Synthesis of analogues of Acyclic nucleoside diphosphates containing a (phosphonomethyl)phosphanyl moiety and studies of their phosphorylation

Article abstract of DOI:10.1002/ejoc.200800911

Acyclic nucleoside diphosphonate derivatives of purines and pyrimidines were prepared by Mitsunobu reaction of suitably protected heterocyclic bases with alcohols containing the (phosphonomethyl)phosphanyl moiety. Furthermore, nonhydrolyzable acyclic analogues of dUDP were prepared as potential inhibitors of dUTPase. Their phosphorylation to analogues of dUTP, however, gave mixtures of linear and branched phosphates. The courses of the phosphorylation reactions were followed by ³¹P NMR spectroscopy, and we discovered that both phosphonate and phosphinate moieties react with 1,1'-carbonyldiimidazole and tri-n-butylammonium phosphate. The pK_a values of the (phosphonomethyl)phosphanyl system were also determined by ³¹P NMR spectroscopy ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009).

Full text of DOI:10.1002/ejoc.200800911

FULL PAPER
DOI: 10.1002/ejoc.200800911
Synthesis of Analogues of Acyclic Nucleoside Diphosphates Containing a
(Phosphonomethyl)phosphanyl Moiety and Studies of Their Phosphorylation
[a]
[a]
[a]
[a]
ˇ
ˇ
Petra Doláková,* Martin Dracínský, Jindrich Fanfrlík, and Antonín Holý
Keywords: Antiviral agents / Nucleotides / Phosphorylation / dUTPase
Acyclic nucleoside diphosphonate derivatives of purines and
pyrimidines were prepared by Mitsunobu reaction of suitably
protected heterocyclic bases with alcohols containing the
(phosphonomethyl)phosphanyl moiety. Furthermore, nonhy-
drolyzable acyclic analogues of dUDP were prepared as po-
actions were followed by 31P NMR spectroscopy, and we dis-
covered that both phosphonate and phosphinate moieties re-
act with 1,1Ј-carbonyldiimidazole and tri-n-butylammonium
phosphate. The pKa values of the (phosphonomethyl)phos-
phanyl system were also determined by 31P NMR spec-
tential inhibitors of dUTPase. Their phosphorylation to ana- troscopy
logues of dUTP, however, gave mixtures of linear and
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
branched phosphates. The courses of the phosphorylation re- Germany, 2009)
phonomethoxy)ethyl side chain by a hydroxymethyl group
Introduction
leads to a third structural type of antiviral compounds:
9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine
(HPMPC, cidofovir, Vistide) possesses general anti-DNA-
viral activity.[9] Cidofovir has been approved for treatment
of cytomegalovirus retinitis in AIDS patients, but was also
successfully used in the treatment of various DNA-virus in-
fections, in particular those caused by pox viruses.[10]
Acyclic nucleoside phosphonates[1] (ANPs) represent a
key class of nucleotide analogues with a broad spectrum of
antiviral and cytostatic activity. Among ANPs, 9-[2-(phos-
phonomethoxy)ethyl]adenine (PMEA, adefovir, Figure 1)
in particular is active against DNA and retroviruses,[2] and
its prodrug, adefovir dipivoxil (Hepsera),[3] has been ap-
proved for hepatitis B therapy.[4] 9-[2-(Phosphonomethoxy)-
ethyl]-2,6-diaminopurine (PMEDAP) is active against DNA
viruses and retroviruses[5] and also exhibits selective antitu-
mor properties,[6] and the guanine derivative bearing the
phosphonomethoxyethyl (PME) side chain possesses
powerful antitumor activity.[7]
These nucleotide analogues each contain a stable isopo-
lar phosphonomethyl ether moiety instead of the nucleotide
phosphate ester group, which blocks their enzymatic degra-
dation and/or eliminates problems with intracellular phos-
phorylation necessary for nucleoside activation. ANPs are
phosphorylated in the cell to di- and triphosphates that in-
hibit viral DNA polymerase and/or reverse transcriptase.[11]
The (phosphonomethyl)phosphanyl unit (P–C–P–C–), in
which the bridging oxygen atoms of the diphosphate moiety
are replaced with methylene groups, can be regarded as a
nonhydrolyzable mimic of diphosphate. Acyclic diphos-
phonate derivatives of adenine and guanine containing
methoxyalkoxy side chains were previously reported to pos-
sess moderate antiviral activity against HSV-1, VZV, and
visna virus.[12] Inhibitory activities of modified triphosphate
analogues of carbocyclic nucleosides containing diphos-
phonate moieties against HIV reverse transcriptase and
DNA polymerase have also been described.[13] Farnesyl di-
phosphate analogues in which the diphosphate unit is re-
placed by a (phosphonomethyl)phosphanyl moiety showed
inhibitory activity against squalene synthetase[14] and
farnesyl diphosphate synthetase.[15] Glutamine synthetase
inhibitors[16] and transcarbamoylase inhibitors[17] contain-
ing diphosphonate moieties have also been described.
Here we describe syntheses of 2-[(hydroxy)(phospho-
nomethyl)phosphorylmethoxy]ethyl purines (A, G) and
Figure 1. Acyclic analogues of nucleotides.
A second class of antiviral compounds is represented by
9-(R)-[2-(phosphonomethoxy)propyl]adenine (PMPA, teno-
fovir), a promising anti-HIV compound; its prodrug Viread
has been approved for treatment of AIDS and chronic
hepatitis B.[8] Substitution at the 2-position in the 2-(phos-
[a] Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic, v.v.i.
Flemingovo nám. 2, 16610 Prague 6, Czech Republic
Fax: +420-220-183-560
E-mail: dolakova@uochb.cas.cz
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Acyclic Nucleoside Diphosphate Analogues
pyrimidines (C, U, T) (Figure 1) – chemically and enzymati- zov reaction between (phosphonomethyl)phosphonite and
cally stable analogues of acyclic nucleoside diphosphates alkyl halide,[12] due to the poor yields of the synthesis of
containing the PME side chain.
the starting phosphonomethyl-phosphonite,[27,15] which was
Furthermore, acyclic analogues of dUDP and dUTP accompanied by formation of side products and compli-
containing the (phosphonomethyl)phosphanyl moiety were cated separation. Isopropyl esters were used instead of ethyl
prepared as potential inhibitors of Mycobacterium tubercu- esters to suppress side Arbuzov reactions with ethyl bro-
losis dUTPase (deoxyuridine 5Ј-triphosphate nucleotidohy- mide.
drolase).[18] dUTPase is essential in both eukaryotes and
Uracil and thymine derivatives 6a and 6b were prepared
prokaryotes;[19] the enzyme catalyzes hydrolysis of dUTP by alkylation of N3-benzoylated uracil 4a and thymine 4b[28]
into dUMP and diphosphate using Mg2+ as a cofactor.[20] with alcohol 3a under Mitsunobu conditions[29] in 79% and
The enzyme supplies the dUMP substrate for dTTP synthe- 52% yields, respectively (Scheme 2). Isopropyl esters were
sis and, by maintaining low dUTP/dTTP ratios in the cell, deprotected with trimethylsilyl bromide in acetonitrile by
minimizes uracil misincorparation into DNA.[21] dUTPase standard procedures. The uracil derivative 5a was converted
has been recognized as a valid target for drug design. Al- into the cytosine analogue 7 by treatment with 2,4,6-triiso-
though some selective inhibitors of parasitic dUTPase have
been described, the high degree of sequence and structural
similarity between parasitic and human dUTPases may
make development of drugs based on inhibition of dUT-
Pase difficult. Previously described inhibitors of dUTPase
include nonhydrolyzable analogues of nucleoside triphos-
phates in which the α,β bridging oxygen atom is replaced
by a methylene[22] or an imido[23] group. 2Ј-Deoxyuridine
derivatives containing either a triphenylmethyl or a tri-
phenylsilyl substituent at their 5Ј-positions[24] and their acy-
clic analogues[25] selectively inhibit Plasmodium falciparum
dUTPase.
Results and Discussion
The diphosphate analogues were prepared by Mitsunobu
reaction of suitably protected heterocyclic bases with the
functionalized alcohols 3a–e, each bearing the (phospho-
nomethyl)phosphanyl unit. Alcohols 3a–e were prepared by
Arbuzov reaction of the alkyl bromides 2a[26] and 2b or
iodides 2c–e with the air- and moisture-sensitive phos-
phonite 1 (Scheme 1).[27,16] The corresponding alkyl chlo-
rides were unreactive at 120 °C, whereas heating to higher
temperature led to the decomposition of the starting materi-
als. Our attempts to perform the reaction under microwave
heating conditions were unsuccessful. The terminal phos-
phonite moieties were oxidized to phosphonates with
DMSO, and the acetyl protecting groups were removed by
hydrolysis with hydrochloric acid. Alcohols 3a–e were pre-
pared in overall yields of 30–40%. This method proved in
our hands to be better than the previously described Arbu-
Scheme 2. Mitsunobu reaction of heterocyclic bases with alcohol
3a. Reagents and conditions: a) Ph3P, DIAD, THF, b) MeONa,
MeOH, c) BrSiMe3, CH3CN, d) 1. TPSCl, CH3CN, 2. NH4OH,
e) HCl, CH2Cl2, f) H2O, THF, g) TFAA, H2O.
Scheme 1. Arbuzov reaction of 1 with alkyl halides 2a–e. Reagents
and conditions: a) 1. 120 °C, 2. DMSO, 3. HCl, EtOH.
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propylbenzenesulfonyl chloride (TPSCl) followed by amin- 2,2Ј-dithiodipyridine and triphenylphosphane,[38] but yields
ation by ammonium hydroxide;[30] treatment with trimethyl- obtained by this method were lower than those of the reac-
silyl bromide afforded 8 in 46% yield. Adenine derivative tion with CDI.
10 was prepared by Mitsunobu reaction between 3a and
the bis-Boc-protected N6-amino adenine 9,[31] followed by
hydrolysis of the bis-Boc protecting group with hydrochlo-
ric acid in dichloromethane.[32] The guanine counterpart 14
was prepared analogously by starting from 2-amino-6-chlo-
ropurine (12) by alkylation with 3a to give predominantly
the N2-triphenylphosphoranylidene derivative 13. Com-
pound 13 was easily hydrolyzed by heating at reflux in a
THF/H2O mixture[33] to afford the 2-amino derivative,
which was subsequently converted into guanine 14 by treat-
ment with trifluoroacetic acid. Deprotection of 10 and 14
with trimethylsilyl bromide gave free acids 11 and 15.
In addition to the 1-{2-[(hydroxy)(phosphonomethyl)-
phosphorylmethoxy]ethyl}uracil (6a) with the methoxyethyl
side chain, the series of uracil derivatives bearing carbon
side chains of four, five, and six carbon atoms, as well as the
ethoxyethyl side chain (Scheme 3), was prepared in order to
study the potential inhibitory activities of these compounds
towards dUTPase. Compounds 16 and 17 were prepared by
the same procedure as described for compounds 5 and 6:
N3-benzoylated uracil 4a was alkylated with alcohols 3b–e
under Mitsunobu conditions, the benzoyl group was re-
moved by treatment with sodium methoxide, and subse-
quent treatment with trimethylsilyl bromide gave diphos-
phonates 17a–d.
Scheme 4. Phosphorylation of compounds 6a and 17a–d. Reagents
and conditions: a) 1. CDI, DMF, 2. (Bu3NH)H2PO4, DMF,
b) Dowex 50ϫ8 (Na+ form).
Treatment of 6a with CDI followed by tri-n-butylammo-
nium phosphate in DMF gave expected triphosphate ana-
logue 18 in 5% yield (Scheme 4). Compound 18 was puri-
fied by anion-exchange chromatography (elution with
TEAB), and the triethylammonium salt was finally con-
verted into the corresponding sodium salt on
DOWEX 50ϫ8 (Na+ form). Its identity was determined by
1H and 31P NMR, and its purity (determined by HPLC)
exceeded 98%.
Compounds 17a–d were similarly converted into their
Scheme 3. Synthesis of analogues of dUDP. Reagents and condi-
phosphate counterparts by the method described above and
tions: a) Ph3P, DIAD, THF, b) MeONa, MeOH, c) BrSiMe3,
isolated as their sodium salts; however, only branched phos-
CH3CN.
phates 19a–d were isolated, as major products, in approxi-
As the natural substrate of dUTPase is 2Ј-deoxyuridine mately 15–30% yields. The branched structures of com-
triphosphate, we tried to convert the diphosphonates 6a and pounds 19a–d were confirmed by their 31P NMR spectra.
17a–d into the corresponding triphosphate analogues In the proton-decoupled spectra, P-β and P-γ appeared as
(Scheme 4). Our attempts to prepare triphosphate mimics doublets with coupling constants to P-α of 2 Hz and 28 Hz,
by the morpholidate method,[34] widely used for conversion respectively (see Table 1, Figure 2). The α position of P-α
of ANPs into phosphates, were unsuccessful, with the mor- was confirmed by its chemical shift having the highest value
pholidate of 6a being formed only in low yield. Synthesis (close to 50 ppm) and by the 31P NMR spectrum without
of triphosphate analogues by methods employing diphenyl proton decoupling, in which P-α exhibited splitting due to
chlorophosphate[35] or benzyl hydrogen phosphoramid- coupling with two neighboring methylene groups.
ate[36] failed as well. Phosphates were finally prepared by
Compound 18 is relatively stable: in neutral aqueous
activation of phosphonate with 1,1Ј-carbonyldiimidazole solution it decomposes into the starting diphosphonate 6a
(CDI), followed by addition of the tri-n-butylammonium and phosphate, but only 5% of diphosphonate 6a had ap-
phosphate in DMF.[37] The imidazolidate of 6a was also peared in neutral aqueous solution over a 24 h period at
prepared by treatment with imidazole in the presence of room temperature. In contrast, very low stabilities of com-
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Acyclic Nucleoside Diphosphate Analogues
Table 1. 31P NMR chemical shifts (ppm) and interaction constants (Hz) of compounds 6a and 17–20.
P-α
P-β
P-γ
6a
36.32 d, J(P-α,P-β) = 9.0
30.98 d, J(P-α,P-β) = 6.2
34.5 dd, J(P-α,P-γ) = 28.6, J(P-α,P-β) = 7.2
49.0 d, J(P-α,P-β) = 7.0
46.2 dd, J(P-α,P-γ) = 28.3, J(P-α,P-β) = 6.0
49.8 d, J(P-α,P-β) = 6.7
50.1 dd, J(P-α,P-γ) = 28.7, J(P-α,P-β) = 2.1
50.53 d, J(P-α,P-β) = 7.8
50.4 dd, J(P-α,P-γ) = 28.4, J(P-α,P-β) = 2.0
44.3 d, J(P-α,P-β) = 9.2
18.18 d, J(P-β,P-α) = 9.0
6.92 dd, J(P-β,P-α) = 6.2, J(P-β,P-γ) = 25.2 –5.51 d, J(P-γ,P-β) = 25.2
–
18
20
11.4 d, J(P-β,P-α) = 7.2
15.6 d, J(P-β,P-α) = 7.0
10.9 d, J(P-β,P-α) = 6.0
14.56 d, J(P-β,P-α) = 6.7
9.61 d, J(P-β,P-α) = 2.1
14.80 d, J(P-β,P-α) = 7.8
9.7 d, J(P-β,P-α) = 2.0
15.8 d, J(P-β,P-α) = 9.2
9.5 d, J(P-β,P-α) = 1.8
–5.4 d, J(P-γ,P-α) = 28.7
17a
19a
17b
19b
17c
19c
17d
19d
–
–5.1 d, J(P-γ,P-α) = 28.3
–
–4.4 d, J(P-γ,P-α) = 28.7
–
–4.3 d, J(P-γ,P-α) = 28.4
–
49.3 dd, J(P-α,P-γ) = 27.3, J(P-α,P-β) = 1.8
–4.4 d, J(P-γ,P-α) = 27.3
the formation of a mixture of products in the reaction of
6a with CDI. No intramolecular migration of the phos-
phate group was observed.
Figure 2. Labeling of phosphorus atoms for 31P NMR spec-
troscopy.
pounds 19a–d were observed; we managed to prepare them
in 90–98% purities (the compounds tend to decompose into
the starting diphosphonate and phosphate during purifica-
tion and concentration).
To investigate whether the branched phosphates are
formed immediately during the reaction or are formed by
intramolecular migration of the terminal phosphate in its
linear counterpart, we followed the course of the reaction
of 6a with CDI and tri-n-butylammonium phosphate by 31
P
Figure 3. pH dependence of the chemical shifts δ (ppm) of P-α of
compound 6a.
NMR spectroscopy. The signals of the starting material dis-
appeared immediately after addition of CDI, but an un-
identifiable mixture of several products was observed. After
2 h the spectrum was no longer changing, and tri-n-butyl-
ammonium phosphate was added to the reaction mixture
(the imidazolide of 6a was not isolated because of its insta-
bility). It was found that after 6 h both phosphates
(branched and linear) had been formed in approximately
2:1 ratio. We used 31P–31P-COSY to identify the signals of
linear and branched triphosphate analogues. Finally, the re-
action mixture was separated, and the linear phosphate 18
was isolated in 7% yield together with the branched phos-
phate 20 in 13% yield (Scheme 4).
Identical experiments with compounds 19a and 19d
showed the same results, with both branched and linear
phosphates being formed. The ratio of branched and linear
phosphate was from 2:1 to 10:1.
From NMR titration studies of 6a, pKa values for the
(phosphonomethyl)phosphanyl residue were determined
(Figures 3 and 4). The pKa value of the phosphinate (P-
Figure 4. pH dependence of the chemical shifts δ (ppm) of P-β of
compound 6a.
The stabilities of linear and branched triphosphate ana-
α, Figure 3) and the pKa1 value of the phosphonate (P-β, logues were also studied with ab initio quantum chemical
Figure 4) are around 1.8 and 2.7, respectively. These pKa calculations. We compared the thermodynamic stabilities of
values indicate that both groups are acidic enough to un- linear and branched phosphates (Figure 5) with carbon side
dergo reaction with CDI and phosphate and explain the chains (21a and 21b) and methoxyethyl side chains (22a and
formation of mixtures of branched and linear phosphates. 22b). The triphosphate analogues were taken to be proton-
This explanation is also supported by our observation of ated for calculations (the molecule 21b corresponds to com-
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pound 19a, compound 22a corresponds to 18, and 22b cor- the phosphonate and the phosphinate residue with CDI. In
responds to compound 20). In the both cases the branched addition, thermodynamic stability calculations showed the
phosphates were more stable (see Table 2).
branched phosphates to be more stable.
All the prepared compounds were tested for antiviral and
cytostatic activity and dUTPase inhibitory activity, but
none exhibited any appreciable biological activity. These
data indicate that the (phosphonomethyl)phosphanyl sys-
tem is not an optimal analogue of the natural diphosphate
in nucleotides. This effect may be due to the differences be-
tween the pKa values of the (phosphonomethyl)phosphanyl
analogues and the normal diphosphates, small geometric
differences between C–P and O–P bonds, and differences in
metal ion binding properties.[39,22]
Figure 5. Molecules studied by ab initio quantum chemical calcula-
tions.
Table 2. Relative gas-phase energies (∆E) and relative hydration
free energies (∆∆GHYD) in kcalmol–1.
Experimental Section
Molecule
∆E
∆∆GHYD
∆E + ∆∆GHYD
General: Solvents were dried by standard procedures. Tetra-
hydrofuran (THF) was freshly distilled from sodium/benzophenone
under argon. NMR spectra were recorded with Bruker Avance 500
21a
21b
0.0
–9.1
0.0
4.9
0.0
–4.2
1
(500 MHz for H and 125.8 MHz for 13C) and Bruker Avance 400
22a
22b
0.0
–10.5
0.0
4.5
0.0
–6.0
(1H at 400, 13C at 100.6 MHz) spectrometers in CDCl3, [D6]-
DMSO, or D2O. Chemical shifts (in ppm, δ scale) were referenced
to TMS (for 1H NMR spectra in CDCl3) and/or to the solvent
signal (CDCl3 δ = 7.26 ppm for 1H NMR and δ = 77.0 ppm for
13C NMR; [D6]DMSO δ = 2.5 ppm for 1H NMR and δ = 39.7 ppm
for 13C). Chemical shifts in D2O were referenced to 1,4-dioxane: δ
= 3.75 ppm for 1H NMR and δ = 67.19 ppm for 13C NMR. Chemi-
cal shifts for 31P NMR spectra were referenced to H3PO4 (δ =
Compounds 6a, 6b, 8, 11, 15, and 17a–d were screened
for cytostatic and antiviral activities: none of the tested
compounds exhibited any significant biological activity or
cytotoxicity. The dUDP and dUTP analogues 6a, 17a–d,
18, 19a–d, and 20 were tested for their potency to inhibit
Mycobacterium tuberculosis dUTPase, but none of the ana- 0 ppm). For 31P NMR spectroscopic data see Table 1. Melting
points were determined on a Büchi Melting Point B-545 apparatus
and are uncorrected. TLC was performed on plates of Kieselgel
60 F254 (Merck). Mass spectra were measured on a ZAB-EQ (VG
Analytical) spectrometer by FAB (ionization with Xe, accelerating
voltage 8 kV, glycerol matrix) or on a LCQ classic spectrometer by
logues inhibited the enzyme.
Conclusions
In conclusion, a series of acyclic nucleoside diphosphate electrospray ionization (ESI). Preparative HPLC purification was
performed on a column packed with 10 µm C18 reversed phase
(Luna), 250ϫ21 mm; in ca 300 mg portions of mixtures with a
linear gradient of triethylammonium hydrogen carbonate (TEAB,
0.1 ) in water and in 50% MeOH (linear gradient of TEAB in
50% MeOH, 0–100%). Preparative HPLC purification of triphos-
phate analogues was performed on a column packed with POROS®
HQ 50 µm (50 mL) with use of a gradient of TEAB in water (0–
0.4 ).
analogues of purine and pyrimidine nucleotides containing
a stable (phosphonomethyl)phosphanyl unit were prepared
by improved versions of previously described methods.
Alcohols 3a–e were prepared by Arbuzov reaction of di-
phosphonite 1 with acetyl alkyl bromides and alkyl iodides
in overall 30–40% yields. Suitably protected heterocyclic
bases were coupled with functionalized alcohols 3a–e by
Mitsunobu reaction and finally deprotected by standard
procedures.
Tetraisopropyl methylenediphosphonite (1) was prepared by the
previously described procedure[16] or purchased from Sigma–
Aldrich. 5-Chloropentyl acetate, 4-bromobutyl acetate, and 6-chlo-
rohexan-1-ol were purchased from Aldrich. 2-(2-Chloroethoxy)-
ethanol was purchased from Janssen Chimica.
(Phosphonomethyl)phosphinates 6a and 17a–d were suc-
cessfully transformed into their phosphate counterparts by
conversion of (phosphonomethyl)phosphinates into the
corresponding imidazolides by treatment with CDI and
subsequently with tri-n-butylammonium phosphate. Inter-
estingly, the branched phosphates 19a–d and 20 were iso-
lated as major products rather than the expected linear
Method of Calculation: Gas-phase geometries and energies of the
studied molecules were obtained with RI-MP2/cc-pVDZ.[40] These
calculations were performed with Turbomole5.8.[41] Hydration free
energies were calculated with the C-PCM implicit solvent model[42]
implemented in the Gaussian03 code.[43] The recommended HF/6–
31G* level combined with the united atom radii (UAHF) model
was used.
phosphates, which were present in lower yields. Detailed 31
P
NMR studies of the courses of the reactions showed that
both branched and linear phosphates are formed immedi-
ately during the reactions. No intramolecular phosphate mi-
gration was observed. The pKa values of the phosphonate
and the phosphinate moieties in compound 6a were deter-
mined by 31P NMR titration studies and are around 2.7
General Procedure 1 (GP1). Arbuzov Reaction: An alkyl halide (2a–
e, 1 mmol) was added dropwise at room temp. to tetraisopropyl
methylenediphosphonite (1, 1 mmol), and the resulting mixture was
heated at 120 °C under argon for 6 h. The reaction mixture was
and 1.8, respectively; this explains the reactivities of both allowed to cool, DMSO (0.15 mL) was added, and the mixture was
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Acyclic Nucleoside Diphosphate Analogues
heated at 60 °C for 2 h. The mixture was partitioned between water
and CHCl3. The organic fraction was washed with three portions
of water and taken down in vacuo. The residue in EtOH (3 mL)
was treated with HCl (2 , 1.2 mL) and heated under reflux for
2 h. The mixture was cooled to room temp. and neutralized with
aqueous ammonia (25%), and the solvents were evaporated. The
residue in CHCl3 was washed with water, dried with MgSO4 and
purified by chromatography on silica gel (CHCl3/MeOH 0–5%).
2-(2-Iodoethoxy)ethyl Acetate: Pale yellow oil, yield 65%. 1H NMR
(CDCl3): δ = 4.22 (m, 2 H, 1-H), 3.74 (m, 2 H, 3-H), 3.69 (m, 2 H,
2-H), 3.25 (m, 2 H, 4-H), 2.08 (s, 3 H, CH3) ppm. MS (ESI): m/z
(%) = 280.9 (65) [M + Na]+. HRMS (ESI) calcd. for C6H11INaO3
[M + Na]+ 280.9645; found 280.9644.
Diisopropyl [2-Hydroxyethoxymethyl(isopropoxy)phosphoryl]meth-
yl-phosphonate (3a): This compound was prepared by GP1, color-
less oil, yield 41%. 1H NMR (CDCl3): δ = 4.63–4.77 (m, 3 H,
CHiPr), 3.97 [dd, Jgem = 12.9, J(H,P) = 7.6 Hz, 1 H] and 3.84 [dd,
Jgem = 12.9, J(H,P) = 8.7 Hz, 1 H, OCH2P], 3.57–3.72 (m, 4 H, 3-
General Procedure 2 (GP2). Mitsunobu Reaction: Diisopropyl azo-
dicarboxylate (DIAD, 273 µL, 1.4 mmol) was added to a suspen-
sion of triphenylphosphane (394 mg, 1.6 mmol) in dry THF
(6 mL), and the solution was stirred at 0 °C for 0.5 h. The prepared
complex was added dropwise at –40 °C under argon to a suspen-
sion of the purine or pyrimidine base (4a, 4b, 9, or 12, 1.1 mmol)
and the appropriate alcohol (3a–e, 0.5 mmol) in dry THF (3 mL).
The reaction mixture was allowed to warm to room temp. and
stirred overnight, and the solvent was removed.
H, 4-H), 2.30–2.51 (m,
2 H, 1-H), 1.25–1.32 (m, 18 H,
CH3iPr) ppm. 13C NMR (CDCl3): δ = 74.83 [d, J(C,P) = 6.5 Hz]
and 71.24 [d, J(C,P) = 6.6 Hz] and 70.56 [d, J(C,P) = 5.6 Hz,
CHiPr], 66.42 [d, J(C,P) = 119.2 Hz, C-2], 60.39 (C-4), 27.11 [dd,
J(C,P) = 137.0, 83.3 Hz, PCH2P], 23.9 (m, CH3iPr) ppm. MS
(ESI): m/z (%) = 359 (100) [M – H]–. HRMS (ESI) calcd. for
C13H30NaO7P2 [M + Na]+ 383.1359; found 383.1359.
Diisopropyl
[4-Hydroxybutyl(isopropoxy)phosphoryl]methylphos-
General Procedure 3 (GP3). Deprotection of Triisopropyl Esters:
Me3SiBr (3 mL) was added to a solution of the diphosphonate tri-
isopropyl ester (1 mmol) in dry CH3CN (30 mL) and the mixture
was stirred at room temp. overnight. The solvent was removed in
vacuo, and the residue was codistilled with water. The crude prod-
uct was purified by preparative HPLC (linear gradient from H2O
to 50% CH3OH in H2O).
phonate (3b): This compound was prepared by GP1, colorless oil,
yield 28%. 1H NMR (CDCl3): δ = 4.62–4.74 (m, 3 H, CHiPr), 3.61
[t, J(4,3) = 5.9 Hz, 2 H, 4-H], 2.22–2.35 (m, 2 H, PCH2P), 1.85–
1.98 (m, 2 H, 1-H), 1.74 (br. s, 1 H, OH), 1.66–1.73 (m, 2 H, 3-H),
1.53–1.64 (m, 2 H, 2-H), 1.26–1.29 (m, 18 H, CH3iPr) ppm. 13C
NMR (CDCl3): δ = 71.73 [d, J(C,P) = 6.5 Hz, CHiPr], 71.27 [d,
J(C,P) = 6.7 Hz, CHiPr], 69.70 [d, J(C,P) = 6.8 Hz, CHiPr], 60.30
(C-4), 32.58 [d, J(C,P) = 16.3 Hz, C-2], 28.74 [dd, J(C,P1) = 135.7,
J(C,P2) = 77.6 Hz, PCH2P], 28.42 [d, J(C,P) = 98.2 Hz, C-1], 24.44
[d, J(C,P) = 3.6 Hz, CH3iPr], 24.25 [d, J(C,P) = 4.2 Hz, CH3iPr],
24.05 [d, J(C,P) = 3.6 Hz, CH3iPr], 23.98 [d, J(C,P) = 4.6 Hz,
2ϫCH3iPr], 23.84 [d, J(C,P) = 5.1 Hz, CH3iPr], 17.64 [d, J(C,P) =
3.6 Hz, C-3] ppm. MS (ESI): m/z (%) = 381 (100) [M + Na]+, 358.9
(52) [M + H]+. HRMS (ESI) calcd. for C14H32NaO6P2 [M +
Na]+ 381.1566; found 381.1567.
5-Iodopentyl Acetate (2c): 5-Chloropentyl acetate (10 g, 60 mmol)
in acetone (500 mL) was treated with NaI (45 g, 0.3 mol) and
heated under reflux for 32 h.[44] The mixture was evaporated to half
of its volume and partitioned between H2O and CHCl3, the organic
fraction was washed with H2O, saturated Na2S2O3, and H2O and
dried with MgSO4, and the solvents were evaporated. Pale yellow
1
oil, yield 11.4 g (73%). H NMR ([D6]DMSO): δ = 3.99 [t, J(1,2)
= 6.57 Hz, 2 H, 1-H], 3.28 [t, J(5,4) = 6.88 Hz, 2 H, 5-H], 1.99 (s,
3 H, CH3), 1.77 (m, 2 H, CH2), 1.58 (m, 2 H, CH2), 1.38 (m, 2 H,
CH2) ppm. MS (ESI): m/z (%) = 279.0 (26) [M + Na]+. HRMS
(ESI) calcd. for C7H13INaO2 [M + Na]+ 278.9858; found 278.9852.
Diisopropyl [5-Hydroxypentyl(isopropoxy)phosphoryl]methyl-phos-
phonate (3c): This compound was prepared by GP1, colorless oil,
yield 30%. H NMR (CDCl3): δ = 4.68 (m, 3 H, CHiPr), 3.58 [t,
1
J(1,2) = 6.4 Hz, 2 H, 1-H], 2.27 (m, 2 H, PCH2P), 1.87 (m, 2 H,
5-H), 1.60 (m, 2 H, 4-H), 1.53 (m, 2 H, 2-H), 1.44 (m, 2 H, 3-H),
1.25–1.30 (m, 18 H, CH3iPr) ppm. 13C NMR (CDCl3): δ = 71.41
[d, J(C,P) = 6.7 Hz], 71.15 [d, J(C,P) = 6.5 Hz] and 69.66 [d, J(C,P)
= 6.9 Hz, CHiPr], 62.32 (C-1), 31.94 (C-2), 29.82 [d, J(C,P) =
98.5 Hz, C-5], 29.32 [dd, J(C,P) = 7.69, 135.8 Hz, PCH2P], 26.80 [d,
J(C,P) = 16.3 Hz, C-3], 23.22–24.45 (m, CH3iPr), 21.28 [d, J(C,P) =
6.6 Hz, C-4] ppm. MS (ESI): m/z (%) = 395.1 (82) [M + Na]+.
HRMS (ESI) calcd. for C15H34NaO6P2 [M + Na]+ 395.1723; found
395.1721.
6-Iodohexyl Acetate (2d): Acetic anhydride (10 mL) was added
dropwise at 0 °C to 6-chlorohexan-1-ol (10 mL, 71.5 mmol) in pyr-
idine (30 mL), and the mixture was allowed to warm slowly to
room temp. and stirred for 4 h. EtOH (20 mL) was added, the mix-
ture was concentrated, and the residue was codistilled with EtOH,
diluted with CHCl3, and washed with HCl (1 ), saturated
NaHCO3, and H2O. The organic fraction was dried with MgSO4
and concentrated to give 6-chlorohexyl acetate, yield 12.8 g (99%).
1H NMR (CDCl3): δ = 4.06 [t, J(1,2) = 6.67 Hz, 2 H, 1-H], 3.53
[t, J(6,5) = 6.67 Hz, 2 H, 6-H], 2.04 (s, 3 H, CH3), 1.78 (m, 2 H,
CH2), 1.64 (m, 2 H, CH2), 1.47 (m, 2 H, CH2), 1.38 (m, 2 H,
CH2) ppm. MS (ESI): m/z (%) = 179 (15) [M + H]+.
Diisopropyl [6-Hydroxyhexyl(isopropoxy)phosphoryl]methyl-phos-
phonate (3d): This compound was prepared by GP1, colorless oil,
yield 40%. 1H NMR (CDCl3): δ = 4.70–4.80 (m, 3 H, CHiPr), 3.62
[t, J(1,2) = 6.5 Hz, 2 H, 1-H], 2.33 (m, 2 H, PCH2P), 1.86–2.0 (m,
2 H, 6-H), 1.65 (m, 2 H, 5-H), 1.57 (m, 2 H, 2-H), 1.37–1.48 (m, 4
H, 3-H, 4-H), 1.32–1.36 (m, 18 H, CH3iPr) ppm. 13C NMR
(CDCl3): δ = 71.32 [d, J(C,P) = 6.6 Hz], 71.09 [d, J(C,P) = 6.5 Hz]
and 69.60 [d, J(C,P) = 6.8 Hz, CHiPr], 62.44 (C-1), 32.29 (C-2),
30.19 [d, J(4,P) = 16.4 Hz, C-4], 29.68 [d, J(6,P) = 98.5 Hz, C-6],
29.30 [dd, J(C,P) = 77.0, 135.9 Hz, PCH2P], 25.01 (C-3), 23.82–
24.40 (m, CH3iPr), 21.42 [d, J(5,P) = 4.6 Hz, C-5] ppm. MS (ESI):
m/z (%) = 387 (6) [M + H]+, 409.1 (55) [M + H]+. HRMS (ESI)
calcd. for C16H36NaO6P2 [M + Na]+ 409.1879; found 409.1880.
6-Chlorohexyl acetate was converted into its 6-iodo congener 2d by
1
the method described for 2c, pale yellow oil, yield 90%. H NMR
(CDCl3): δ = 4.05 [t, J(1,2) = 6.67 Hz, 2 H, 1-H], 3.18 [t, J(6,5) =
6.97 Hz, 2 H, 6-H], 2.04 (s, 3 H, CH3), 1.84 (m, 2 H, CH2), 1.63
(m, 2 H, CH2), 1.46–1.32 (m, 4 H, 2ϫCH2) ppm. MS (ESI): m/z
(%) = 293.1 (63) [M + Na]+. HRMS (ESI) calcd. for C8H15INaO2
[M + Na]+ 293.0977; found 293.0976.
2-(2-Iodoethoxy)ethyl Acetate (2e): This compound was prepared
from 2-(chloroethoxy)ethanol by the procedure described for 2d.
2-(2-Chloroethoxy)ethyl Acetate: Colorless oil, yield 88%. 1H NMR
(CDCl3): δ = 4.36 (m, 2 H, 1-H), 3.88 [t, J(3,4) = 5.76 Hz, 2 H, 3-
Diisopropyl [2-Hydroxyethoxyethyl(isopropoxy)phosphoryl]methyl-
H], 3.85 (m, 2 H, 2-H), 3.76 [t, J(4,3) = 5.76 Hz, 2 H, 4-H], 2.08 phosphonate (3e): This compound was prepared by GP1, colorless
1
(s, 3 H, CH3) ppm. MS (ESI): m/z (%) = 189.0 (94) [M + Na+].
oil, yield 37%. H NMR (CDCl3): δ = 4.76 (m, 3 H, CHiPr), 3.85
Eur. J. Org. Chem. 2009, 1082–1092
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1087

P. Doláková, M. Dracˇínský, J. Fanfrlík, A. Holý
FULL PAPER
(m, 2 H, 3-H), 3.71 (m, 2 H, 1-H), 3.59 (m, 2 H, 2-H), 2.51 (m, 2 [dd, J(C,P1) = 128.8, J(C,P2) = 80.5 Hz, PCH2P] ppm. MS (ESI):
H, PCH2P), 2.44 and 2.18 (m, 2 H, 4-H), 1.33–1.37 (m, 18 H, m/z (%) = 351.1 (66) [M + Na]+. C8H14N2O8P2 (328.15): calcd. C
CH3iPr) ppm. 13C NMR (CDCl3): δ = 72.41 (C-2), 71.45 [d, J(C,P) 29.28, H 4.30, N 8.54, O 39.00, P 18.88; found C 29.62, H 4.44, N
= 6.6 Hz], 71.18 [d, J(C,P) = 6.6 Hz], 70.15 [d, J(C,P) = 7.0 Hz,
CHiPr], 64.19 [d, J(C,P) = 5.9 Hz, C-3], 61.12 (C-1), 30.85 [dd,
J(C,P) = 80.8, 135.9 Hz, PCH2P], 29.36 [d, J(C,P) = 98.2 Hz, C-4],
23.82–24.34 (m, CH3iPr) ppm. MS (ESI): m/z (%) = 375 (48) [M +
8.23, P 18.73.
1-{2-[(Hydroxy)(phosphonomethyl)phosphorylmethoxy]ethyl}-
thymine (6b): This compound was prepared by GP3 and freeze
dried, white solid, yield 82%, m.p. 238 °C. 1H NMR (D2O): δ =
7.52 [q, J(6,CH3) = 1.2 Hz, 1 H, 6-H], 3.97 (m, 2 H, 1Ј-H), 3.81–
3.83 (m, 4 H, 2Ј-H, OCH2P), 2.38 [dd, J(H,P) = 16.9, J(H,P) =
20.5 Hz, 2 H, PCH2P], 1.88 [d, J(CH3,6) = 1.2 Hz, 3 H, 5-
CH3] ppm. 13C NMR (D2O): δ = 167.68 (C-4), 152.90 (C-2), 144.49
(C-6), 110.91 (C-5), 71.20 [d, J(C,P) = 12.6 Hz, C-2Ј], 68.38 [d,
J(C,P) = 118.2 Hz, OCH2P], 48.68 (C-1Ј), 27.20 [dd, J(C,P) = 81.2,
J(C,P) = 129.2 Hz, PCH2P], 11.89 (5-CH3) ppm. MS (ESI): m/z
(%) = 341 (100) [M – H]–. C10H18N2O7P2 (342.18): calcd. C 31.59,
H 4.71, N 8.19, O 37.41, P 18.10; found C 31.39, H 4.72, N 7.96,
P 18.41.
H]+, 397.1 (100) [M
+
Na]+. HRMS (ESI) calcd. for
C14H32NaO7P2 [M + Na]+ 397.1515; found 397.1514.
1-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphorylmeth-
oxy]ethyl}uracil (5a): This compound was prepared by GP2, the
crude product in dry MeOH (5 mL) was treated with MeONa/
MeOH (1 , 1 mL) at room temp. overnight and neutralized with
acetic acid, and the solvents were evaporated. The product was
purified by flash chromatography (CHCl3/MeOH 0–10%) gave a
1
colorless oil (180 mg, 79%). H NMR (CDCl3): δ = 9.29 (br. s, 1
H, NH), 7.42 [d, J(6,5) = 7.9 Hz, 1 H, 6-H], 5.65 [dd, J(5,6) = 7.9,
J(5,NH) = 1.9 Hz, 1 H, 5-H], 4.79 [octet, J(CH,CH3) = J(CH,P) =
6.3 Hz, 1 H, CHiPr], 4.78 [dh, J(CH,CH3) = 6.2, J(CH,P) = 7.0 Hz,
1 H, CHiPr], 4.74 [dh, J(CH,CH3) = 6.2, J(CH,P) = 8.0 Hz, 1 H,
CHiPr], 4.01 [ddd, Jgem = 14.5, J(1Јa,2Јa) = 5.8, J(1Јa,2Јb) =
3.2 Hz, 1 H, 1Јa-H], 4.00 [dd, Jgem = 13.3, J(H,P) = 6.9 Hz, 1 H,
OCH2Pa], 3.92 (m, 2 H, 1Јb, OCH2Pb), 3.84 (m, 2 H, -H2Ј), 2.40
(m, 2 H, PCH2P), 1.36 [d, J(CH3,CH) = 6.3 Hz, 3 H, CH3iPr], 1.35
[d, J(CH3,CH) = 6.3 Hz, 3 H, CH3iPr], 1.35 [d, J(CH3,CH) =
6.3 Hz, 3 H, CH3iPr], 1.34 [d, J(CH3,CH) = 6.3 Hz, 3 H, CH3iPr],
1.31 [d, J(CH3,CH) = 6.3 Hz, 3 H, CH3iPr] ppm. 13C NMR
(CDCl3): δ = 163.68 (C-4), 150.79 (C-2), 145.80 (C-6), 101.50 (C-
5), 71.83 [d, J(C,P) = 6.4 Hz, CHiPr], 71.36 [d, J(C,P) = 6.5 Hz,
CHiPr], 71.16 [d, J(2Ј,P) = 12.3 Hz, C-2Ј], 70.92 [d, J(C,P) =
6.7 Hz, CHiPr], 67.63 [d, J(C,P) = 117.3 Hz, OCH2P], 48.11 (C-
1Ј), 27.33 [dd, J(C,P) = 136.6, 82.9 Hz, PCH2P], 24.29 [d, J(C,P) =
3.8 Hz, CH3iPr], 24.19 [d, J(C,P) = 3.5 Hz, CH3iPr], 24.07 [d,
J(C,P) = 3.1 Hz, CH3iPr], 23.97 [d, J(C,P) = 4.1 Hz, 2 C, CH3iPr],
23.83 [d, J(C,P) = 5.0 Hz, CH3iPr] ppm. MS (ESI): m/z (%) = 477.0
(100) [M + Na]+, 454.9 (19) [M + H]+. C17H32N2O8P2 (454.39):
calcd. C 44.94, H 7.10, N 6.17, O 28.17, P 13.63; found C 45.19,
H 7.38, N 5.81, P 13.58.
1-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl-meth-
oxy]ethyl}cytosine (7): Compound 5a (310 mg, 0.68 mmol), Et3N
(0.3 mL), and TPSCl (0.63 g, 2.04 mmol) were stirred in CH3CN
(10 mL) at room temp. for 48 h. NH4OH (25%, 5 mL) was added,
the mixture was stirred for 24 h, and the solvents were evaporated.
The residue in ethyl acetate was washed with brine, the aqueous
fraction was than washed with five portions of CHCl3, and the
organic fractions were dried with MgSO4 and concentrated under
reduced pressure. The crude product was purified by flash
chromatography to give a pale yellow foam (240 mg, 78%). 1H
NMR ([D6]DMSO): δ = 7.60 [d, J(6,5) = 7.25 Hz, 1 H, 6-H], 7.49
(br. s, 1 H, NH-a), 7.23 (br. s, 1 H, NH-b), 5.67 [d, J(5,6) = 7.25 Hz,
1 H, 5-H], 4.60 (m, 6 H, CHiPr), 3.83 (m, 4 H, 1Ј-H, 3Ј-H), 3.69
(m, 2 H, 2Ј-H), 2.40 (m, 2 H, PCH2P), 1.24 (m, 9 H, CH3iPr), 1.18
[d, J(CH3,CH) = 6.14 Hz, 3 H, CH3iPr], 1.16 [d, J(CH3,CH) =
6.19 Hz, 3 H, CH3iPr], 1.10 [d, J(CH3,CH) = 6.19 Hz, 3 H,
CH3iPr] ppm. MS (ESI): m/z (%) = 454.0 (70) [M + H]+, 496.0 (84)
[M + Na]+. C17H33N3O7P2 (453.41): calcd. C 45.03, H 7.34, N 9.27,
O 24.70, P 13.66; found C 45.12, H 7.31, N 9.12, P 13.52.
1-{2-[(Hydroxy)(phosphonomethyl)phosphorylmethoxy]ethyl}-
cytosine (8): This compound was prepared by GP3 and freeze dried,
white foam, yield 69 %. 1H NMR (D2O): δ = 7.90 [d, J(6,5) =
7.6 Hz, 1 H, 6-H], 6.17 [d, J(5,6) = 7.7 Hz, 1 H, 5-H], 4.08 [t,
J(1Ј,2Ј) = 4.7 Hz, 2 H, 1Ј-H], 3.83 [t, J(2Ј,1Ј) = 4.7 Hz, 2 H, 2Ј-H],
3.78 [d, J(H,P) = 7.6 Hz, 2 H, OCH2P], 2.31 [dd, J(H,P) = 6.8,
10.3 Hz, 2 H, PCH2P] ppm. 13C NMR (D2O): δ = 160.16 (C-4),
151.04 (C-6), 149.69 (C-2), 94.67 (C-5), 70.49 [d, J(2Ј,P) = 13.1 Hz,
C-2Ј], 68.80 [d, J(C,P) = 119.9 Hz, OCH2P], 49.87 (C-1Ј), 27.79
[dd, J(C,P) = 79.1, 125.9 Hz, PCH2P] ppm. MS (ESI): m/z (%) =
326 (100) [M – H]–. C8H15N3O7P2 (327.17): calcd. C 29.37, H 4.62,
N 12.84, O 34.23, P 18.93; found C 29.31, H 4.82, N 12.83, P 18.78.
1-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphorylmeth-
oxy]ethyl}thymine (5b): This compound was prepared by GP2, the
crude product in dry MeOH (5 mL) was treated with MeONa/
MeOH (1 , 1 mL) at room temp. overnight and neutralized with
acetic acid, and the solvents were evaporated. The product was
purified by flash chromatography (CHCl3/MeOH 0–10%) to give
a colorless oil (122 mg, 52%). 1H NMR (CDCl3): δ = 8.16 (br. s, 1
H, NH), 7.18 [d, J(6,CH3) = 1.20 Hz, 1 H, 6-H], 4.77 (m, 3 H,
CHiPr), 3.95 (m, 4 H, 1Ј-H, 2Ј-H), 3.80 (m, 2 H, 3Ј-H), 2.37 (m, 2
H, PCH2P), 1.36 [d, J(CH3,CH) = 6.2 Hz, 3 H, CH3iPr], 1.34 [d,
J(CH3,CH) = 6.2 Hz, 3 H, CH3iPr], 1.33 [d, J(CH3,CH) = 6.2 Hz,
3 H, CH3iPr], 1.32 [d, J(CH3,CH) = 6.2 Hz, 3 H, CH3iPr], 1.31 [d,
J(CH3,CH) = 6.2 Hz, 3 H, CH3iPr] ppm. MS (ESI): m/z (%) =
491.0 (100) [M + Na]+, 469.0 (35) [M + H]+. C18H34N2O8P2
(454.39): calcd. C 46.15, H 7.32, N 5.98, O 27.32, P 13.22; found
C 45.98, H 7.41, N 5.86, P 13.41.
9-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl-meth-
oxy]ethyl}adenine (10): This compound was prepared by GP2, and
the residue in dichloromethane (40 mL) was treated with HCl (6 ,
40 mL) and heated under reflux for 6 h. The pH of the aqueous
phase was adjusted to 8 with a saturated aqueous solution of
NaHCO3. The mixture was then extracted with five portions of
dichloromethane, the combined organic fractions were dried with
MgSO4, and the solvent was evaporated under reduced pressure.
The crude product was purified by flash chromatography (CHCl3/
1-{2-[(Hydroxy)(phosphonomethyl)phosphorylmethoxy]ethyl}uracil
(6a): This compound was prepared by GP3, white solid, yield 83%,
m.p. 195 °C. 1H NMR (D2O): δ = 7.68 [d, J(6,5) = 7.9 Hz, 1 H,
H-6], 5.82 [d, J(5,6) = 7.8 Hz, 1 H, 5-H], 4.01 (m, 2 H, H-1Ј), 3.81– MeOH). Crystallization from ethyl acetate/light petroleum gave a
1
3.84 (m, 4 H, H-2Ј, OCH2P), 2.40 [dd, J(H,P1) = 20.3, J(H,P2) =
white, crystalline product (28%), m.p. 82 °C. H NMR (CDCl3): δ
17.0 Hz, 2 H, PCH2P] ppm. 13C NMR (D2O): δ = 167.54 (C-4), = 8.35 (s, 1 H, 2-H), 7.97 (s, 1 H, 8-H), 5.84 (br. s, 2 H, NH2),
152.87 (C-2), 148.64 (C-6), 101.77 (C-5), 71.07 [d, J(C,P) = 12.5 Hz,
C-2Ј], 68.40 [d, J(C,P) = 117.6 Hz, OCH2P], 48.93 (C-1Ј), 27.33
4.68–4.80 (m, 3 H, CHiPr), 4.42 (m, 2 H, 1Ј-H), 4.01 [dd, Jgem
13.3, J(H,P) = 6.4 Hz, 1 H, OCH2Pa], 3.98 (m, 2 H, 2Ј-H), 3.90
=
1088
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1082–1092

Acyclic Nucleoside Diphosphate Analogues
1
[dd, Jgem = 13.3, J(H,P) = 7.7 Hz, 1 H, OCH2Pb], 2.31–2.42 (m, 2 190 °C with dec. H NMR (D2O): δ = 7.88 (s, 1 H, 8-H), 4.25 [t,
H, PCH2P), 1.31–1.35 (m, 15 H, CH3iPr) and 1.24 [d, J(CH3,CH) J(1Ј,2Ј) = 5.1 Hz, 2 H, 1Ј-H], 3.92 [t, J(2Ј,1Ј) = 5.1 Hz, 2 H, 2Ј-H],
= 6.2 Hz, 3 H, CH3iPr] ppm. 13C NMR (CDCl3): δ = 155.39 (C- 3.71 [d, J(H,P) = 6.9 Hz, 2 H, OCH2P], 2.02 [dd, J(H,P) = 17.0,
6), 152.89 (C-2), 149.90 (C-4), 141.37 (C-8), 119.39 (C-5), 71.71 [d,
J(C,P) = 6.5 Hz, CHiPr], 71.31 [d, J(2Ј,P) = 12.1 Hz, C-2Ј], 71.28 154.28 (C-2), 152.03 (C-4), 141.12 (C-8), 116.30 (C-5), 71.31 [d,
19.1 Hz, 2 H, PCH2P] ppm. 13C NMR (D2O): δ = 159.54 (C-6),
[d, J(C,P) = 7.0 Hz, CHiPr], 70.81 [d, J(C,P) = 6.8 Hz, CHiPr],
67.55 [d, J(C,P) = 116.8 Hz, OCH2P], 43.31 (C-1Ј), 27.22 [dd,
J(C,P) = 136.4, J(C,P) = 83.1 Hz, PCH2P], 23.99 (m, CH3iPr) ppm.
MS (ESI): m/z (%) = 500.1 (100) [M + Na]+. C18H33N5O6P2
(477.43): calcd. C 45.28, H 6.97, N 14.67, O 20.11, P 12.98; found
C 45.36, H 6.92, N 14.30, P 12.89.
J(2Ј,P) = 10.9 Hz, C-2Ј], 69.82 [d, J(C,P) = 112.9 Hz, OCH2P],
43.70 (C-1Ј), 30.33 [dd, J(C,P) = 80.1, 119.6 Hz, PCH2P] ppm. MS
(ESI): m/z (%) = 366.0 (100) [M – H]–. C9H15N5O7P2·H2O (385.20):
calcd. C 28.06, H 4.45, N 18.18, O 33.23, P 16.08; found C 28.11,
H 4.39, N 17.77, P 15.77.
1-{4-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl]-
butyl}uracil (16a): This compound was prepared by GP2, the crude
product in dry MeOH (5 mL) was treated with MeONa/MeOH
(1 , 1 mL) at room temp. overnight and neutralized with acetic
acid, and the solvents were evaporated. Purification by flash
chromatography (CHCl3/MeOH 0–10%) afforded a colorless oil
9-{2-[(Hydroxy)(phosphonomethyl)phosphorylmethoxy]ethyl}adenine
(11): This compound was prepared by GP3 and crystallized from
water, white crystals, yield 78%, m.p. 196 °C. 1H NMR (D2O +
NaOD): δ = 8.22 (s, 1 H, 8-H), 8.14 (s, 1 H, 2-H), 4.41 [t, J(1Ј,2Ј)
= 5.1 Hz, 2 H, 1Ј-H], 3.95 [t, J(2Ј,1Ј) = 5.1 Hz, 2 H, 2Ј-H], 3.71 [d,
J(H,P) = 6.5 Hz, 2 H, PCH2O], 1.96 [t, J(H,P) = 18.0 Hz, 2 H,
PCH2P] ppm. 13C NMR (D2O + NaOD): δ = 155.91 (C-6), 152.75
(C-2), 149.27 (C-4), 143.63 (C-8), 118.76 (C-5), 71.15 [d, J(2Ј,P) =
10.4 Hz, C-2Ј], 69.99 [d, J(C,P) = 111.8 Hz, PCH2O], 44.06 (C-1Ј),
30.98 [dd, J(C,P) = 80.9, J(C,P) = 118.4 Hz, PCH2P] ppm. MS
(ESI): m/z (%) = 350.1 (100) [M – H]–. C9H15N5O6P2·H2O (369.21):
calcd. C 29.28, H 6.64, N 18.97, O 30.33, P 16.78; found C 29.60,
H 6.65, N 18.98, P 17.06.
1
(225 mg, 96%). H NMR (CDCl3): δ = 9.16 [d, J(NH,5) = 6.5 Hz,
1 H, NH], 7.18 [d, J(6,5) = 7.9 Hz, 1 H, 6-H], 5.61 [dd, J(5,6) =
7.9, J(5,NH) = 2.0 Hz, 1 H, 5-H], 4.64–4.73 (m, 3 H, CHiPr), 3.64–
3.73 (m, 2 H, 1Јa,1Јb-H), 2.22–2.36 (m, 2 H, PCH2P), 1.86–2.02
(m, 2 H, 4Ј-H), 1.74–1.79 (m, 2 H, 2Ј-H), 1.60–1.66 (m, 2 H, 3Ј-
H), 1.25–1.29 (m, 18 H, CH3iPr) ppm. 13C NMR (CDCl3): δ =
163.68 (C-4), 150.71 (C-2), 144.57 (C-6), 102.06 (C-5), 71.57 [d,
J(C,P) = 6.6 Hz, CHiPr], 71.19 [d, J(C,P) = 6.7 Hz, CHiPr], 69.93
[d, J(C,P) = 6.8 Hz, CHiPr], 48.41 (C-1Ј), 29.63 [d, J(C,P) =
15.4 Hz, C-2Ј], 29.43 [dd, J(C,P) = 135.3, J(C,P) = 177.5 Hz,
PCH2P], 29.07 [d, J(C,P) = 99.2 Hz, C-4Ј], 24.42 [d, J(C,P) =
3.7 Hz, CH3iPr], 24.16 [d, J(C,P) = 4.3 Hz, CH3iPr], 24.05 [d,
J(C,P) = 3.7 Hz, CH3iPr], 23.97 [d, J(C,P) = 4.5 Hz, 2ϫCH3iPr],
23.86 [d, J(C,P) = 5.2 Hz, CH3iPr], 18.58 [d, J(C,P) = 4.4 Hz, C-
3Ј] ppm. MS (ESI): m/z (%) = 452.9 (100) [M + H]+, 475.1 (67) [M
+ Na]+. C18H34N2O7P2 (452.42): calcd. C 47.79, H 7.57, N 6.19, O
24.75, P 13.69; found C 47.71, H 7.53, N 5.92, P 13.60.
N2-Triphenylphosphoranylidene 2-Amino-6-chloro-9-{2-[(diisopro-
poxyphosphorylmethyl)(isopropoxy)phosphorylmethoxy]ethyl}purine
(13): This compound was prepared by GP2, and the residue was
separated by flash chromatography (CHCl3/MeOH), white foam,
1
yield 24%. H NMR (CDCl3): δ = 7.87 (m, 5 H, arom.), 7.77 (s, 1
H, 8-H), 7.54 (m, 3 H, arom.), 7.44 (m, 7 H, arom.), 4.71 (m, 3 H,
CHiPr), 4.20 (m, 2 H, 2Ј-H), 3.94 [dd, Jgem = 13.3, J(H,P) = 6.2 Hz,
1 H, PCH2a], 3.79 [dd, Jgem = 13.2, J(H,P) = 7.7 Hz, 1 H, PCH2b],
3.72 (m, 2 H, 1Ј-H), 2.35 (m, 2 H, PCH2P), 1.31 (m, 15 H, CH3iPr),
1.23 [d, J(CH3,CH) = 6.2 Hz, 3 H, CH3iPr] ppm. MS (ESI): m/z
(%) = 772.2 (100) [M + H]+. MS (ESI): m/z (%) = 770.2 (100) [M –
H]–.
1-{5-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl]-
pentyl}uracil (16b): This compound was prepared by GP2, the
crude product in dry MeOH (5 mL) was treated with MeONa/
MeOH (1 , 1 mL) at room temp. overnight and neutralized with
acetic acid, and the solvents were evaporated. The product was
isolated by flash chromatography (CHCl3/MeOH 0–10%) as a col-
9-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl-meth-
oxy]ethyl}guanine (14): Compound 13 (350 mg, 0.45 mmol) in THF
(10 mL) was treated with water (3 mL) and heated under reflux for
2 d. The mixture was evaporated and the crude product was treated
with TFA in water (75%, 10 mL) at room temp. overnight. The
solvent was evaporated and the residue was codistilled with water.
The product was purified by flash chromatography (CH3Cl/MeOH)
and crystallized from EtOH/Et2O mixture to afford white crystals,
yield (two steps) 167 mg (74 %), m.p. 108 °C. 1H NMR ([D6]-
DMSO): δ = 10.56 (br. s, 1 H, NH), 7.65 (s, 1 H, 8-H), 6.44 (br. s,
2 H, NH2), 4.53–4.63 (m, 3 H, CHiPr), 4.12 (m, 2 H, 1Ј-H), 3.85
[d, J(H,P) = 6.7 Hz, 2 H, OCH2P], 3.81 (m, 2 H, 2Ј-H), 2.38–2.51
(m, 2 H, PCH2P), 1.23 (d, J = 6.2 Hz, 6 H), 1.22 (d, J = 6.2 Hz, 6
H), 1.20 (d, J = 6.2 Hz, 3 H) and 1.12 (d, J = 6.2 Hz, 3 H,
CH3iPr) ppm. 13C NMR ([D6]DMSO): δ = 157.05 (C-6), 153.74
(C-2), 151.38 (C-4), 137.93 (C-8), 116.61 (C-5), 70.88 [d, J(2Ј,P) =
11.3 Hz, C-2Ј], 70.61 [d, J(C,P) = 6.2 Hz], 70.44 [d, J(C,P) = 6.2 Hz]
and 69.80 [d, J(C,P) = 6.5 Hz, CHiPr], 66.85 [d, J(C,P) = 115.7 Hz,
OCH2P], 42.44 (C-1Ј), 25.99 [dd, J(C,P) = 81.7, 133.9 Hz], 23.78–
24.24 (m, CH3iPr) ppm. MS (ESI): m/z (%) = 494.1 (34) [M +
H]+, 516.1 (100) [M + Na]+. C18H33N5O7P2 (493.43): calcd. C
43.81, H 6.74, N 14.19, O 22.70, P 12.55; found C 43.87, H 6.77,
N 13.76, P 12.59.
1
orless oil (186 mg, 79%). H NMR (CDCl3): δ = 9.15 (br. s, 1 H,
NH), 7.21 [d, J(6,5) = 7.9 Hz, 1 H, 6-H], 5.68 [dd, J(5,6) = 7.9,
J(5,NH) = 1.5 Hz, 1 H, 5-H], 4.70–4.80 (m, 3 H, CHiPr), 3.73 (m,
2 H, 1Ј-H), 2.35 (m, 2 H, PCH2P), 1.95 (m, 2 H, 5Ј-H), 1.65–1.76
(m, 4 H, 2Ј-H, 4Ј-H), 1.46 (m, 2 H, 3Ј-H), 1.32–1.36 (m, 18 H,
CH3iPr) ppm. 13C NMR (CDCl3): δ = 163.65 (C-4), 150.71 (C-2),
144.49 (C-6), 102.04 (C-5), 71.46 [d, J(C,P) = 6.7 Hz], 71.13 [d,
J(C,P) = 6.4 Hz] and 69.76 [d, J(C,P) = 7.0 Hz, CHiPr], 48.47 (C-
1Ј), 29.37 [dd, J(C,P) = 77.7, 135.2 Hz, PCH2P], 29.59 [d, J(C,P) =
99.0 Hz, C-5Ј], 28.37 (C-2Ј), 27.26 [d, J(3Ј,P) = 16.5 Hz, C-3Ј],
23.84–24.44 (m, CH3iPr), 21.10 [d, J(C,P) = 4.5 Hz, C-4Ј] ppm. MS
(ESI): m/z = 489.1 [M + Na]+ . HRMS (ESI) calcd. for
C19H36N2NaO7P2 [M + Na]+ 489.1895; found 489.1891.
C19H36N2O7P2 (466.44): calcd. C 48.92, H 7.78, N 6.01, O 24.01,
P 13.28; found C 48.81, H 7.72, N 5.83, P 13.11.
1-{6-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl]-
hexyl}uracil (16c): This compound was prepared by GP2, the prod-
uct in dry MeOH (5 mL) was treated with MeONa/MeOH (1 ,
1 mL) at room temp. overnight and neutralized with acetic acid,
and the solvents were evaporated. Flash chromatography (CHCl3/
9-{2-[(Hydroxy)(phosphonomethyl)phosphorylmethoxy]ethyl}- MeOH 0–10%) gave 16c as a colorless oil (187 mg, 78%). 1H NMR
guanine (15): This compound was prepared by GP3; DMF (1 mL)
was added to the reaction mixture. White solid, yield 52%, m.p.
(CDCl3): δ = 9.45 (br. s, 1 H, NH), 7.19 [d, J(6,5) = 7.9 Hz, 1 H,
6-H], 5.70 [d, J(5,6) = 7.9 Hz, 5-H], 4.70–4.82 (m, 3 H, CHiPr),
Eur. J. Org. Chem. 2009, 1082–1092
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1089

P. Doláková, M. Dracˇínský, J. Fanfrlík, A. Holý
FULL PAPER
1
3.72 (m, 2 H, 1Ј-H), 2.36 (m, 2 H, PCH2P), 1.86–2.02 (m, 2 H, 6Ј- 185–186 °C. H NMR (D2O): δ = 7.64 [d, J(6,5) = 7.8 Hz, 1 H, 6-
H), 1.69 (m, 2 H, 2Ј-H), 1.64 (m, 2 H, 5Ј-H), 1.45 (m, 2 H, 4Ј-H), H], 5.81 [d, J(5,6) = 7.8 Hz, 1 H, 5-H], 3.77 (t, 2 H, 1Ј-H), 2.33
1.37 (m, 2 H, 3Ј-H), 1.32–1.36 (m, 18 H, CH3iPr) ppm. 13C NMR [dd, J(H,P) = 16.7, 19.8 Hz, 2 H, PCH2P], 1.89 (m, 2 H, 6Ј-H),
(CDCl3): δ = 163.72 (C-4), 150.76 (C-2), 144.38 (C-6), 102.02 (C-
5), 71.34 [d, J(C,P) = 6.6 Hz], 71.07 [d, J(C,P) = 6.5 Hz] and 69.65
1.69 (m, 2 H, 2Ј-H), 1.56 (m, 2 H, 5Ј-H), 1.43 (m, 2 H, 4Ј-H), 1.35
(m, 2 H, 3Ј-H) ppm. 13C NMR (D2O): δ = 167.54 (C-4), 152.98
[d, J(C,P) = 6.8 Hz, CHiPr], 48.61 (C-1Ј), 30.04 [d, J(4Ј,P) = (C-2), 148.13 (C-6), 101.92 (C-5), 49.57 (C-1Ј), 30.09 [d, J(4Ј,P) =
16.2 Hz, C-4Ј], 29.70 [d, J(6Ј,P) = 99.6 Hz, C-6Ј], 29.32 [dd, J(C,P) 16.4 Hz, C-4Ј], 29.81 [dd, J(C,P) = 78.1, 123.4 Hz, PCH2P], 29.70
= 77.8, 134.3 Hz, PCH2P], 28.68 (C-2Ј), 25.83 (C-3Ј), 23.83–24.43
(m, CH3iPr), 21.36 [d, J(5Ј,P) = 4.6 Hz, C-5Ј] ppm. MS (ESI): m/z
[d, J(5Ј,P) = 95.7 Hz, C-6Ј], 28.42 (C-2Ј), 25.67 (C-3Ј), 21.51 [d,
J(5Ј,P) = 4.3 Hz, C-5Ј] ppm. MS (ESI): m/z (%) = 353 (100) [M –
( % ) = 5 0 3 ( 1 0 0 ) [ M + N a ] + . H R M S ( E S I ) c a l c d . fo r H]–. C11H20N2O7P2 (354.23): calcd. C 37.30, H 5.69, N 7.91, O
C20H38N2NaO7P2 [M + Na]+ 503.2051; found 503.2045.
C20H38N2O7P2 (480.47): calcd. C 50.00, H 7.97, N 5.83, O 23.31,
P 12.89; found C 50.26, H 7.99, N 5.68, P 12.79.
31.62, P 17.49; found C 37.69, H 5.71, N 7.96, P 17.20.
1-{2-[(Hydroxy)(phosphonomethyl)phosphorylethoxy]ethyl}uracil (17d):
This compound was prepared by GP3, white solid, yield 72%, m.p.
1
1-{2-[(Diisopropoxyphosphorylmethyl)(isopropoxy)phosphoryl-
ethoxy]ethyl}uracil (16d): This compound was prepared by GP2,
the crude product in dry MeOH (5 mL) was treated with MeONa/
MeOH (1 , 1 mL) at room temp. overnight and neutralized with
acetic acid, and the solvents were evaporated. The product was
purified by flash chromatography (CHCl3/MeOH 0–10%), color-
less oil (171 mg, 73%). 1H NMR (CDCl3): δ = 9.26 (br. s, 1 H,
NH), 7.43 [d, J(6,5) = 7.9 Hz, 1 H, 6-H], 5.66 [dd, J(5,6) = 7.9,
J(5,NH) = 1.8 Hz, 1 H, 5-H],4.72–4.79 (m, 3 H, CHiPr), 3.92 (m,
195 °C. H NMR (D2O): δ = 7.65 [d, J(6,5) = 7.9 Hz, 1 H, 6-H],
5.81 [d, J(5,6) = 7.9 Hz, 1 H, 5-H], 3.99 (m, 2 H, 1Ј-H), 3.80 [dt,
J(3Ј,4Ј) = 6.9, J(3Ј,P) = 15.5 Hz, 2 H, 3Ј-H], 3.76 (m, 2 H, 2Ј-H),
2.45 [dd, J(H,P) = 17.1, 20.3 Hz, 2 H, PCH2P], 2.23 [dt, J(H,P) =
14.7, J(4Ј,3Ј) = 6.9 Hz, 2 H, 4Ј-H] ppm. 13C NMR (D2O): δ =
167.53 (C-4), 152.92 (C-2), 148.45 (C-6), 101.87 (C-5), 68.40 (C-
2Ј), 65.17 [d, J(C,P) = 2.7 Hz, C-3Ј], 48.84 (C-1Ј), 30.68 [d, J(C,P)
= 96.3 Hz, C-4Ј], 30.42 [dd, J(C,P) = 81.1, 126.6 Hz, PCH2P] ppm.
MS (ESI): m/z (%) = 341 (100) [M – H]–. C9H16N2O8P2 (342.18):
2 H, 1Ј-H), 3.66–3.85 (m, 4 H, 2Ј-H, 3Ј-H), 2.35–2.46 (m, 3 H, calcd. C 31.59, H 4.71, N 8.19, O 37.41, P 18.10; found C 31.48,
PCH2P, 4a-H), 2.21 (m, 1 H, 4b-H), 1.32–1.36 (m, 18 H, H 4.83, N 8.15, P 17.82.
CH3iPr) ppm. 13C NMR (CDCl3): δ = 163.76 (C-4), 150.85 (C-2),
Triphosphate Analogues 18–20. General Procedure: Diphosphonate
146.06 (C-6), 101.48 (C-5), 71.48 [d, J(C,P) = 6.5 Hz], 71.26 [d,
6a or 17a–d (0.061 mmol) in MeOH (5 mL) was treated with tri-n-
J(C,P) = 6.5 Hz] and 70.19 [d, J(C,P) = 6.9 Hz, CHiPr], 68.63 (C-
butylamine (29 µL, 0.122 mmol) and the mixture was heated until
2Ј), 64.90 [d, J(C,P) = 2.4 Hz, C-3Ј], 48.21 (C-1Ј), 30.57 [dd, J(C,P)
clear solution was obtained. The solvent was evaporated and the
= 80.3, 135.4 Hz, PCH2P], 30.51 [d, J(C,P) = 97.5 Hz, C-4Ј], 23.84–
residue was dried with P2O5 in vacuo. CDI (49 mg, 0.305 mmol) in
24.37 (m, CH3iPr) ppm. MS (ESI): m/z (%) = 491.1 (100) [M +
DMF (1 mL) was added dropwise under argon at 0 °C to the solu-
Na]+. HRMS (ESI) calcd. for C18H34N2NaO8P2 [M + Na]+
tion of the prepared bis(tri-n-butylammonium) salt in DMF (1 mL)
491.1688; found 491.1686. C18H34N2O8P2 (468.42): calcd. C 46.15,
and the resulting mixture was stirred at room temp. for 3 h. Meth-
H 7.32, N 5.98, O 27.32, P 13.22; found C 45.91, H 7.29, N 5.79,
anol (10 µL, 0.244 mmol) was added, and after the system had been
P 13.12.
stirred for 1 h tri-n-butylammonium phosphate (0.5  in DMF)
1-{4-[(Hydroxy)(phosphonomethyl)phosphoryl]butyl}uracil
(17a):
was added and the reaction mixture was stirred for 6–7 h at room
temp. The reaction solution was diluted with TEAB (2 mL,
0.025 ), placed on a column of POROS® HQ, and eluted with a
linear gradient of TEAB (0–0.4 ). The fractions containing prod-
uct were concentrated, the residue was applied onto DOWEX
50ϫ8 (Na+ form), eluted with water, and concentrated, and the
residue in water (2 mL) was freeze-dried. The structures were eluci-
dated by 1H and 31P NMR and the purities were determined by
HPLC. For 31P NMR spectroscopic data see Table 1.
This compound was prepared by GP3, white solid, yield 74%,
1
182 °C. H NMR (D2O): δ = 7.65 [d, J(6,5) = Hz 7.9, 1 H, 6-H],
5.82 [d, J(5,6) = 7.8 Hz, 1 H, 5-H], 3.81 [t, J(1Ј,2Ј) = 7.2 Hz, 2 H,
1Ј-H], 2.47 [dd, J(H,P) = 16.8, J(H,P) = 20.4 Hz, 2 H, PCH2P],
1.94–1.99 (m, 2 H, 4Јa, 4Јb-H), 1.78–1.83 (m, 2 H, 2Јa, 2Јb-H),
1.58–1.65 (m, 2 H, 3Јa, 3Јb-H) ppm. 13C NMR (D2O): δ = 167.55
(C-4), 152.98 (C-2), 147.98 (C-6), 102.09 (C-5), 48.96 (C-1Ј), 29.63
[d, J(C,P) = 16.5 Hz, C-2Ј], 29.32 [d, J(C,P) = 96.4 Hz, C-4Ј], 29.25
[dd, J(C,P1) = 80.0, J(C,P2) = 127.0 Hz, PCH2P], 18.71 [d, J(C,P)
= 4.1 Hz, C-3Ј] ppm. MS (ESI): m/z (%) = 325 (100) [M – H]–.
C9H16N2O7P2·1/2H2O (335.19): calcd. C 32.25, H 5.11, N 8.36, O
35.80, P 18.48; found C 32.07, H 5.29, N 8.02, P 18.37.
1
Compound 18: White powder, yield 5%. H NMR (D2O): δ = 7.84
[d, J(6,5) = 7.9 Hz, 1 H, H-6], 5.93 [d, J(5,6) = 7.8 Hz, 1 H, H-5],
4.11 [t, J(1Ј,2Ј) = 5.0 Hz, 2 H, 1Ј-H], 3.93 [t, J(2Ј,1Ј) = 5.0 Hz, 2Ј-
H], 3.83 [d, J(CH2,P) = 6.8 Hz, PCH2], 2.37 [dd, J(H,P1) = 20.3,
J(H,P2) = 17.2 Hz, 2 H, PCH2P] ppm.
1-{5-[(Hydroxy)(phosphonomethyl)phosphoryl]pentyl}uracil (17b):
This compound was prepared by GP3, white solid, yield 90%, m.p.
198–200 °C. H NMR (D2O): δ = 7.64 [d, J(6,5) = 7.9 Hz, 1 H, 6-
Compound 19a: White powder, yield 16%. 1H NMR (D2O): δ = 7.8
[d, J(6,5) = 7.8 Hz, 1 H, 6-H], 5.93 [d, J(5,6) = 7.8 Hz, 1 H, 5-H],
3.9 [t, J(1Ј,2Ј) = 7.3 Hz, 2 H, 1Ј-H], 2.6 (m, 2 H, PCH2P), 2.25–
2.18 (m, 2 H, 4Ј-H), 1.96–1.88 (m, 2 H, 2Ј-H), 1.81–1.75 (m, 2 H,
3Ј-H) ppm.
1
H], 5.81 [d, J(5,6) = 7.8 Hz, 1 H, 5-H], 3.79 [t, J(1Ј,2Ј) = 7.2 Hz, 2
H, 1Ј-H], 2.46 [dd, J(H,P) = 16.7, 20.3 Hz, 2 H, PCH2P], 1.92 (m,
2 H, 5Ј-H), 1.72 (m, 2 H, 2Ј-H), 1.62 (m, 2 H, 4Ј-H), 1.43 (m, 2 H,
3Ј-H) ppm. 13C NMR (D2O): δ = 167.57 (C-4), 153.01 (C-2), 148.09
(C-6), 101.98 (C-5), 49.37 (C-1Ј), 29.54 [d, J(C,P) = 95.8 Hz, C-5Ј],
29.25 [dd, J(C,P) = 79.6, 126.6 Hz, PCH2P], 28.13 (C-2Ј), 27.24 [d,
J(3Ј,P) = 16.7 Hz, C-3Ј], 21.24 [d, J(4Ј,P) = 4.1 Hz, C-4Ј] ppm. MS
(ESI): m/z (%) = 339 (100) [M – H]–. C10H18N2O7P2 (340.21): calcd.
C 35.30, H 5.33, N 8.23, O 32.92, P 18.21; found C 35.36, H 5.39,
N 8.08, P 18.29.
Compound 19b: White powder, yield 22%. 1H NMR (D2O): δ =
7.74 [d, J(6,5) = 7.7 Hz, 1 H, 6-H], 5.9 [d, J(5,6) = 7.7 Hz, 1 H, 5-
H], 3.89 [t, J(1Ј,2Ј) = 7.2 Hz, 2 H, 1Ј-H], 2.38 (dd, J1 = J2
=
17.7 Hz, PCH2P), 2.17 (m, 2 H, 5Ј-H), 1.84–1.82 (m, 4 H, 2Ј-H, 4Ј-
H), 1.54 (m, 2 H, 3Ј-H) ppm.
Compound 19c: White powder, yield 19%. 1H NMR (D2O): δ =
7.72 [d, J(6,5) = 7.7 Hz, 1 H, 6-H], 5.9 [d, J(5,6) = 7.7 Hz, 1 H, 5-
1-{6-[(Hydroxy)(phosphonomethyl)phosphoryl]hexyl}uracil
(17c):
This compound was prepared by GP3, white solid, yield 73%, m.p.
H], 3.87 [t, J(1Ј,2Ј) = 6.6 Hz, 2 H, 1Ј-H], 2.38 (dd, J1 = J2
=
1090
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 1082–1092

Acyclic Nucleoside Diphosphate Analogues
[8]
a) C. C. Tsai, K. E. Follis, T. W. Beck, A. Sabo, N. Bischof-
berger, P. J. Dailey, AIDS Res. Hum. Retroviruses 1997, 13,
707–712; b) R. V. Srinivas, A. Fridland, Antimicrob. Agents
Chemother. 1998, 42, 1484–1487; c) Y. Suruga, M. Makino, Y.
Okada, H. Tanaka, E. De Clercq, M. Baba, J. Acquired Im-
mune Defic. Syndr. Hum. Retrovirol. 1998, 18, 316–322; d)
K. K. Van Rompay, M. D. Miller, M. L. Marthas, N. A. Mar-
got, P. J. Dailey, D. R. Canfield, P. R. Tarara, J. M. Cherring-
ton, N. L. Aguirre, N. Bischofberger, N. C. Pedersen, J. Virol.
2000, 74, 1767–1774; e) S. A. Grim, F. Romanelli, Annals Phar-
macother. 2003, 37, 849–859.
a) E. De Clercq, Collect. Czech. Chem. Commun. 1998, 63,
480–506; b) S. J. Safrin, H. S. Jaffe, Adv. Exp. Med. Biol. 1999,
458, 111–120.
K. P. Meadows, S. K. Tyring, A. T. Pavia, T. M. Rallis, Arch.
Dermatol. 1997, 133, 987–990.
E. De Clercq, Biochem. Pharmacol. 2007, 73, 911–922.
A. Parkin, J. Chem. Soc. Perkin Trans. 1 1991, 2983–2990.
N. Dyatkina, E. Shirokova, F. Theil, S. M. Roberts, A. Krayev-
sky, Bioorg. Med. Chem. Lett. 1996, 6, 2639–2642.
a) M. Prashad, J. C. Tomesh, J. R. Wareing, T. Scallen, Eur. J.
Med. Chem. 1993, 28, 527–531; b) L. H. Cohen, E. Pieterman,
R. E. W. van Leeuwen, J. Du, P. Negre-Aminou, A. R. P. M.
Valentijn, M. Overhand, G. A. van der Marel, J. H. van Boom,
Biochem. Pharmacol. 1999, 57, 365–373; c) L. H. Cohen,
A. R. P. M. Valentijn, L. Roodenburg, R. E. W. van Leeuwen,
R. H. Huisman, R. J. Lutz, G. A. van der Marel, J. H.
van Boom, Biochem. Pharmacol. 1995, 49, 839–845; d) S. A.
Biller, M. J. Sofia, B. DeLange, C. Foster, E. M. Gorgon, T.
Harrity, L. C. Rich, C. P. Ciosek Jr., J. Am. Chem. Soc. 1991,
113, 8522–8524.
17.7 Hz, 2 H, PCH2P), 2.16 (m, 2 H, 6Ј-H), 1.81–1.78 (m, 4 H, 2Ј-
H, 5Ј-H), 1.54–1.44 (m, 4 H, 4Ј-H, 3Ј-H) ppm.
Compound 19d: White powder, yield 28%. 1H NMR (D2O): δ =
7.81 [d, J(6,5) = 7.9 Hz, 1 H, 6-H], 5.94 [d, J(5,6) = 7.9 Hz, 1 H,
5-H], 4.11 (m, 2 H, 1Ј-H), 4.0–3.95 (m, 2 H, 3Ј-H), 3.9 (m, 2 H, 2Ј-
H), 2.45 [dd, J(H,P) = 18.2, 19.0 Hz, 2 H, PCH2P], 2.23 (m, 2 H,
4Ј-H) ppm.
Compound 20: White powder, yield 13%. 1H NMR (D2O): δ = 7.84
[d, J(6,5) = 7.9 Hz, 1 H, H-6], 5.93 [d, J(5,6) = 7.8 Hz, 1 H, 5-H],
4.04 [t, J(1Ј,2Ј) = 4.8 Hz, 2 H, 1Ј-H], 3.91 [t, J(2Ј,1Ј) = 4.88 Hz, 2Ј-
H], 3.66 [d, J(CH2,P) = 7.0 Hz, 2 H, PCH2O], 2.56 [dd, J(H,P1) =
19.7, J(H,P2) = 17.8 Hz, 2 H, PCH2P] ppm.
[9]
[10]
Determination of pKa: Compound 6a (10 mg) was dissolved in ace-
tate buffer (0.025 , 0.5 mL) and acidified with HCl (2 ), the pH
was measured, and then the 31P NMR spectrum was acquired.
NaOH (0.1 , one drop) was then added repeatedly, the sample
was shaken, and pH and 31P NMR spectrum were measured. The
pH dependence of the 31P chemical shifts was plotted and pKa was
estimated to be at the pH at which the phosphorus chemical shift
is just in the middle between the chemical shifts of the protonated
and unprotonated forms.
[11]
[12]
[13]
[14]
Acknowledgments
This work was part of a research project OZ40550506 of the Insti-
tute of Organic Chemistry and Biochemistry, Czech Academy of
Science, v.v.i. This study was supported by the Czech Ministry of
Education, Youth and Sports, Centre of new antivirals and anti-
neoplastics, 1M0508, by Gilead Sciences and IOCB Research Cen-
tre, by a grant of the U.S. National Institutes of Heatlth (NIH)
1UC1AI062540–01 and by the Czech Academy of Sciences, Pro-
gram of targeted projects, #1QS400550501. The authors’ thanks
are due to Prof. E. De Clercq and his group in the Rega Institute,
Katholic University Leuven (Belgium) for the evaluation of antivi-
ral activity; and to Dr I. Pichová and Dr I. Votruba of the Institute
of Organic Chemistry and Biochemitry for the evaluation of cyto-
static activity and dUTPase inhibitory activity.
[15]
[16]
[17]
[18]
R. W. McClard, T. S. Fujita, K. E. Stremler, C. D. Poulter, J.
Am. Chem. Soc. 1987, 109, 5544–5545.
G. K. Farrington, A. Kumar, F. C. Wedler, J. Med. Chem. 1987,
30, 2062–2067.
A. Flohr, A. Aemissegger, D. Hilvert, J. Med. Chem. 1999, 42,
2633–2640.
a) S. S. Helt, M. Thymark, P. Harris, C. Aagaard, J. Ditrich, S.
Larsen, M. Willemoes, J. Mol. Biol. 2008, 376, 554–569; b) S.
Chan, B. Segelke, T. Lekin, H. Krupka, U. S. Cho, M. Kim,
M. So, Ch. Kim, C. M. Naranjo, Y. C. Rogers, M. S. Park,
G. S. Waldo, I. Pashkov, D. Cascio, J. L. Perry, M. R. Sawaya,
J. Mol. Biol. 2004, 341, 503–517.
[19]
[20]
a) H. H. El-Hajj, H. Zhang, B. Weis, J. Bacteriol. 1988, 170,
1069–1075; b) M. H. Gadsen, E. M. McIntosh, J. C. Game,
P. J. Wilson, R. H. Haynes, EMBO J. 1993, 12, 4425–4431.
a) L. E. Bertani, A. Häggmark, P. Reichard, J. Biol. Chem.
1961, 236, 67–68; b) J. Shlomai, A. Kornberg, J. Biol. Chem.
1987, 253, 3305–3312.
[1] a) E. De Clercq, A. Holý, Nature Rev. Drug Discov. 2005, 4,
928–940; b) A. Holý, Curr. Pharm. Des. 2003, 9, 2567–2592; c)
A. Khandazhinskaya, M. Yasko, E. Shirokova, Curr. Med.
Chem. 2006, 13, 2953–2980; d) E. De Clercq, A. Holý, I. Ro-
senberg, T. Sakuma, J. Balzarini, P. C. Maudgal, Nature 1986,
323, 464–467.
[2] a) L. Naesens, R. Snoeck, G. Andrei, J. Balzarini, J. Neyts, E.
De Clercq, Antivir. Chem. Chemother. 1997, 8, 1–23; b) C. Ying,
E. De Clercq, J. Neyts, J. Viral Hepat. 2000, 7, 79–83.
[3] J. E. Starrett Jr., D. R. Tortolani, M. J. Hitchcock, J. C. Martin,
M. M. Mansuri, Antiviral Res. 1992, 19, 267–273.
[4] a) R. Perrillo, E. Schiff, E. Yoshida, A. Statler, K. Hirsch, T.
Wright, K. Gutfreund, P. Lamy, A. Murray, Hematology 2000,
32, 129–134; b) T. M. Dando, G. L. Plosker, Drugs 2003, 63,
2215–2234.
[5] L. Naesens, J. Neyts, J. Balzarini, A. Holý, I. Rosenberg, E.
De Clercq, J. Med. Virol. 1993, 39, 167–172.
[6] a) B. Otová, Z. Zídek, A. Holý, I. Votruba, M. Sladká, I. Mari-
nov, V. Lesková, In Vivo 1997, 11, 163–167; b) B. Otová, K.
Francová, F. Franeˇk, P. Koutník, I. Votruba, A. Holý, M.
Sladká, J. Schramlová, Anticancer Res. 1999, 19, 3173–3182; c)
W. C. Rose, A. R. Crosswell, J. J. Bronson, J. C. Martin, J. Natl.
Cancer Inst. 1990, 82, 510–512.
[7] a) F. Franeˇk, A. Holý, I. Votruba, T. Eckschlager, Int. J. On-
cology 1999, 14, 745–752; b) P. Kramata, K. M. Downey, Mol.
Pharmacol. 1999, 56, 1262–1270.
[21]
[22]
T. L. Richie, A. Saul, Nature 2002, 415, 694–701.
a) P. Zalud, W. O. Wachs, P. O. Nyman, M. Zeppezauer, Adv.
Exp. Med. Biol. 1995, 370, 135–138; b) J. Kovári, O. Barabás,
B. Varga, A. Békési, F. Tölgyesi, J. Fidy, J. Nagy, B. G.
Vértessy, Proteins 2008, 71, 308–319.
T. Persson, G. Larsson, P. O. Nyman, Bioorg. Med. Chem.
1996, 4, 553–556.
a) J. L. Whittingham, I. Leal, C. Nguyen, G. Kasinathan, E.
Bell, A. F. Jones, C. Berry, A. Benito, J. P. Turkenburg, E. J.
Dodson, L. M. Ruiz Perez, A. J. Wilkinson, N. G. Johansson,
R. Brun, I. H. Gilbert, D. G. Pacanowska, K. S. Wilson, Struc-
ture 2005, 13, 329–338; b) C. Nguyen, G. Kanisathan, I. Leal-
Cortijo, A. Musso-Buendia, M. Kaiser, R. Brun, M. Ruiz-
Pérez, N. G. Johansson, D. González-Pacanowska, I. H. Gil-
bert, J. Med. Chem. 2005, 48, 5942–5954.
C. Nguyen, G. F. Ruda, A. Schipani, G. Kasinathan, I. Leal, A.
Musso-Buendia, M. Kaiser, R. Brun, L. M. Ruiz-Pérez, B. L.
Sahlberg, N. G. Johansson, D. González-Pacanowska, I. H.
Gilbert, J. Med. Chem. 2006, 49, 4183–4195.
[23]
[24]
[25]
[26]
M. J. Robins, P. W. Hatfield, Can. J. Chem. 1982, 60, 547–553.
Eur. J. Org. Chem. 2009, 1082–1092
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1091

P. Doláková, M. Dracˇínský, J. Fanfrlík, A. Holý
FULL PAPER
[27] a) Z. S. Novikova, A. A. Prishchenko, I. F. Lutsenko, Zh.
Obshch. Khim. 1977, 47, 775–781; b) Z. S. Novikova, A. A.
Prishchenko, S. Ya. Skorobogatova, V. I. Martynov, I. F. Lut-
senko, Zh. Obshch. Khim. 1980, 50, 989–992.
[28] K. A. Cruickshank, J. Jiricny, C. B. Reese, Tetrahedron Lett.
1984, 25, 681–684.
[39]
a) R. Engel, Chem. Rev. 1977, 77, 349–367; b) G. N. Blackburn,
D. A. England, F. Kolkmann, J. Chem. Soc., Chem. Commun.
1981, 930–932; c) G. M. Blackburn, Chem. Ind. 1981, 134–138;
d) M. Larsen, R. Willett, R. G. Yount, Science 1969, 166,
1510–1511; e) R. G. Yount, D. Babcock, W. Ballantyne, D.
Ojala, Biochemistry 1971, 10, 2484–2489; f) H. Sigel, Chem.
Soc. Rev. 2004, 33, 191–200.
M. Feyereisen, G. Fitzgerald, A. Komornicki, Chem. Phys.
Lett. 1993, 208, 359–363.
R. Ahlrichs, M. Bar, M. Haser, H. Horn, C. Kolmel, Chem.
Phys. Lett. 1989, 162, 165–169.
[29] a) O. Mitsunobu, Synthesis 1981, 1, 1–28; b) O. R. Ludek, Ch.
Meier, Eur. J. Org. Chem. 2006, 941–946.
[40]
[41]
[30] a) H. Komatsu, K. Morizane, T. Kohno, H. Tanikawa, Org.
Process Res. Dev. 2004, 8, 564–567; b) H. Zhang, R. F. Schin-
azi, C. K. Chu, Bioorg. Med. Chem. 2006, 14, 8314–8322.
[31] a) S. Dey, P. Garner, J. Org. Chem. 2000, 65, 7697–7699; b) X.
Yin, W. Li, S. W. Schneller, Tetrahedron Lett. 2006, 47, 9187–
9189.
[42] M. Cossi, V. Barove, R. Cammi, J. Tomasi, Chem. Phys. Lett.
1996, 255, 327–335.
[43] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, J. A. Montgomery Jr., T.
Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N.
Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.
Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P.
Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pom-
elli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich,
A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D.
Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui,
A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.
Wong, C. Gonzalez, J. A. Pople, Gaussian, Inc., Pittsburgh PA,
2003.
[32] N. M. Howarth, W. E. Lindsell, E. Murray, P. N. Preston, Tet-
rahedron 2005, 61, 8875–8887.
[33] D. Zhou, I. M. Lagoja, A. Van Aerschot, P. Herdewijn, Collect.
Czech. Chem. Commun. 2006, 71, 15–34.
[34] a) J. G. Moffatt, H. G. Khorana, J. Am. Chem. Soc. 1961, 83,
649–658; b) A. Holy, I. Rosenberg, Collect. Czech. Chem. Com-
mun. 1987, 52, 2801–2809; c) W. G. H. Laux, C. Périgaud, J. L.
Imbach, G. Gosselin, Nucleos. Nucleot. 1999, 18, 1003–1004.
[35] a) L. Schmitt, R. Tampé, J. Am. Chem. Soc. 1996, 118, 5532–
5543; b) I. Zgani, Ch. Menut, M. Seman, V. Gallois, V. Laffont,
J. Liautard, J. P. Liautard, M. Criton, J. L. Montero, J. Med.
Chem. 2004, 47, 4600–4612.
[36] a) C. Flader, J. Liu, R. F. Borch, J. Med. Chem. 2000, 43, 3157–
3167; b) T. Ueda, E. Ohtsuka, Chem. Pharm. Bull. 1959, 7,
740–743.
[37] a) D. E. Hoard, D. G. Ott, J. Am. Chem. Soc. 1965, 87, 1785–
1788; b) A. L. Khandazhinsaya, E. A. Shirokova, Y. S. Skoblov,
L. S. Victorova, L. Y. Goryunova, R. S. Beabealashivilli, T. R.
Pronyaeva, N. V. Fedyuk, V. V. Zolin, A. G. Pokrovsky, M. K.
Kukhanova, J. Med. Chem. 2002, 45, 1284–1291; c) Q. F. Ma,
I. C. Barhurst, P. J. Barr, G. L. Kenyon, J. Med. Chem. 1992,
35, 1938–1941.
[44] G. A. Molander, G. A. Brown, I. S. de Garcia, J. Org. Chem.
2002, 67, 3459–3463.
Received: September 17, 2008
Published Online: January 14, 2009
[38] M. Kalek, J. Jemielity, Z. M. Darzynkiewicz, E. Bojarska, J.
Stepinski, R. Stolarski, R. E. Davis, E. Darzynkiewicz, Bioorg.
Med. Chem. 2006, 14, 3223–3230.
1092
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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