisopropoxyphosphono)methoxy]-3-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)propanoic acid (9f). Yellowish crystals,
dec >150 ꢁC, yield 80%; 1H NMR (DMSO-d6), 13C NMR (DMSO-d6),
MS and FTIR spectra are identical with compound 9e. [
0.584, MeOH).
a]
20 þ68.1 (c
D
4.6.7. (S)-3-(2-Benzamido-6-oxo-1H-purin-9(6H)-yl)-2-[(diisopro-
poxyphosphono)methoxy]propanoic acid (9i). Colourless oil, yield
4.8.3. (S)-3-(6-Amino-9H-purin-9-yl)-2-(phosphonomethoxy)prop-
78%; 1H NMR (DMSO-d6)
d
: 10.59 (br s, 1H, NH), 8.01 (s, 1H, H-8),
anoic acid (7a). Method A: white solid (68%), dec >250 ꢁC; method
7.95 (m, 2H, H-200), 7.58 (m, 1H, H-400), 7.49 (m, 2H, H-300), 7.43 (br s,
2H, NH2), 4.35e4.54 (m, 5H, H-20, CHipr, H-10), 3.97 (dd,
Jgem¼13.7 Hz, JHeCeP¼8.9 Hz, 1H, CH2Pa), 3.77 (dd, Jgem¼13.7 Hz,
JHeCeP¼9.7 Hz, 1H, CH2Pb), 1.09e1.20 (m, 12H, CH3ipr); 13C NMR
B: white crystals (46%), dec >250 ꢁC; 1H NMR (D2O)
d: 8.17 (s,1H, H-
0
0
2), 8.16 (s, 1H, H-8), 4.55 (dd, Jgem¼14.8 Hz, J1 ae2 ¼4.0 Hz, 1H, H-
0
10a), 4.48 (dd, Jgem¼14.8 Hz, J1 be2 ¼6.0 Hz, 1H, H-1 b), 4.20 (dd,
0
0
J2 e1 b¼6.0 Hz, J2 e1 a¼4.0 Hz, 1H, H-20), 3.71 (dd, Jgem¼12.8 Hz,
0
0
0
0
(DMSO-d6) d
: 170.59 (C-30),165.88 (CON),152.42 (C-2 or C-6),150.61
JHeCeP¼8.6 Hz, 1H, CH2Pa), 3.45 (dd, Jgem¼12.8 Hz, JHeCeP¼9.8 Hz,
(C-4),141.37 (C-8),134.65 (C-100), 131.99 (C-400),128.50 (C-300), 128.18
1H, CH2Pb); 13C NMR (D2O)
d
: 177.20 (C-30), 155.86 (C-6), 149.62 (C-
(C-200), 116.23 (C-5), 77.98 (d, J2 -P¼13.5 Hz, C-20), 70.54e70.63 (m,
4), 143.74 (C-8), 118.57 (C-5), 81.75 (d, J2 eP¼12.4 Hz, C-20), 67.33 (d,
JCeP¼155.0 Hz, CH2P), 46.12 (C-10); MS-ESIþ m/z (%) 318 (100,
MþHþ), 340 (83, MþNaþ), 362 (65, Mþ2Naþ); HRMS-ESIþ: m/z
calcd for C9H13O6N5P (MþHþ) 318.0598, found 318.0598; FTIR (KBr,
0
0
CHipr), 64.16 (d, JCeP¼164.4 Hz, CH2P), 44.25 (C-10), 23.68e23.95
(m, CH3ipr); MS-ESIþ m/z (%) 522 (75, MþHþ), 544 (100, MþNaþ);
HRMS-ESIþ: m/z calcd for C22H29O8N5P (MþHþ): 522.1748, found:
522.1747; FTIR (KBr, cmꢀ1
1529, 1453, 1443, 1387, 1255, 955. [
)
n
: 3318, 3143, 2981, 2500, 1674, 1602,
cmꢀ1
) n: 3178, 3033, 2838, 1647, 1598, 1474, 1417, 1056. Anal. Calcd
a
]
20 ꢀ40.7 (c 0.194, MeOH).
for C9H12N5O6P 0.75H2O: C, 32.69; H, 4.11; N, 21.18; P, 9.37. Found:
D
C, 32.86; H, 3.99; N, 20.95; P, 9.61. [
a
]
20 ꢀ6.1 (c 0.379, H2O).
D
4.6.8. (S)-3-(N2,N6-Dibenzoyl-2,6-diamino-9H-purin-9-yl)-2-[(diiso-
propoxyphosphono)methoxy]propanoic acid (9j). Colourless oil,
yield 80%; MS-ESIþ m/z (%) 625 (7, MþHþ), 647 (15, MþNaþ) 669
(100, Mþ2Naþ); HRMS-ESIþ: m/z calcd for C29H34O8N6P (MþHþ):
625.2170, found: 625.2171.
4.8.4. (R)-3-(6-Amino-9H-purin-9-yl)-2-(phosphonomethoxy)prop-
anoic acid (7b). Method A: white crystals (50%), dec >250 ꢁC; 1H
NMR (D2O)
d: 8.17 (s, 1H, H-2), 8.16 (s, 1H, H-8), 4.55 (dd,
0
0
0
Jgem¼14.8 Hz, J1 ae2 ¼4.0 Hz, 1H, H-1 a), 4.48 (dd, Jgem¼14.8 Hz,
0
0
0
0
0
0
0
J1 be2 ¼6.0 Hz, 1H, H-1 b), 4.20 (dd, J2 e1 b¼6.0 Hz, J2 e1 a¼4.0 Hz,
4.7. (S)-3-(2-Amino-6-oxo-1H-purin-9(6H)-yl)-2-
((diisopropoxyphosphoryl)methoxy)propanoic acid (9g)
1H, H-20), 3.71 (dd, Jgem¼12.8 Hz, JHeCeP¼8.6 Hz, 1H, CH2Pa), 3.45
(dd, Jgem¼12.8 Hz, JHeCeP¼9.8 Hz, 1H, CH2Pb); 13C NMR (D2O)
d:
177.20 (C-30), 155.86 (C-6), 149.62 (C-4), 143.74 (C-8), 118.57 (C-5),
81.75 (d, J2 -P¼12.4 Hz, C-20), 67.33 (d, JCeP¼155.0 Hz, CH2P), 46.12
0
To a solution of compound 9i (1 mmol, 521 mg) in MeOH
(10 mL) was added 1.0 M MeONa (2 mmol, 2 mL). After stirring at
room temperature for 24 h, the reaction mixture was neutralized
with glacial AcOH. The solvents were evaporated and the residue
was codistilled with toluene (2ꢂ10 mL) and ethanol (2ꢂ10 mL).
Chromatography on silica gel column (gradient 5e20% MeOH in
chloroform) followed by crystallization from EtOAc afforded
296 mg (71%) of compound 9g as a yellowish oil: 1H NMR (DMSO-
(C-10); MS-ESIþ m/z (%) 318 (100, MþHþ), 340 (83, MþNaþ), 362
(65, Mþ2Naþ); HRMS-ESIþ: m/z calcd for C9H13O6N5P (MþHþ)
318.0598, found 318.0598; FTIR (KBr, cmꢀ1
) n: 3178, 3033, 2838,
1647,1598,1474,1417,1056. Anal. Calcd for C9H12N5O6P$0.75H2O: C,
32.69; H, 4.11; N, 21.18; P, 9.37. Found: C, 32.86; H, 3.99; N, 20.97; P,
20
9.63. [
a
]
þ6.2 (c 0.324, H2O).
D
d6)
d
: 12.32 (br s, 1H, H-1), 7.56 (s, 1H, H-8), 7.22 (br s, 2H, NH2),
4.8.5. (S)-3-(4-Amino-2-oxopyrimidin-1(2H)-yl)-2-(phosphonome-
thoxy)propanoic acid (7c). Method A: yellowish crystals (62%), dec
>250 ꢁC; method B: yellowish crystals (39%), dec >250 ꢁC; 1H NMR
0
0
4.41e4.53 (m, 2H, CHipr), 4.33 (dd, Jgem¼13.5 Hz, J1 ae2 ¼1.9 Hz, 1H,
H-10a), 4.10 (dd, Jgem¼13.7 Hz, JHeCeP¼8.1 Hz,1H, CH2Pa), 3.88e3.97
(m, 2H, H-10b, H-20), 3.56 (dd, Jgem¼13.7 Hz, JHeCeP¼9.0 Hz, 1H,
CH2Pb), 1.19 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr),1.15 (d, JCH3eCH¼6.2 Hz,
3H, CH3ipr), 1.14 (d, JCH3eCH¼6.2 Hz, 3H, CH3ipr), 1.11 (d,
(D2O)
d
: 7.64 (d, J6e5¼7.4 Hz,1H, H-6), 5.98 (d, J5e6¼7.3 Hz,1H, H-5),
4.21 (dd, Jgem¼14.0 Hz, J1 ae2 ¼3.9 Hz, 1H, H-10a), 4.05 (dd,
0
0
J2 e1 b¼7.7 Hz, J2 e1 a¼03.9 Hz, 1H, H-20), 3.89 (dd, Jgem¼14.0 Hz,
0
0
0
0
JCH3eCH¼6.2 Hz, 3H, CH3ipr); 13C NMR (DMSO-d6)
d
: 171.53 (C-30),
J1 be2 ¼7.7 Hz, 1H, H-1 b), 3.71 (dd, Jgem¼12.9 Hz, JHeCeP¼8.7 Hz, 1H,
0
0
157.95 (C-6), 154.61 (C-2), 151.56 (C-4), 137.78 (C-8), 116.26 (C-5),
CH2Pa), 3.42 (dd, Jgem¼12.8 Hz, JHeCeP¼10.2 Hz, 1H, CH2Pb); 13C
81.36 (d, J2 -P¼13.0 Hz, C-20), 70.24e70.38 (m, CHipr), 63.28 (d,
NMR (D2O)
d
: 177.57 (C-30), 166.01 (C-4), 157.42 (C-2), 148.94 (C-6),
0
JCeP¼163.7 Hz, CH2P), 45.54 (C-10), 23.74e23.98 (m, CH3ipr); MS-
ESIꢀ m/z (%) 416 (100, MꢀHþ), 438 (10, MþNaþ); HRMS-ESIꢀ: m/z
calcd for C15H23O7N5P (MꢀHþ) 416.1340, found 416.1339; FTIR (KBr,
95.69 (C-5), 81.62 (d, J2 -P¼13.1 Hz, C-20), 67.07 (d, JCeP¼156.4 Hz,
CH2P), 51.87 (C-10); MS-ESIꢀ m/z (%) 292 (100, MꢀHþ), 314 (50,
MþNaþ), 336 (25, Mþ2Naþ); HRMS-ESIꢀ: m/z calcd for C8H11O7N3P
0

M.M. Kaiser et al. / Tetrahedron 68 (2012) 4003e4012
4011
(MꢀHþ) 292.0340, found 292.0340; FTIR (KBr, cmꢀ1
)
n
: 3385, 3171,
>250 ꢁC; 1H NMR (D2O)
d
: 8.11 (s, 1H, H-8), 7.97 (m, 2H, H-200), 7.62
2795, 1650, 1613, 1494, 1400, 1051. [
a
]
20 ꢀ7.0 (c 0.339, H2O).
(m, 1H, H-400), 7.53 (m, 2H, H-300), 4.45e4.52 (m, 2H, H-10), 4.16 (m,
D
1H, H-20), 3.59 (m, 1H, CH2P), 3.40 (m, 1H, CH2P); 13C NMR (D2O)
d:
4.8.6. (S)-3-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-
(phosphonomethoxy)propanoic acid (7d). Method A: white crystals
(64%), dec >250 ꢁC; method B: white crystals (44%), dec >250 ꢁC;
177.97 (C-30), 172.77 (CON), 162.48 (C-6), 153.12 (C-2), 151.42 (C-4),
142.76 (C-8), 135.04 (C-100), 133.13 (C-40), 129.28 (C-30), 128.54 (C-
20), 119.73 (C-5), 81.83 (d, J2 -P¼12.9 Hz, C-20), 68.76 (d,
0
1H NMR (D2O)
d
: 7.50 (q, J6-CH3¼1.2 Hz, 1H, H-6), 4.10 (dd,
JCeP¼150.8 Hz, CH2P), 45.76 (C-10); MS-ESIþ m/z (%) 438 (100,
MþHþ), 460 (78, MþNaþ); HRMS-ESIꢀ: m/z calcd for C16H15O8N5P
0
0
0
0
0
Jgem¼13.9 Hz, J1 ae2 ¼4.1 Hz, 1H, H-1 a), 4.05 (dd, J2 e1 ¼6.6 and
4.1 Hz, 1H, H-20), 3.96 (dd, Jgem¼13.9 Hz, J1 be2 ¼6.6 Hz, 1H, H-1 b),
3.69 (dd, Jgem¼12.8 Hz, JHeCeP¼8.7 Hz, 1H, CH2Pa), 3.42 (dd,
Jgem¼12.8 Hz, JHeCeP¼9.9 Hz, 1H, CH2Pb), 1.87 (d, JCH3-6¼1.2 Hz, 3H,
(MꢀHþ) 436.0664, found 436.0664; FTIR (KBr, cmꢀ1
) n: 3408, 3135,
0
0
0
3033, 1665, 1604, 1402, 1269, 1154, 1061. [
a
]
20 ꢀ31.9 (c 0.244, H2O).
D
5-CH3); 13C NMR (D2O)
d: 177.59 (COOH), 167.78 (C-4), 152.84 (C-2),
Acknowledgements
144.62 (C-6), 110.71 (C-5), 81.61 (d, J2 -P¼12.6 Hz, C-20), 67.24 (d,
JCeP¼157.1 Hz, CH2P), 50.34 (C-10), 11.93 (5-CH3); MS-ESIꢀ m/z (%)
307 (100, MꢀHþ), 329 (60, MþNaþ), 351 (15, Mþ2Naþ); HRMS-
ESIꢀ: m/z calcd for C9H12O8N2P (MꢀHþ) 307.0326, found 307.0327;
0
This study was performed as a part of research project RVO:
61388963 of the Institute of Organic Chemistry and Biochemistry,
v.v.i. This study was supported by Ministry of the Interior of the
Czech Republic (VG20102015046), and by Gilead Sciences and IOCB
Research Centre.
FTIR (KBr, cmꢀ1
) n: 3191, 3036, 2833, 1691, 1601, 1475, 1432, 1359,
20
1110, 1062. [
a]
ꢀ2.7 (c 0.401, H2O).
D
4.8.7. (S)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(phospho-
Supplementary data
nomethoxy)propanoic acid (7e). Method B: white crystals (42%), dec
>250 ꢁC; 1H NMR (D2OþNaOD)
: 7.59 (d, J6e5¼7.5 Hz,1H, H-6), 5.74
d
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2012.03.066. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
(d, J5e6¼7.5 Hz, 1H, H-5), 3.97e4.05 (m, 3H, H-10, 20), 3.53 (dd,
Jgem¼11.8 Hz, JHeCeP¼9.1 Hz, 1H, CH2P), 3.30 (dd, Jgem¼11.8 Hz,
JHeCeP¼10.2 Hz, 1H, CH2P); 13C NMR (D2OþNaOD)
d
: 178.47 (C-30),
0
174.43 (C-4), 158.01 (C-2), 147.85 (C-6), 102.05 (C-5), 81.99 (d, J2 -
¼13.6 Hz, C-20), 68.91 (d, JCeP¼150.7 Hz, CH2P), 51.03 (C-10); MS-
P
ESIꢀ m/z (%) 293 (100, MꢀHþ); HRMS-ESIꢀ: m/z calcd for C8H10O8N2P
References and notes
(MꢀHþ): 293.0180, found: 293.0180; FTIR (KBr, cmꢀ1
) n: 3428, 3168,
ꢁ
1. (a) De Clercq, E.; Holy, A.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P. C.
20
2976, 1692, 1617, 1402, 1356, 1070, 1050. [
a
]
365 ꢀ50.0 (c 0.346, H2O).
ꢁ
Nature 1986, 323, 464; (b) De Clercq, E. J. Med. Microbiol. 1998, 47, 1; (c) Holy, A.
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Curr. Pharm. Des. 2003, 9, 2567; (d) De Clercq, E.; Holy, A. Nat. Rev. Drug Discov.
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2005, 4, 928; (e) Holy, A. Antiviral Res. 2006, 71, 248; (f) De Clercq, E. Antiviral
4.8.8. (R)-3-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(phos-
Res. 2007, 75, 1; (g) De Clercq, E. Biochem. Pharmacol. 2007, 73, 911.
phonomethoxy)propanoic acid (7f). Method B: white crystals (48%),
ꢀ
ꢁ
ꢀ
ꢁ
ꢁ
2. (a) Holy, A.; Votruba, I.; Tloustova, E.; Masojídkova, M. Collect. Czech. Chem.
dec >250 ꢁC; 1H NMR (D2OþNaOD), 13C NMR (D2OþNaOD), MS
Commun. 2001, 66, 1545; (b) Zídek, Z.; Potmesil, P.; Holy, A. Toxicol. Appl.
Pharmacol. 2003, 192, 246; (c) Reiser, H.; Wang, J.; Chong, L.; Watkins, W. J.; Ray,
A. S.; Shibata, R.; Birkus, G.; Cihlar, T.; Wu, S.; Li, B.; Liu, X.; Henne, I. N.;
Wolfgang, G. H. I.; Desai, M.; Rhodes, G. R.; Fridland, A.; Lee, W. A.; Plunkett, W.;
Vail, D.; Thamm, D. H.; Jeraj, R.; Tumas, D. B. Clin. Cancer. Res. 2008, 14, 2824.
ꢀꢀ
ꢁ
20
and FTIR spectra are identical with compound 7e. [
0.336, H2O).
a
]
þ46.0 (c
365
ꢁ
ꢁ
3. (a) Keough, D. T.; Hockova, D.; Holy, A.; Naesens, L. M. J.; Skinner-Adams, T. S.;
4.8.9. (S)-3-(2-Amino-6-oxo-1H-purin-9(6H)-yl)-2-(phosphonome-
thoxy)propanoic acid (7g). Method A: yellowish crystals (61%), dec
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ꢁ
de Jersey, J.; Guddat, L. W. J. Med. Chem. 2009, 52, 4391; (b) Hockova, D.; Holy,
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A.; Masojídkova, M.; Keough, D. T.; de Jersey, J.; Guddat, L. W. Bioorg. Med. Chem.
>250 ꢁC; method B: white solid (37%), dec >250 ꢁC; 1H NMR (D2O)
d
:
2009, 17, 6218.
0
ꢁ
ꢁ
4. (a) Zídek, Z.; Frankova, D.; Holy, A. Int. J. Immunopharmacol. 2000, 22, 1121; (b)
0
0
7.84 (s,1H, H-8), 4.40 (dd, Jgem¼14.7 Hz, J1 ae2 ¼4.1 Hz,1H, H-1 a), 4.32
ꢀꢀ
ꢀ
ꢁ
ꢁ
ꢁ
ꢀ
Zídek, Z.; Potmesil, P.; Kmoníckova, E.; Holy, A. Eur. J. Pharmacol. 2003, 475, 149;
0
0
0
0
0
(dd, Jgem¼14.7 Hz, J1 be2 ¼6.1 Hz, 1H, H-1 b), 4.17 (dd, J2 e1 ¼6.1 and
ꢀꢀ
ꢀ
ꢁ
ꢁ
(c) Potmesil, P.; Krecmerova, M.; Kmoníckova, E.; Holy, A.; Zídek, Z. Eur. J.
4.1 Hz, 1H, H-20), 3.73 (dd, Jgem¼13.0 Hz, JHeCeP¼8.7 Hz, 1H, CH2Pa),
Pharmacol. 2006, 540, 191.
3.51 (dd, Jgem¼13.0 Hz, JHeCeP¼9.8 Hz, 1H, CH2Pb); 13C NMR (D2O)
d:
5. (a) De Clercq, E. Intervirology 1997, 40, 295; (b) De Clercq, E. Clin. Microbiol. Rev.
2003, 16, 569; (c) De Clercq, E. Expert Rev. Anti-infect. Ther. 2003, 1, 21.
177.21 (C-30), 159.57 (C-6), 154.24 (C-2), 152.37 (C-4), 141.38 (C-8),
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6. (a) De Clercq, E.; Holy, A. Antimicrob. Agents Chemother. 1991, 35, 701; (b) Neyts,
116.08 (C-5), 81.73 (d, J2 -P¼12.5 Hz, C-20), 66.85 (d, JCeP¼156.1 Hz,
0
J.; Snoeck, R.; Balzarini, J.; De Clercq, E. Antiviral Res. 1991, 16, 41; (c) De Clercq,
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CH2P), 45.82 (C-10); MS-ESIꢀ m/z (%) 332 (100, MꢀHþ), 354 (50,
MþNaþ), 376 (15, Mþ2Naþ); HRMS-ESIꢀ: m/z calcd for C9H11O7N5P
ꢁ
ꢁ
7. (a) Holy, A.; Votruba, I.; Masojídkova, M.; Andrei, G.; Snoeck, R.; Naesens, L.; De
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) n: 3405, 3128,
Clercq, E.; Balzarini, J. J. Med. Chem. 2002, 45, 1918; (b) Balzarini, J.; Pan-
necouque, C.; De Clercq, E.; Aquaro, S.; Perno, C.-F.; Egberink, H.; Holy, A. An-
20 ꢀ20.2(c 0.316, H2O).
ꢁ
1693, 1607, 1540, 1481, 1409, 1108, 1069, 914. [
a]
D
ꢁ
ꢁ
timicrob. Agents Chemother. 2002, 46, 2185; (c) Hockova, D.; Holy, A.;
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Masojídkova, M.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem.
4.8.10. (S)-3-(2,6-Diamino-9H-purin-9-yl)-2-(phosphonomethoxy)
propanoic acid (7h). Method B (from 9j): white crystals (38%), dec
ꢁ
ꢁ
ꢁ
2003, 46, 5064; (d) Hockova, D.; Holy, A.; Masojídkova, M.; Andrei, G.; Snoeck,
R.; De Clercq, E.; Balzarini, J. Bioorg. Med. Chem. 2004, 12, 3197; (e) Ying, C.;
>250 ꢁC; 1H NMR (D2OþNaOD)
d
: 7.94 (0s, 1H, H-8), 4.33e4.39 (m,
ꢁ
ꢁ
Holy, A.; Hockova, D.; Havlas, Z.; De Clercq, E.; Neyts, J. Antimicrob. Agents
Chemother. 2005, 49, 1177.
2H, H-10), 4.13 (t, J2 e1 ¼5.1 Hz, 1H, H-2 ), 3.58 (dd, Jgem¼12.1 Hz,
0
0
8. Wagner, C. R.; Iyer, V. V.; McIntee, E. J. Med. Res. Rev. 2000, 20, 417.
JHeCeP¼8.9 Hz, 1H, CH2P), 3.32 (dd, Jgem¼12.1 Hz, JHeCeP¼9.8 Hz,
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1H, CH2P); 13C NMR (D2OþNaOD)
d
: 178.02 (C-30), 160.60 (C-2),
ꢁ
10. (a) Holy, A.; Rosenberg, I. Collect. Czech. Chem. Commun. 1987, 52, 2775; (b)
ꢁ
Rosenberg, I.; Holy, A. Collect. Czech. Chem. Commun. 1987, 52, 2792; (c)
0
456.66 (C-6), 151.92 (C-4), 141.68 (C-8), 113.20 (C-5), 81.90 (d, J2 -
ꢀ
ꢁ
ꢁ
ꢁ
Krecmerova, M.; Masojídkova, M.; Holy, A. Collect. Czech. Chem. Commun. 2004,
¼12.7 Hz, C-20), 68.82 (d, JCeP¼150.5 Hz, CH2P), 45.75 (C-10); MS-
ꢁ
ꢁ
P
69, 1889; (d) Janeba, Z.; Masojídkova, M.; Holy, A. Collect. Czech. Chem. Commun.
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ESIþ m/z (%) 333 (100, MþHþ), 355 (25, MþNaþ), 377 (15,
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ꢁ
ꢀꢁ
ꢁ
Mþ2Naþ); HRMS-ESIþ: m/z calcd for C9H14O6N6P (MþHþ)
11. (a) Jindrich, J.; Holy, A.; Dvorakova, H. Collect. Czech. Chem. Commun. 1993, 58,
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ꢀ
ꢀꢁ
ꢁ
1645; (b) Balzarini, J.; Holy, A.; Jindrich, J.; Dvorakova, H.; Hao, Z.; Snoeck, R.;
333.0707, found 333.0707; FTIR (KBr, cmꢀ1
) n: 3194, 2470, 1605,
Herdewijn, P.; John, D. G.; De Clercq, E. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4961;
20 ꢀ17.4 (c 0.259, H2O).
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ꢀ
(c) Balzarini, J.; Holy, A.; Jindrich, J.; Naesens, L.; Snoeck, R.; Schols, D.; De
1560, 1551, 1482, 1407, 1119, 1087, 916. [a]
D
Clercq, E. Antimicrob. Agents Chemother. 1993, 37, 332.
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4.8.11. (S)-3-(2-Benzamido-6-oxo-1H-purin-9(6H)-yl)-2-(phospho-
nomethoxy)propanoic acid (7i). Method A: white crystals (52%), dec
ꢁ
De Clercq, E.; Holy, A. J. Med. Chem. 1979, 22, 510.
ꢁ
13. Holy, A.; Votruba, I.; De Clercq, E. Collect. Czech. Chem. Commun. 1982, 47, 1392.

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