Novel nucleotide analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases

Article abstract of DOI:10.1016/j.tet.2016.12.046

A novel family of acyclic nucleoside phosphonates (ANPs) bearing a (1H-1,2,3-triazol-4-yl)phosphonic acid group in the acyclic side chain have been prepared in order to study the influence of the hetaryl rigidizing element on the biological properties of

Full text of DOI:10.1016/j.tet.2016.12.046

Accepted Manuscript
Novel nucleotide analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as
inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases
Miloš Lukáč, Dana Hocková, Dianne T. Keough, Luke W. Guddat, Zlatko Janeba
PII:
S0040-4020(16)31333-3
10.1016/j.tet.2016.12.046
TET 28338
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 21 October 2016
Revised Date: 7 December 2016
Accepted Date: 19 December 2016
Please cite this article as: Lukáč M, Hocková D, Keough DT, Guddat LW, Janeba Z, Novel nucleotide
analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as inhibitors of Plasmodium and human
6-oxopurine phosphoribosyltransferases, Tetrahedron (2017), doi: 10.1016/j.tet.2016.12.046.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel nucleotide analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as
inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases
Miloš Lukáča,‡, Dana Hockováa, Dianne T. Keoughb, Luke W. Guddatb, Zlatko Janebaa*
aInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,
Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic
bThe School of Chemistry and Molecular Biosciences, The University of Queensland,
Brisbane 4072, QLD, Australia
Abstract
A novel family of acyclic nucleoside phosphonates (ANPs) bearing a (1H-1,2,3-triazol-4-
yl)phosphonic acid group in the acyclic side chain have been prepared in order to study the
influence of the hetaryl rigidizing element on the biological properties of such compounds.
The key synthetic step consisted of a copper(I)-catalyzed azide-alkyne cycloaddition
(CuAAC) between diethyl ethynylphosphonate and the corresponding azidoalkyl precursor.
Two ANPs in this family, bearing a guanine base, exhibited the highest potency for the human
6-oxopurine phosphoribosyltransferase irrespective of the stereochemistry on the C-2’ atom.
Four compounds inhibited Plasmodium falciparum 6-oxopurine phosphoribosyltransferase
with little differences in their Ki values irrespective of whether the base was guanine,
hypoxanthine or xanthine but only two, with guanine as base, inhibited PvHGPRT.
Keywords: acyclic nucleoside phosphonates; 6-oxopurine; hypoxanthine-guanine-(xanthine)
phosphoribosyltransferase, copper(I)-catalyzed azide-alkyne cycloaddition
Introduction
Acyclic nucleoside phosphonates (ANPs)1 represent an important class of antimetabolites that
mimic the naturally occurring nucleoside monophosphates. Extensive structure-activity
relationship (SAR) studies have been carried out and several distinct classes of ANPs with
diverse biological activities have been identified. 2-(Phosphonomethoxy)ethyl or PME (e.g.
PMEA, Fig. 1), 2-(phosphonomethoxy)propyl or PMP, and 3-hydroxy-2-
*Corresponding author. Tel.: +420 220 183 143.
E-mail addresses: janeba@uochb.cas.cz (Z. Janeba).
‡ Current address: Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
Kalinčiakova 8, 832 32 Bratislava, Slovakia
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(phosphonomethoxy)propyl or HPMP (e.g. (S)-HPMPC, Fig. 1) analogues have antiviral
properties,2-4 and 2-(phosphonoethoxy)ethyl or PEE derivatives (such as PEEHx and PEEG,
Fig. 1), bisphosphonates (Fig. 1) and aza-ANPs (Fig. 1) have antimalarial5-9 and/or
antimycobacterial10-11 activity. Several different chemical types of ANPs, including modified
PMEA analogues, have also been studied as potent inhibitors of bacterial adenylate cyclases,
namely adenylate cyclase toxin from Bordetella pertussis and edema factor from Bacillus
anthracis.12-14 Such analogues may have potential for treatment or prevention of toxaemia
caused by the invasion of these bacteria into the human host.
NH2
N
NH2
N
O
N
N
N
N
N
N
N
HN
O
Y
OH
O
O
O
O
PMEA
PEEHx: Y = H
PEEG: Y= NH2
(S)-HPMPC
O
O
P
P
P
HO
HO
OH
OH
HO
OH
O
O
OH
nucleobase
N
N
N
HN
HN
P
OH
O
OH
O
*
N
H2N
N
H2N
N
N
A
N
O
N
O
O
O
N
R
P
P
OH
P
bisphosphonate
derivative
OH
HO
OH
aza-ANP
HO
HO
Figure 1. Examples of chemical structures of biologically active acyclic nucleoside
phosphonates (ANPs). Top row: The antiviral compounds PMEA and (S)-HPMPC, and
antimalarial compounds PEEHx and PEEG. Bottom row: an antiplasmodial bisphosphonate,
the general structure of the aza-ANPs, and the general scaffold of the newly designed (1H-
1,2,3-triazol-4-yl)phosphonates A.
Inhibition of plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferases
(HG(X)PRTs) by the ANPs is well-correlated with their antimalarial activity.5-9 These
enzymes catalyze the formation of the 6-oxopurine mononucleotides from the 6-oxopurine
nucleobases and 5-phospho-α-D-ribosyl-1-pyrophosphate (Fig. 2).15 HG(X)PRTs are key
enzymes of the purine salvage pathway and since malarial parasites lack the de novo pathway
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for purine nucleotide synthesis, this enzyme is a validated target for the development of new
antimalarials.5-9 Importantly, the mode of action of the ANPs is different from the currently
used drugs, so represents a new approach to developing antimalarial therapeutics.
Figure 2. Reaction catalyzed by the HG(X)PRTs. The naturally occurring bases are
hypoxanthine (R = H), guanine (R = NH2) and xanthine (R = OH).
Novel types of ANPs bearing (1H-1,2,3-triazol-4-yl)phosphonic acid group attached to
the acyclic side chain (general structure A, Fig. 1) have been synthesized as a continuation of
the extended SAR studies carried out by our group. Compounds A (Fig. 1) bearing 6-
oxopurine bases were designed as potential inhibitors of plasmodial HG(X)PRTs since it has
been reported16 that the optimal length of the aliphatic linker between the nucleobase and the
phosphonate group is 5 or 6 atoms (in contrast to antiviral ANPs with 4-atom-linkers). In
comparison to the flexible PEE or modified PEE analogues which are potent HG(X)PRTs
inhibitors, derivatives A (Fig. 1) have the 1H-1,2,3-triazol-4-yl moiety integrated into the
acyclic chain to rigidify the linker, thus, possibly leading to increased affinity.
An efficient synthetic methodology to access the desired 6-oxopurine ANPs with the
(1H-1,2,3-triazol-4-yl)phosphonic acid moiety has been developed and optimized. To show
the full scope of this synthetic approach, the whole series of ANPs with various purine or
pyrimidine bases attached were prepared in good overall yields.
Results and Discussion
Chemistry.
Since the designed compounds A (Fig. 1) contain a stereogenic centre at the C-2’ atom, both
(R)- and (S)-enantiomers were synthesized side by side for their subsequent biological
evaluations. The key synthetic step involved the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) between diethyl ethynylphosphonate17 and suitable intermediate
bearing an azido group. In general, two strategies could be utilized for the introduction of the
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1,2,3-triazole ring into target ANPs: a) using the “click” CuAAC chemistry between diethyl
ethynylphosphonate and acyclic nucleoside analogue having the azido group in the side
aliphatic chain or b) the preparation of suitable intermediate bearing diethyl (1H-1,2,3-triazol-
4-yl)phosphonate moiety for its subsequent attachment to purine or pyrimidine nucleobases.
The first approach was applied for the synthesis of adenine ANPs by analogy to previously
reported procedures.18,19 The synthesis started with alkylation of adenine with commercially
available enantiomerically pure tritylated (R)-(+)- and (S)-(–)-glycidols, (R)-2 and (S)-2
(Scheme 1), to give compounds (R)-3 and (S)-3, respectively.18 Intermediates (R)-3 and (S)-3
were then benzoylated at the exocyclic amino group to form derivatives (R)-4 and (S)-4,18
which were further converted into their mesylate derivetives and, subsequently, to azido
derivatives (R)-5 and (S)-5, respectively, using NaN3 (Scheme 1).19
Scheme 1. Reagents and conditions: a) NaH, DMF, 105 °C, 16 h; b) Me3SiCl, Py, rt, 2h and
then PhCOCl, Py, rt, 2 h; c) MsCl, Py, rt, 4 h; d) NaN3, DMF/HMPA, 100 °C, 12 h; e)
HC≡C(P)(O)(OEt)2, CuI, DiPEA, DMF; f) MeNH2 in MeOH (8M solution), toluene, rt, 4 h;
g) 80% aq. CH3COOH, 90 °C, 4 h; h) Me3SiBr, MeCN, rt, 24 h, then aq. MeOH.
The CuAAC cycloaddition between azido compounds (R)-5 or (S)-5 and diethyl
ethynylphosphonate,17 using CuI and DiPEA in DMF,20 provided the desired 1,4-substituted
triazole derivatives (R)-6 or (S)-6 in good yields (Scheme 1). The removal of benzoyl and
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trityl groups using methylamine in toluene21 and 80% aq. acetic acid, respectively, followed
by removal of phosphonate ethyl ester moieties with Me3SiBr/MeCN with ensuing
hydrolysis,22 afforded final products (R)-8 or (S)-8 (Scheme 1).
The second synthetic strategy seems to be more efficient and more broadly applicable since
the preformed aliphatic precursor bearing (1H-1,2,3-triazol-4-yl)phosphonate group can be
directly attached to suitably modified purines or pyrimidines. At first, the starting tritylated
(R)-(+)- and (S)-(–)-glycidols, compounds (R)-2 and (S)-2 (Scheme 2), were successively
treated with freshly prepared sodium benzyloxide in DMF (without isolation of the
products),23 with MsCl in pyridine (to give (R)-9 and (S)-9, respectively), and finally with
NaN3 in a DMF/HMPA mixture to afford azido derivatives (R)-10 and (S)-10, respectively, in
overall yields higher than 60% (Scheme 2).
Scheme 2. Reagents and conditions: a) NaH, DMF, PhCH2OH, 100 °C, 2 h; b) MsCl, Py, rt, 4
h c) NaN3, DMF/HMPA; 100 °C, 5 h; d) NaBrO3, Na2S2O4, EtOAc, H2O; e)
HC≡C(P)(O)(OEt)2, CuI, DiPEA, DMF.
To prepare the desired 1,2,3-triazole intermediates (R)-12 and (S)-12 (Scheme 2), azides (R)-
10 and (S)-10 can be either first debenzylated and then cyclized under the CuAAC
cycloaddition conditions or first cyclized and then debenzylated. Both approaches were
tentatively tested and the former approach was selected as it gave higher yields. Thus,
removal of the benzyl group from compounds (R)-10 and (S)-10, using a NaBrO3/Na2S2O4
reagent under two-phase conditions (a method compatible with the present azido group),24
afforded hydroxyl derivatives (R)-11 and (S)-11, respectively, which were then converted by
the above described CuAAC cycloaddition20 with diethyl ethynylphosphonate17 to the desired
precursors (R)-12 and (S)-12 in satisfactory overall yields (Scheme 2).
The next crucial synthetic step consisted of the attachment of 1,2,3-triazole intermediates (R)-
12 and (S)-12 to appropriate purine or pyrimidine nucleobases via Mitsunobu reaction. First,
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6-chloropurine and 2-amino-6-chloropurine were reacted with compounds (R)-12 and (S)-12
in the presence of PPh3 and DIAD in dioxane at room temperature to give 6-chloropurine
derivatives (R)-13 and (S)-13, and 2-amino-6-chloropurine analogues (R)-16 and (S)-16 in
good yields (Scheme 3). The final free phosphonates (R)-15, (S)-15, (R)-18 and (S)-18 were
obtained by simultaneous hydrolysis of the 6-chloropurine group and detritylation using 75%
aq. CF3COOH at room temperature (to give 6-oxopurine intermediates 14 and 17), followed
by the standard removal of the phosphonate ethyl ester moieties (Scheme 3).
Scheme 3. Reagents and conditions: a) 6-chloropurine, PPh3, DIAD, dioxane, rt, 48 h; b) 75%
aq. CF3COOH, rt, 48 h; c) TMSBr, MeCN, rt, 24 h, then aq. MeOH; d) 2-amino-6-
chloropurine, PPh3, DIAD, dioxane, rt, 48 h; e) isoamyl nitrite, 80% aq. CH3COOH, rt, 12 h;
f) NH3, EtOH, 100 °C, 24 h; g) 80% aq. CH3COOH, 90 °C, 2 h.
The amino group at C-2 position of compounds (R)-18 and (S)-18 was replaced with an oxo
group using standard diazotization-hydroxydediazoniation approach (treatment with isoamyl
6

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nitrite in 80% acetic acid) to afford the corresponding xanthine derivatives (R)-19 and (S)-19,
respectively, in 45% yields (Scheme 3).
Ammonolysis of 2-amino-6-chloropurine intermediates (R)-16 and (S)-16 gave 2,6-
diaminopurine derivatives (R)-20 and (S)-20 which, after detritylation (to yield 21) and
subsequent removal of the ethyl ester groups, afforded the final phosphonates (R)-22 and (S)-
22 (Scheme 3).
Next, the corresponding ANPs containing pyrimidine nucleobases were synthesized. The
Mitsunobu procedure25 (compounds (R)-12 or (S)-12, PPh3, DIAD, dioxane, room
temperature), followed by nucleobase N-debenzoylation with propylamine in dioxane, was
employed for alkylation of 3-benzoyluracil25 and 3-benzoylthymine25 to form pyrimidine
intermediates (R)-23, (S)-23 and (R)-26, (S)-26, respectively (Scheme 4).26 Detritylation of
compounds 23 and 26 (to yield hydroxy derivatives 24 and 27) and subsequent removal of the
ethyl ester groups gave final phosphonates (R)-25, (S)-25 and (R)-28, (S)-28, respectively
(Scheme 4).
Scheme 4. Reagents and conditions: a) N3-benzoyluracil, PPh3, DIAD, dioxane, rt, 48 h; b)
propylamine, dioxane, rt, 12 h; c) 80% aq. CH3COOH, 90 °C, 2 h; d) TMSBr, MeCN, rt, 24 h,
then aq. MeOH; e) N3-benzoylthymine, PPh3, DIAD, dioxane, rt, 48 h; f) N4-benzoylcytosine,
PPh3, DIAD, dioxane, rt, 48 h.
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Analogously, N4-benzoylcytosine27 was treated with (R)-12 or (S)-12 under reported
Mitsunobu reaction conditions,25 followed by a subsequent removal of the benzoyl and trityl
groups to give cytosine derivatives (R)-29 and (S)-29, respectively (Scheme 4). Standard
removal of the ethyl ester groups from (R)-29 and (S)-29 produced the final compounds (R)-
30 and (S)-30, respectively (Scheme 4).
Biological activity
Considering the important biological properties of ANPs in general, free phosphonates (R)-8,
(S)-8, (R)-15, (S)-15, (R)-18, (S)-18, (R)-19, (S)-19, (R)-22, (S)-22, (R)-25, (S)-25, (R)-28, (S)-
28, (R)-30 and (S)-30 were tested in standard antiviral assays,28 but no significant antiviral
activity was observed against viruses tested, i.e. against human immunodeficiency virus 1
(HIV-1), human rhinovirus (HRV) and hepatitis C virus (HCV).
ANPs bearing 6-oxopurine bases, namely analogues with hypoxanthine (compounds 15),
guanine (compounds 18) or xanthine (compounds 19) as the purine base have been designed
as potential inhibitors of hypoxanthine-guanine-(xanthine) phosphoribosyltransferases
[HG(X)PRTs].5-9 Thus, the compounds were tested as potential inhibitors of human HGPRT,
Plasmodium falciparum HGXPRT and Plasmodium vivax HGPRT. The hypoxanthine
derivatives (R)-15 and (S)-15 showed an inhibition of the human HGPRT and PfHGXPRT
(only isomer (R)-15) with Ki values as low as 3 µM range (Table 1). These compounds did
not inhibit the Pv enzyme. The most potent ANPs in this series are the guanine analogues (R)-
18 and (S)-18 which are submicromolar inhibitors of human HGPRT and low micromolar
inhibitors of both PfHGXPRT and PvHGPRT (Table 1). Interestingly, the potent inhibitory
properties of compounds 18 did not depend distinctly on the stereochemistry on the C-2’
atom, as both (R)-18 and (S)-18 isomers exhibited similar Ki values against all three
HG(X)PRTs tested (Table 1). On the other hand, in the case of xanthine analogues only
isomer (R)-19 (similarly as in the hypoxanthine series) was a potent inhibitor and selective for
PfHGXPRT (Table 1). This is because xanthine is not a substrate for the human or Pv
enzymes and compounds containing this base cannot, in consequence, bind in the active site.
However, if the orientation of the compound varies so that the location of the base is not
identical to that of with hypoxanthine or guanine, there remains a possibility that they could
bind to these two enzymes.
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Table 1. Ki values of the ANPs bearing three different 6-oxopurine nucleobases for human
HGPRT, PfHGXPRT and PvHGPRT.
Ki [µM]
Compound
nucleobase
human
8
Pf
Pv
(R)-15
(S)-15
(R)-18
(S)-18
(R)-19
(S)-19
hypoxanthine
hypoxanthine
guanine
>100
3
>50 >100
7
0.1
1
2
2
6
2
guanine
0.4
xanthine
>500
>500
>200
xanthine
>50 >500
Conclusions
A methodology for the synthesis of acyclic nucleoside phosphonates (ANPs) bearing (1H-
1,2,3-triazol-4-yl)phosphonate moiety in the acyclic side chain has been developed and a
series of 16 compounds in both (R)- and (S)-conformations was prepared for their biological
evaluation. Six of these compounds were investigated as inhibitors of the human, Pf and Pv
HG(X)PRTs. Two of these ANPs with guanine as the purine base were found to be
submicromolar inhibitors of human HGPRT, i.e. with lower Ki values than when
hypoxanthine is the base. This is consistent with the fact that the Km for guanine for this
enzyme is lower than for hypoxanthine. The guanine analogues also inhibited the two
plasmodial enzymes but with higher Ki values. Guanine bearing ANPs were the most potent
HG(X)PRTs inhibitors, irrespectively of the stereochemistry on the C-2’ atom of the aliphatic
linker, but they lack selectivity for the plasmodial enzymes. The xanthine compound (R)-19
with the Ki value of 2 µM against PfHGXPRT is analogue with best selectivity (no inhibition
of human HGPRT) and, thus, represents the most promising compound for the future
synthesis of suitable prodrugs and their further biological evaluations as potential antimalarial
agents.
Experimental section
Starting compounds and reagents were purchase from commercial suppliers or were prepared
according to the published procedures. Solvents were dried by standard procedures. Solvents
were evaporated at 40 °C/2 kPa. Analytical TLC was performed on plates of Kieselgel 60 F254
from Merck. NMR spectra were recorded on Bruker Avance 400 spectrometer (1H at 400
13
31
1
MHz, C at 100.6 MHz, P at 161.9 MHz) with TMS or dioxane (3.75 ppm for H, 67.19
9

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ppm for 13C NMR) as internal standard or referenced to the residual solvent signal. Mass
spectra were measured on UPLC-MS (Waters SQD-2). HR MS were taken on a LTQ Orbitrap
XL spectrometer. The purity of the tested compounds was determined by HPLC (H2O-
CH3CN, linear gradient) and was higher than 95%.
General procedure 1 (GP1). Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).20
Diisopropylethylamine (1.7 ml, 10 mmol) and CuI (190 mg, 1 mmol) were added to the
mixture of starting azido derivative (5 mmol) and diethyl ethynylphosphonate17 (970 mg, 6
mmol) in anhydrous DMF (30 mL) under argon at room temperature. The reaction mixture
was stirred at room temperature for 4 h and then diluted with EtOAc (50 mL). Organic phase
was washed with water (3 × 50 mL) water, dried over anhydrous MgSO4, filtered,
concentrated in vacuo, and purified by chromatography over silica gel (CHCl3
→
CHCl3/MeOH, 100/3, v/v) to give the corresponding (1H-1,2,3-triazol-4-yl)phosphonic acid
derivative.
General procedure 2 (GP2). Synthesis of free phosphonic acids.22 A mixture of the
corresponding diethyl ester phosphonate (1.0 mmol), acetonitrile (5 mL), and TMSBr (1.0
mL) was stirred at room temperature for 24 h. After evaporation and codistillation with
acetonitrile (2 × 5 mL), the residue was treated with aqueous methanol (2:1, 10 mL) for 30
min, evaporated to dryness and crystallized/precipitated to afford the final phosphonic acids
as crystals/solid.
General procedure 3 (GP3). Mitsunobu reaction.25 Diisopropylazadicarboxylate (DIAD,
14 mmol) was added dropwise to the solution of triphenylphosphine (15 mmol), alcohol (R)-
12 or (S)-12 (5 mmol), and the corresponding heterocyclic base (7.5 mmol) in dry dioxane or
THF (100 mL). The reaction mixture was stirred at room temperature for 48 h, evaporated in
vacuo, and purified by column chromatography over silica gel.
General procedure 4 (GP4). Detritylation. A mixture of a trityl derivative (1.0 mmol) in
80% aq. acetic acid (2.5 mL) was heated at 90 °C for 2 h. Volatiles were evaporated and the
crude product was purified by chromatography over silica gel.
(R)-1-(6-Amino-9H-purin-9-yl)-3-(trityloxy)propan-2-ol (R)-3 and (S)-1-(6-Amino-9H-
purin-9-yl)-3-(trityloxy)propan-2-ol (S)-3. In analogy to the reported procedure,18 a mixture
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of adenine (1, 4.73 g, 35 mmol) and NaH (60% in mineral oil, 280 mg, 7 mmol) in DMF (75
mL) was stirred at room temperature for 2 h. Tritylated glycidol (R)-2 or (S)-2 (9.5 g, 30
mmol) in DMF (100 mL) was added, and the resulting solution was heated at 105 °C for 16 h.
Solvent was evaporated, the residue was extracted with EtOAc (2 × 50 mL), and purified by
chromatography over silica gel (EtOAc → EtOAc/MeOH, 30/2, v/v) to give (R)-3 (51%) and
1
(S)-3 (69%) as white foams. Compound (R)-3: H NMR (400 MHz, CDCl3) 3.06 (dd, 3JH,H
=
6.3 Hz, 2JH,H = 9.6 Hz, 1H), 3.28 (dd, 3JH,H = 5.4 Hz, 2JH,H = 9.6 Hz, 1H), 4.15 – 4.23 (m, 1H),
4.28 (dd, 3JH,H = 6.8 Hz, 2JH,H = 14.2 Hz, 1H), 4.39 (dd, 3JH,H = 2.2 Hz, 2JH,H = 14.2 Hz, 1H),
3
5.85 (br s, 2H), 7.16 – 7.32 (m, 9H), 7.40 (d, JH,H = 7.2 Hz, 6H), 7.68 (s, 1H), 8.22 (s, 1H);
13C NMR (100 MHz, CDCl3) 48.5, 64.7, 69.7, 87.2, 119.5, 127.4, 128.1, 128.6, 141.8, 143.6,
152.5, 155.5; HRMS m/z calcd for C27H26N5O2 [M+H]+ 452.20810, found 452.20799.
1
Compound (S)-3: H and 13C NMR spectra are identical with (R)-3. HRMS m/z calcd for
C27H26N5O2 [M+H]+ 452.20810, found 452.20800.
(R)-N-{9-[2-Hydroxy-3-(trityloxy)propyl]-9H-purin-6-yl}benzamide (R)-4 and (S)-N-{9-
[2-Hydroxy-3-(trityloxy)propyl]-9H-purin-6-yl}benzamide (S)-4. In analogy to the
reported procedure,18 compound (R)-3 or (S)-3 (6.77 g, 15 mmol) was disolved in anhydrous
pyridine (100 mL) and trimethylsilyl chloride (8 mL, 63 mmol) was added at room
temperature. After stirring for 2 h, the mixture was cooled to 0 °C and benzoyl chloride (2.6
mL, 22.5 mmol) was added dropwise. The mixture was allowed to warm to room temperature
and was stirred for additional 2 h. Water (20 mL) was added at 0 °C with stirring, and after 15
min, 25% aq. NH4OH (40 mL) was added. After stirring for another 30 min, the mixture was
extracted with EtOAc (2 × 100 mL). Solvents were partially removed in vacuo and the
products were precipitated to give (R)-4 (51%) and (S)-4 (41%) as white powders. Compound
3
2
(R)-4: 1H NMR (400 MHz, CDCl3) 3.16 (dd, JH,H = 5.7 Hz, JH,H = 9.7 Hz, 1H), 3.25 (dd,
3JH,H = 5.5 Hz, 2JH,H = 9.7 Hz, 1H), 4.16 – 4.24 (m, 1H), 4.32 (dd, 3JH,H = 7.1 Hz, 2JH,H = 14.2
Hz, 1H), 4.49 (dd, 3JH,H = 2.2 Hz, 2JH,H = 14.2 Hz, 1H), 7.18 – 7.33 (m, 9H), 7.40 (d, 3JH,H
=
3
3
7.3 Hz, 6H), 7.51 (t, JH,H = 7.5 Hz, 2H), 7.61 (t, JH,H = 7.4 Hz, 1H), 7.99 – 8.06 (m, 3H),
13
8.71 (s, 1H); C NMR (100 MHz, CDCl3) 48.0, 64.9, 69.4, 87.3, 122.6, 127.4, 128.0, 128.1,
128.6, 128.9, 132.9, 133.8, 143.5, 144.3, 149.5, 152.1, 152.3 164.9; HRMS m/z calcd for
C34H30N5O3 [M+H]+ 556.23432, found 556.23435. Compound (S)-4: 1H and 13C NMR spectra
are identical with (R)-4. HRMS m/z calcd for C34H30N5O3 [M+H]+ 556.23432, found
556.23435.
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(R)-N-{9-[2-Azido-3-(trityloxy)propyl]-9H-purin-6-yl}benzamide (R)-5 and (S)-N-{9-[2-
Azido-3-(trityloxy)propyl]-9H-purin-6-yl}benzamide (S)-5. In analogy to reported
procedure,19 compounds (R)-4 or (S)-4 (4.17 g, 7.5 mmol) was dissolved in anhydrous
pyridine (70 mL). The mixture was cooled to 0 °C and methanesulfonyl chloride (1.2 mL, 15
mmol) was added drop-wise. The reaction mixture was then warmed up to room temperature
and stirred for 4 h. Methanol (5 mL) was added at 0 °C and the solvents were evaporated in
vacuo. The crude product was dissolved in CH2Cl2 (200 mL), the solution was washed with
saturated aq. NaHCO3 (2 × 100 mL), with brine (1 × 100 mL) and dried (anhydrous Na2SO4).
After filtration the solvent was evaporated in vacuo. The resulting foam was dissolved in a
DMF/HMPA mixture (1:1, 20 mL) and sodium azide (2.3 g, 35 mmol) was added. The
reaction mixture was stirred at 100 °C for 12 h, cooled down to room temperature and poured
into EtOAc (200 mL). The organic layer was washed with water (2 × 200 mL), dried over
anhydrous Na2SO4, filtered, evaporated in vacuo, and purified by silica gel chromatography
(CHCl3 to CHCl3/MeOH, 98/2, v/v) to afford (R)-5 (80%) and (S)-5 (51%) as white foams.
Compound (R)-5: 1H NMR (400 MHz, CDCl3) 3.30 (dd, 3JH,H = 6.3 Hz, 2JH,H = 10.2 Hz, 1H),
3
3
3.45 (dd, JH,H = 4.2 Hz, 2JH,H = 10.2 Hz, 1H), 3.95 – 4.03 (m, 1H), 4.18 (dd, JH,H = 8.6 Hz,
3
2
3
2JH,H = 14.3 Hz, 1H), 4.41 (dd, JH,H = 4.1 Hz, JH,H = 14.3 Hz, 1H), 7.26 (t, JH,H = 7.2 Hz,
3
3
3
3H), 7.33 (t, JH,H = 7.4 Hz, 6H), 7.45 (d, JH,H = 7.2 Hz, 6H), 7.52 (t, JH,H = 7.5 Hz, 2H),
3
3
7.60 (t, JH,H = 7.4 Hz, 1H), 7.98 (s, 1H), 8.03 (d, JH,H = 7.2 Hz, 2H), 8.76 (s, 1H); 13C
NMR (100 MHz, CDCl3) 44.8, 60.9, 64.0, 87.8, 122.8, 127.5, 128.0, 128.2, 128.6, 129.0,
132.9, 133.8, 143.3, 143.5, 149.7, 152.1, 152.7, 164.8; HRMS m/z calcd for C34H29N8O2
1
[M+H]+ 581.24080, found 581.24086. [α]20 +9.5 (c 0.59, MeOH). Compound (S)-5: H and
D
13C NMR spectra are identical with (R)-5. HRMS m/z calcd for C34H29N8O2 [M+H]+
581.24080, found 581.24086. [α]20D –8.6 (c 0.50, MeOH).
Diethyl (R)-1-[1-(6-benzamido-9H-purin-9-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-
4-ylphosphonate (R)-6
and Diethyl (S)-1-[1-(6-benzamido-9H-purin-9-yl)-3-
(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-6. Compounds (R)-5 and (S)-
5 were treated by GP1 to give (R)-6 (68%) and (S)-6 (83%), respectively, as white powders.
1
Compound (R)-6: H NMR (400 MHz, CDCl3) 1.28 and 1.30 (2 × t, 3JH,H = 7.1 Hz, 2 × 3H),
3.67 (dd, 3JH,H = 6.3 Hz, 2JH,H = 10.4 Hz, 1H), 3.71 (dd, 3JH,H = 4.9 Hz, 2JH,H = 10.4 Hz, 1H),
3
3
4.15 – 4.35 (m, 4H), 4.86 (dd, JH,H = 4.6 Hz, 2JH,H = 14.5 Hz, 1H), 5.01 (dd, JH,H = 9.4 Hz,
2JH,H = 14.5 Hz, 1H), 5.13 – 5.22 (m, 1H), 7.17 – 7.34 (m, 15H), 7.50 (t, 3JH,H = 7.6 Hz, 2H),
3
3
7.59 (t, JH,H = 7.4 Hz, 1H), 7.71 (s, 1H), 7.95 (s, 1H), 8.03 (d, JH,H = 7.2 Hz, 2H), 8.74 (s,
12

ACCEPTED MANUSCRIPT
13
1H); C NMR (100 MHz, CDCl3) 16.3 (d, 3JC,P = 6.4 Hz), 44.6, 60.7, 63.2 and 63.3 (2 × d,
2JC,P = 5.8 Hz), 63.9, 88.0, 122.7, 127.7, 128.0, 128.3, 128.4, 129.0, 131.6 (d, 2JC,P = 32.8 Hz),
133.0, 133.7, 137.9 (d, 1JC,P = 238.5 Hz), 142.9, 143.1, 149.8, 151.9, 152.8, 164.8; 31P (161.9
MHz, CDCl3) 6.57; HRMS m/z calcd for C40H39N8NaO5P [M+Na]+ 765.26732, found
1
765.26705. [α]20 +36.0 (c 0.56, MeOH). Compound (S)-6: H and 13C NMR spectra are
D
identical with (R)-6. HRMS m/z calcd for C40H39N8NaO5P [M+Na]+ 765.26732, found
765.26727. [α]20D –34.0 (c 0.52, MeOH).
Diethyl
(R)-1-[1-(6-amino-9H-purin-9-yl)-3-hydroxypropan-2-yl)-1H-1,2,3-triazol-4-
ylphosphonate (R)-7 and Diethyl (S)-1-[1-(6-amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl)-1H-1,2,3-triazol-4-ylphosphonate (S)-7. In analogy to the reported procedure,21
methylamine in MeOH (4 mL, 8M solution) was added dropwise at room temperature to a
solution of (R)-6 or (S)-6 (1.11 g, 1.5 mmol) in anhydrous toluene (20 mL). The reaction
mixture was stirred for 4 h at room temperature, volatiles were evaporated and the residue
was disolved in 80% aq. acetic acid (20 mL). The mixture was heated at 90 °C for 4 h, cooled
down to room temperature and solvents were evaporated. Column chromatography over silica
gel (CHCl3 → CHCl3/MeOH, 100/20, v/v) afforded (R)-7 (74%) and (S)-7 (70%) as
colourless solid. Compound (R)-7: 1H NMR (400 MHz, DMSO-d6) 1.18 and 1.19 (2 × t, 3JH,H
3
2
= 6.9 Hz, 2 × 3H), 3.84 – 4.02 (m, 6H), 4.67 (dd, JH,H = 4.8 Hz, JH,H = 14.5 Hz, 1H), 4.75
3
2
3
(dd, JH,H = 9.7 Hz, JH,H = 14.5 Hz, 1H), 5.27 – 5.37 (m, 1H), 5.44 (t, JH,H = 5.4 Hz, 1H),
7.22 (br s, 2H), 7.85 (s, 1H), 8.06 (s, 1H), 8.68 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 28.4
and 28.5 (2 × d, 3JC,P = 6.2 Hz), 56.1, 73.2, 74.6 and 74.7 (2 × d, 2JC,P = 5.6 Hz), 74.9, 130.8,
2
1
31
143.5 (d, JC,P = 33.6 Hz), 148.5 (d, JC,P = 236.6 Hz), 152.9, 161.9, 164.9, 168.4; P NMR
(161.9 MHz, CDCl3) 8.31; HRMS m/z calcd for C14H22N8O4P [M+H]+ 397.14961, found
397.14949. [α]20D +101.9 (c 0.59, MeOH). Compound (S)-7: 1H, 13C and 31P NMR spectra are
identical with (R)-7. HRMS m/z calcd for C14H22N8O4P [M+H]+ 397.14961, found 397.14944.
[α]20D –102.7 (c 0.59, MeOH).
(R)-1-[1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonic acid (R)-8 and (S)-1-[1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]-
1H-1,2,3-triazol-4-ylphosphonic acid (S)-8. Compounds (R)-7 and (S)-7 were treated by
GP2 to give (R)-8 (80%) and (S)-8 (70%), respectively, as white powders. Compound (R)-8:
1H NMR (400 MHz, DMSO-d6) 3.88 (d, 3JH,H = 5.5 Hz, 2H), 4.69 (dd, 3JH,H = 4.9 Hz, 2JH,H
=
14.5 Hz, 1H), 4.78 (dd, 3JH,H = 9.5 Hz, 2JH,H = 14.5 Hz, 1H), 5.25 – 5.35 (m, 1H), 7.44 (br s,
13

ACCEPTED MANUSCRIPT
2H), 7.79 (s, 1H), 8,15 (s, 1H), 8.44 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 43.9, 61.2,
61.6, 118.4, 128.6 (d, 2JC,P = 32.4 Hz), 140.8, 141.6 (d, 1JC,P = 228.7 Hz), 149.4, 151.9, 155.5;
31P NMR (161.9 MHz, CDCl3) 3.17; HRMS m/z calcd for C10H12N8O4P [M-H]- 339.07246,
found 339.07190. [α]20D +95.2 (c 0.52, H2O). Compound (S)-8: 1H, 13C and 31P NMR spectra
are identical with (R)-8. HRMS m/z calcd for C10H12N8O4P [M-H]- 339.07246, found
339.07224. [α]20D –89.2 (c 0.51, H2O).
(R)-1-Benzyl-2-mesyl-3-tritylglycerol (R)-9 and (S)-1-Benzyl-2-mesyl-3-tritylglycerol
(S)-9. 60% NaH in mineral oil (190 mmol, 7.6 g) was suspended in anhydrous DMF (200
mL) and benzyl alcohol (20 mL, 190 mmol) was added dropwise. The reaction mixture was
stirred at room temperature for 20 min and a mixture of tritylated glycidol (R)-2 or (S)-2 (158
mmol, 50 g) in DMF (100 mL) was added. The mixture was stirred at 100 °C for 2 h, cooled
down to room temperature and diluted with water (50 mL). The mixture was extracted with
EtOAc (3 × 250 mL). Joint organic portions were dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to afford (R)-1-benzyl-3-tritylglycerol and (S)-1-benzyl-3-
tritylglycerol, respectively. Each crude intermediate was dissolved in anhydrous pyridine (200
mL) and methanesulfonyl chloride (40 mL) was added dropwise at 0 °C. The reaction mixture
was then stirred at room temperature for 4 h. Methanol (50 mL) was added at 0 °C and
solvents were evaporated in vacuo. The residue was dissolved in CH2Cl2 (500 mL) and
washed with H2O (200 mL), solution of sat. NaHCO3 (2 × 200 mL), and brine (50 mL). The
organic solution was dried (anhydrous Na2SO4), filtered, and concentrated in vacuo.
Precipitation from a Et2O/hexane (10/3, v/v) mixture gave (R)-9 (68%) and (S)-9 (63%) as
white powders. Compound (R)-9: 1H NMR (400 MHz, CDCl3) 3.03 (s, 3H), 3.37 (dd, 3JH,H
5.6 Hz, 2JH,H = 10.6 Hz, 1H), 3.43 (dd, 3JH,H = 4.3 Hz, 2JH,H = 10.6 Hz, 1H), 3.68 (dd, 3JH,H
=
=
3.9 Hz, 2JH,H = 10.8 Hz, 1H), 3.75 (dd, 3JH,H = 6.8 Hz, 2JH,H = 10.8 Hz, 1H), 4.53 (s, 2H), 4.86
13
– 4.93 (m, 1H), 7.23 – 7.48 (m, 20H); C NMR (100 MHz, CDCl3) 38.7, 63.3, 69.6, 73.5,
80.8, 87.3, 127.4, 127.9, 128.0, 128.1, 128.6, 128.7, 137.6, 143.5; HRMS m/z calcd for
C30H30NaO5S [M+Na]+ 525.17062, found 525.17078. [α]20D +6.7 (c 1.12, CHCl3). Compound
1
13
(S)-9: H and C NMR spectra are identical with (R)-9. HRMS m/z calcd for C30H30NaO5S
[M+Na]+ 525.17062, found 525.17078. [α]20D –5.1 (c 1.65, CHCl3).
(R)-2-Azido-3-benzyloxy-1-(trityloxy)propane (R)-10 and (S)-2-Azido-3-benzyloxy-1-
(trityloxy)propane (S)-10. According to the reported procedure,19 sodium azide (300 mmol,
19.5 g) was added to the mixture of compound (R)-9 or (S)-9 (0.1 mol, 50.3 g) in
14

ACCEPTED MANUSCRIPT
HMPA/DMF (200 mL, 1:1) and the mixture was stirred at 100 °C for 5h and then diluted with
EtOAc (500 mL). The organic phase was washed with water (2 × 400 mL), dried (anhydrous
Na2SO4), filtered, and evaporated in vacuo. A column chromatography over silica gel (hexane
→ hexane/EtOAc, 90/10, v/v) afforded (R)-10 (99%) and (S)-10 (97%) as colourless oils.
Compound (R)-10: 1H NMR (400 MHz, CDCl3) 3.26 (dd, 3JH,H = 6.2 Hz, 2JH,H = 9.8 Hz, 1H),
3
2
3
2
3.29 (dd, JH,H = 4.8 Hz, JH,H = 9.8 Hz, 1H), 3.57 (dd, JH,H = 6.8 Hz, JH,H = 9.9 Hz, 1H),
3
3.61 (dd, JH,H = 4.5 Hz, 2JH,H = 9.9 Hz, 1H), 4.53 (s, 2H), 7.22 – 7.38 (m, 15H), 7.42 – 7.48
(m, 6H); 13C NMR (100 MHz, CDCl3) 61.4, 63.5, 70.0, 73.5, 87.3, 127.3, 127.7, 127.9, 128.0,
128.5, 128.8, 137.9, 143.8; HRMS m/z calcd for C29H27N3NaO2 [M+Na]+ 472.19955, found
1
472.19951. [α]20 –1.4 (c 2.58, MeOH). Compound (S)-10: H and 13C NMR spectra are
D
identical with (R)-10. HRMS m/z calcd for C29H27N3NaO2 [M+Na]+ 472.19955, found
472.19953. [α]20D +1.4 (c 2.19, MeOH).
Diethyl
(R)-1-[-1hydroxy-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate
(R)-12 and Diethyl (S)-1-[-1hydroxy-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonate (S)-12. In analogy to the reported procedure,24 to a solution of compound (R)-
10 or (S)-10 (27.0 g, 60.0 mmol) in EtOAc (200 mL) was added a solution of NaBrO3 (27.2 g,
0.18 mol) in water (200 mL). A solution of Na2S2O4 (31.3 g, 0.18 mol) in water (200 mL) was
added to the reaction mixture over 1 h and the mixture was vigorously stirred for 2.5 h at
room temperature. The mixture was diluted with EtOAc (150 mL), quenched with 10%
Na2S2O3 (50 mL) and extracted with water (3 × 150 mL). The combined organic layers were
dried (MgSO4) and concentrated in vacuo. The residue was purified by flash chromatography
(hexane → hexane/EtOAc, 80/20, v/v) to give (R)-11 (89%) and (S)-11 (91%), respectively,
as yellowish oils. Treatment of crude compound (R)-11 or (S)-11 by GP1 afforded
1
compounds (R)-12 (87%) and (S)-12 (73%) as colourless solid. Compound (R)-12: H (400
3
3
2
MHz, CDCl3) 1.27 and 1.28 (2 × t, JH,H = 7.1 Hz, 2 × 3H), 3.58 (dd, JH,H = 5.1 Hz, JH,H
=
10.1 Hz, 1H), 3.64 (dd, 3JH,H = 7.1 Hz, 2JH,H = 10.1 Hz, 1H), 3.96 – 4.19 (m, 7H), 4.76 – 4.86
(m, 1H), 7.17 – 7.30 (m, 15H), 8.20 (s, 1H); 13C NMR (100 MHz, CDCl3) 16.3 (2 × d, 3JC,P
=
2
6.2 Hz), 61.9, 63.0, 63.3 and 63.5 (2 × d, JC,P = 5.9 Hz), 63.4, 87.4, 127.5, 128.1, 128.5,
2
1
131.4 (d, JC,P = 33.4 Hz), 136.6 (d, JC,P = 239.7 Hz), 143.2; HRMS m/z calcd for
C28H32N3NaO5P [M+Na]+ 544.19718, found 544.19706. [α]20 –10.1 (c 1.32, MeOH).
D
1
13
Compound (S)-12: H and C NMR spectra are identical with (R)-12. HRMS m/z calcd for
C28H32N3NaO5P [M+Na]+ 544.19718, found 544.19705. [α]20D +10.9 (c 1.44, MeOH).
15

ACCEPTED MANUSCRIPT
Diethyl (R)-1-[1-(6-chloro-9H-purin-9-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonate (R)-13 and Diethyl (S)-1-[1-(6-chloro-9H-purin-9-yl)-3-(trityloxy)propan-
2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-13. Treatment of (R)-12 or (S)-12 with 6-
chloropurine by GP3, followed by purification by column chromatography over silica gel
(CHCl3/MeOH, 98/2, v/v), afforded (R)-13 (64%) and (S)-13 (67%), respectively, as
1
3
colourless foams. Compound (R)-13: H NMR (400 MHz, CDCl3) 1.29 and 1.30 (2 × t, JH,H
= 7.1 Hz, 2 × 3H), 3.62 – 3.73 (m, 2H), 4.04 – 4.22 (m, 4H), 4.87 (dd, 3JH,H = 4.8 Hz, 2JH,H
=
14.4 Hz, 1H), 5.02 (dd, 3JH,H = 9.2 Hz, 2JH,H = 14.4 Hz, 1H), 5.08 – 5.17 (m, 1H), 7.19 – 7.35
(m, 15H), 7.82 (s, 1H), 7.94 (s, 1H), 8.69 (s, 1H); 13C NMR (100 MHz, CDCl3) 16.4 (d, 3JC,P
= 6.5 Hz), 44.6, 60.5, 63.3 (d, 2JC,P = 5.7 Hz), 63.6, 88.0, 127.7, 128.3, 128.4, 131.5 (d, 2JC,P
=
1
33.0 Hz), 131.6, 138.0 (d, JC,P = 238.3 Hz), 142.8, 145.4, 147.0, 151.6, 152.2; 31P NMR
(161.9 MHz, CDCl3) 6.23; HRMS m/z calcd for C33H33ClN7NaO4P [M+Na]+ 680.19124,
1
found 680.19119. [α]20 +30.0 (c 0.26, MeOH). Compound (S)-13: H, 13C and 31P NMR
D
spectra are identical with (R)-13. HRMS m/z calcd for C33H33ClN7NaO4P [M+Na]+
680.19124, found 680.19125. [α]20D –26.3 (c 0.32, MeOH).
Diethyl (R)-1-[1-hydroxy-3-(6-oxo-1H-purin-9(6H)-yl)propan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonate (R)-14 and Diethyl (S)-1-[1-hydroxy-3-(6-oxo-1H-purin-9(6H)-yl)propan-
2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-14. A mixture of (R)-13 or (S)-13 (2.3 mmol) in
75% aq. trifluoroacetic acid (20 mL) was stirred at room temperature for 48 h. Solvents were
evaporated and the crude product was purified by chromatography over silica gel
(CHCl3/MeOH, 95/5, v/v → CHCl3/MeOH, 80/20, v/v). Crystallization from a mixture of
MeOH/Et2O (1:1) gave (R)-14 (80%) and (S)-14 (91%) as colourless crystals. Compound (R)-
14: mp 185 °C; 1H NMR (400 MHz, DMSO-d6) 1.19 and 1.20 (2 × t, 3JH,H = 7.0 Hz, 2 × 3H),
3
3.86 – 4.06 (m, 6H), 4.63 – 4.78 (m, 2H), 5.20 – 5.30 (m, 1H), 5.44 (t, JH,H = 5.4 Hz, 1H),
7.82 (s, 1H), 7.95 (s, 1H), 8.66 (s, 1H), 12.30 (br s, 1H); 13C NMR (100 MHz, DMSO-d6)
3
2
16.0 (d, JC,P = 6.2 Hz), 44.1, 60.6, 62.1 and 62.2 (2 × d, JC,P = 5.6 Hz), 62.6, 123.6, 131.1
1
31
(d, 2JC,P = 33.6 Hz), 136.1 (d, JC,P = 236.9 Hz), 140.0, 145.5, 148.3, 156.4; P NMR (161.9
MHz, DMSO-d6) 8.32; HRMS m/z calcd for C14H20N7NaO5P [M+Na]+ 420.11557, found
1
420.11561. [α]20 +101.1 (c 0.27, MeOH). Compound (S)-14: mp 185 °C; H, 13C and 31P
D
NMR spectra are identical with (R)-14. HRMS m/z calcd for C14H20N7NaO5P [M+Na]+
420.11557, found 420.11547. [α]20D –97.0 (c 0.36, MeOH).
16

ACCEPTED MANUSCRIPT
(R)-1-[1-Hydroxy-3-(6-oxo-1H-purin-9(6H)-yl)propan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonic acid (R)-15 and (S)-1-[1-Hydroxy-3-(6-oxo-1H-purin-9(6H)-yl)propan-2-
yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-15. Treatment of (R)-14 or (S)-14 by PG2
afforded (R)-15 (54%) and (S)-15 (59%), respectively, as white amorphous powders.
1
3
Compound (R)-15: H NMR (400 MHz, DMSO-d6) 3.91 (d, JH,H = 5.6 Hz, 2H), 4.69 (dd,
2
3
2
3JH,H = 4.8 Hz, JH,H = 14.5 Hz, 1H), 4.76 (dd, JH,H = 9.5 Hz, JH,H = 14.5 Hz, 1H), 5.18 –
5.28 (m, 1H), 7.79 (s, 1H), 8.00 (s, 1H), 8.43 (s, 1H), 12.35 (br s, 1H); 13C NMR (100 MHz,
2
1
DMSO-d6) 44.2, 61.0, 61.9, 123.5, 128.8 (d, JC,P = 32.2 Hz), 140.0, 141.3 (d, JC,P = 229.4
Hz), 145.7, 148.3, 156.4; 31P NMR (161.9 MHz, DMSO-d6) 3.40; HRMS m/z calcd for
C10H11N7O5P [M-H]- 340.05648, found 340.05634. [α]20 +80.5 (c 0.33, H2O). Compound
D
1
(S)-15: H, 13C and 31P NMR spectra are identical with (R)-15; HRMS m/z calcd for
C10H11N7O5P [M-H]- 340.05648, found 340.05665. [α]20D –79.3 (c 0.24, H2O).
Diethyl
(R)-1-[1-(2-amino-6-chloro-9H-purin-9-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonate (R)-16 and Diethyl (S)-1-[1-(2-amino-6-chloro-9H-purin-9-yl)-3-
(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-16. Treatment of (R)-12 or
(S)-12 with 2-amino-6-chloropurine by GP3, followed by purification by column
chromatography over silica gel (hexane/CHCl3/MeOH, 6/4/0.1 → hexane/CHCl3/MeOH,
6/4/0.3), afforded (R)-16 (31%) and (S)-16 (46%), respectively, as whitish foams. Compound
1
3
(R)-16: H NMR (400 MHz, CDCl3) 1.30 and 1.31 (2 × t, JH,H = 7.2 Hz, 2 × 3H), 3.65 (d,
3JH,H = 5.6 Hz, 2H), 4.06 – 4.22 (m, 4H), 4.63 (dd, 3JH,H = 4.9 Hz, 2JH,H = 14.6 Hz, 1H), 4.79
(dd, 3JH,H = 9.0 Hz, 2JH,H = 14.6 Hz, 1H), 5.02 – 5.11 (m, 1H), 5.21 (br s, 2H), 7.21 – 7.32 (m,
15H), 7.45 (s, 1H), 8.08 (s, 1H); 13C NMR (100 MHz, CDCl3) 16.4 (d, 3JC,P = 6.5 Hz), 44.5,
2
2
60.6, 63.3 (d, JC,P = 5.7 Hz), 63.6, 88.0, 125.1, 127.7, 128.3, 128.5, 131.6 (d, JC,P = 33.0
1
31
Hz), 137.8 (d, JC,P = 238.1 Hz), 142.1, 142.9, 151.8, 153.6, 159.2; P NMR (161.9 MHz,
CDCl3) 6.58; HRMS m/z calcd for C33H34N8NaO4P [M+Na]+ 695.20214, found 695.20209.
1
13
31
[α]20 +36.2 (c 0.41, MeOH). Compound (S)-16: H, C and P NMR spectra are identical
D
with (R)-16. HRMS m/z calcd for C33H34N8NaO4P [M+Na]+ 695.20214, found 695.20212.
[α]20D –36.3 (c 0.38, MeOH).
Diethyl
(R)-1-[1-(2-amino-6-oxo-1H-purin9(6H)-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonate (R)-17 and Diethyl (S)-1-[1-(2-amino-6-oxo-1H-purin9(6H)-yl)-
3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-17. A mixture of (R)-16 or
(S)-16 (2.3 mmol) in 75% aq. trifluoroacetic acid (20 mL) was stirred at room temperature for
17

ACCEPTED MANUSCRIPT
48 h. Solvents were evaporated and the crude product was purified by chromatography over
silica gel (CHCl3/MeOH, 98/2 → CHCl3/MeOH, 80/30) to give (R)-17 (90%) and (S)-17
1
(91%), respectively, as white amorphous solids. Compound (R)-17: H NMR (400 MHz,
DMSO-d6) 1.20 and 1.21 (2 × t, 3JH,H = 7.1 Hz, 2 × 3H), 3.86 – 4.05 (m, 6H), 4.48 (dd, 3JH,H
=
2
3
5.0 Hz, JH,H = 14.5 Hz, 1H), 4.56 (dd, JH,H = 9.7 Hz, 2JH,H = 14.5 Hz, 1H), 5.18 – 5.28 (m,
1H), 5.36 – 5.57 (m, 1H), 6.55 (br s, 2H), 7.34 (s, 1H), 8.66 (s, 1H), 10.67 (br s, 1H); 13C
3
NMR (100 MHz, DMSO-d6) 16.0 (d, JC,P = 6.2 Hz), 43.4, 60.7, 62.1, 62.2 and 62.3 (2 × d,
2
1
2JC,P = 5.6 Hz), 116.2, 131.1 (d, JC,P = 33.7 Hz), 136.0 (d, JC,P = 236.8 Hz), 136.9, 151.1,
31
153.7, 156.6; P NMR (161.9 MHz, DMSO-d6) 8.39; HRMS m/z calcd for C14H21N8NaO5P
1
[M+Na]+ 435.12647, found 435.12651. [α]20 +72.7 (c 0.97, MeOH). Compound (S)-17: H,
D
13C and 31P NMR spectra are identical with (R)-17; HRMS m/z calcd for C14H21N8NaO5P
[M+Na]+ 435.12647, found 435.12644. [α]20D –82.4 (c 0.95, MeOH).
(R)-1-[1-(2-Amino-6-oxo-1H-purin9(6H)-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonic
acid
(R)-18
and
(S)-1-[1-(2-Amino-6-oxo-1H-purin9(6H)-yl)-3-
hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-18. Treatment of (R)-17 or
(S)-17 by PG2 afforded (R)-18 (59%) and (S)-18 (86%), respectively, as white amorphous
1
3
powders. Compound (R)-18: H NMR (400 MHz, DMSO-d6) 3.82 (d, JH,H = 5.5 Hz, 2H),
4.48 (dd, 3JH,H = 5.9 Hz, 2JH,H = 14.5 Hz, 1H), 4.57 (dd, 3JH,H = 9.3 Hz, 2JH,H = 14.5 Hz, 1H),
5.14 – 5.23 (m, 1H), 5.30 – 5.50 (m, 1H), 6.48 (br s, 1H), 7.30 (s, 1H), 8.45 (s, 1H), 10.59 (br
2
s, 1H); 13C NMR (100 MHz, DMSO-d6) 43.9, 61.0, 61.3, 116.3, 128.9 (d, JC,P = 32.1 Hz),
1
137.1, 141.4 (d, JC,P = 229.4 Hz), 150.9, 154.0, 155.9; 31P NMR (161.9 MHz, DMSO-d6)
3.56; HRMS m/z calcd for C10H12N8O5P [M-H]- 355.06738, found 355.06726. [α]20D +92.7 (c
0.23, H2O). Compound (S)-18: 1H, 13C and 31P NMR spectra are identical with (R)-18; HRMS
m/z calcd for C10H12N8O5P [M-H]- 355.06738, found 355.06726. [α]20D –87.7 (c 0.25, H2O).
(R)-1-[1-(2,6-Dioxo-2,3-dihydro-1H-purin-9(6H)-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonic acid (R)-19 and (S)-1-[1-(2,6-Dioxo-2,3-dihydro-1H-purin-9(6H)-
yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-19. Isoamyl nitrite
(1.74 g, 2.0 mL) was added to a solution of (R)-18 or (S)-18 (0.4 mmol) in 80% acetic acid
(50 mL) and the reaction mixture was stirred at room temperature overnight. Solvents were
evaporated and the residues were crystallized from a water-methanol-acetone mixture to offer
(R)-19 (45%) and (S)-19 (45%), respectively, as yellowish crystals. Compound (R)-19: not
1
3
melting up to 265 °C; H NMR (400 MHz, DMSO-d6) 3.76 – 3.90 (m, 2H), 4.51 (dd, JH,H
=
18

ACCEPTED MANUSCRIPT
2
3
4.9 Hz, JH,H = 14.8 Hz, 1H), 4.68 (dd, JH,H = 9.9 Hz, 2JH,H = 14.8 Hz, 1H), 5.02 – 5.10 (m,
1H), 7.19 (s, 1H), 8.43 (s, 1H), 10.84 (br s, 1H), 12.02 (br s, 1H); 13C NMR (100 MHz,
2
1
DMSO-d6) 44.2, 60.7, 61.6, 115.2, 129.2 (d, JC,P = 32.1 Hz), 136.5, 140.2, 141.3 (d, JC,P
=
228.6 Hz), 150.7, 157.7; 31P NMR (161.9 MHz, DMSO-d6) 3.38; HRMS m/z calcd for
C10H11N7O6P [M-H]- 356.05139, found 356.05141. [α]20 +70.0 (c 0.08, H2O). Compound
D
1
(S)-19: not melting up to 265 °C; H, 13C and 31P NMR spectra are identical with (R)-19;
HRMS m/z calcd for C10H11N7O6P [M-H]- 356.05139, found 356.05109. [α]20D –81.5 (c 0.07,
H2O).
Diethyl (R)-1-[1-(2,6-diamino-9H-purin-9-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-
4-ylphosphonate
(R)-20
and
Diethyl
(S)-1-[1-(2,6-diamino-9H-purin-9-yl)-3-
(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-20. A mixture of (R)-16 or
(S)-16 (1.0 mmol) was dissolved in the ethanolic solution of NH3 (3.5 M, 65 mL) and the
reaction mixture was heated in an autoclave at 100 °C for 24 h. Volatiles were evaporated and
the residues were purified by flash chromatography over silica gel (hexane/CHCl3/MeOH,
6/4/0.2 → hexane/CHCl3/MeOH, 6/4/1) to give (R)-20 (32%) and (S)-20 (34%), respectively,
1
as yellowish foams. Compound (R)-20: H NMR (400 MHz, CDCl3) 1.26 and 1.30 (2 × t,
3
3JH,H = 7.1 Hz, 2 × 3H), 3.57 – 3.67 (m, 2H), 4.00 – 4.21 (m, 4H), 4.54 (dd, JH,H = 4.8 Hz,
2JH,H = 14.4 Hz, 1H), 4.72 (dd, 3JH,H = 9.2 Hz, 2JH,H = 14.4 Hz, 1H), 4.89 (s, 2H), 5.16 – 5.25
(m, 1H), 5.64 (s, 1H), 7.13 (s, 1H), 7.18 – 7.31 (m, 15H), 8.16 (s, 1H); 13C NMR (100 MHz,
3
2
CDCl3) 16.4 (d, JC,P = 6.5 Hz), 44.0, 60.8, 63.2 (d, JC,P = 5.6 Hz), 63.6, 87.8, 114.3, 127.6,
2
1
128.2, 128.5, 132.2 (d, JC,P = 33.2 Hz), 137.4 (d, JC,P = 237.8 Hz), 137.8, 142.9, 151.9,
156.1, 160.1; 31P NMR (161.9 MHz, CDCl3) 6.63; HRMS m/z calcd for C33H36N9NaO4P
1
[M+Na]+ 676.25201, found 676.25163. [α]20 –45.0 (c 0.14, MeOH). Compound (S)-20: H,
D
13C and 31P NMR spectra are identical with (R)-20; HRMS m/z calcd for C33H36N9NaO4P
[M+Na]+ 676.25201, found 676.25168. [α]20D +50.0 (c 0.20, MeOH).
Diethyl (R)-1-[1-(2,6-diamino-9H-purin-9-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonate
(R)-21
and
Diethyl
(S)-1-[1-(2,6-diamino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-21. Treatment of (R)-20 or (S)-
20 by GP4, followed by purification by chromatography over silica gel (CHCl3/MeOH, 98/2
→ CHCl3/MeOH, 70/30) gave (R)-21 (96%) and (S)-21 (97%), respectively, as white
1
amorphous solids. Compound (R)-21: H NMR (400 MHz, DMSO-d6) 1.20 and 1.21 (2 × t,
3
3JH,H = 7.0 Hz, 2 × 3H), 3.83 – 4.07 (m, 6H), 4.49 (dd, JH,H = 5.1 Hz, 2JH,H = 14.5 Hz, 1H),
19

ACCEPTED MANUSCRIPT
4.58 (dd, 3JH,H = 9.5 Hz, 2JH,H = 14.5 Hz, 1H), 5.22 – 5.31 (m, 1H), 5.81 (s, 2H), 6.67 (s, 2H),
7.33 (s, 1H), 8.68 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 16.0 (d, 3JC,P = 6.1 Hz), 43.1, 60.8,
62.0, 62.2 and 62.3 (2 × d, 2JC,P = 5.7 Hz), 112.8, 131.0 (d, 2JC,P = 33.6 Hz), 136.0 (d, 1JC,P
=
31
237.0 Hz), 136.9, 151.6, 156.0, 160,3; P NMR (161.9 MHz, DMSO-d6) 8.18; HRMS m/z
calcd for C14H22N9NaO4P [M+Na]+ 434.14246, found 434.14229. [α]20D +34.0 (c 0.21, H2O).
1
13
31
Compound (S)-21: H, C and P NMR spectra are identical with (R)-21; HRMS m/z calcd
for C14H23N9O4P [M+H]+ 412.16051, found 412.16057. [α]20D –29.0 (c 0.17, H2O).
(R)-1-[1-(2,6-Diamino-9H-purin-9-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonic acid (R)-22 and (S)-1-[1-(2,6-Diamino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-22. Treatment of (R)-21 or (S)-21 by PG2
afforded (R)-22 (74%) and (S)-22 (86%), respectively, as colorless solids. Compound (R)-22:
1H NMR (400 MHz, D2O+NaOD) 4.06 – 4.20 (m, 2H), 4.53 – 4.64 (m, 2H), 5.09 – 5.20 (m,
13
1H), 7.30 (s, 1H), 7.97 (s, 1H); C NMR (100 MHz, D2O+NaOD) 44.7, 61.3, 62.6, 113.3,
2
1
31
127.8 (d, JC,P = 26.5 Hz), 140.2, 148.4 (d, JC,P = 201.6 Hz), 151.5, 156.6, 160.6; P NMR
(161.9 MHz, D2O+NaOD) 0.44; HRMS m/z calcd for C10H13N9O4P [M-H]- 354.08226, found
1
13
354.08260. [α]20 +166.2 (c 0.18, 0.1 M aq. solution of NaOH). Compound (S)-22: H, C
D
and 31P NMR spectra are identical with (R)-22; HRMS m/z calcd for C10H13N9O4P [M-H]-
354.08226, found 354.08194. [α]20D –162.1 (c 0.18, 0.1 M aq. solution of NaOH).
Diethyl
(R)-1-[1-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(trityloxy)propan-2-yl]-
(R)-23 and Diethyl (S)-1-[1-(2,4-dioxo-3,4-
1H-1,2,3-triazol-4-ylphosphonate
dihydropyrimidin-1(2H)-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate
(S)-23. Treatment of (R)-12 or (S)-12 with N3-benzoyluracil by GP3, followed by column
chromatography over silica gel, afforded crude N3-benzoyluracil intermediates. Propylamine
(3 mL) was added to the solutions of the crude intermediates in dioxane (30 mL) and the
reaction mixture was stirred at room temperature for 12 h. Column chromatography over
silica gel (hexane/CHCl3/MeOH, 6/4/0.1 → hexane/CHCl3/MeOH, 6/4/0.35) afforded (R)-23
1
(81%) and (S)-23 (79%), respectively, as white foams. Compound (R)-23: H NMR (400
3
3
2
MHz, CDCl3) 1.30 and 1.32 (2 × t, JH,H = 7.1 Hz, 2 × 3H), 3.52 (dd, JH,H = 6.1 Hz, JH,H
=
3
2
10.2 Hz, 1H), 3.57 (dd, JH,H = 4.4 Hz, JH,H = 10.2 Hz, 1H), 4.06 – 4.30 (m, 5H), 4.44 (dd,
2
3
3JH,H = 4.8 Hz, JH,H = 14.2 Hz, 1H), 5.05 – 5.14 (m, 1H), 5.42 (d, JH,H = 7.9 Hz, 1H), 6.91
3
13
(d, JH,H = 7.9 Hz, 1H), 7.12 – 7.32 (m, 15H), 8.21 (s, 1H), 9.46 (br s, 1H); C NMR (100
MHz, CDCl3) 16.4 (d, 3JC,P = 6.4 Hz), 49.7, 59.8, 63.3 and 63.4 (2 × d, 2JC,P = 5.6 Hz), 63.5,
20

ACCEPTED MANUSCRIPT
87.7, 102.4, 127.3, 127.6, 128.0, 128.1, 128.3, 128.4, 132.3 (d, 2JC,P = 33.0 Hz), 137.8 (d, 1JC,P
= 238.7 Hz), 142.8, 144.7, 150.9, 163.4; 31P NMR (161.9 MHz, CDCl3) 6.86; HRMS m/z
calcd for C32H34N5NaO6P [M+Na]+ 638.21389, found 638.21391. [α]20 +59.7 (c 0.67,
D
1
13
31
MeOH). Compound (S)-23: H, C and P NMR spectra are identical with (R)-23; HRMS
m/z calcd for C32H34N5NaO6P [M+Na]+ 638.21389, found 638.21390. [α]20 –57.3 (c 0.44,
D
MeOH).
Diethyl (R)-1-[1-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]-1H-
1,2,3-triazol-4-ylphosphonate
(R)-24
and
Diethyl
(S)-1-[1-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate
(S)-24. Treatment of (R)-23 or (S)-23 by GP4, followed by flash chromatography over silica
gel (CHCl3/MeOH, 95/5 → CHCl3/MeOH, 90/10) afforded (R)-24 (80%) and (S)-24 (67%),
respectively, as white hygroscopic foams. Compound (R)-24: 1H NMR (400 MHz, DMSO-d6)
3
3
1.22 and 1.23 (2 × t, JH,H = 7.0 Hz, 2 × 3H), 3.82 – 4.09 (m, 6H), 4.14 (dd, JH,H = 9.6 Hz,
2JH,H = 14.3 Hz, 1H), 4.21 (dd, 3JH,H = 4.9 Hz, 2JH,H = 14.3 Hz, 1H), 5.02 – 5.12 (m, 1H), 5.39
3
3
(d, JH,H = 7.9 Hz, 1H), 7.26 (d, JH,H = 7.9 Hz, 1H), 8.74 (s, 1H), 11.27 (br s, 1H); 13C
3
NMR (100 MHz, DMSO-d6) 16.0 (d, JC,P = 6.2 Hz), 48.4, 60.5, 61.3, 62.2 and 62.3 (2 × d,
2
1
2JC,P = 5.4 Hz), 101.0, 131.2 (d, JC,P = 33.6 Hz), 136.2 (d, JC,P = 236.4 Hz), 145.0, 150.7,
163.4; 31P NMR (161.9 MHz, DMSO-d6) 8.14; HRMS m/z calcd for C13H20N5NaO6P
[M+Na]+ 396.10434, found 396.10429. [α]20D +156.5 (c 0.41, MeOH). Compound (S)-24: 1H,
13C and 31P NMR spectra are identical with (R)-24; HRMS m/z calcd for C13H20N5NaO6P
[M+Na]+ 396.10434, found 396.10425. [α]20D –148.5 (c 0.34, MeOH).
(R)-1-[1-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonic acid (R)-25 and (S)-1-[1-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-25. Treatment of (R)-
24 or (S)-24 by GP2 afforded, after crystallization from a water-MeOH (1:5) mixture, (R)-25
(91%) and (S)-25 (98%), respectively, as white crystals. Compound (R)-25: decomposition at
260 °C; 1H NMR (400 MHz, DMSO-d6) 3.78 – 3.87 (m, 2H), 4.14 (dd, 3JH,H = 9.5 Hz, 2JH,H
=
3
2
14.3 Hz, 1H), 4.23 (dd, JH,H = 4.9 Hz, JH,H = 14.3 Hz, 1H), 4.99 – 5.09 (m, 1H), 5.40 (d,
3JH,H = 7.9 Hz, 1H), 7.22 (d, 3JH,H = 7.9 Hz, 1H), 8.44 (s, 1H), 11.31 (s, 1H); 13C NMR (100
2
1
MHz, DMSO-d6) 48.6, 60.8, 60.9, 101.0, 128.9 (d, JC,P = 32.3 Hz), 141.4 (d, JC,P = 229.7
Hz), 145.1, 150.8, 163.5; 31P NMR (161.9 MHz, DMSO-d6) 3.26; HRMS m/z calcd for
C9H11N5O6P [M-H]- 316.04524, found 316.04542. [α]20 +169.9 (c 0.35, H2O). Compound
D
21

ACCEPTED MANUSCRIPT
1
(S)-25: decomposition at 262 °C; H, 13C and 31P NMR spectra are identical with (R)-25;
HRMS m/z calcd for C9H11N5O6P [M-H]- 316.04524, found 316.04554. [α]20D –173.2 (c 0.37,
H2O).
Diethyl
(R)-1-[1-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-
(trityloxy)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (R)-26 and Diethyl (S)-1-[1-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(trityloxy)propan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonate (S)-26. Treatment of (R)-12 or (S)-12 with N3-benzoylthymine by
GP3, followed by column chromatography over silica gel (hexan/CHCl3/MeOH, 6/4/0.1 →
hexan/CHCl3/MeOH, 6/4/0.4), afforded crude N3-benzoylthymine intermediates. Propylamine
(3 mL) was added to the solutions of the crude intermediates in dioxane (30 mL) and the
reaction mixture was stirred at room temperature for 12 h. Column chromatography over
silica gel (hexane/CHCl3/MeOH, 6/4/0.2) afforded (R)-26 (64%) and (S)-26 (65%),
1
respectively, as white foams. Compound (R)-26: H NMR (400 MHz, CDCl3) 1.30 and 1.32
3
(2 × t, JH,H = 7.1 Hz, 2 × 3H), 1.71 (s, 3H), 3.51 – 3.61 (m, 2H), 4.08 – 4.27 (m, 5H), 4.40
3
2
(dd, JH,H = 4.7 Hz, JH,H = 14.2 Hz, 1H), 5.05 – 5.15 (m, 1H), 6.72 (s, 1H), 7.18 – 7.35 (m,
15H), 8.18 (s, 1H), 9.17 (s, 1H); 13C NMR (100 MHz, CDCl3) 12.2, 16.4 (d, 3JC,P = 6.5 Hz),
49.7, 60.0, 63.2 and 63.3 (2 × d, 2JC,P = 5.6 Hz), 63.6, 87.7, 111.1, 127.4, 127.6, 128.0, 128.1,
2
1
128.3, 128.5, 132.1 (d, JC,P = 33.0 Hz), 137.9 (d, JC,P = 238.8 Hz), 140.4, 142.9, 150.9,
31
163.8; P NMR (161.9 MHz, CDCl3) 6.98; HRMS m/z calcd for C33H36N5NaO6P [M+Na]+
1
13
652.22954, found 652.22952. [α]20 +54.7 (c 0.49, MeOH). Compound (S)-26: H, C and
D
31P NMR spectra are identical with (R)-26; HRMS m/z calcd for C33H36N5NaO6P [M+Na]+
652.22954, found 652.22951. [α]20D –47.2 (c 0.50, MeOH).
Diethyl (R)-1-[1-hydroxy-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propan-
2-yl]-1H-1,2,3-triazol-4-ylphosphonate (R)-27 and Diethyl (S)-1-[1-hydroxy-3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate
(S)-27. Treatment of (R)-26 or (S)-26 by GP4, followed by flash chromatography over silica
gel (CHCl3/MeOH, 95/5 → CHCl3/MeOH, 90/10) afforded (R)-27 (82%) and (S)-27 (84%),
respectively, as white hygroscopic foams. Compound (R)-27: 1H NMR (400 MHz, DMSO-d6)
1.22 and 1.23 (2 × t, 3JH,H = 7.0 Hz, 2 × 3H), 1.60 (s, 3H), 3.82 – 4.08 (m, 6H), 4.10 (dd, 3JH,H
= 9.7 Hz, 2JH,H = 14.3 Hz, 1H), 4.17 (dd, 3JH,H = 4.8 Hz, 2JH,H = 14.3 Hz, 1H), 5.02 – 5.12 (m,
3
13
1H), 5.35 (t, JH,H = 5.4 Hz, 1H), 7.14 (s, 1H), 8.73 (s, 1H), 11.26 (s, 1H); C NMR (100
3
2
MHz, DMSO-d6) 11.7, 16.0 (d, JC,P = 6.2 Hz), 48.2, 60.5, 61.4, 62.2 and 62.3 (2 × d, JC,P
=
22

ACCEPTED MANUSCRIPT
5.9 Hz), 108.5, 131.2 (d, 2JC,P = 33.7 Hz), 136.2 (d, 1JC,P = 236.6 Hz), 140.7, 150.7, 164.0; 31P
NMR (161.9 MHz, DMSO-d6) 8.17; HRMS m/z calcd for C14H22N5NaO6P [M+Na]+
1
13
410.11999, found 410.11997. [α]20 +131.0 (c 0.29, MeOH). Compound (S)-27: H, C and
D
31P NMR spectra are identical with (R)-27; HRMS m/z calcd for C14H22N5NaO6P [M+Na]+
410.11999, found 410.12012. [α]20D –136.3 (c 0.57, MeOH).
(R)-1-[1-Hydroxy-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propan-2-yl]-
1H-1,2,3-triazol-4-ylphosphonic acid (R)-28 and (S)-1-[1-Hydroxy-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)propan-2-yl]-1H-1,2,3-triazol-4-ylphosphonic acid
(S)-28. Treatment of (R)-27 or (S)-27 by GP2 afforded, after crystallization from a water-
EtOH-EtOAc-acetone (1:5:30:30) mixture, (R)-28 (88%) and (S)-28 (91%), respectively, as
1
white crystals. Compound (R)-28: decomposition at 250 °C; H NMR (400 MHz, DMSO-d6)
3
2
1.62 (s, 3H), 3.78 – 3.89 (m, 2H), 4.09 (dd, JH,H = 9.4 Hz, JH,H = 14.3 Hz, 1H), 4.18 (dd,
3JH,H = 5.0 Hz, 2JH,H = 14.3 Hz, 1H), 4.99 – 5.09 (m, 1H), 7.10 (s, 1H), 8.43 (s, 1H), 11.28 (s,
2
1H); 13C NMR (100 MHz, DMSO-d6) 11.8, 48.2, 60.8, 60.9, 108.5, 128.9 (d, JC,P = 32.4
Hz), 140.8, 141.4 (d, 1JC,P = 229.9 Hz), 150.8, 164.0; 31P NMR (161.9 MHz, DMSO-d6) 3.62;
HRMS m/z calcd for C10H13N5O6P [M-H]- 330.06089, found 330.06086. [α]20 +151.1 (c
D
1
13
31
0.32, H2O). Compound (S)-28: decomposition at 238 °C; H, C and P NMR spectra are
identical with (R)-28; HRMS m/z calcd for C10H13N5O6P [M-H]- 330.06089, found
330.06120. [α]20D –149.1 (c 0.46, H2O).
Diethyl
(R)-1-[1-(4-amino-2-oxopyrimidin-1(2H)-yl-3-hydroxypropan-2-yl]-1H-1,2,3-
triazol-4-ylphosphonate (R)-29 and Diethyl (S)-1-[1-(4-amino-2-oxopyrimidin-1(2H)-yl-
3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonate (S)-29.
Treatment of (R)-12 or (S)-12 with N4-benzoylcytosine by GP3, followed by column
chromatography over silica gel, afforded crude N4-benzoylcytosine intermediates.
Propylamine (3 mL) was added to the solutions of the crude intermediates in dioxane (30 mL)
and the reaction mixture was stirred at room temperature for 12 h. Column chromatography
over silica gel (hexane/CHCl3/MeOH, 6/4/0.1 → hexane/CHCl3/MeOH, 6/4/0.7) afforded
debenzoylated cytosine intermediates as white foams. Treatment of the cytosine intermediates
by GP4, followed by flash chromatography over silica gel (CHCl3/MeOH, 95/5 →
CHCl3/MeOH, 7/3) afforded (R)-29 (40%) and (S)-29 (38%), respectively, as white
1
hygroscopic foams. Compound (R)-29: H NMR (400 MHz, DMSO-d6) 1.22 and 1.23 (2 × t,
3
3JH,H = 7.0 Hz, 2 × 3H), 3.78 – 3.91 (m, 2H), 3.94 – 4.10 (m, 5H), 4.24 (dd, JH,H = 4.6 Hz,
23

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3
2JH,H = 13.9 Hz, 1H), 5.09 – 5.18 (m, 1H), 5.43 (d, JH,H = 7.2 Hz, 1H), 6.99 (br s, 1H), 7.07
(br s, 1H), 7.12 (d, 3JH,H = 7.2 Hz, 1H), 8.69 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 16.0 (d,
2
2
3JC,P = 6.2 Hz), 49.8, 60.9, 61.4, 62.2 and 62.3 (2 × d, JC,P = 5.5 Hz), 93.3, 131.2 (d, JC,P
=
1
33.4 Hz), 136.1 (d, JC,P = 236.6 Hz), 145.4, 155.5, 165.9; 31P NMR (161.9 MHz, DMSO-d6)
8.49; HRMS m/z calcd for C13H21N6NaO5P [M+Na]+ 395.12033, found 395.12024. [α]20
D
1
13
31
+182.5 (c 0.32, MeOH). Compound (S)-29: H, C and P NMR spectra are identical with
(R)-29; HRMS m/z calcd for C13H21N6NaO5P [M+Na]+ 395.12033, found 395.12031. [α]20
–
D
172.1 (c 0.41, MeOH).
(R)-1-[1-(4-Amino-2-oxopyrimidin-1(2H)-yl-3-hydroxypropan-2-yl]-1H-1,2,3-triazol-4-
ylphosphonic acid (R)-30 and (S)-1-[1-(4-Amino-2-oxopyrimidin-1(2H)-yl-3-
hydroxypropan-2-yl]-1H-1,2,3-triazol-4-ylphosphonic acid (S)-30. Treatment of (R)-29 or
(S)-29 by GP2 afforded, after crystallization from a water-EtOH (1:1) mixture, (R)-30 (71%)
and (S)-30 (87%), respectively, as white crystals. Compound (R)-30: decomposition at 265
1
°C; H NMR (400 MHz, DMSO-d6) 3.82 (d, 3JH,H = 5.2 Hz, 2H), 4.07 – 4.21 (m, 1H), 4.23 –
4.35 (m, 1H), 5.01 – 5.13 (m, 1H), 5.66 (d, 3JH,H = 7.2 Hz, 1H), 7.33 (d, 3JH,H = 7.2 Hz, 1H),
8.15 (br s, 2H), 8.38 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 49.8, 60.6, 61.0, 93.5, 128.5 (d,
1
2JC,P = 31.8 Hz), 142.3 (d, JC,P = 226.5 Hz), 147.1, 152.5, 163.3; 31P NMR (161.9 MHz,
DMSO-d6) 2.94; HRMS m/z calcd for C9H12N6O5P [M-H]- 315.06123, found 315.06119.
1
[α]20 +204.4 (c 0.45, H2O). Compound (S)-30: decomposition at 265 °C; H, 13C and 31P
D
NMR spectra are identical with (R)-30; HRMS m/z calcd for C9H12N6O5P [M-H]- 315.06123,
found 315.06125. [α]20D –198.2 (c 0.53, H2O).
Determination of Ki values for human HGPRT, PfHGXPRT and PvHGPRT.8
Human HGPRT and PvHGPRT were stored in 0.1 M Tris-HCl, 0.01 M MgCl2, pH 7.4, 200
ꢀM PRib-PP, 1 mM dithiothreitol (DTT), at −80 °C, while PfHGXPRT in 0.01 M phosphate,
60 ꢀM hypoxanthine, 200 ꢀM PRib-PP, at pH 7.2, 1 mM DTT as previously described.29 The
buffer was 0.1 M Tris-HCl, 0.01 M MgCl2, pH 7.4 for the enzyme assays. For the Pf and Pv
enzymes, the assays were performed in this buffer and also in 0.01 M phosphate, 5 mM DTT
as described by Hazelton and colleagues.30 The Ki values were calculated by Hanes’ plots at a
fixed concentration of guanine (60 ꢀM) and at variable concentrations of PRib-PP (14−1000
ꢀM) depending on the Km(app) in the presence of the inhibitor.
24

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This work was supported by the subvention for development of Institute of Organic
Chemistry and Biochemistry (RVO 61388963), by Czech Science Foundation (16-06049S),
by Gilead Sciences (Foster City, CA, USA) and by the Australian NHMRC (Grant No.
1030353).
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