Highly convergent synthesis and antiviral activity of (E)-but-2-enyl nucleoside phosphonoamidates

Article abstract of DOI:10.1016/j.ejmech.2018.01.086

Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs) were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These compounds were evaluated against a large spectrum of DNA and RNA viruses. Among them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against varicella-zoster virus (VZV) with EC₅₀ values of 0.33 and 0.39 μM for wild-type and thymidine kinase deficient strains, respectively, and a selectivity index ≥200 μM. This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate analogs.

Full text of DOI:10.1016/j.ejmech.2018.01.086

Accepted Manuscript
Highly convergent synthesis and antiviral activity of (E)-but-2-enyl nucleoside
phosphonoamidates
Maxime Bessières, Vincent Hervin, Vincent Roy, Agnès Chartier, Robert Snoeck,
Graciela Andrei, Jean-François Lohier, Luigi A. Agrofoglio
PII:
S0223-5234(18)30108-9
10.1016/j.ejmech.2018.01.086
EJMECH 10163
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 November 2017
Revised Date: 11 January 2018
Accepted Date: 26 January 2018
Please cite this article as: M. Bessières, V. Hervin, V. Roy, Agnè. Chartier, R. Snoeck, G. Andrei,
Jean.-Franç. Lohier, L.A. Agrofoglio, Highly convergent synthesis and antiviral activity of (E)-but-2-
enyl nucleoside phosphonoamidates, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.01.086.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Highly convergent synthesis and antiviral activity of (E)-but-2-enyl
nucleoside phosphonoamidates
a
a
a,
a
b
Maxime Bessières, Vincent Hervin, Vincent Roy, * Agnès Chartier, Robert Snoeck,
b
c
a,
Graciela Andrei, Jean-François Lohier, Luigi. A. Agrofoglio *
a
Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France
b
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
c
Univ. Caen, CNRS, LCMT, UMR 6507, F-14050 Caen, France
Corresponding Authors
Vincent Roy : Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France Phone :
33-2-3849-4572, E-mail : vincent.roy@univ-orleans.fr and Luigi A. Agrofoglio : Univ.
+
Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France, E-mail : luigi.agrofoglio@univ-
orleans.fr
Abstract
Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs)
were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound
irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These
compounds were evaluated against a large spectrum of DNA and RNA viruses. Among
them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against
varicella-zoster virus (VZV) with EC values of 0.33 and 0.39 µM for wild-type and thymidine
50
kinase deficient strains, respectively, and a selectivity index ≥200µM. This breakthrough
approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate
analogs.
Keywords: ultrasound, aqueous cross-metathesis, phosphonoamidate, acyclic nucleoside
phosphonates, DNA viruses
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;
ACN, acetonitrile; DCM, dichloromethane

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1. Introduction
Modified nucleosides represent a major class of therapeutics for cancer and viral
diseases.[1] Among them, acyclic nucleoside phosphonates (ANPs) pioneered with (S)-9-[3-
hydroxy-2-(phosphonomethoxy)propyl]adenine ((S)-HPMPA) [2] in 1986 by Antonín Holý and
Erik De Clercq, forms a key class of drugs active against various DNA viruses as well as
against retroviruses. However, those compounds suffer from limitations such as their
reduced cell penetration (the free phosphonic acid form is negatively charged at
physiological pH) as well as from nephrotoxicity. This has led to extensive search for new
ANPs as well as to the development of prodrug approaches [3,4] for enhanced bioavailability
and cell internalisation.[5-8] Several ANP prodrugs were marketed, such as adevofir
dipivoxyl (bis-POM PMEA) [9,10] for the treatment of hepatitis B virus (HBV), tenefovir
disoproxyl (bis-POC PMPA) [11] or the newly tenofovir alafenamide [12-14] for the treatment
of human immunodeficiency virus (HIV) and HBV.[15,16]
Over the last decade, our group has developed a new family of ANPs based on a
trans-but-2-enyl phosphonate scaffold.[17] Compounds were directly obtained as prodrugs
by a highly convergent and modular approach based on the powerful olefin acyclic cross
metathesis (CM) between various allylphosphonate synthons bearing biolabile groups and
N1- (or N9) crotyl (or allyl) pyrimidines or purines. This approach showed a remarkable
breakthrough for the synthesis of nucleoside prodrugs compared to the known linear
approaches, which suffer from low yields. It is also clear from the literature that the choice of
a prodrug has a direct impact on its targeting and cell release and greatly influences the
overall outcome and efficiency of the parent drug.[18-20] Thus, following this synthetic
pathway, we have obtained several prodrugs [21-25] including the most commonly used
carbonyloxymethyl
isopropyloxycarbonyloxymethyl- or POC), but also the alkoxyalkyl monoester
hexadecyloxypropyl or HDP, octadecyloxyethyl or ODE).[26] Several of these (E)-but-2-enyl
pronucleotides
(pivaloyloxymethyl-
or
POM,
(
ANPs prodrugs exhibited remarkable antiviral activity against DNA and RNA viruses in
submicromolar concentrations. The bis-(POM)-(E)-TbutP (1) and the prodrugs 2-4 were all
very active against several herpesviruses [i.e. herpes simplex virus 1 (HSV-1) and 2 (HSV-
2), and varicella-zoster virus (VZV)], representing a new potential antiviral lead.
Despite a significant amount of research and development on aryl phosphoramidate
prodrugs reported by McGuigan,[27,28] the development of aryl phosphonoamidates,
especially in the field of ANPs, has been very sparely investigated.[29] Thus, in this article,
we describe the in-water ultrasound promoted synthesis and antiviral evaluation of hitherto
unknown (E)-but-2-enyl nucleoside phosphonoamidates with high yields, (Figure 1).
[
Figure 1]
2
. Results and discussion
.1 Chemistry
Aryl phosphonoamidates are generally obtained by treatment of the parent
2
dimethylphosphonate nucleoside with TMSBr into the corresponding silyl esters, followed by
a subsequent treatment with an excess of phenol and L-alanine-O-alkyl ester in the presence
of triphenylphosphine and Aldrithiol. The desired products are often isolated with poor yields
(
< 3%) to traces as stated for the preparation of phosphonoamidate prodrug of tenofovir

ACCEPTED MANUSCRIPT
(
TAF) [30,31] In order to avoid these low yields, our strategy to the targeted phenyl
phosphonoamidate of (E)-but-2-enyl ANPs, was to react the allyl phenylphosphonoamidate
with a N1-crotylated nucleobase under acyclic cross metathesis (CM), which is quite
challenging taking into account the poisoning effect of nitrogens on CM ruthenium
catalysts.[32] Any attempt to obtain the desired allylic phosphonoamidates, by the shortest
strategy involving the introduction of allyl group on the phosphorodichloridate 5 to give the
desired monoalkenylated compound 6, failed. In our hands as, despite several conditions,
only the product of dialkylation was obtained, when observed (Schema 1).
[
Scheme 1.]
Therefore, we decided to turn our attention to the H-phosphonate chemistry. The
dimethylphosphite 7 was reacted with allyl bromide under Michaelis-Becker conditions to
give the dimethylallylphosphonate 8 with 78% yield. It is important to quote than this reaction
was scaled-up to 50 g. After substitution of a OMe group of 8 by a chlorine in presence of
oxalyl chloride, this position was then substituted by a phenolate generated in situ to give 9
(
as
a
mixture of enantiomers) in 65% yield. Compound
9
was treated by
bromotrimethylsilane in dichloromethane for 24h at room temperature, to give the
phosphonic acid monoester derivative 10 in excellent 91% yield (scheme 2).
[
Scheme 2.]
Following a procedure described by Gajda et al.,[33] phosphonate 10 was then
converted to various methyl-, isopropyl- and benzyl-L-alanine esters 11-15, in order to
compare the influence of the ester group or the phosphorus chirality, on the activity and
toxicity of final ANPs. Compounds 11,14 and 15 were obtained as a mixture of diastereomers
(from 6:4 to 1:1). Only the diastereomers of compound 11 (R = Bn) were separated by
careful column chromatography on silica gel (twice) and compounds 12 and 13 were isolated
31
as single isomers, respectively. P-NMR spectroscopy confirmed the isolation of each
products with the presence of single peak, while a mixture of isomers provide two peaks. The
sitting drop crystallization allows to obtain a crystal of both molecules and their structures
were unambiguously determined by X-ray to be P(R) for 12 and P(S) for 13, respectively,
(
Figure 2).
[
Figure 2.]
Next, the silylation of thymine was obtained in 5 minutes at room temperature in
presence of bistrimethylsilylacetamide BSA. The intermediate was directly engaged in
nucleophilic substitution reaction with crotyl bromide, chlorotrimethylsilane and sodium
iodide.[34] This reaction is performed under ultrasonic activation to afford after seven hours
the desired compound 16 in quantitative yield. 16 was then converted to the N3-Boc thymine
derivative 17 in quantitative yield, (Scheme 3).[35]
[
Scheme 3.]

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With all partners in hand (16, 17 and 11-15), we turned our attention to the olefin CM
nd
reaction using either the 2 generation Grubbs catalyst [36] (G-II), the more reactive
Hoveyda-Grubbs (HG-II) catalyst [37] or its derivative, the Zhan catalyst-1B, (Table 1).
[
Table 1.]
This specific CM reaction with a phosphonoamidate is challenging and needs
optimization since, as stated previously, it is well established in the literature that compounds
containing basic nitrogen atoms can poison the CM ruthenium catalysts and are thus
problematic substrates for olefin metathesis. It was shown that the presence of electron
withdrawing groups next to the nitrogen decreases the electron density and the deactivation
of the catalyst can be attenuated as well as the use of microwave irradiations.[38] It was
shown also that the presence of Lewis acid or Cu(I) salt can improved the yields of RCM of
amino acids.[39] The influence of ultrasonication was also tested. For this optimization, we
used the allylphosphonoamidate 11 and the crotylthymine 16 or 17, taken as model.
When N1 alkylated thymines 16 or 17 were reacted in DCM with diastereomeric
mixture of phosphonoamidate synthon 11 under classical heat activation (∆) (entries 1-3), no
desired compounds were found in all conditions tested (equivalents, substrate, Ru catalyst,
co-catalyst (Lewis acid, entry thymine derivatives). The use of microwaves irradiation (MW)
(
entries 4, 5) and sonication ()))) (entries 6 to 10) failed also. Only the use of water on
sonication led the expected phosphonoamidate ANP 18 in moderate 41% yield (entry
1).[40] The modulation of the conditions in a sealed tube and with an addition of a
1
surfactant (2.5% of polyoxyethanyl-α-tocopheryl sebacate) (entries 12 and 13) does not
improve significantly the yield of the reaction.[41]
Thanks to this breakthrough approach, some (E)-but-2-enyl C5-substituted thymidine
phosphonoamidates 19-22 were obtained in yields ranging from 38% to 44%, (scheme 4).
Based on our previous data [22], the 5-fluoro- (23) and 5-chloro- (24) analogs, which are
bioisosteres of methyl group, were obtained, in 35% and 36% yield, respectively.
[
Scheme 4.]
2.2 Biological evaluation
Among all the tested compounds, bearing different biolabile group (POM, POC, HDP,
phosphonoamidate), the diastereomeric single phosphonoamidate forms 19 and 20 were the
+
-
most potent and selective against both wild-type (TK ) and thymidine kinase deficient (TK )
varicella-zoster (VZV) strains with EC50 (50% effective concentration) values in the range of
0
.3-0.6 µM (Table 2). The cytostatic activity (CC ) decreased by a factor 2 when comparing
50
compound 19 with the bis-(POM)-(E)-TbutP (1) resulting in a selectivity index (SI, ratio CC50
to EC ) superior to 200. The selectivity of 20 against VZV was about half of that calculated
50
-
for 19. These prodrugs showed also activity against herpes simplex virus 1 (HSV-1), TK
HSV-1 and herpes simplex virus 2 (HSV-2) strains (EC in the range of 3-12 µM), which was
50
comparable to the EC ’s obtained for cidofovir (Table 3). The diastereomeric single
50
phosphonoamidate forms 19 and 20 had weak activity against human cytomegalovirus

ACCEPTED MANUSCRIPT
(
HCMV) or no activity at the higher concentration tested (100 µM) against vaccinia virus and
adenovirus.
The (E)-but-2-enyl C5-substituted pyrimidine phosphonoamidates 21 and 22 inhibited VZV
replication with EC ’s in the range of 1-8 µM and did not affect cell growth or morphology at
50
the highest tested concentration (100 µM). In contrast to 22, compound 21 had some anti-
HSV activity while both were able to reduce HCMV multiplication.
The introduction of these biolabile prodrugs revealed the potential of our ANPs to inhibit
HCMV replication (EC ’s in the range of 13-70 µM for compounds 19, 20, 21, 22), hitherto
50
undetected under other pronucleotide forms. Several hypotheses can support these results,
as a better bioavailability of these molecules under the phosphonoamidate form compared to
the other prodrug forms. The activity can also be increased by a better half-life and less toxic
side-products. However, the newly synthesized prodrugs did not showed activity against
vaccinia virus in contrast to bis-(POM)-(E)-TbutP (1).
The phosphonamidate ANPs 23 and 24 were not active against various viral strains; only the
chlorine analog 24 shown a moderate activity against human coronavirus (EC 8.9 µM).
50
The compounds were also evaluated against different RNA viruses, but no activity was
found.
[
Table 2.]
[
Table 3.]
3. Conclusion
We have described herein the synthesis of (E)-but-2-enyl nucleoside
phosphonoamidates using the cross-metathesis in water-under ultrasound irradiation. The
overall yield obtained from commercial dimethylallylphosphonate is > 15%, well above the
datas reported in the literature for the preparation of phosphonoamidates (~3%). Two
diastereoisomers were formally identified by X-ray diffraction. All those compounds were
evaluated against various DNA viruses for their antiviral properties. Among them, the
thymine analogue 19 showed to be the best prodrug tested against VZV with an EC = 0.33-
5
0
0.39 µM and a selectivity index increased up to ≥200, compared to its other prodrugs 1-4.
This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl
phosphonoamidates.
4. Experimental section
4.1 Chemistry
General.
Commercially available chemicals were of reagent grade and used as received. All reactions
requiring anhydrous conditions were carried out using oven-dried glassware and under an
atmosphere of dry Ar or N2. All reactions under microwave irradiation were performed using
the Microwave Biotage Initiator in 2-5mL.sealed tubes. The reactions under ultrasound were
carried out with Elmasonic P30H apparatus with a frequency of 80 kHz and effective power

ACCEPTED MANUSCRIPT
of 100 W. 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 60 M (0.040-0.063 mm, E. Merck). The 1H and 13C NMR spectra were recorded
on Bruker Avance DPX 250 or Bruker Avance 400 Spectrometers using deuterated solvents
as internal standard. Chemical shifts are given in ppm and multiplicities are reported as s
(
singlet), d (doublet), t (triplet), q (quartet), bs (broad signal) and m (multiplet). High
Resolution Mass spectra were performed on a Bruker Q-TOF MaXis mass spectrometer by
the “Fédération de Recherche” ICOA/CBM (FR2708) platform. LC-MS data was acquired on
a Thermo-Fisher UHPLC-MSQ system equipped with an electron spray ionization source
(
ESI). The temperature of the source was maintained at 350°C. Initially, the cone voltage
was set at 35 V and after 5 minutes was increased to 75V. In full scan mode, data was
acquired between 100 and 1000 m/z in the positive mode with a 1.00 S scan time. In addition
a UV detection was performed with a Diode array detector at three wavelengths 273, 254
and 290nm, respectively. A water/methanol (70%/30%) solution mixture with 0.1% formic
acid was used as mobile phase. The composition of the mobile phase was increased to
100% methanol with 0.1% formic acid with a 7% ramp. The flow rate was set at 0.300
mL.min-1. Samples diluted in the mobile phase were injected (3µL) on a C18 column (X-
terra, Waters), 2.1mm internal diameter, 100mm length placed into an oven at 40°C. The
conversion of compound 11 is equal to 67%. LC-MS analysis of allylphosphonoamidate 11
was optimized in terms of separation and identification. The reaction with crotylthymine was
followed by LC-MS in Electron Spray Ionization (ESI), in positive mode. After 20h, three
compounds were observed the allylphosphonoamidate 11, ((M+H)+,360), its homodimer
form ((M+H)+,690), and phosphonoamidate ANP 18 product ((M+H)+,498). Electronic
extraction of ions was performed and the subsequent areas under the corresponding
chromatographic peaks determined. The conversion yield was determined as the ratio of the
concentration of the allylphosphonoamidate 11 transformed to its initial concentration.
4.1.1. Dimethyl allylphosphonate (8). Under inert atmosphere, allyl bromide (51 mL, 1.25
eq.,0.57 mol) was dissolved in THF (400 mL). To this mixture potassium carbonate (94 g, 1.5
eq., 0.68mol), tert-butylammonium bromide (2.9 g, 2 mol%, 9.1 mmol) and finally
dimethylphosphite 7 (41.2 mL, 1 eq., 0.45 mol) were added. The resulting solution was
stirred for 36 hours at room temperature, followed by the filtration of all solids present in the
flask. The filtrate was then evaporated under reduced pressure, and the crude product was
then distilled at 130°C under 40 mm/Hg. After collection of the different fractions, the clean
1
product 8 was obtained as a colorless liquid. (53 g, 75%). H NMR (250 MHz, CDCl ) δ 5.80
3
(
m, 1H, CH=CH2), 5.23 (m, 2H, CH2=CH), 3.77 (s, 3H, OMe), 3.72 (s, 3H, OMe), 2.62 (ddt, J
=
22.0, 7.4, 1.3 Hz, 2H, CH2-P). CAS # 757-54-0
4.1.2. Methoxyphenoxy allylphosphonate (9). To a mixture of dimethylallylphosphonate 8 (4.9
g, 1 eq., 32.5 mmol) and DCM (150 mL), oxalyl chloride (8.6 mL, 3 eq., 97.5 mmol) was
added. The reaction was stirred 24h at reflux, followed by the removal of the volatiles in
vacuo to obtain the methyl allylphosphonochloridate. In another flask, a solution of phenol
(
6.12 g, 2 eq., 65 mmol), triethylamine (8.8 mL, 2 eq., 65 mmol) and DCM (0.2 M) was stirred
at room temperature. The phosphonate residue was then dissolved in DCM (0.2 M), and
slowly added to this solution, and refluxed during 48h. After evaporation of all volatiles, the
residue was purified by silica gel column chromatography, eluting Petroleum ether/Ethyl
1
acetate 8/2, to afford 9 as a colorless oil. (4.5 g, 65%). H NMR (400 MHz, CDCl ) δ 7.29 (m,
3
2H, Aromatic H), 7.15 (m, 3H, Aromatic H), 5.80 (m, 1H, CH=CH2), 5.24 (m, 2H, CH2=CH),

ACCEPTED MANUSCRIPT
13
3
.76 (d, J = 11.1 Hz, 3H, OMe), 2.73 (dd, J = 22.0, 7.3 Hz, 2H, CH2-P). C NMR (101 MHz,
CDCl ) δ 150.46 (d, J = 8.5 Hz), 129.72 (Aromatic C), 126.59 (d, J = 11.6 Hz, CH=CH2),
3
1
24.95 (d, J = 1.3 Hz, Aromatic C), 120.67 (d, J = 14.8 Hz, CH2=CH), 120.42 (d, J = 4.4 Hz,
31
Aromatic C), 53.16 (d, J = 5.1 Hz, OMe), 31.21 (d, J = 139.9 Hz, CH2-P). P NMR (162
MHz, CDCl ) δ 25.06. HRMS (ESI) m/z [M+H]+ calcd for C10H14O3P : 213.0681, found :
3
213.0675.
4.1.3. Phenyloxy allylphosphonic acid (10). Bromotrimethylsilane (8.9 mL, 6 eq.,61.8 mmol)
was slowly added to a solution of 9 (2.2 g, 1 eq., 10.3 mmol) in DCM (110 mL). After 24h at
room temperature and evaporation of all volatiles, the crude product was co-evaporated 5
times with methanol (5 x 15 mL). The residue was then purified by flash column
chromatography (DCM / MeOH 95/5) to obtain desired product 10 as an amorphous white
1
solid. (1.85 g, 91%). H NMR (400 MHz, CDCl ) δ 8.12 (s, 1H, OH), 7.28 (m, 2H, Aromatic
3
H), 7.14 (m, 3H, Aromatic H), 5.79 (m, 1H, CH=CH2), 5.22 (m, 2H, CH2=CH), 2.67 (dd, J =
13
2
2.5, 7.3 Hz, 2H, CH2-P). C NMR (101 MHz, CDCl ) δ 150.18 (d, J = 8.9 Hz), 129.61
3
(
Aromatic C), 126.65 (d, J = 11.4 Hz, CH=CH2), 124.92 (Aromatic C), 120.79 (d, J = 4.4 Hz,
31
Aromatic C), 120.66 (d, J = 14.9 Hz,CH2=CH), 31.55 (d, J = 141.5 Hz, CH2-P). P NMR
162 MHz, CDCl ) δ 26.26. HRMS (ESI) m/z [M+H]+ calcd for C9H12O3P : 199.0525, found :
(
3
199.0518.
4.2. General procedure for the synthesis of allylphosphonoamidates 11-15
Under inert atmosphere,phenyloxy allylphosphonic acid (10) (1 eq.) was dissolved in DCM. A
catalytical amount of DMF (0.3 eq.) was then introduced, followed by the addition of oxalyl
chloride (2 eq.). After 30 min. at 0°C and 1.5h at room temperature, the volatiles were
removed, and the residue diluted with 20 mL of DCM to afford solution A. A second solution
was prepared with a L-alanine ester chlorhydrate (1.2 eq.), freshly distilled triethylamine (8
eq.) and DCM. To this mixture, the solution A was slowly added at 0°C, and stirred 24 h at
room temperature. For 1 g. of starting phosphonate, the solution was successively washed
with 10 mL of water, 10mL NaOH 1M, 10 mL water, 10 mL HCl 10%, 10 mL NaHCO and 10
3
mL water. After this work-up, the aqueous phase was extracted with 4 x 10 mL with ethyl
acetate, the organic phases were washed with brine (20 mL), dried over MgSO4, filtered and
evaporated. The residue was purified twice by flash column chromatography to afford the
desired allylphosphonoamidates 11, 14 and 15 as diastereomeric mixture. Diastereomers of
compound 11 were separated as single diastereomers 12 and 13, respectively.
4.2.1. Benzyl 2-[(S)-[allyl(phenoxy)phosphoryl]amino] propanoate (11). Titled compound was
obtained following general procedure starting from compound 10 (1 g, 1 eq., 5.05 mmol). The
residue was purified twice by flash column chromatography, first eluting PE/EA (7/3) and
then pentane/diethyl ether (55/45); the 6:4 diastereomeric mixture of 11 was obtained as a
white powder (1.14 g, 63%).
Each diastereoisomers were crystallized from a
toluene/pentane mixture, as colorless needles, respectively, and their structures were
established by X-ray.

ACCEPTED MANUSCRIPT
Diastereoisomer 1: Benzyl 2-[(S)-[(R)-allyl(phenoxy)phosphoryl]amino] propanoate
12): H NMR (400 MHz, (CD ) CO) δ 7.37 (m, 7H, Aromatic H), 7.24 (m, 2H, Aromatic H),
3 2
1
(
7
4
.14 (t, J = 7.4 Hz, 1H, Aromatic H), 5.89 (m, 1H, CH=CH2), 5.18 (m, 4H, CH2=CH, CH2-O),
.60 (t, J = 11.2 Hz, 1H, NH), 4.16 (m, 1H, CH-NH), 2.76 (ddd, J = 20.7, 7.1, 2.5 Hz, 2H,
13
CH2-P), 1.22 (d, J = 7.2 Hz, 3H, CH3). C NMR (101 MHz, (CD ) CO) δ 174.42 (d, J = 4.8
3
2
Hz, C=O), 151.94 (d, J = 9.1 Hz), 137.12, 130.19 (Aromatic C), 129.46 (d, J = 11.0 Hz,
CH=CH2), 129.28 (Aromatic C), 128.92 (Aromatic C), 128.88 (Aromatic C), 125.09 (d, J =
1
.1 Hz, Aromatic C), 121.82 (d, J = 4.6 Hz, Aromatic C), 119.86 (d, J = 14.3 Hz, CH2=CH),
7.09 (CH2-O), 50.47 (CH-N), 35.03 (d, J = 128.7 Hz, CH2-P), 20.88 (d, J = 4.8 Hz, CH3).
P NMR (162 MHz, (CD ) CO) δ 26.70. HRMS (ESI) : m/z [M+H]+ calcd for C19H23O4NP :
6
31
3
2
360.1365, found : 360.1355.
Diastereoisomer 2 : Benzyl 2-[(S)-[(S)-allyl(phenoxy)phosphoryl]amino] propanoate
1
(
13): H NMR (400 MHz, CDCl ) δ 7.19 (m, 10H, Aromatic H), 5.79 (m, 1H, CH=CH2), 5.17
3
(
2
m, 2H, CH2=CH), 5.04 (s, 2H, CH2-O), 4.06 (m, 1H, CH-NH), 3.49 (t, J = 10.6 Hz, 1H, NH),
.69 (dd, J = 21.5, 7.4 Hz, 2H, CH2-P), 1.27 (d, J = 7.1 Hz, 3H, CH3). C NMR (101 MHz,
13
CDCl ) δ 173.56 (d, J = 5.3 Hz, C=O), 150.52 (d, J = 9.0 Hz), 135.25, 129.65 (Aromatic C),
3
128.62 (Aromatic C), 128.48 (Aromatic C), 128.21 (Aromatic C), 127.45 (d, J = 11.2 Hz,
CH=CH2), 124.67 (Aromatic C), 120.60 (d, J = 14.3 Hz, CH2=CH), 120.56 (d, J = 4.7 Hz,
Aromatic C), 67.12 (CH2-O), 49.61 (CH-NH), 34.25 (d, J = 128.8 Hz, CH2-P), 21.57 (d, J =
31
4
.3 Hz, CH3). P NMR (162 MHz, CDCl ) δ 26.47. HRMS (ESI) : m/z [M+H]+ calcd for
3
C19H23O4NP : 360.1365, found : 360.1354.
4.2.2. Methyl 2-[(S)-[allyl(phenoxy)phosphoryl]amino] propanoate (14). Titled compound was
obtained following general procedure 1, starting from compound 10 (1.3 g, 1 eq., 6.56 mmol).
The obtained residue was purified twice by flash column chromatography, eluting PE/EA
(
(
55:45 to 5:5) to afford a non-separable 6:4 mixture of diastereoisomers 14 as a colorless oil,
1
960 mg, 52%). H NMR (400 MHz, CDCl ) δ 7.30 (m, 2H, Aromatic H), 7.18 (m, 3H,
3
Aromatic H), 5.87 (m, 1H, CH=CH2), 5.27 (m, 2H, CH2=CH), 4.08 (m, 1H, CH-NH), 3.66 (d,
J = 10.6 Hz, 3H, OMe), 3.32 (2 x t, J = 10.1 Hz, 1H, NH), 2.77 (m, 2H, CH2-P), 1.28 (2 x d, J
13
=
7.2 Hz, 3H, CH3-CH). C NMR (101 MHz, CDCl ) δ 174.53 (d, J = 6.1 Hz, C=O), 174.20 (d,
3
J = 5.3 Hz, C=O), 150.53 (d, J = 9.5 Hz), 150.45 (d, J = 9.9 Hz), 129.65 (Aromatic C), 129.60
(
(
Aromatic C), 127.68 (d, J = 11.4 Hz, CH=CH2), 127.52 (d, J = 11.2 Hz, CH=CH2), 124.72
d, J = 1.2 Hz, Aromatic C), 124.68 (d, J = 1.1 Hz, Aromatic C), 120.70 (d, J = 4.8 Hz,
Aromatic C), 120.60 (d, J = 14.4 Hz, CH2=CH), 120.57 (d, J = 4.8 Hz, Aromatic C), 52.35
(
1
CH3-O), 49.62 (CH-NH), 49.45 (CH-NH), 34.32 (d, J = 128.3 Hz, CH2-P), 34.30 (d, J =
28.3 Hz, CH2-P), 21.60 (d, J = 4.3 Hz, CH3), 21.46 (d, J = 3.8 Hz, CH3). P NMR (162
31
MHz, CDCl ) δ26.78, 26.34.HRMS (ESI) m/z [M+H]+ calcd for C13H19NO4P : 284.1052,
3
found : 284.1045.
4.2.3. Isopropyl 2-[(S)-[allyl(phenoxy)phosphoryl]amino] propanoate (15). Titled compound
was obtained following general procedure 1, starting from compound 10 (1.3 g, 1 eq., 6.56
mmol). The obtained residue was purified by flash column chromatography, eluting PE/EA
(
(
65:35) to afford a non-separable 6:4 mixture of two diastereoisomers 15 as a colorless oil.
1
1.16 g, 57%). H NMR (400 MHz, CDCl ) δ 7.32 (m, 2H, Aromatic H), 7.19 (m, 3H, Aromatic
3

ACCEPTED MANUSCRIPT
H), 5.92 (m, 1H, CH=CH2), 5.28 (m, 2H, CH2=CH), 4.99 (pd, J = 6.3, 4.9 Hz, 1H, CH-iPr),
4
.04 (m, 1H, CH-NH), 3.38 (2 x t, J = 10.1 Hz, 1H, NH), 2.78 (m, 2H, CH2-P), 1.26 (m, 9H,
13
CH3-CH-NH, CH3 iPr). C NMR (101 MHz, CDCl ) δ 173.24 (d, J = 5.7 Hz, C=O), 150.58 (d,
3
J = 9.5 Hz), 150.45 (d, J = 9.9 Hz), 129.65 (Aromatic C), 129.59 (Aromatic C), 127.71 (d, J =
11.4 Hz, C2'), 127.53 (d, J = 11.4 Hz, C2'), 124.69 (Aromatic C), 124.65 (Aromatic C), 120.75
(
C3'/Aromatic C), 120.70 (C3'/Aromatic C), 120.68 (C3'/Aromatic C), 120.64 (C3'/Aromatic
C), 120.59 (C3'/Aromatic C), 120.55 (C3'/Aromatic C), 120.50 (C3'/Aromatic C), 69.09 (CH
iPr), 69.07 (CH iPr), 49.77 (CH-NH), 49.62 (CH-NH), 34.30 (d, J = 129.3 Hz, CH2-P), 21.71
31
(
CH3), 21.70 (CH3), 21.67 (CH3), 21.62 (CH3), 21.60 (CH3), 21.57 (CH3). P NMR (162
MHz, CDCl ) δ 26.77, 26.35. HRMS (ESI) m/z [M+H]+ calcd for C15H23NO4P : 312.1365,
3
found : 312.1359.
4.3. General procedure for in water-ultrasound-promoted convergent synthesis of (E)-
but-2-enyl nucleoside phosphonoamidates
To a 2.5% Polyoxyethanyl-α-tocopheryl Sebacate (PTS) solution of water (0.5 M) of
phosphonoamidate (1 eq.) and crotylnucleobase (2 eq.), second generation Grubbs catalyst
(
(
6 mol%) was added. The reaction was irradiated under ultrasound activation (55°C, 80 KHz)
two additional portions of 6 mol% Grubbs catalyst were respectively added at 2h and 4h).
The progress of the reaction was followed by LC/MS and reaction was stopped after 20
hours of ultrasouns activation. A maximum of 67 % of phosphonoamidate conversion was
observed. Volatiles were removed under reduced pressure, and the residue was submitted
twice to flash chromatography purification, firstly DCM/MeOH 95/5 followed by a second one
eluting toluene/acetone 6/4.
4.3.1.
Benzyl
2-[(S)-[(R)-[(E)-4-(thymin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino]
propanoate (19). Titled compound was obtained following general procedure, starting from
single diastereomer phosphonoamidate 12 (68 mg, 1 eq., 0.19 mmol) and crotylthymine (68
mg, 0.38 mmol, 2 eq.). The obtained residue was purified by twice flash chromatography
1
(
DCM/MeOH 95/5 then toluene/acetone 6/4), to afford 19 as a white solid (36 mg, 38%). H
NMR (400 MHz, CDCl ) δ 8.35 (s, 1H, NH), 7.32 (m, 7H, Aromatic H), 7.15 (m, 3H, Aromatic
3
H), 6.97 (d, J = 1.3 Hz, 1H, H6), 5.78 (dt, J = 13.7, 7.3 Hz, 1H, H3'), 5.67 (dt, J = 15.5, 4.8
Hz, 1H, H2'), 5.10 (d, AB system, J = 13.0 Hz, 2H, CH2-O), 4.27 (t, J = 4.8 Hz, 2H, H1'), 4.15
(
2
dq, J = 9.9, 7.3 Hz, 1H, CH-NH), 3.38 (d, J = 10.9 Hz, 1H, NH), 2.76 (dt, J = 21.0, 7.3 Hz,
H, CH2-P), 1.89 (d, J = 1.3 Hz, 3H, CH3-C), 1.25 (s, 3H, CH3-CH). C NMR (101 MHz,
13
CDCl ) δ 173.86 (d, J = 5.9 Hz, C=O), 163.77 (C=O), 150.59 (C=O), 150.24 (d, J = 9.1 Hz),
3
1
1
1
2
39.73 (C6), 135.28, 129.68 (Aromatic C), 129.07 (d, J = 14.0 Hz, C2'), 128.64 (Aromatic C),
28.49 (Aromatic C), 128.18 (Aromatic C), 125.32 (d, J = 11.5 Hz, C3'), 124.90 (Aromatic C),
20.4 (d, J = 4.6 Hz, Aromatic C), 110.97 (C5), 67.19 (CH2-O), 49.75 (CH-NH), 49.53 (d, J =
.2 Hz, C1'), 32.88 (d, J = 128.9 Hz, CH2-P), 21.31 (d, J = 4.0 Hz, CH3-CH), 12.27 (CH3-C).
3
1
P NMR (162 MHz, CDCl ) δ 26.00. HRMS (ESI) m/z [M+H]+ calcd for C25H29N3O6P
3
:
498.1794, found : 498.1786.
4.3.2.
Benzyl
2-[(S)-[(S)-[(E)-4-(thymin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino]
propanoate (20). Titled compound was obtained following general procedure, starting from
phosphonoamidate 13 (125 mg, 1 eq., 0.35 mmol) and crotylthymine (125 mg, 2 eq., 0.70

ACCEPTED MANUSCRIPT
mmol). The obtained residue was purified by twice flash chromatography (DCM/MeOH 95/5
1
and toluene/acetone 6/4), to afford 20 as a white solid (70 mg, 41%). H NMR (400 MHz,
CDCl ) δ 8.79 (bs, 1H, NH), 7.27 (m, 7H, Aromatic H), 7.12 (m, 3H, Aromatic H), 6.94 (d, J =
3
1
=
1
.1 Hz, 1H), 5.67 (m, 2H, H2', H3'), 5.07 (s, 2H), 4.23 (t, J = 4.9 Hz, 2H, CH2-O), 4.03 (dq, J
9.6, 7.6 Hz, 1H, CH-NH), 3.38 (d, J = 10.3 Hz, 1H, NH), 2.70 (dt, J = 20.3, 5.7 Hz, 2H),
13
.84 (d, J = 1.1 Hz, 3H), 1.27 (s, 3H). C NMR (101 MHz, CDCl ) δ 173.46 (d, J = 5.3 Hz,
3
C=O), 163.95 (C=O), 150.68 (C=O), 150.40 (d, J = 9.1 Hz), 139.57 (C6), 135.19, 129.74
(
(
Aromatic C), 129.17 (d, J = 14.4 Hz, C2'), 128.66 (Aromatic C), 128.56 (Aromatic C), 128.25
Aromatic C), 125.08 (d, J = 11.1 Hz, C3'), 124.84 (Aromatic C), 120.37 (d, J = 4.9 Hz,
Aromatic C), 111.00 (C5), 67.25 (CH2-O), 49.67 (CH-NH), 49.17 (d, J = 2.2 Hz, C1'), 32.73
31
(
d, J = 128.2 Hz), 21.46 (d, J = 4.3 Hz, CH3-CH), 12.27 (CH3-C) P NMR (162 MHz, CDCl )
3
δ 25.60. HRMS (ESI) m/z [M+H]+ calcd for C25H29N3O6P : 498.1794, found : 498.1789.
4.3.3. Methyl 2-[(S)-[[(E)-4-(thymin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino] propanoate
(
21). Titled compound was obtained following general procedure, starting from
phosphonoamidate 14 (50 mg, 1 eq., 0.18 mmol) and crotylthymine (64 mg, 2 eq., 0.35
mmol). The obtained residue was purified by twice flash chromatography (DCM/MeOH 95/5
1
and toluene/acetone 6/4), to afford 21 as a white solid (30 mg, 41%). H NMR (400 MHz,
CDCl ) δ 8.06 (bs, 1H, NH), 7.27 (m, 2H, Aromatic H), 7.15 (m, 3H, Aromatic H), 6.97 (bs,
3
1
3
H, H6), 5.73 (m, 2H, CH=CH), 4.28 (t, J = 4.9 Hz, 2H, CH2-N), 4.00 (m, 1H, CH-NH), 3.72 –
.59 (m, 3H), 3.42 (m, 1H, NH), 2.74 (dd, J = 20.9, 6.8 Hz, 2H, CH2-P), 1.88 (m, 3H, CH3
31
thym), 1.26 (m, 3H, CH3-CH). P NMR (162 MHz, CDCl ) δ 26.01, 25.51. HRMS (ESI) : m/z
3
[
M+H]+ calcd for C19H25N3O6P : 422.1481 found : 422.1480.
4.3.4. Isopropyl 2-[(S)-[[(E)-4-(thymin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino] propanoate
(
22). Titled compound was obtained following general procedure, starting from
phosphonoamidate 15 (50 mg, 1 eq., 0.16 mmol) and crotylthymine (58 mg, 2 eq., 0.32
mmol). The obtained residue was purified by twice flash chromatography (DCM/MeOH 95/5
and toluene/acetone 6/4), to afford 22 as a white solid (32 mg, 44%). 1H NMR (400 MHz,
CDCl ) δ 8.38 (bs, 1H, NH), 7.30 (m, 2H, Aromatic H), 7.16 (m, 3H, Aromatic H), 7.00 (s, 1H,
3
H6), 5.75 (m, 2H, CH=CH), 4.96 (d sept., J = 6.3, 2.1 Hz, 1H, CH-iPr), 4.31 (t, J = 4.7 Hz, 1H,
H1'), 3.99 (m, 1H, CH-NH), 3.45 (2 x t, J = 10.8 Hz, 1H, NH), 2.77 (m, 2H, CH2-P), 1.88 (s,
13
3
H, CH3-C) 1.26 (m, 9H, CH3-CH-NH, CH3 iPr). C NMR (101 MHz, CDCl ) δ 173.17
3
(
1
C=O), 163.76 (C=O), 150.60 (C=O), 150.54, 139.69, 139.59 (C6), 129.74 (Aromatic C),
29.67 (Aromatic C), 129.12 (d, J = 14.0 Hz, C2'), 125.20 (d, J = 11.1 Hz, C3'), 124.85
(
(
Aromatic C), 120.65 (Aromatic C), 120.61 (Aromatic C), 120.44 (Aromatic C), 120.40
Aromatic C), 111.03 (CH-iPr), 69.09 (CH iPr), 69.07 (CH iPr), 49.77 (CH-NH), 49.64 (CH-
NH), 49.19 (CH2-O), 32.70 (d, J = 129.4 Hz, CH2-P), 21.68 (CH3), 21.60 (CH3), 21.58
(
(
CH3), 21.54 (CH3), 21.57 (CH3-C). 31P NMR (162 MHz, CDCl3) δ 26.02, 25.61. HRMS
ESI) m/z [M+H]+ calcd for C21H29N3O6P : 450.1795, found : 450.1789.
4.3.5.
Benzyl
2-[(S)-[(E)-4-(5-fluorouridin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino]
propanoate (23). Titled compound was obtained following general procedure, starting from
phosphonoamidate 11 (64 mg, 1 eq., 0.18 mmol) and N1-crotyl-5-fluorouracile (66 mg, 2 eq.,
0.36 mmol). The obtained residue was purified by twice flash chromatography (DCM/MeOH

ACCEPTED MANUSCRIPT
1
9
5/5 and toluene/acetone 6/4), to afford 23 as a white solid (32 mg, 36%). H NMR (400
MHz, CDCl ) δ 9.26 (bs, 1H, NH), 7.34 (m, 7H, Aromatic H), 7.17 (m, 3H, Aromatic H),5.75
3
(
9
m, 2H, CH=CH), 5.13 (s, 1H, CH2 benzylic), 5.09 (t, J = 5.0 Hz, 1H, H1'), 3.99 (2 x dq, J =
.3, 6.9 Hz, 1H, CH-NH), 3.72 (t, J = 10.8 Hz, 1H, major NH-P), 3.45 (t, J = 10.8 Hz, 1H,
13
minor NH-P), 2.77 (m, 2H, CH2-P), 1.29 (s, 3H, CH3-CH). C NMR (101 MHz, CDCl ) δ
3
1
1
73.49 (d, J = 5.3 Hz, C=O), 159.14 (C=O), 150.38 (C=O), 150.27 (d, J = 9.1 Hz, minor C),
49.75 (d, J = 9.1 Hz, major C), 140.57, 135.21 (C6), 129.79 (Aromatic C), 129.72 (Aromatic
C), 128.68 (Aromatic C), 128.62 (d, J = 4.7 Hz, Aromatic C), 128.49 (Aromatic C), 128.30 (d,
J = 13.9 Hz, C3'), 128.28 (Aromatic C), 128.18 (Aromatic C), 126.50 (d, J = 10.1 Hz, minor
C2'), 126.28 (d, J = 10.1 Hz, major C2'), 124.93 (Aromatic C), 120.66 (d, J = 4.6 Hz, minor
Aromatic C), 120.38 (d, J = 4.6 Hz, major Aromatic C), 67.30 (CH2-O), 49.73 (CH-NH), 49.19
(
CH2-O), 32.51 (d, J = 128.8 Hz, CH2-P), 21.36 (d, J = 4.6 Hz, major CH3), 21.28 (d, J = 4.6
Hz, minor CH3). 31P NMR (162 MHz, CDCl3) δ 25.95, 25.54. HRMS (ESI) m/z [M+H]+ calcd
for C24H26FN3O6P : 502.1544, found : 502.1537.
4.3.6.
Benzyl
2-[(S)-[[(E)-4-(5-chlorouridin-1-yl)but-2-enyl]-phenoxyphosphoryl]amino]
propanoate (24). Titled compound was obtained following general procedure 2, starting from
phosphonoamidate 13* (50 mg, 1 eq., 0.14 mmol) and N1-crotyl-5-chlorouracile (56 mg, 2
eq., 0.28 mmol). The obtained residue was purified by twice flash chromatography
(
DCM/MeOH 95/5 and toluene/acetone 6/4), to afford compound 24* as a white solid (33 mg,
35%). 1H NMR (400 MHz, CDCl3) δ 9.26 (bs, 1H, NH), 7.33 (m, 7H, Aromatic H), 7.18 (m,
3H, Aromatic H), 5.74 (m, 2H, CH=CH), 5.13 (s, 1H, CH2 benzylic), 5.09 (s, 1H, H1'), 4.07 (2
x dq, J = 8.8, 7.0 Hz, 1H, CH-NH), 3.68 (t, J = 10.8 Hz, 1H, major NH-P), 2.77 (m, 2H, CH2-
P), 1.29 (s, 3H, CH3-CH). 13C NMR (101 MHz, CDCl3) δ 173.47 (d, J = 5.2 Hz, C=O),
1
1
59.14 (C=O), 150.34 (d, J = 9.1 Hz), 149.12 (C=O), 141.69, 139.33, 135.28, 135.21 (C6),
29.79 (Aromatic C), 128.68 (Aromatic C), 128.60 (Aromatic C), 128.49 (Aromatic C), 128.28
(
(
d, J = 20.6 Hz, C3'), 128.28 (Aromatic C), 128.17 (Aromatic C), 127.62 (Aromatic C),126.23
d, J = 11.0 Hz, C2'), 124.94 (Aromatic C),120.35 (d, J = 4.8 Hz, Aromatic C), 67.31 (CH2-O),
49.73 (CH-NH), 49.71 (CH2-O), 32.48 (d, J = 128.9 Hz, CH2-P), 21.35 (d, J = 4.6 Hz, CH3).
31P NMR (162 MHz, CDCl3) δ 25.91, 25.53. HRMS (ESI) m/z [M+H]+ calcd for
C24H26ClN3O6P : 518.1248,found : 518.1242.
4.4. Antiviral activity assays
The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-
induced cytopathicity or plaque formation in HEL [herpes simplex virus 1 (HSV-1) (KOS),
HSV-2 (G), vaccinia virus, vesicular stomatitis virus, human cytomegalovirus (HCMV),
varicella-zoster virus (VZV) and Human Coronavirus (229E)], 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 CCID of virus (1 CCID being the virus dose to infect
50
50
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 cytopathic effect (CPE)
or plaque formation (VZV) 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 EC or compound concentration required reducing virus-induced CPE
50

ACCEPTED MANUSCRIPT
or viral plaque (VZV) plaque 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
3
rate of 5 x 10 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 with a Coulter counter. The
cytostatic concentration was calculated as the CC , or the compound concentration required
50
to reduce cell proliferation by 50% relative to the number of cells in the untreated controls.
The antiviral activity of the compounds was evaluated against HIV-1 in activated primary
human PBM cells.[42] Cytotoxicity was evaluated in normal PBM cells, along with CEM and
Vero cells.[43]
Acknowlegment
MB is grateful to MESR for a PhD scholarship. We thank the LABEX SynOrg (ANR-11-
LABX-0029) for VH PhD fellowship and partial financial support.
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DRAWING CAPTION
Figure 2. (E)-but-2-enyl ANPs prodrugs developed by Agrofoglio et al. and targeted
phosphonoamidates
Scheme 1. Reagents and conditions : (a) allylmagnesium bromide, diethyl ether or THF, -
78°C to RT (failed)
Scheme 2. Reagents and conditions : (a) Allyl bromide, K CO , TBAB, THF, 90°C, 12h,
2
3
7
8%, (b) 1) (COCl) , DCM, 50°C, 24h, 2) Et N, Phenol, DCM, 50°C, 48h, 65%, (c) TMSBr,
2
3
DCM, RT, 24h, 91%, (d) 1) (COCl) , DMF cat., DCM, RT, 1.5h, 2) Et N, DCM, L-alanine
2
3

ACCEPTED MANUSCRIPT
benzyl ester hydrochloride, RT, 24h, 63% (for 11, 12 and 13). 2) Et N, DCM, L-alanine
3
methyl ester hydrochloride, RT, 24h, 52% (for 14). 2) Et N, DCM, L-alanine isopropyl ester
3
hydrochloride, RT, 24h, 57% (for 15).
Figure 2. ORTEP crystal structure for allyl phosphonoamidates 12 (P ) and 13 (P )
(R)
(S)
Scheme 3. Reagents and conditions : a) BSA, ACN, RT, 5 min., b) crotyl bromide, TMSCl,
NaI, ))), 55°C, 7h, > 98% c) Boc O, DMAP, THF, MW, 70°C, 10 min, > 98%.
2
Table 3. Cross-metathesis optimization
Scheme 4. Reagents and conditions : (a) N1-crotylated thymine, G-II catalyst (3 x 6 mol%),
H O (2.5% PTS), ))), 55°C, 20h or N1-crotylated 5-fluorouracile, G-II catalyst (3 x 6 mol%),
2
H O (2.5% PTS), ))), 55°C, 20h or, or N1-crotylated 5-bromouracile G-II catalyst (3 x 6
2
mol%), H O (2.5% PTS), ))), 55°C, 20h
2
Table 4. Antiviral properties against HCMV and VZV
Table 3. Antiviral properties against HSV, vaccinia virus and adenovirus

ACCEPTED MANUSCRIPT
Equivalents
Entry
Solvent
(of 11 and
Catalyst
Activation
R1
Yield
nucleobase)
1
2
3
4
5
6
7
8
9
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
1 - 2
1.3 - 1
1 - 1
HG-II
HG-II
∆, 50°C, 24h
∆, 50°C, 24h
∆, 50°C, 24h
MW, 100°C, 1h
MW, 100°C, 1h
))), 55°C, 20h
))), 55°C, 20h
))), 55°C, 20h
))), 55°C, 20h
))), 55°C, 20h
))), 55°C, 20h
))), 55°C, 20h
H
H
/
/
HG-II, Cy BCl
Boc
H
/
2
1.3 - 1
1.3 - 1
1.3 - 1
1.3 - 1
1.3 - 1
1.3 - 1
1 - 2
G-II
/
G-II, CuI
HG-II
H
/
H
/
G-II, CuI
H
/
G-II, BCl .SMe
H
/
3
2
HG-II
Boc
Boc
H
/
10
11
12
Zhan 1B
/
c
H
O
2
1 - 2
G-II
41%
40%
35%
a
c
H
O
O
1 - 2
G-II
H
2
b
a
c
13
H
2
1 - 2
G-II
))), 55°C, 20h
H
a
b
c
2
.5% of Polyoxyethanyl-α-tocopheryl Sebacate PTS; Sealed tube; Catalyst introduced
in 3 x 6 mol%
Table 1.

ACCEPTED MANUSCRIPT
HCMV
VZV
Cytotoxicity (µM)
a
+
-
Compounds
EC50 (µM)
TK (OKA)
TK (07/1)
b
c
d
(
AD-169)
(Davis)
EC (µM)
SI
EC (µM)
SI
MCC
CC50
5
0
50
1
9
0
29.91
34.2
44.72
52.53
0.39 ± 0.21
0.59 ± 0.08
≥213
0.33 ± 0.05
0.39 ±0.14
≥252
> 100
> 100
≥83 ± 24
2
93
141
55 ± 3
21
22
23
24
≥72 ± 39
25.9 ± 8.3
>20
≥70 ± 42
13.5 ± 2.5
>20
8.13 ±1.15
4.59 ± 3.18
>100
12
22
-
1.82 ± 0.33
1.03 ± 0.59
>100
55
97
-
>100
>100
≥ 100
≥ 100
>100
>100
ND
>20
>20
44.72
-
20
-
ND
1
2
(R1=Me)
(R1=Me)
>41
102
>41
83
1.91 ± 1.32
1.26 ± 0.11
19
29
0.43 ± 0.21
0.45 ± 0.32
85
80
≥100
36.4 ± 2.3
36
>200
0
3 (R1=Me)
4 (R1=Me)
>6
>6
>6
-
-
>6
-
-
30
14.7
76
0
>39
13.5
19.4
20.5
184
e
Acyclovir
Brivudine
ND
ND
2.93 ± 1.25
>150
54.4 ± 27.8
>8
>440
>440
ND
ND
0.014 ± 0.012
>21,429
≥ 93.8 ± 58.0
>3
-
>300
≥350
>300
>300
>350
>300
Ganciclovir
Cidofovir
5.63 ± 3.84
0.77 ±0.41
4.05 ± 1.35
0.90 ±0.33
ND
ND
-
-
ND
ND
-
a
b
c
d
e
Effective concentration required to reduce virus plaque formation (VZV) or viral cytopathic effect (HCMV) by 50%.
Selectivity index: ratio CC50 to EC50
Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
Cytostatic concentration required to reduce cell growth by 50%.
Not done.
Table 2.

ACCEPTED MANUSCRIPT
a
EC50 (µM)
Cytotoxicity (µM)
HSV-1
Human
Coronavirus
(229E)
Compounds
HSV-2
(G)
Vaccinia
virus
-
(
TK
b
c
(
KOS)
r
MCC
>100
>100
>100
>100
>100
>100
≥100
>200
>152
184
CC50
KOS ACV )
1
2
2
2
2
2
9
0
1
2
3
4
9.6 ± 4.7
11.5 ± 4.7
59.0 ± 19.8
>100
3.2 ± 3.2
5.3 ± 4.2
2.9 ± 3.4
31.0 ±4.1
>100
>100
>100
24.6± 18.5
60± 35
>100
>83 ± 24
55 ± 3
>100
>100
ND
4.1 ± 5.1
23.5 ± 16.3
>100
>100
>100
>100
>100
79.0 ± 29.7
24 ± 6
>100
>100
45±0
43 ± 21
3.1 ± 1.4
4.2 ± 0.7
>152
40 ± 8
>100
8.9±0
ND
d
1
2
(R1=Me)
(R1=Me)
9.2 ± 7.2
2.8 ± 1.2
>152
6.5 ± 3.4
6.1 ± 2.9
>152
30.7 ± 14.5
>200
ND
36.4 ± 2.3
36
ND
3
(R1=Me) (
ND
ND
14.7
4
(R1=Me)
Acyclovir
Brivudine
Cidofovir
UDA
>37
>37
>37
>37
ND
76
1.2 ± 1.1
0.081 ± 0.034
6.5 ± 3.7
-
94 ± 25
420 ± 280
4.6 ± 2.7
-
0.7 ± 0.7
169 ± 116
4.1 ± 2.7
-
>250
-
>440
>300
>300
>100
>440
>300
>300
-
10.8 9.4
21.3 ± 3.8
-
-
-
5.4± 5.0 µg/mL
a
b
c
d
Effective concentration required to viral cytopathic effect by 50%.
Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
Cytostatic concentration required to reduce cell growth by 50%.
Not done.
Table 3.

ACCEPTED MANUSCRIPT
Figure 1.
Figure 2.
O
O
O
P
P
P
O
Cl
Cl
Cl
a
O
O
NH
O
OR
5
6
11-15
Scheme 1.

ACCEPTED MANUSCRIPT
Scheme 2.
Scheme 3.
Scheme 4.

ACCEPTED MANUSCRIPT
Highlights
Phosphonoamidate prodrugs acyclic nucleosides were synthesized by convergent
approach;
Metathesis reaction in water was used between pyrimidic bases and a new
phosphonoamidate synthons
EC values of any molecules were in (sub)micromolar range against DNA viruses
50