Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of novel acyclic nucleosides

Article abstract of DOI:10.1016/j.ejmech.2013.06.053

A series of seventeen hitherto unknown ANP analogs bearing the (E)-but-2-enyl aliphatic side chain and modified heterocyclic base such as cytosine and 5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3- carboxamide or 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as key synthetic step. All novel compounds were evaluated for their antiviral activities against a large number of DNA and RNA viruses including herpes simplex virus type 1 and 2, varicella zoster virus, feline herpes virus, human cytomegalovirus, hepatitis C virus (HCV), HIV-1 and HIV-2. Among these molecules, only compound 31 showed activity against human cytomegalovirus in HEL cell cultures with at EC ₅₀ of ～ 10 μM. Compounds 8a, 13, 14, and 24 demonstrated pronounced anti-HCV activity without significant cytotoxicity at 100 μM.

Full text of DOI:10.1016/j.ejmech.2013.06.053

Accepted Manuscript
Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of novel
acyclic nucleosides
Manabu Hamada, Vincent Roy, Tamara R. McBrayer, Tony Whitaker, Cesar Urbina-
Blanco, Steven P. Nolan, Jan Balzarini, Robert Snoeck, Graciela Andrei, Raymond F.
Schinazi, Luigi A. Agrofoglio
PII:
S0223-5234(13)00421-2
10.1016/j.ejmech.2013.06.053
EJMECH 6281
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 April 2013
Revised Date: 14 June 2013
Accepted Date: 20 June 2013
Please cite this article as: M. Hamada, V. Roy, T.R. McBrayer, T. Whitaker, C. Urbina-Blanco, S.P.
Nolan, J. Balzarini, R. Snoeck, G. Andrei, R.F. Schinazi, L.A. Agrofoglio, Synthesis and broad spectrum
antiviral evaluation of bis(POM) prodrugs of novel acyclic nucleosides, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.06.053.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of
novel acyclic nucleosides
M. Hamada, V. Roy, T.R. McBrayer, T. Whitaker, C.Urbina-Blanco, S. P. Nolan, J. Balzarini, R. Snoeck, G. Andrei, R. F.
Schinazi and L. A. Agrofoglio*
Seventeen ANP bearing the (E)-but-2-enyl side chain with cytosine, 5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-
triazole-3- carboxamide and 4-substituted-1,2,3-triazoles were prepared through challenging olefin acyclic cross
metathesis. Some pronounced anti-HCV activity were found.
Target Acyclic Nucleoside Phosphonates
where B are modified heterocyclic bases such as :

ACCEPTED MANUSCRIPT
Synthesis and broad spectrum antiviral evaluation of bis(POM) prodrugs of
novel acyclic nucleosides
Manabu Hamadaa, Vincent Roya, Tamara R. McBrayerb,Tony Whitakerb, Cesar
Urbina-Blancoc, Steven P. Nolanc, Jan Balzarinid, Robert Snoeckd, Graciela Andreid,
Raymond F. Schinazie and Luigi A. Agrofoglioa*
a Institut de Chimie Organique et Analytique, UMR 7311 CNRS, Université d’Orléans, 45067 Orléans –
France
b RFS Pharma, LLC, 1860 Montreal road, Tucker, GA 30084, USA
c EaStCHEM School of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
d Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
e
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory
University School of Medecine, Atlanta, GA, 30322, USA
Abstract
A series of seventeen hitherto unknown ANP analogs bearing the (E)-but-2-enyl aliphatic side chain and
modified heterocyclic base such as cytosine and 5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-
carboxamide or 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic
cross metathesis as key synthetic step.
All novel compounds were evaluated for their antiviral activities against a large number of DNA and RNA
viruses including herpes simplex virus type 1 and 2, varicella zoster virus, feline herpes virus, human
cytomegalovirus, hepatitis C virus (HCV), HIV-1 and HIV-2. Among these molecules, only compound 31
showed activity against human cytomegalovirus in HEL cell cultures with at EC50 of ~10 µM. Compounds 8a,
13, 14, and 24 demonstrated pronounced anti-HCV activity without significant cytotoxicity at 100 µM.
* Corresponding author. Tel. : +33 2 3849 4582
E-mail address : luigi.agrofoglio@univ-orleans.fr
1
Abbreviations: VZV, varicella zoster virus; VV, vaccinia virus; HSV, herpes simplex virus; VSV, vesicular
stomatitis virus; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; CC50, compound concentration affording
50% inhibition of cell growth; EC50, compound concentration affording 50% inhibition of the viral cytopathicity;
MCC, minimum cytotoxic concentration required to afford a microscopically detectable alteration of cell
morphology; MDCK, Madin-Darby canine kidney;
1

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1. Introduction
The discovery by A. Holý and E. De Clercq in 1986 of broad-spectrum antiviral activity of (S)-HPMPA
[9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and PMEA [9-[2-(phosphonomethoxy)ethyl]adenine] led
to a new family of nucleotides designated as acyclic nucleoside phosphonates (ANP), [1-4]. ANPs are
nucleotide analogs that are characterized by the presence of a phosphonate group linked to a pyrimidine or
purine base through an aliphatic linker. Three of these are approved drugs for the treatment of severe/fatal
infectious diseases and represent three different types of ANPs: (i) HPMP derivatives such as
(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir (Vistide®)] which is approved for
the treatment of cytomegalovirus (CMV) retinitis in AIDS patients [5]; (ii) PME derivatives such as PMEA
[adefovir (in its oral prodrug form, adefovir dipivoxil (Hepsera®)] for the treatment of hepatitis B virus infections
[6], and (iii) PMP derivatives such as PMPA [tenofovir (in its oral prodrug form, tenofovir disoproxil fumarate
(Viread®)] is used for the treatment of HIV infections (AIDS) and hepatitis B virus [7]. From these data, it
appears that small chemical alterations in the acyclic side-chain lead to marked differences in antiviral activity
and the spectrum of activity of acyclic nucleoside phosphonates against various classes of viral agents [1].
Thus, the synthesis and biological evaluation of a large panel of ANPs were systematically investigated
as potential antiviral compounds [1]. In our search for antiviral compounds, we synthesized a new class of
acyclic nucleoside phosphonates based on a 4-phosphono-but-2-en-1-yl base motif in which the oxygen
heteroatom has been replaced with a double bond having trans stereochemistry [8]. We have shown that
this modification allows mimicry of the three-dimensional geometry provided by the backbone of PMEA,
PMPA, and CDV while maintaining an electronic contribution similar to that brought by the oxygen atom [8].
Several new derivatives are efficiently activated by human thymidylate kinase (hTMPK), and the best
substrates were converted to bis-(pivaloyloxymethyl)ester phosphonate prodrugs and found to be active
against several herpes viruses in cell culture.
On the basis of these findings, it was interesting to design and synthesize hitherto unknown ANP analogs
bearing the biolabile phosphonate (E)-but-2-enyl aliphatic side-chain and a series of modified heterobases
selected from the literature as lead nucleobases with antiviral properties, such as cytosine and
5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide or 4-substituted-1,2,3-triazoles (Fig.
1). From a chemical synthesis point of view, the strategy based on olefin cross metathesis we have
developed to obtain a large library of (E)-4-phosphono-but-2’-en-1’-yl pyrimidine nucleosides [9-11].
[Fig. 1]
2. Results and discussion
2.1 Chemistry
First, we turned our attention to the synthesis of cytosine derivatives as cytosine modified nucleosides
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form a prolific family of antitumor and antiviral agents [12]. As it could be excepted, even if the ruthenium
carbene complex 3 (Grubbs-Nolan catalyst 2nd generation) is less affected by free amine and
nitrogen-containing groups than the Grubbs’s 1st generation catalyst [13], the cross-metathesis reaction
between unprotected N1-crotylated cytosine 2 and bis-(POM) allylphosphonate 1 failed (Scheme 1).
[Scheme 1]
The successful cross-metathesis occurred with protected N1-crotylated cytosines 6a,b. Thus, cytosine
4a and 5-fluorocytosine 4b were converted to their N4-bis-Boc cytosine derivatives 5a,b, respectively, through
a N-peracylation followed by subsequent and regioselective N1 deprotection by a saturated solution of
NaHCO3 in methanol. [14, 15] Crotylation of the N1 position of 5a,b using Cs2CO3 and crotyl bromide
afforded the desired compounds 6a (85%) and 6b (81%). Compound 6a,b were then engaged in the olefin
cross metathesis reaction with bis(POM)-allylphosphonate 1 using 5 mol% of the (NHC)Ru=CHR Nolan’s
catalyst,
Cl2(PCy3)(IMes)Ru(CHPh)
(3),
in
dry
CH2Cl2
(0.1M)
at
reflux
to
afford
(E)-N1-(4’-bis(POM)-phosphinyl-2’-butenyl)-bis-Boc-cytosine 7a,b in moderate yields (43% (for R = H) and
26% (for R = F)). The removal of the Boc group requires oftentimes harsh conditions (e.g. trifluoroacetic acid,
trimethylsilyl iodide, hydrochloric acid in ethyl acetate, potassium carbonate, etc.) that are not compatible with
the POM moiety. However, Hwu et al. [16] have reported an efficient and milder selective Boc deprotection
under neutral conditions using ceric ammonium nitrate (CAN) that is consistent with the stability of the
phosphonate biolabile group. Thus, protected ANP 7a,b were reacted with a catalytic amount of ceric
ammonium nitrate (CAN) (20 mmol%) in CH3CN-MeOH (1:1) to give the expected compounds 8a,b,
respectively, in moderate yield, with no observed removal of the POM moiety (Scheme 2).
[Scheme 2]
We turned then our attention to the synthesis of the pyrazinecarboxamide derivative 14 since, among the
modified nucleobases, a series of pyrazinecarboxamide derivatives (including T-705, favipiravir) developed by
Furuta et al. [17,18] have demonstrated good activity in various RNA viral infections. In a first attempt, we
struggled to introduce the pyrazine moiety through direct N-alkylation of 10 with the corresponding
(E)-4-phosphono-but-2’-en-1’-yl bromide (9), in the presence of K2CO3 in anhydrous DMF, (Pathway A).
Unfortunately only the bis(POM)-but-1,3-dienyl phosphonate 9’ resulting from undesired bromine elimination
was obtained, (Scheme 3). Thus, we decided to reach the pyrazine phosphonate analogues 13, 14 following
the same strategy developed for the cytosine derivatives, (Pathway B). Starting from the
3-hydroxypyrazine-2-carboxamide 10, the N1-Crotyl-3-oxo-pyrazine-N3-bis-Boc-carboxamide 11 was obtained
in
49%
yield,
via
the
two
step
crotylation
/
N-Boc-protection.
Next,
N1-crotyl-3-oxo-pyrazine-N3-bis-(Boc)-carboxamide 11 in CH2Cl2 was treated in the presence of
bis(POM)-allylphosphonate 1 and (NHC)Ru=CHR Nolan’s catalyst 3 to give the desired product 12 in 22%
yield. To obtain the carboxamide 14, we first applied our previous described methods, using ceric ammonium
nitrate and CH3CN-MeOH (1:1). Surprisingly, the methylester 13 was isolated as the major compound in 47%
yield. Thinking that the presence of the undesired product 13 was due to the use of methanol as co-solvent,
the preparation of the desired amide 14, was achieved in CH3CN using ceric ammonium nitrate in 25% yield.
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[Scheme 3]
Based on the above results, we extended this approach to the formation of a ribavirin analogue bearing
the 1,2,4-triazole derivative [19, 20]. Starting from 15, the protection of both nitrogens provided compound 16,
in quantitative yield, which is directly used in the next step. An attempt to selectively Boc deprotect at the N1
position of 16 to 17 failed and gave the unexpected free amide derivative 18. Alternatively, we decided to
successively alkylate the N1 position of the 1,2,4-triazole-3-carboxamide in the presence of crotyl bromide and
Cs2CO3 , followed by the protection of the free nitrogen in dry THF in the presence of Boc2O and DMAP at
room temperature to give the desired triazole 19 in 34% in two steps (Scheme 4).
N1-crotyl-1,2,4-triazole-3-bis-Boc-carboxamide 19 was then subjected to the olefin cross metathesis reaction,
with bis(POM)-allylphosphonate in CH2Cl2 to obtain the desired compound 20 in 16% yield. The bis-Boc
groups were cleaved according to the previous procedure, by treatment with CAN in a mixture of
CH3CN-MeOH (1:1) to give the free ANP analog 21 in 42% yield (Scheme 4).
[Scheme 4]
To complete our investigation, we elaborated a small library of ANPs in their prodrug form bearing the
substituted 1,2,3-triazolyl moiety 24-33. The triazole derivatives were obtained using Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) using selected alkynes and phosphonate azide 22, [21-24]. The
introduction of the azide group could be easily performed from the key intermediate previously described. The
olefin cross metathesis of bis(POM)-allylphosphonate with (E)-1,4-dibromobut-2-ene catalyzed by catalyst 3
afforded (E)-4-bromo-bis(POM)-allylphosphonate (9) in 88% yield. Then, the introduction of azido on 9 with
sodium azide in DMSO-THF-H2O (5:2:1) afforded (E)-4-azide-bis(POM)-allylphosphonate 22 in 69% yield
(Scheme 5).
Among the modifications on the base moiety, carboxylic acid is an unavoidable function. In a
first attempt, following our previously described method C, in the presence of propiolic acid under microwave
irradiation, we observed the formation and isolation of the unexpected decarboxylated product 23 in 16% yield.
[25, 26] To circumvent this decarboxylation, Hall et al. reported boronic acid catalysis (BAC) for the activation
of carboxylic acids, to lead to a classic dipolar [3+2] cycloaddition with several azides. [27] To obtain the
desired (E)-4-(4-carboxylic acid-[1,2,3]-triazol-1-yl)methyl- bis(POM)-but-2-enylphosphonate derivative 24,
we selected the BAC of the azide-alkyne cycloaddition, which was converted in 58% yield in the presence of
2-nitrophenylboronic acid at room temperature.
[Scheme 5]
Finally, the copper-catalyzed azide-alkynes 1,3-dipolar cycloaddition (CuAAC) affording
chemo-selectively and complete regioselectively the (1,4) substituted-1,2,3-triazoles, [22] that permitted the
synthesis of
a
number of ANPs analogs, (Table 1).
A
first series of bis(POM)-
(1,4-disubstituted-1,2,3-triazol)-but-2-enyl-phosphonate congeners bearing a substituted 1,2,3-triazole by
phenylacetylene moieties (chosen from apolar to bulky substituents) was obtained by CuAAC reaction with
Cu(0)/CuSO4·5H2O as catalyst in the presence of substituted phenylacetylenes, ethynylthiophene and
non-aromatic alkynes, in moderate to good yields ranging from 35 to 93% (Table 1, entries 1-6) at room
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temperature (method A. The reaction between our synthon 22 and 2-ethynylthiophene or prop-2-ynol at room
temperature only led to trace amounts of the expected products 31 and 32. These were, however, isolated.
The cycloaddition was then carried out at 60 °C Tab le 1, entries 7 and 8, method B) to afford compounds 31
and 32 in 45 and 95% yield, respectively. However, the alkyne listed in Table 1, entry 9 did not allow the
formation of the triazole product 33 under thermal conditions. Microwave heating is known as a powerful tool
that can produce a variety of nucleoside products. [23] Following our previous work, [24] the microwave
irradiation allowed to obtain in moderate yields the desired ANPs 33 and 34 in 44% and 34% yield
respectively (Table 1, entry 9 and 10, method C).
[Table 1]
2.2 Antiviral evaluation
The title bis(POM) (E)-4-phosphono-but-2-en-1-yl acyclic nucleosides were subjected to an in vitro
antiviral screening using a wide spectrum of viruses, in MDKC cell cultures for anti-influenza virus activity, in
Vero cell cultures for an antiviral activity against Para-influenza-3 virus, Reovirus-1, Sindbis virus, Coxsackie
B4 virus, Punta Toro virus, in CRFK cell cultures for their anti-geline corona virus and anti-feline herpes virus
activity, in HEL cell cultures for vaccinia virus, herpes simplex virus-1, herpes simplex virus-2, vesicular
stomatitis virus, varicella-zoster virus (VZV TK+ and TK-), human cytomegalovirus (AD-169 strain and Davis
strain) inhibitory activity and in HeLa cell cultures for Coxsackie B4 and respiratory syncytial virus inhibitory
activity. Among the tested final molecules against human cytomegalovirus in Hel cell cultures (data not
shown) only compound 31 showed activity at an EC50 of ~10 µM (AD-169 strain) with no observed cytotoxicity
at 100 µM (Table 2).
[Table 2]
The compounds were also evaluated for inhibition of hepatitis C virus (HCV) in the subgenomic HCV
replicon system in Huh 7 cells [28], and cytotoxicity testing was performed in PBMC, human lymphoblastoid
CEM and Vero cells [29,30]. Data for anti-HCV activity and anti-HIV activity, and the cytoxicity are shown in
Table 3. Many of the compounds were found to be moderately cytotoxic, which must be considered when
interpreting the anti-HCV data. Compounds 8a, 13, 14, and 24 demonstrated anti-HCV activity without
significant cytotoxicity at 100 µM. The other molecules showed pronounced inhibition at 10 µM, but at
compound concentrations that were close to their cytostatic activity.
[Table 3]
3. Conclusion
We have efficiently synthesized a series of seventeen hitherto unknown ANP analogs bearing the
E-but-2-enyl aliphatic side chain and a series of modified heterocyclic bases such as cytosine,
5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide and 4-substituted-1,2,3-triazoles and
5

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evaluated their antiviral activities. Among the tested molecules only compound 31 showed activity against
human cytomegalovirus at an EC50 of ~10 µM (AD-169 strain) at subtoxic concentrations with no observed
cytotoxicity up to 100 µM. Compounds 8a, 13, 14, and 24 demonstrated pronounced anti-HCV activity at 10
µM without significant cytotoxicity at 100 µM. Further structural optimization of both the (E)-but-2-enyl
aliphatic side chain and the heterocycle is well under way, alongside more detailed biological testing of the
most active compounds, with the aim of improving their antiviral potency.
Acknowledgment:
MH received a Post-doctoral fellowship from the Scientific Council of the University of Orleans. The
antiviral/cytostatic tests were performed by the financial support of the KU Leuven (GOA 10/14). We thank L.
Persoons, F. De Meyer, L. Ingels, A. Camps, S. Carmans and L. Van den Heurck for the antiviral evaluations.
4. Experimental section
4.1. Chemistry
Commercially available chemicals were of reagent grade and used as received. Solvents were dried following
standard procedures. The reactions were monitored by thin layer chromatography (TLC) analysis using silica
gel plates (Kieselgel 60F254, E. Merck). Column chromatography was performed on Silica Gel 60M
1
(0.040-0.063 mm, E. Merck). The H, 31P and 13C NMR spectra were recorded on a Varian InovaUnity 400
spectrometer (400MHz) in (d4) methanol, CDCl3, shift values in parts per million relative to SiMe4 as internal
reference. High Resolution Mass spectra (HRMS) were performed on a Bruker maXis mass spectrometer by
the “Fédération de Recherche ICOA/CBM (FR 2708) platform.
4.1.1.
Bis(POM)-allyl phosphonate (1). Physico-chemical data are in agreement with reported information
[9]. CAS number: 1258789-63-7
4.1.2. N1-crotyl cytosine (2). 1H NMR (400 MHz, CD3OD) δ 7.56 (d, J = 7.2 Hz, 1H minor), 7.54 (d, J = 7.2 Hz,
1H major), 5.88 (d, J = 7.2 Hz, 1H), 5.82 – 5.72 (m, 1H), 5.63 – 5.47 (m, 1H), 4.47 (d, J = 7.2 Hz, 2H, CH2 minor),
4.32 (d, J = 6.0 Hz, 2H, CH2 major), 1.79 (dd, J = 6.4, 1.2 Hz, CH3 minor), 1.69 (dd, J = 6.4, 1.3 Hz, 2H, CH3 major).
13C NMR (100 MHz, CD3OD) δ 158.7, 146.9, 131.4, 130.2, 126.5, 125.6, 95.9, 51.8, 46.7, 17.8, 13.1. HRMS
(ESI): m/z [M+H]+ calcd for C8H12N3O: 166.09746, found: 166.09748.
4.1.3.
N3,N3-Bis-Boc-cytosine (5a). To the stirred suspension of cytosine in an argon atmosphere (444.0
mg, 4.0 mmol, 1.0 equiv.) in dry THF (13 mL), DMAP (44.0 mg, 0.4 mmol, 0.1 equiv.) Boc2O (3.60 g, 16 mmol,
4.0 equiv.) were added. After 20 h stirring at room temperature, the mixture was diluted with EtOAc and then
extracted with (2 x 30 mL) EtOAc. The combined organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting tri-Boc-cytosine was used for the next step without further purification. To
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a solution of tri-Boc-cytosine in methanol (40 mL) was added saturated NaHCO3 aq. solution (18 mL) at room
temperature. After stirring 2 h at 60 °C, confirmin g the complete consumption of substrate by examining a TLC
developed with petroleum ether-EtOAc (1:1). After removal of methanol in vacuo, the mixture was diluted
with EtOAc (30 mL), quenched with water (20 mL) and finally extracted with EtOAc (2 X 20 mL). The
combined organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography with petroleum ether-EtOAc (1:1 to 1:20) to give 5a (465 mg,
37%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 12.88 (s, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 7.1 Hz,
1H), 1.54 (s, 17H). 13C NMR (100 MHz, CDCl3) δ 163.9, 158.6, 149.7, 145.7, 96.9, 85.2, 27.9. CAS number :
1108637-28-0
4.1.4.
N4,N4-Bis-Boc-5-fluoro-cytosine (5b). In an analogous manner to the preparation of 5a, 5b was
prepared from 5-fluoro-cytosine (158.0 mg, 24%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J =
3.5 Hz, 1H), 1.46 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 157.7, 156.8, 149.0, 143.6, 141.2, 134.0, 133.7, 85.2,
27.9. 19F NMR (376 MHz, CDCl3) δ -154.8. HRMS (ESI): m/z [M+H]+ calcd for C14H21FN3O5: 330.1463,
found: 330.1459.
4.1.5. N4,N4-Bis-Boc-N1-crotyl-cytosine (6a). To a solution of 5a (90.0 mg, 0.29 mmol, 1.0 equiv.) in dry DMF
(1 mL) was added Cs2CO3 (104.3 mg, 0.32 mmol, 1.1 equiv.) and crotyl bromide (43.0 mg, 0.32 mmol, 1.1
equiv.) at room temperature and stirred under an argon atmosphere for 2 hours. The resulting mixture was
then diluted with EtOAc (2 X 20 mL), quenched with water and extracted with EtOAc. The combined organic
layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica
gel column chromatography with petroleum ether-EtOAc (1:1) to give a mixture of Z (minor)/E (major)
N4,N4-bis-Boc-N1-crotyl-cytosine 6a (90.0 mg, 85%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J =
7.3 Hz, 1H), 6.95 (d, J = 7.3 Hz, 1H), 5.86 – 5.68 (m, 1H), 5.58 – 5.43 (m, 1H), 4.48 (d, J = 7.1 Hz, 2H, CH2
minor), 4.37 (d, J = 6.4 Hz, 2H, CH2 major), 1.73 (dd, J = 7.0, 3.0 Hz, 3H, CH3 minor), 1.69 (d, J = 6.4 Hz, 3H, CH3
major), 1.52 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 162.4, 162.3, 155.2, 149.8, 147.2, 146.9, 132.1, 131.2,
124.6, 123.5, 96.5, 85.0, 51.8, 46.2, 27.9, 24.0, 18.0, 13.3. HRMS (ESI): m/z [M+H]+ calcd for C18H28N3O5:
366.2026, found: 366.2023.
4.1.6.
N4,N4-Bis-Boc-N1-crotyl-5-fluoro-cytosine (6b). In a similar manner as described for 6a, a solution of
bis-Boc-5-fluoro-cytosine 5b (139.0 mg, 0.42 mmol) in dry DMF (1.6 mL) was treated with crotyl bromide (61.8
mg, 0.46 mmol) and Cs2CO3 (150.0 mg, 0.46 mmol), to give a mixture of Z (minor)/E (major)
N4,N4-bis-Boc-N1-crotyl-5-fluoro-cytosine 6b (130.1 mg, 81%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ
7.60 (d, J = 4.5Hz, 1H major), 7.59 (d, J = 3.1 Hz, 1H minor), 6.02 – 5.74 (m, 1H), 5.62 – 5.46 (m, 1H), 4.53 (d, J =
7.3 Hz, 2H, CH2 minor), 4.42 (d, J = 6.6 Hz, 2H, CH2 major), 1.76 – 1.73 (m, 3H), 1.45 (s, 18H). 13C NMR (100
MHz, CDCl3) δ 154.0, 149.1, 134.0, 133.1, 132.7, 123.6, 122.3, 84.9, 52.2, 46.5, 27.9, 24.1, 18.1, 13.4. 19F
NMR (376 MHz, CDCl3) δ -156.54 (d, J = 4.5 Hz), -156.66 (d, J = 4.5 Hz). HRMS (ESI): m/z [M+H]+ calcd for
C18H27FN3O5: 384.1932, found: 384.1929.
4.1.7.
(E)- N4,N4-bis-Boc-N1-(4’-Bis(POM)phosphinyl-2’-butenyl)cytosine (7a). To
a
solution of
bis(POM)-allylphosphonate 1 (77.0 mg, 0.22 mmol, 1.0 equiv.) and compound 6a (90.0 mg, 0.25 mmol, 1.1
equiv.) in dry CH2Cl2 (2 mL, 0.1 M) was added (NHC)Ru=CHR Nolan’s catalyst 3 (17.0 mg, 0.02 mmol, 0.1
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equiv.). The reaction mixture was stirred at 45 °C under an argon atmosphere for 16 h. After evaporation of all
volatiles, the crude was purified by silica gel chromatography with petroleum ether-EtOAc (1:2 to 1:4) to give
7a (63.0 mg, 43%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 7.4 Hz, 1H), 7.01 (d, J = 7.4 Hz,
1H), 5.79 – 5.70 (m, 1H), 5.69 – 5.61 (m, 5H), 4.23 (t, J = 4.8 Hz, 2H), 2.68 (dd, J = 22.6, 6.9, 2H), 1.53 (s,
18H), 1.20 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 176.8, 162.3, 154.9, 149.5, 146.9, 129.8, 129.7, 124.1,
123.9, 96.6, 84.9, 81.6, 81.5, 51.2, 38.7, 31.6, 30.2, 27.7, 26.8. 31P NMR (162 MHz, CDCl3) δ 26.6. HRMS
(ESI): m/z [M+H]+ calcd for C30H49N3O12P: 674.3050, found: 674.3048.
4.1.8.
(E)- N4,N4- bis-Boc-N1-(4’-Bis(POM)phosphinyl-2’-butenyl)-5-fluoro-cytosine (7b). In a similar
manner as described for 7a, a solution of bis(POM)-allylphosphonate (77.0 mg, 0.20 mmol) and 6b (85.0 mg,
0.22 mmol) in dry CH2Cl2 (2 mL, 0.1 M) were treated with (NHC)Ru=CHR Nolan’s catalyst 3 (17.0 mg, 0.02
mmol.), to give 7b (35.5 mg, 26%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 4.4 Hz, 1H), 5.82 –
5.69 (m, 2H), 5.67 (dd, J = 13.8, 5.2 Hz, 2H), 5.63 (dd, J = 13.8, 5.2 Hz, 2H), 4.47 (t, J = 4.4 Hz, 2H), 2.71 (dd,
J = 22.9, 5.3 Hz, 2H), 1.45 (s, 18H), 1.20 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 177.1, 155.9, 153.8, 149.1,
142.0, 139.6, 133.1, 132.8, 128.9, 128.7, 126.2, 126.1, 85.0, 81.9, 81.8, 51.8, 38.9, 31.8, 30.4, 27.9, 27.0. 19
F
NMR (376 MHz, CDCl3) δ -155.90 (d, J = 4.3 Hz). 31P NMR (162 MHz, CDCl3) δ 26.2. HRMS (ESI): m/z
[M+H]+ calcd for C30H48FN3O12P: 692.29539, found: 692.29541.
4.1.9.
(E)-N1-(4’-Bis(POM)phosphinyl-2’-butenyl)-cytosine (8a). To a solution of 7a (44.0 mg, 0.07 mmol,
1.0 equiv.) in 0.7 mL of CH3CN-MeOH (1:1) was added ceric ammonium nitrate (7.2 mg, 0.013 mmol, 0.2
equiv.) and the reaction solution was stirred at 60 °C for 16 h. After evaporation of all volatiles, t he residue
was
purified
by
silica
gel
chromatography
with
EtOAc
to
give
(E)-N1-(4’-bis(POM)phosphinyl-2’-butenyl)-cytosine 8a (18.2 mg, 55%) as a solid. 1H NMR (400 MHz, CDCl3)
δ 7.55 (d, J = 7.2 Hz, 1H), 5.91 (d, J = 7.2 Hz, 1H), 5.89 – 5.76 (m, 2H), 5.66 (m, 4H), 4.39 (t, J = 5.2 Hz, 2H),
2.81 (dd, J = 22.5, 7.2 Hz, 2H), 1.23 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 178.3, 147.1, 132.4, 132.3, 123.6,
123.5, 83.3, 83.2, 51.6, 39.9, 32.0, 30.6, 27.4. 31P NMR (162 MHz, CDCl3) δ 29.8. HRMS (ESI): m/z [M+H]+
calcd for C20H33N3O8P: 474.20045, found: 474.19997.
4.1.10. (E)-N1-(4’-Bis(POM)phosphinyl-2’-butenyl)- 5-fluoro-cytosine
(8b). In a similar manner as
described for 8a, a solution of 7b (35.5mg, 0.05 mmol) in 0.8 mL of CH3CN-MeOH (1:1) was treated with ceric
ammonium nitrate (5.5 mg, 0.01 mmol), to give (E)-N1-(4’-bis(POM)phosphinyl-2’-butenyl)-5-fluoro-cytosine
8b (8.2 mg, 35%) as a colorless oil. 1H NMR (400 MHz, CD3OD) δ 7.72 (d, J = 6.2 Hz, 1H), 5.89 – 5.77 (m,
1H), 5.66 (m, 5H), 4.35 (t, J = 5.2 Hz, 2H), 2.82 (dd, J = 22.5, 7.2 Hz, 2H), 1.23 (s, 18H). 13C NMR (100 MHz,
MeOD) δ 178.3, 132.1, 132.0, 131.2, 130.9, 124.0, 83.3, 83.3, 51.8, 39.9, 32.0, 30.6, 27.4. 19F NMR (376
MHz, CD3OD) δ -170.68 (d, J = 5.4 Hz). 31P NMR (162 MHz, CD3OD) δ 25.7. HRMS (ESI): m/z [M+H]+ calcd
for C20H32FN3O8P: 492.19057, found: 492.19055.
4.1.11. (E)-4-Bromo-bis(POM)-but-2-enylphosphonate (9). To a solution of bis(POM)-allylphosphonate 1
(560 mg, 1.60 mmol, 1.0 equiv.) in 24 mL of dry CH2Cl2 was added (E)-1,4-dibromobut-2-ene (1.37 g, 6.40
mmol, 4.0 equiv.) and (NHC)Ru=CHR Nolan’s catalyst 3 (68.6 mg, 0.08 mmol, 0.05 equiv.). After 16 h of
stirring at 45 °C under an argon atomsphere, all vo latiles were evaporated and the residue was purified by
silica
gel
chromatography
eluting
with
petroleum
ether-EtOAc
(4:1)
to
give
8

ACCEPTED MANUSCRIPT
(E)-4-bromo-bis(POM)-but-2-enylphosphonate 9 (623 mg, 88%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ
5.93-5.83 (m, 1H), 5.74-5.62 (m, 5H), 3.92 (dd, J = 7.5, 3.5 Hz, 2H), 2.70 (dd, J = 22.7, 7.3 Hz, 2H), 1.23 (s,
18H). 13C NMR (100 MHz, CDCl3) δ 176.3, 132.1, 131.9, 122.7, 122.6, 81.2, 38.3, 31.2, 30.9, 29.5, 26.5. 31
P
NMR (162 MHz, CDCl3) δ 26.8. HRMS (ESI): m/z [M+Na]+ calcd for C16H28O7NaBrP: 465.0654, found:
465.0671. CAS: 1365350-40-8
4.1.12. 4-bis(POM)-but-1,3-diene phosphonate (9’). 1H NMR (400 MHz, CDCl3) δ 7.10 (ddd, J = 22.2, 16.9,
10.6 Hz, 1H), 6.38 (tdd, J = 16.9, 10.6, 1.8 Hz, 1H), 5.81–5.50 (m, 7H), 1.52 (s, 18H). 13C NMR (100 MHz,
CDCl3) δ 176.8, 149.7, 149.6, 135.4, 135.1, 126.2, 117.9, 116.0, 81.5, 38.7, 26.8. 31P NMR (164 MHz, CDCl3)
δ 18.86.
4.1.13. N1-crotyl-3-oxo-pyrazine-N,N-bis-Boc-carboxamide (11). In a similar manner as described for 6a, a
solution of 3-hydroxypyrazine-2-carboxamide 10 (139.1 mg, 1.0 mmol) in dry DMF (3 mL) was treated with
Cs2CO3 (358.4 mg, 1.1 mmol) and crotyl bromide (148.5 mg, 1.1 mmol) for 4 h at 70 °C, to give a crude
mixture of (E/Z) N1-crotyl-3-oxo-pyrazine-2-carboxamide. After evaporation of all volatiles and rapid silica gel
chromatography, the residue (134.0 mg) was treated with DMAP (7.7 mg, 0.07 mmol) and Boc2O (602.4 mg,
2.76 mmol) in dry THF (3 mL), to give N1-crotyl-3-oxo-pyrazine-2-bis-Boc-carboxamide 11 (134.0 mg, 49%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 4.2 Hz, 1H), 7.22 (d, J = 4.2 Hz, 1H), 5.87 – 5.70
(m, 1H), 5.53 – 5.38 (m, 1H), 4.52 (t, J = 7.3 Hz, 2H, CH2 minor), 4.40 (t, J = 7.5 Hz, 2H, CH2 major), 1.70 (dd, J =
7.0, 1.7 Hz, 3H, CH3 minor), 1.65 dd, J = 7.0, 1.7 Hz, 3H, CH3 major), 1.41 (s, 18H). 13C NMR (100 MHz, CDCl3) δ
171.2, 164.4, 153.4, 150.1, 149.4, 133.2, 132.1, 130.6, 130.4, 123.4, 122.8, 122.4, 85.1, 60.5, 50.7, 45.0,
27.7, 21.1, 17.9, 14.3, 13.3. HRMS (ESI): m/z [M+H]+ calcd for C19H28N3O6: 394.19724, found: 394.19726.
4.1.14. (E)-N1-(4’-Bis(POM)- phosphinylbut-2’-enyl)-3-oxo-pyrazine-N,N-bis-Boc-carboxamide (12). In a
similar manner as described for 7a, a solution of bis(POM)-allylphosphonate 1 (273.5 mg, 0.78 mmol) and
N1-crotyl-3-oxo-pyrazine-N,N-bis-Boc-carboxamide 11 (338.2 mg, 0.86 mmol) in dry CH2Cl2 (8 mL) were
treated with (NHC)Ru=CHR Nolan’s catalyst 3 (66.2 mg, 0.078 mmol), to give 12 (122.8 mg, 22%) as a yellow
oil. 1H NMR (400 MHz, CDCl3) δ 7.35 (d, J = 4.3 Hz, 1H), 7.22 (d, J = 4.3 Hz, 1H), 5.80 – 5.69 (m, 2H), 5.64
(m, 4H), 4.49 (t, J = 4.8 Hz, 2H), 2.69 (dd, J = 22.2, 5.2 Hz, 2H), 1.47 (s, 18H), 1.21 (s, 18H). 13C NMR (100
MHz, CDCl3) δ 177.1, 153.4, 149.9, 149.4, 130.1, 128.8, 128.6, 125.8, 125.7, 123.1, 85.3, 82.2, 81.9, 81.8,
50.2, 39.0, 31.9, 28.3, 27.8, 27.1. 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+ calcd for
C31H49N3O13P: 702.29871, found: 702.29975.
4.1.15. (E)-N1-(4’-Bis(POM)- phosphinylbut-2’-enyl)-3-oxo-pyrazine-2-methylester (13). In a similar manner
as described for 8a, a solution of 12 (84.1 mg, 0.12 mmol) in 2 mL of CH3CN-MeOH (1:1) was treated with
1
ceric ammonium nitrate (13.1 mg, 0.024 mmol), to give 13 (38.6 mg, 47%) as a colorless oil. H NMR (400
MHz, CD3OD) δ 7.80 (d, J = 4.1 Hz, 1H), 7.49 (d, J = 4.1 Hz, 1H), 5.94 – 5.83 (m, 1H), 5.81 – 5.73 (m, 1H),
5.71 – 5.61 (m, 4H), 4.66 – 4.61 (m, 2H), 3.91 (s, 3H), 2.83 (dd, J = 22.7, 7.1 Hz, 2H), 1.22 (s, 18H). 13C NMR
(100 MHz, CD3OD) δ 178.3, 155.4, 135.1, 130.4, 130.3, 126.1, 126.0, 124.7, 83.4, 83.3, 53.3, 52.0, 39.9, 32.1,
30.7, 27.4. 31P NMR (162 MHz, CD3OD) δ 26.9. HRMS (ESI): m/z [M+H]+ calcd for C22H34N2O10P: 517.19480,
found: 517.19455.
9

ACCEPTED MANUSCRIPT
4.1.16. (E)-N1-(4’-bis(POM)- phosphinylbut-2’-enyl)-3-oxo-pyrazine-2-carboxamide (14). In a similar manner
as described for 8a, a solution of 12 (54.3 mg, 0.08 mmol) in CH3CN (1 mL) was treated with ceric ammonium
nitrate (11.0 mg, 0.02 mmol), to give 14 (10.0 mg, 25%) as a colorless oil. 1H NMR (400 MHz, CD3OD) δ 8.01
(d, J = 4.0 Hz, 1H), 7.74 (d, J = 4.0 Hz, 1H), 5.96 – 5.76 (m, 2H), 5.69 -5.62 (m, 4H), 4.73 (t, J = 5.2 Hz, 2H),
2.85 (dd, J = 22.7, 6.9 Hz, 2H), 1.22 (s, 18H). 13C NMR (100 MHz, CD3OD) δ 178.3, 154.1, 136.6, 130.1,
130.0, 126.6, 126.1, 125.2, 83.4, 83.3, 53.4, 39.9, 32.0, 30.6, 27.4. 31P NMR (162 MHz, CD3OD) δ 26.9.
HRMS (ESI): m/z [M+H]+ calcd for C21H33N3O9P: 502.19498, found: 502.19489.
4.1.17.
N1-Crotyl-1,2,4-triazole-N,N-bis-Boc-carboxamide
(15).
To
a
solution
of
1,2,4-triazole-3-carboxamide 15 (224.5 mg, 2.0 mmol, 1.0 equiv.) in dry DMF (6 mL) was added Cs2CO3
(716.8 mg, 2.2 mmol, 1.1 equiv.) and crotyl bromide (270.0mg, 2.2 mmol, 1.1 equiv.) and the reaction solution
was stirred at room temperature under an argon atmosphere for 2 h and then warmed at 70 °C for 12 h.
After concentration to dryness in vacuo, the residue was subjected to silica gel chromatography with
CH2Cl2-MeOH (5:1) and employed in the next step without further purification. The residue (330 mg) was
suspended in THF (10 mL), DMAP (44 mg, 0.2 mmol, 0.1 equiv.) and Boc2O (1.31 g, 6.0 mmol, 3.0 equiv.)
were added under an argon atmosphere. The solution was stirred for 20 h at room temperature and then the
mixture was diluted with EtOAc and then extracted with EtOAc. The combined organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with petroleum ether-EtOAc (4:1) to give a mixture of Z (minor)/E (major)
1
N1-crotyl-1,2,4-triazole-3-bis-Boc-carboxamide 19 (248.0 mg, 34%) as a colorless oil. H NMR (400 MHz,
CDCl3) δ 8.09 (s, 1H), 5.88 – 5.74 (m, 1H ), 5.72 – 5.63 (m, 1H), 4.82 (d, J = 7.1 Hz, 2H, CH2 minor), 4.71 (d, J =
6.6 Hz, 2H, CH2 major), 1.71 (dd, J = 6.5, 1.3 Hz, 3H, CH3 minor), 1.67 (dd, J = 6.5, 1.3 Hz, 3H, CH3 major), 1.36 (s,
18H). 13C NMR (100 MHz, CDCl3) δ 160.6, 157.1, 149.7, 144.0, 133.4, 131.8, 123.3, 122.0, 84.5, 52.6, 47.1,
27.7, 17.8, 13.2. HRMS (ESI): m/z [M+H]+ calcd for C17H27N4O5: 367.19775, found: 367.19759.
4.1.18.
(E)-N1-(4’-Bis(POM)-phosphinylbut-2’-enyl)-1,2,4-triazole-N,N-bis-Boc-carboxamide (20). In
a
similar manner as described for 7a, a solution of bis(POM)-allylphosphonate (173.1 mg, 0.45 mmol) and
N1-crotyl-1,2,4-triazole-3-bis-Boc-carboxamide (185.0 mg, 0.50 mmol) in dry CH2Cl2 (4.5 mL, 0.1 M) was
treated
with
(NHC)Ru=CHR
Nolan’s
catalyst
3
(38.1
mg,
0.045
mmol),
to
give
(E)-N1-(4’-bis(POM)phosphinyl-2’-butenyl)-1,2,4-triazole-3-bis-Boc-carboxamide 20 (48.8 mg, 16%) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 5.92 – 5.72 (m, 2H), 5.67 (dd, J = 13.6, 5.2 Hz, 4H), 5.63
(dd, J = 13.6, 5.2 Hz, 2H), 4.81 (t, J = 4.8 Hz, 2H), 2.71 (dd, J = 22.6, 6.6 Hz, 2H), 1.42 (s, 18H), 1.20 (s, 18H).
13C NMR (100 MHz, CDCl3) δ 177.1, 160.6, 157.4, 149.8, 144.0, 128.3, 128.2, 126.1, 125.9, 84.7, 81.9, 81.8,
52.3, 38.9, 31.7, 30.3, 28.2, 27.8, 27.0. 31P NMR (162 MHz, CDCl3) δ 26.0. HRMS (ESI): m/z [M+H]+ calcd
for C29H48N4O12P: 675.29986, found: 675.30008.
4.1.19. (E)-N1-(4’-Bis(POM)-phosphinylbut-2’-enyl)-1,2,4-triazole-3-carboxamide (21). In a similar manner
as
described
for
8a,
a
solution
of
(E)-N1-(4’-bis(POM)-phosphinyl-2’-butenyl)-1,2,4-triazole-3-bis-Boc-carboxamide 20 (44.3 mg, 0.065 mmol)
in 1 mL of CH3CN-MeOH (1:1) was treated with ceric ammonium nitrate (7.2 mg, 0.013 mmol), to give
(E)-N1-(4’-bis(POM)phosphinyl-2’-butenyl)-1,2,4-triazole-3-carboxamide 21 (14.0 mg, 42%) as a colorless oil.
1H NMR (400 MHz, CD3OD) δ 8.47 (s, 1H), 6.02 – 5.92 (m, 1H), 5.80 – 5.71 (m, 1H), 5.66 (m, 4H), 4.91 (t, J =
10

ACCEPTED MANUSCRIPT
4.8 Hz, 2H), 2.85 (dd, J = 22.7, 7.3 Hz, 2H), 1.22 (s, 18H). 13C NMR (100 MHz, CD3OD) δ 178.3, 163.5, 158.1,
146.4, 131.1, 130.9, 125.4, 125.3, 83.3, 83.3, 52.6, 39.9, 32.0, 30.6, 27.4. 31P NMR (162 MHz, CD3OD) δ 27.0.
HRMS (ESI): m/z [M+H]+ calcd for C19H32N4O8P: 475.19513, found: 475.19522.
4.1.20.
(E)-4-azido-bis(POM)-but-2-enylphosphonate
(22).
To
a
solution
of
(E)-4-bromo-bis(POM)-but-2-enylphosphonate 9 (222.0 mg, 0.50 mmol, 1.0 equiv.) in mixture of DMSO (5
mL), THF (2 mL) and H2O (1 mL) was added sodium azide (163.0 mg, 2.50 mmol, 5.0 equiv.). After 24 h
stirring at room temperature, the mixture was diluted with EtOAc and water and then extracted with EtOAc.
The combined organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography, eluting with petroleum ether-EtOAc (4:1) to give
(E)-4-azide-bis(POM)-but-2-enylphosphonate 22 (140.0 mg, 69%). 1H NMR (400 MHz, CDCl3) δ 5.70-5.61 (m,
6H), 3.74 (dd, J = 8.8, 3.5 Hz, 2H), 2.71 (dd, J = 22.4, 6.0 Hz, 2H), 1.21 (s, 18H). 13C NMR (100 MHz, CDCl3)
δ 177.1, 129.8, 123.4, 81.8, 81.7, 38.9, 31.7, 30.3, 27.1. 31P NMR (162 MHz, CDCl3) δ 27.7. IR νmax 3456.4,
2974.2, 2098.6, 1747.5, 1263.4, 1136.1, 960.6 cm-1. HRMS (ESI): m/z [M+Na]+ calcd for C16H28N3NaO7P:
428.15549, found: 428.15571.
4.1.21.
General Procedures for Huisgen 1,3-dipolar cycloaddition
Procedure A: To a solution of alkyne (1.3 equiv.) and (E)-4-azido-bis(POM)-but-2-enylphosphonate 22 (0.11
mmol, 1.0 equiv.) in t-BuOH/H2O (1:1 ratio, 400 µL) were added Cu powder (11.6 mg, 0.40 mmol, 5.0 equiv.)
and CuSO4 (5.0 mg, 0.020 mmol, 0.25 equiv). The resulting suspension was stirred 8 h at room temperature,
then the crude mixture was diluted in EtOAc (1 mL), and directly transferred on a preparative thin layer silica
plate to give (E)-4’-(1,2,3-triazol-1-yl)-bis(POM)-but-2’-enylphosphonate.
Procedure B: Following the procedure described above, the resulting suspension was stirred 16 h at 60 °C.
Procedure C: In a similar procedure, the mixture was stirred 1 h under microwave conditions at 125 °C.
4.1.21.1. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-1,2,3-triazole (23). The title compound was prepared
from 22 with procedure C to give 23 (16%) as a colorless oil. 1H NMR (250 MHz, CD3OD) δ 7.95 (d, J = 0.9 Hz,
1H), 7.74 (d, J = 0.9 Hz, 1H), 6.04 – 5.87 (m, 1H), 5.83 – 5.58 (m, 5H), 5.06 (t, J = 5.0 Hz, 2H), 2.85 (dd, J =
22.7, 7.2 Hz, 2H), 1.23 (s, 18H). 13C NMR (100 MHz, CD3OD) δ 178.3, 134.7, 131.3, 131.2, 125.8, 125.2,
125.1, 83.3, 83.2, 52.6, 39.9, 31.9, 30.5, 27.4. 31P NMR (162 MHz, CD3OD) δ 27.1. HRMS (ESI): m/z [M+H]+
calcd for C19H31N3O9P: 476.17894, found: 476.17924.
4.1.21.2. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(carboxylic acid)-1,2,3-triazole (24). To a solution of
propiolic acid (14.5 mg, 0.21 mmol, 1.1 equiv.) and (E)-4-azido-bis(POM)-but-2-enylphosphonate 22 (79.1 mg,
0.19 mmoL, 1.0 equiv.) in 1,2-dichloroethane (500 µL) was added 2-nitrophenylboronic acid (6.7 mg, 0.04
mmol, 0.21 equiv.) at room temperature. The solution was stirred for for 30 h. After evaporation of solvent, the
residue was purified by silica gel chromatography with EtOAc-MeOH (6:1) to give (E)-4-(4-carboxylic
1
acid-[1,2,3]-triazol-1-yl)methyl-bis(POM)-but-2’-enylphosphonate 24 (52.7 mg, 58%) as a colorless oil. H
NMR (400 MHz, CD3OD) δ 8.10 (s, 1H), 5.93-6.01 (m, 1H), 5.80 – 5.73 (m, 1H), 5.68 (dd, J = 13.2, 5.2 Hz,
4H), 5.64 (dd, J = 13.2, 5.2 Hz, 4H), 5.03 (t, J = 5.3 Hz, 2H), 2.85 (dd, J = 22.7, 7.3 Hz, 2H), 1.23 (s, 18H). 13
C
NMR (100 MHz, CD3OD) δ 178.3, 131.2, 127.1, 125.1, 83.3, 83.3, 52.7, 39.9, 31.9, 30.6, 27.4. 31P NMR (162
MHz, CD3OD) δ 27.1. HRMS (ESI): m/z [M+H]+ calcd for C19H31N3O9P: 476.17897, found: 476.17924.
11

ACCEPTED MANUSCRIPT
4.1.21.3. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-phenyl-1,2,3-triazole (25). The title compound was
prepared from 22 with procedure A to give 25 (70%) as a yellow oil. 1H NMR (400MHz, CDCl3) δ 7.84 (d, J =
7.6 Hz, 2H), 7.80 (s, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H) 5.97-5.88 (m, 1H), 5.84-5.75 (m, 1H),
5.71-5.64 (m, 4H), 5.00 (dd, J = 10.4, 5.2 Hz, 2H), 2.75 (dd, J = 22.7, 7.2 Hz, 2H), 1.21 (s, 18H). 13C NMR
(100 MHz, CDCl3) δ 177.0, 148.2, 130.7, 129.4, 129.3, 128.9, 128.3, 125.9, 124.8, 124.7, 119.5, 81.8, 81.7,
51.9, 38.9, 31.7, 30.3, 27.0. 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+ calcd for
C24H35N3O7P: 508.22099, found: 508.22071.
4.1.21.4. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(4-npropylphenyl)-1,2,3-triazole (26). The title
1
compound was prepared from 22 using procedure A to give 26 (88%) as a yellow oil. H NMR (400 MHz,
CDCl3) δ 7.73 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 5.97-5.88 (m, 1H), 5.84-5.70 (m, 1H),
5.71 – 5.64 (m, 4H), 4.97 (t, J = 5.2 Hz, 2H), 2.73 (dd, J = 22.7, 7.2 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.63 (m,
2H), 1.19 (s, 18H), 0.92 (t, J = 7.5 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.0, 148.4, 143.0, 129.5, 129.4,
129.0, 128.1, 125.8, 124.7, 124.6, 119.1, 81.8, 81.7, 51.9, 38.9, 38.0, 31.7, 30.3, 26.9, 24.6, 13.9. 31P NMR
(162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+ calcd for C27H41N3O7P: 550.26727, found: 550.26766.
4.1.21.5. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(4-nhexylphenyl)-1,2,3-triazole (27). The title
1
compound was prepared from 22 using procedure A to give 27 (93%) as a yellow oil. H NMR (400MHz,
CDCl3) δ 7.75 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 5.96-5.88 (m, 1H), 5.82-5.75 (m, 1H),
5.64-5.61 (m, 4H), 4.99 (t, J = 5.2 Hz, 2H), 2.74 (dd, J = 22.7, 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.59 (m,
2H), 1.37 – 1.19 (m, 8H), 1.20 (s, 18H), 0.84 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 177.0, 148.3,
143.2, 129.5, 129.3, 128.9, 128.0, 125.7, 124.6, 124.5, 119.1, 81.7, 81.6, 51.9, 38.8, 35.8, 31.8, 31.6, 31.4,
30.2, 29.0, 26.9, 22.7, 14.2. 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+ calcd for
C30H47N3O7P: 592.31497, found: 592.31461.
4.1.21.6. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(4-trifluoromethoxyphenyl)-1,2,3-triazole (28). The
1
title compound was prepared from 22 using procedure A to give 28 (52%) as a colorless oil. H NMR (400
MHz, CDCl3) δ 7.87 (d, J = 8.6 Hz, 2H), 7.82 (s, 1H), 7.26 (d, J = 8.6 Hz, 2H), 5.94-5.89 (m, 1H), 5.82-5.77 (m,
1H), 5.71-5.64 (m, 4H), 5.01 (t, J = 5.2 Hz 2H), 2.75 (dd, J = 22.7, 7.2, 2H), 1.18 (s, 18H). 13C NMR (100 MHz,
CDCl3) δ 177.0, 171.3, 147.0, 129.5, 129.2, 129.0, 127.3, 125.1, 125.0, 121.4, 119.7, 81.8, 81.7, 51.9, 38.9,
31.7, 30.3, 27.0, 14.3. 31P NMR (162 MHz, CDCl3) δ 26.2. HRMS (ESI): m/z [M+H]+ calcd for C25H34F3N3O8P:
592.20335, found: 592.20301.
4.1.21.7.. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(4-fluorophenyl)-1,2,3-triazole (29). Prepared from
azide 22 using procedure A, compound 29 was isolated in 65% as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ 7.81 (dd, J = 8.6, 5.4 Hz, 2H), 7.77 (s, 1H), 7.09 (t, J = 8.6 Hz, 2H), 5.96-5.88 (m, 1H), 5.84-5.75 (m, 1H),
5.71-5.64 (m, 4H), 4.99 (t, J = 5.2 Hz, 2H), 2.75 (dd, J = 22.7, 7.2 Hz, 2H), 1.20 (s, 18H). 13C NMR (100 MHz,
CDCl3) δ 177.0, 164.0, 161.5, 147.4, 129.3, 129.1, 127.6, 127.5, 126.9, 126.8, 124.9, 124.8, 119.2, 116.0,
115.8, 81.7, 81.6, 51.9, 38.8, 31.7, 30.3, 26.9. 19F NMR (376 MHz, CDCl3) δ -113.68 (ddd, J = 10.5, 6.8, 4.3
Hz). 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+ calcd for C24H34FN3O7P: 526.21159, found:
526.21129. HRMS (ESI): m/z [M+Na]+ calcd for C24H33FN3NaO7P: 548.19361, found: 548.19324.
12

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4.1.21.8. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(3,5-dimethoxyphenyl)-1,2,3-triazole (30). Prepared
1
from azide 22 following procedure A, compound 30 was isolated in 35% as a yellow oil. H NMR (400MHz,
CDCl3) δ 7.78 (s, 1H), 7.01 (d, J = 2.2 Hz, 2H), 6.44 (t, J = 2.2 Hz, 1H), 5.96-5.88 (m, 1H), 5.83-5.76 (m, 1H),
5.71-5.64 (m, 4H), 5.00 (t, J = 5.2 Hz, 2H), 3.84 (s, 6H), 2.75 (dd, J = 22.7, 7.2 Hz, 2H), 1.21 (s, 18H). 13C
NMR (100 MHz, CDCl3) δ 177.0, 161.3, 148.2, 132.5, 129.4, 129.3, 124.8, 124.7, 119.8, 103.8, 100.8, 81.8,
81.7, 55.6, 51.9, 38.9, 31.7, 30.3, 29.8, 27.0. 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+
calcd for C26H39N3O7P: 568.24210, found: 568.24184.
4.1.21.9. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(hydroxymethyl)-1,2,3-triazole (31). The title
compound was prepared from 22 using procedure B to give 31 (44%) as a colorless oil. 1H NMR (250 MHz,
CDCl3) δ 7.54 (s, 1H), 5.87-5.62 (m, 6H), 4.93 (m, 2H), 4.77 (brs, 2H), 2.71 (dd, J = 22.8, 7.0 Hz, 2H), 2.46
(brs, 1H), 1.20 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 177.3, 148.5, 129.4, 129.2, 124.9, 124.8, 121.6, 81.8,
81.7, 56.9, 51.9, 39.0, 31.8, 30.4, 27.0. 31P NMR (162 MHz, CDCl3) δ 26.4. HRMS (ESI): m/z [M+H]+ calcd
for C19H33N3O8P: 462.20003, found: 462.19998.
4.1.21.10. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(thiophenyl)-1,2,3-triazole (32). The title compound
was prepared from 22 with typical procedure B to give 32 (95%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ
7.68 (s, 1H), 7.36 (dd, J = 3.6, 1.1 Hz, 1H), 7.25 (dd, J = 5.0, 1.1 Hz, 1H), 7.03 (dd, J = 5.0, 3.6 Hz, 1H), 5.94 –
5.69 (m, 2H), 5.71-5.64 (m, 4H), 4.99 (dd, J = 5.2, 4.8 Hz, 2H), 2.72 (dd, J = 22.7, 7.1 Hz, 2H), 1.18 (s, 18H).
13C NMR (100 MHz, CDCl3) δ 177.1, 143.4, 133.0, 129.3, 129.1, 127.8, 125.2, 125.0, 124.9, 124.4, 119.0,
81.8, 81.8, 52.0, 52.0, 38.9, 31.7, 30.3, 27.0. 31P NMR (162 MHz, CDCl3) δ 26.3. HRMS (ESI): m/z [M+H]+
calcd for C22H33N3O7PS: 514.17714, found: 514.17713.
4.1.21.11. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(octyl)-1,2,3-triazole (33). The title compound was
1
prepared from 22 with typical procedure C to give 33 (44%) as a colorless oil. H NMR (400 MHz, CDCl3) δ
8.03 (s, 1H), 5.90 – 5.72 (m, 2H), 5.71 – 5.59 (m, 4H), 4.99 (m, 2H), 3.08 (t, J = 7.5 Hz, 2H), 2.72 (dd, J = 22.7,
6.8 Hz, 2H), 1.75 – 1.68 (m, 2H), 1.39 – 1.10 (m, 28H), 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
177.1, 148.4, 125.9, 125.3, 100.2, 81.9, 81.8, 52.2, 39.8, 39.0, 31.9, 31.9, 30.4, 29.5, 29.3, 27.1, 24.2, 22.8,
14.3. 31P NMR (162 MHz, CDCl3) δ 26.0. HRMS (ESI): m/z [M+H]+ calcd for C26H47N3O7P: 544.31454, found:
544.31461.
4.1.21.12. (E)-1-(4’-bis(POM)- phosphinylbut-2’-enyl)-4-(carboxamide)-1,2,3-triazole (34). The title compound
was prepared from 22 with typical procedure C to give 34 (34%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ
8.34 (s, 1H), 6.02-5.91 (m, 1H), 5.79 – 5.72 (m, 1H), 5.69-5.64(m, 4H), 5.08 (t, J = 5.3 Hz, 2H), 2.85 (dd, J =
22.7, 7.3, 2H), 1.22 (s, 18H). 13C NMR (100 MHz, CDCl3) δ 178.3, 131.0, 130.9, 127.5, 125.6, 125.5, 83.4,
83.3, 52.9, 39.9, 31.9, 30.5, 27.4. 31P NMR (162 MHz, CDCl3) δ 27.0. HRMS (ESI): m/z [M+H]+ calcd for
C19H32N4O8P: 475.19537, found: 475.19522.
4.2. Antiviral activity assays
The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced cytopathicity or
13

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plaque formation in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, vesicular
stomatitis virus, cytomegalovirus (HCMV), and varicella-zoster virus (VZV)], Vero (parainfluenza-3, reovirus-1,
Sindbis virus and Coxsackie B4), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus) or MDCK [influenza A (H1N1; H3N2) and influenza B] cell cultures. Confluent cell cultures (or
nearly confluent for MDCK cells) in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (for VZV)
in the presence of varying concentrations (100, 20, … µM) of the test compounds. Viral cytopathicity was
recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with
the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required
reducing virus-induced cytopathogenicity or viral plaque (VZV) formation by 50%. The minimal cytotoxic
concentration (MCC) of the compounds was defined as the compound concentration that caused a
microscopically visible alteration of cell morphology. Alternatively, the cytostatic activity of the test compounds
was measured based on inhibition of cell growth. HEL cells were seeded at a rate of 5 x 103 cells/well into
96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations
of the test compounds was added. After 3 days of incubation at 37 °C, the cell number was determined w ith a
Coulter counter. The cytostatic concentration was calculated as the CC50, or the compound concentration
required to reduce cell proliferation by 50% relative to the number of cells in the untreated controls. The
methodology of the anti-HIV assays was as follows: human CEM ( 3 x 105 cells/cm3) cells were infected with
100 CCID50 of HIV(IIIB) or HIV-2(ROD)/ml and seeded in 200 µL wells of a microtiter plate containing
appropriate dilutions of the test compounds. After 4 days of incubation at 37 °C, HIV-induced CEM gian t cell
formation was examined microscopically.
Hepatitis C antiviral activity was evaluated as previously described [28]. Huh 7 Clone B cells containing HCV
Replicon RNA were seeded in a 96-well plate at 3000 cells/well, and the compounds were tested at 10 µM in
triplicate immediately after seeding. Following five days incubation (37oC, 5% CO2), total cellular RNA was
isolated using the RNeasy96 well extraction kit from Qiagen. Replicon RNA and an internal control (TaqMan
rRNA control reagents, Applied Biosystems) were amplified in a single step multiplex Real Time RT-PCR
Assay. The antiviral effectiveness of the compounds was calculated by subtracting the threshold RT-PCR
cycle of the test compound from the threshold RT-PCR cycle of the no-drug control (ꢀCtHCV). A ꢀCt of 3.3
equals a 1-log reduction (equal to 90% less starting material) in Replicon RNA levels. The cytotoxicity of the
compounds was also calculated by using the ꢀCt rRNA values. RS-446 (2’-C-Me-C) was used as the control
and tested at 10 µM. To determine EC50 and EC90 values [31], ꢀCt values were first converted into fraction
of starting material and then were used to calculate the % inhibition.
Appendix. Supplementary data
Supplementary data associated with this article can be found, in the online version, at :
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DRAWING CAPTIONS
Fig 1. Structure of selected ANPs and target derivatives
Scheme 1. Attempted cross-metathesis reaction with bis-(POM)-allylphosphonate 1 with unprotected
cytosine derivatives.
Scheme 2. Reagents and conditions: (a) i) Boc2O, DMAP, dry THF then ii) saturated NaHCO3 aq. solution,
methanol, 60 °C, 37% (for R = H) and 24% (for R = F ) (yields for two steps); (b) crotyl bromide, Cs2CO3, dry
DMF, 85% (for R = H) and 81% (for R = F); (c) bis-(POM)-allylphosphonate (1), 10 mol% [Ru]= catalyst 3, dry
CH2Cl2, 45 °C, 43% (for R = H) and 26% (for R = F); (d) c eric ammonium nitrate, CH3CN:MeOH (1:1), 55%
(for R = H) and 35% (for R = F).
Scheme 3. Reagents and conditions: (a) (E)-1,4-dibromobut-2-ene, 10 mol% [Ru]= catalyst 3, dry CH2Cl2,
45 °C, 88%; (b) heterocycle 10, K2CO3, dried DMF; (c) 1) crotyl bromide, Cs2CO3, dry DMF then ii) Boc2O,
DMAP, dry THF, 49% (for two steps); (d) 10 mol% [Ru]= catalyst 3, dry CH2Cl2, 45 °C, 22%; (e) ceric
ammonium nitrate, CH3CN-MeOH (1:1), 47%; (f) ceric ammonium nitrate, CH3CN, 25%.
Scheme 4. Reagents and conditions: (a) Boc2O, DMAP, dry THF then (b) saturated NaHCO3 aq. solution,
methanol, 60 °C, 40% (for two steps); (c) i) crotyl bromide, Cs2CO3, dry DMF then ii) Boc2O, DMAP, dry THF,
r.t., 34% (for two steps); (d) 10 mol% [Ru]= catalyst 3, dry CH2Cl2, 45 °C, 16%; (e) ceric ammonium nitrate,
CH3CN-MeOH (1:1), 42%.
Scheme 5. Reagents and conditions: (a) (E)-1,4-dibromobut-2-ene, 10 mol% [Ru]= catalyst 3, dry CH2Cl2,
45 °C, 88%; (b) sodium azide, DMSO-THF-H 2O (5:2:1), r.t., 69%; (c) (a) Cu(0), CuSO4·5H2O, t-BuOH/H2O
(1:1), MW (125 °C), 16%; (d) 2-nitrophenylboronic a cid, DCE, r.t., 69%.
Table 1. Cycloaddition of CuAAC reactiona
Table 2. Anti-cytomegalovirus activity and cytotoxicity of compound 31 in human embryonic lung (Hel) cells.
Table 3. Anti- HCV activity, anti-HIV activity and cytotoxicity of synthesized compounds in cellular assays
17

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Table 1.
R
N
N
N
O
P
O
P
CuAAc
+
POMO
POMO
R
N3
POMO
POMO
25-34
22
entry
1
Product
Method
A
yield (%)b
70
25
2
3
26
27
A
A
88
93
4
5
28
29
A
A
52
65
OCF3
F
OMe
6
30
A
35
OMe
7
8
31
32
B
B
44
95
OH
S
9
33
34
C
C
44
34
10
O
NH2
a All reactions were performed with 1.0 equiv of (E)-4-azide-bis(POM)-but-2-enylphosphonate, 1.3 equiv of alkyne,
and 5.0 equiv of Cu (0), and 0.25 equiv of CuSO4·5H2O, t-BuOH/H2O (1:1).
b Isolated yield.
Method A: room temperature during 8h.
Method B: 60 °C during 16h.
Method C: 125 °C during 1 h under microwave irradia tion (MW).

ACCEPTED MANUSCRIPT
Table 2.
EC50a (µM)
AD-169 strain
Compound
CC50b (µM)
31
9.9±1.0
8.2±0.3
1.0±0.14
100
Ganciclovir
Cidofovir
> 350
> 350
a 50% Effective concentration or compound concentration required to inhibit virus-induced
cytopathicity by 50%.
b 50% Cytotoxic concentration or compound concentration required to reduce the viability of
Hel cells by 50%.
Table 3.
Compound
Anti-HCV activity:
% inhibition
10µM in Huh7 cells
Anti-HIV activity
(µM)
Cytotoxicity in CC50 (µM)
HCV
EC50
EC90
PBM
CEM
Vero
8a
8b
13
14
21
23
24
25
26
27
28
29
30
31
32
33
34
80.4
73.6
76.9
60.0
73.9
85.9
53.8
83.1
95.6
95.0
60.6
84.3
20.8
78.1
0.2
70.4
nd
>100
nd
>100
nd
>100
nd
>100
nd
>100
>100
>100
>100
>100
35.8
31.6
43.9
55.2
>100
>100
>100
62.3
>100
70.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
58.3
93.1
>100
16.0
35.9
5.9
19.6
61.9
>100
73.2
32.9
19.8
62.4
>100
>100
41.8
81.8
>100
31.6
55.3
16.1
37.6
31.6
68.2
57.4
31.6
16.8
52.9
>100
>100
86.8
87.9
>100
15.6
41.0
10.4
12.7
47.0
86.8
87.1
17.1
10.3
80.6
88.7
86.5

ACCEPTED MANUSCRIPT
Figure 1.
HPMPC, Cidofovir
R1 = H, PMEA
R1 = POM, Adefovir dipivoxil
R2 = H, PMPA
R2 = POC, Tenofovir disoproxil
POM = CH2OC(O)tBu; POC = CH2OCO2iPr
where B are modified heterocyclic bases such as :
Target Acyclic Nucleoside Phosphonates
Scheme 1
NH2
N
NH2
N
5 mol% [Ru]= catalyst (3)
O
P
CH2Cl2, rfx
O
O
P
N
POMO
+
N
O
POMO
POMO
POMO
2
1
free amino group likely to poison [Ru]= catalyst
N
N
Cl
Cl
Ru
PCy3
Ph
Cl2(PCy3)(IMes)Ru(CHPh)
[Ru]= Grubbs-Nolan's catalyst (3)
Scheme 2
NBoc2
NBoc2
N
NBoc2
R
NH2
N
NH2
N
R
R
R
R
b
a
d
c
N
N
N
H
N
O
O
P
N
O
O
P
N
O
O
N
H
O
POMO
POMO
POMO
POMO
4a R = H
b R = F
5a,b
6a,b
7a,b
8a,b

ACCEPTED MANUSCRIPT
Scheme 3
N
N
CONH2
O
pathway A
O
P
O
P
O
P
a
b
POMO
POMO
POMO
POMO
Br
POMO
POMO
POMO
1
9
14
O
P
pathway B
O
O
POMO
N
N
N
NBoc2
NH2
9'
c
O
N
H
O
O
O
N
N
11
10
R
d
O
P
POMO
POMO
12 R = NBoc2
13 R = OCH3
14 R = NH2
e
f
Scheme 4
O
O
O
b
a
N
N
N
NBoc2
NBoc2
17
NH2
15
N
N
N
N
N
N
N
H
H
16
Boc
O
N
NH2
c
N
18
Boc
O
O
N
N
N
NR2
NBoc2
N
N
O
d
N
P
POMO
POMO
19
20 R = Boc
21 R = H
e

ACCEPTED MANUSCRIPT
Scheme 5
O
O
P
O
P
a
b
P
POMO
POMO
POMO
Br
N3
POMO
POMO
POMO
22
9
1
N
N
c
O
P
N
POMO
POMO
23
24
CO2H
CO2H
N
N
N
O
P
d
POMO
POMO

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
The synthesis of new acyclonucleoside phosphonates is proposed.
Challenging olefin acyclic cross metathesis is the key synthetic step.
CuAAC reaction under microwave activation led to substituted-1,2,3-triazoles.
Compounds exhibited pronounced anti-HCV activity.
Compounds were also evaluated on a wide panel of other viral strains.

ACCEPTED MANUSCRIPT
Synthesis and broad spectrum antiviral evaluation of bis(POM) produgs of novel acyclic
nucleosides
Manabu Hamadaa, Vincent Roya, Tamara R. McBrayerb,Tony Whitakerb, Cesar Urbina-Blancoc, Steven P.
Nolanc, Jan Balzarinid, Robert Snoeckd, Graciela Andreid, Raymond F. Schinazie and Luigi A. Agrofoglioa*
a Institut de Chimie Organique et Analytique, UMR 7311 CNRS, Université d’Orléans, 45067 Orléans, France
b RFS Pharma, LLC, 1860 Montreal road,
c
EaStCHEM School of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
d Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
e
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory
University School of Medecine, Atlanta, GA, 30322, USA
Corresponding author: luigi.agrofoglio@univ-orleans.fr
Supplementary Information
Copies of 1H and 13C and 31P NMR spectra of some representative molecules.

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3) 6a
13C NMR (100 MHz, CDCl3) 6a

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3) 7a
13C NMR (100 MHz, CDCl3) 7a

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3) 12
13C NMR (100 MHz, CDCl3) 12

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CD3OD) 14
13C NMR (100 MHz, CD3OD) 14