Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives

Article abstract of DOI:10.1007/s00706-019-2351-y

Abstract: To study the influence of a linker rigidity and changes in donor–acceptor properties, three series of nucleotide analogs containing a P–X–HN–C(O)– residue (X=CH(OH)CH₂, CH(OH)CH₂CH₂, CH₂CH(OH)CH_2<

Full text of DOI:10.1007/s00706-019-2351-y

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-2351-y
ORIGINAL PAPER
Acyclic nucleoside phosphonates containing the amide bond: hydroxy
derivatives
Iwona E. Głowacka1 · Dorota G. Piotrowska1 · Graciela Andrei2 · Dominique Schols2 · Robert Snoeck2 ·
Andrzej E. Wróblewski1
Received: 26 June 2018 / Accepted: 3 January 2019
© The Author(s) 2019
Abstract
To study the infuence of a linker rigidity and changes in donor–acceptor properties, three series of nucleotide analogs
containing a P–X–HN–C(O)– residue (X=CH(OH)CH2, CH(OH)CH2CH2, CH2CH(OH)CH2) as a replacement for the
P–CH2–O–CHR– fragment in acyclic nucleoside phosphonates, e.g., adefovir, cidofovir, were synthesized. EDC proved to
provide good yields of the analogs from the respective ω-amino-1- or -2-hydroxyalkylphosphonates and nucleobase-derived
acetic acids. New phosphorus–nucleobase linkers are characterized by two fragments of the restricted rotation within amide
bonds and in four-atom units (P–CH(OH)–CH2–N, P–CH(OH)–CH2–C and P–CH2–CH(OH)–C) in which antiperiplanar
disposition of P and N/C atoms was deduced from 1H and 13C NMR spectral data. The synthesized analogs P–X–HNC(O)–
CH2B [X=CH(OH)CH2, CH(OH)CH2CH2, CH2CH(OH)CH2] appeared inactive in antiviral assays on a wide variety of
DNA and RNA viruses at concentrations up to 100 μM, while two phosphonates showed cytostatic activity towards myeloid
leukemia (K-562) and multiple myeloma cells (MM.1S) with IC50 of 28.8 and 40.7 μM, respectively.
Graphical abstract
Keywords Nucleotides · Amide bond formation · NMR spectroscopy · Phosphonates
Introduction
nucleoside phosphonates (ANPs) belong to the most impor-
tant [1–3].
Despite numerous eforts, highly efective antiviral drugs
without side efects are not yet available. A search for such
compounds appeared even more difficult, since various
viruses can undergo fast mutations. Several antiviral medi-
cations are at physicians disposal and among them acyclic
Formulations delivering adefovir (1), tenofovir (2), (S)-
HMPA (3), and cidofovir (4) (Fig. 1) have been used for
several decades and their structural frameworks stimulated
chemists to create modifcations that are more active. Link-
ers connecting nucleobase and phosphonic acid moieties in
1–4 seem to be the frst choice for modifcations but this
approach so far did not lead to discovery of more active
compounds.
* Andrzej E. Wróblewski
Based on the commonly accepted mechanism of action
of ANPs [3, 4], a structure of new analogs has to contain
at both termini a P(O)–CH2 fragment which is responsible
for the stability towards phosphate-cleaving enzymes and
nucleobases or their close analogs which facilitate interac-
tions with nucleobases of viral nucleic acids. Preferably, a
andrzej.wroblewski@umed.lodz.pl
1
Bioorganic Chemistry Laboratory, Faculty of Pharmacy,
Medical University of Lodz, Muszyńskiego 1, 90-151 Lodz,
Poland
2
Rega Institute for Medical Research, KU Leuven, Herestraat
49, 3000 Louvain, Belgium
Vol.:(0123456789)
1 3

I. E. Głowacka et al.
linkages X (CH2, CH2CH2, and CH2CH2CH2) or ethereal
interconnection (CH2OCH2CH2) including the methylene
group which secured the separation of nitrogen atoms (N1
or N9) in nucleobases and the phosphorus atom by four
bonds and resulted in compounds structurally closest to
the drugs 1–4 that appeared inactive. Hence, we reasoned
that installation of additional polar functionalities in the
aliphatic linker could improve acyclic nucleoside–enzyme
interactions, and thus signifcantly increase the activity.
Herein, we wish to describe our studies on the synthesis
and the biological activity of the new amides 7–9 having
the hydroxy groups within the aliphatic linker. To syn-
thesize the fnal acyclic nucleoside phosphonates 7–9,
the approach involving the formation of the amide bond
between the respective acetic acid derivatives 13a–13h
and ω-aminophosphonates 10–12 was followed (Scheme 1)
[5].
Fig. 1 Structures of adefovir (1), tenofovir (2), (S)-HMPA (3), and
cidofovir (4)
four-atom linker capable of hydrogen bonding should inter-
connect these units.
Recently, we put forward an idea of replacing a
P–CH2–O–CHR– fragment in 1–4 with the amide bond
[5]. This was inspired by the successful application of the
isosteric replacement of this bond by a methylene ether
[–CH2–O] moiety in studies on the biological activity of
natural peptides [6–8]. However, the introduction of the
amide [P–CH2–HN–C(O)–] residue signifcantly increases
donor–acceptor interactions within a new linker. It also
modifes its conformational fexibility due to the restricted
rotation around the amide bond. In our frst approach [5],
four series of the phosphonate amides of general formulae
5 and 6 were synthesized (Fig. 2).
For many years, the biological activity of phospho-
nate nucleoside analogs has been studied employing free
phosphonic acids but recently, they are administered in
the form of prodrugs (esters or amides) to signifcantly
improve bioavailability of very polar acids [2, 3, 9–12].
We opted to test phosphonate diethyl esters 7–9, since they
to some extent resemble the lipophilic prodrugs in ability
to permeable cell membranes. Our recent experience is in
line with this strategy as we discovered examples of the
biologically active phosphonate diethyl esters substituted
with various heterocyclic motives, while the respective
free acids appeared inactive [13, 14].
In this paper, we continue our eforts to identify struc-
tural features in acyclic nucleoside phosphonates con-
taining the amide bond 7–9 responsible for their antiviral
activity. The phosphonates 5 and 6 containing the aliphatic
Furthermore, we were afraid of possible dehydration
of our hydroxyalkylphosphonates under harsh conditions
associated with the application of, for example, acids,
bases or even iodotrimethylsilane.
Fig. 2 Structures of previously described and newly designed phosphonates as acyclic nucleoside phosphonates (B=canonical nucleobases;
B′=nucleobase analogs: see Scheme 1)
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
Scheme 1
2,3-epoxypropanephosphonate framework [17] assured us
that phosphonates 10 and 12 of the highest purity could
be obtained from the epoxides 14 and 15, respectively,
when dibenzylamine will be applied instead of ammonia
followed by hydrogenolysis (Scheme 2) [18].
Results and discussion
To accomplish the synthesis of the second series of the
amides 7a–7h to 9a–9h, pure ω-aminophosphonates
10–12 have to be efciently prepared (Schemes 2 and
3). The phosphonate 10 is a known compound [15,
16] and it was prepared by the ammonolysis of diethyl
1,2-epoxyethanephosphonate 14, but the authors failed
to provide a full characterization of the material they
obtained. The phosphonate 12 is also known [15] and it
was obtained in a similar manner from diethyl 2,3-epoxy-
propanephosphonate 15, though the authors were unable
to prove the purity of the product. Our experience with the
Available synthetic strategies to the phosphonate 11 take
advantage of the addition of diethyl phosphite derivatives to
N-protected 3-aminopropanal (Scheme 3) [19, 20].
In our hands, N-Cbz protection of 3-aminopropan-1-ol
followed by the Swern oxidation proved optimal for the
preparation of the aldehyde 16 which was later subjected to
a triethylamine-catalyzed phosphorylation to provide a pro-
tected hydroxyphosphonate 17. Final hydrogenolysis gave
pure phosphonate 11 in 25% overall yield.
Scheme 2
Reagents and conditions: a. Bn2NH, 60 °C, 20 h, b. H2, 10% Pd-C, EtOH, 72 h.
1 3

I. E. Głowacka et al.
Scheme 3
The known uracil- 13a, 5-fuorouracil- 13b, 5-bromoura-
cil- 13c, thymine- 13d and benzouracil-containing 13f acetic
acids were prepared by alkylation of the respective nucle-
obases with chloro- or bromoacetic acid [21–25]. Synthesis
of amides 7e, 8e, 9e and 7g, 8g, 9g containing cytosine- and
adenine-acetyl subunits required prior protection of the start-
ing acids as N-Cbz- and N-diBoc-derivatives 13e [26] and
13g [27] to signifcantly increase lipophilicity of the fnal
products. Following the literature precedences [28] later
confrmed by our experience [5], we anticipated to prepare
guanine analogs 7i–9i (Fig. 3) from the 2-amino-6-chloro-
purine precursors 7h–9h (Scheme 1) using 2-amino-6-chlo-
ropurin-9-yl)acetic acid 13h [29] as a substrate.
into guanine phosphonates 7i–9i appeared fruitless leading
to complex mixtures of unidentifed products.
Conformational analysis
We were also interested in the conformational mobility of
the amides 7a–7h to 9a–9h within new acyclic linkers to
provide important information regarding structure–activ-
1
ity analysis. Since H NMR spectra of all nucleotide ana-
logs 7a–7h exhibited almost identical patterns regarding
a P–CH(OH)–CH2–NHC(O)–CH2 fragment, we concluded
that in a methanolic solution they exist in the same confor-
1
mation. Detailed analysis of a H NMR spectrum of 7d,
1
With the ω-aminophosphonates 10–12 of high purity
secured, we turned to the coupling with a series of nucle-
obase-acetic acids (Scheme 1). As established earlier [5],
all syntheses of amides 7a–7h to 9a–9h were best per-
formed in the presence of EDC×HCl as a coupling reagent
(Scheme 1) to give products of high purity in good yields.
Although we succeeded in purifcation of the phosphonate
7g on a silica gel column, under the same conditions both
homologs 8g and 9g were obtained as inseparable mixtures
containing various amounts of triethylamine hydrochloride.
When HPLC technique was applied, in both cases, partial
deprotection of 8g and 9g occurred to provide pure mono-
N-Boc protected phosphonates 8j and 9j (Fig. 3) in addition
to mixtures of 8j and 8g as well as 9j and 9g free from tri-
ethylamine hydrochloride. Several attempts at transforming
the 2-amino-6-chloropurine moiety in phosphonates 7h–9h
including a H{31P} one, clearly revealed an antiperipla-
nar disposition of the phosphoryl and amide groups pro-
jected as 18 (Fig. 4). Vicinal couplings H1–H2a (3.6 Hz)
and H1–H2b (9.2 Hz) ft well into the ranges expected for
the gauche and antiperiplanar arrangement of these protons
[30]. Furthermore, values of both 3J(P–H2a/H2b) coupling
constants are close (7.2 and 8.5 Hz). These values were
well correlated with those characteristic of the gauche
hydrogen–phosphorus relationship [31, 32]. The stability
of the conformation 18 primarily results from the steric
bulkiness of the substituents at C1 and C2 but may also
be enforced by a C2–N–H·······O(H)–C1 hydrogen bond
within a fve-membered ring as depicted in 19 (Fig. 4). This
suggestion fnds support in a large downfeld shift of the
H2a proton (3.73 ppm) as compared with the H2b proton
(3.38 ppm) which can only be observed when an amide C=O
Fig. 3 Guanine phosphonate
analogs 7i, 8i, 9i and NH-Boc
adenine phosphonates 8j and 9j
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
a P–CH2–CH(OH)–CH2–NHC(O)–CH2 portion. The
values of 3J(P–C3) = 15.4 Hz [33, 34] together with
3
3J(H1a–H2) = 4.6 Hz and J(H1b–H2) = 8.2 Hz [31, 32]
1
found in 13C in H NMR spectra of 9a again suggest that
the diethoxyphosphoryl and CH2–NHC(O) groups for steric
reasons prefer to exist in the anti conformation 21 (Fig. 5).
This is commonly observed for β-hydroxyphosphonates
[35]. This conclusion is further supported by the lack of
the vicinal phosphorus–HC2 coupling as expected for the
gauche relationship within a H-C2–C1-P unit [31, 32]. As
in 8c, a free rotation within a CH2–NHC(O) moiety in 9a
is evident from the values of vicinal H2–H3a/3b couplings.
Fig. 4 Preferred conformations 18/19 of nucleotide analogs 7a–7h
Antiviral evaluation
All phosphonates, i.e., 7a–7h, 8a–8f, 8h, 8j, 9a–9f, 9h,
and 9j were evaluated for inhibitory activity against a wide
variety of DNA and RNA viruses, using the following cell-
based assays. The antiviral assays were performed in: (a)
human embryonic lung (HEL) cells [herpes simplex virus-1
(KOS strain), herpes simplex virus-2 (G strain), thymidine
kinase defcient (acyclovir resistant) herpes simplex virus-1
(TK– KOS ACVr strain), vaccinia virus, adenovirus-2,
human coronavirus (229E), cytomegalovirus (AD-169
and Davis strains), varicella-zoster virus (TK+ VZV and
TK– VZV strains)], (b) HeLa cell cultures (vesicular sto-
matitis virus, Coxsackie virus B4 and respiratory syncytial
virus), (c) Vero cell cultures [para-infuenza-3 virus, reovi-
rus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus,
yellow fever virus], and (d) MDCK cell cultures [infuenza
A virus (H1N1 and H3N2 subtypes) and infuenza B virus].
Ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine,
zanamivir, alovudine, amantadine, rimantadine, ribavi-
rin, dextran sulfate (molecular weight 10,000, DS-10000),
mycophenolic acid and Urtica dioica agglutinin (UDA) were
used as the reference compounds. The antiviral activity was
expressed as the EC50: the compound concentration required
to reduce virus plaque formation (VZV) by 50% or to reduce
virus-induced cytopathogenicity by 50% (other viruses).
None of the tested compounds showed appreciable antiviral
activity toward any of the tested DNA and RNA viruses at
concentrations up to 100 μM, nor afected cell morphology
of HEL, HeLa, Vero, and MDCL cells.
Fig. 5 Preferred conformations 20 and 21 of nucleotide analogs 8a–
8h and 9a–9h
acts as a deshielding group. This is only possible when a
C2–N–H·······O(H)–C1 hydrogen bond is strong enough to
enable a free rotation around a C2–NH bond.
Also, all 1H NMR spectra of nucleotide analogs
8a–8f and 8h showed almost identical patterns for a
P–CH(OH)–CH2–CH2–NHC(O)–CH2 chain and we rea-
soned that in methanol they adopt the same conformation
20 (Fig. 5) which resembles 18 in terms of antiperiplanar
1
location of the phosphoryl and H2C3 groups. From H,
1H{31P}, and 13C NMR spectra of 8c, the following diagnos-
tic coupling constants were extracted: 3J(H1–H2a)=3.1 Hz,
3J(H1–H2b) = 10.7 Hz [30], 3J(P–H2a) = 6.7 Hz,
3
3J(P–H2b)=9.5 Hz [31, 32], and J(P–C3)=16.1 Hz [33,
34] which fully confrm our conclusion.
Thus, again the steric requirements of the diethoxyphos-
phoryl group and a H2C3–NHC(O) substituent are respon-
sible for stability of the antiperiplanar conformation 20.
However, although all vicinal proton–proton couplings were
successfully calculated for a HaHbC2–C3HaHb subunit in 8c,
their values can only be interpreted in favor of a free rotation
around a C2–C3 bond and thus preclude any intramolecular
H-bonding for amide moieties in 8a–8h.
Cytostatic evaluation
The 50% cytostatic inhibitory concentration (IC50) causing
a 50% decrease in cell proliferation was determined for all
phosphonates, i.e., 7a–7h, 8a–8f, 8h, 8j, 9a–9f, 9h, and 9j
toward 9 cancerous cell lines [Capan-1 (pancreatic adenocarci-
noma), Hap1 (chronic myeloid leukemia), HCT-116 (colorec-
tal carcinoma), NCI-H460 (lung carcinoma), DND-41 (acute
lymphoblastic leukemia), HL-60 (acute myeloid leukemia),
1
Similar to H NMR spectra of nucleotide analogs
7a–7h and 8a–8f and 8h, the spectra of the analogs 9a–9f
and 9h in methanol displayed very similar patterns for
1 3

I. E. Głowacka et al.
K-562 (chronic myeloid leukemia), MM.1S (multiple mye-
loma), Z-138 non-Hodgkin lymphoma)] as well as normal
retina (non cancerous) cells (hTERT RPE-1). Docetaxel and
stauroporine were used as the reference compounds. Among
all screened compounds, phosphonates 7c, 8c, and 9d were
slightly cytostatic against multiple myeloma cells (MM.1S)
with IC50 values 78.8, 91.3, and 40.7 μM, respectively, how-
ever, lower than reference drugs (IC50 values 3.6 and 16.6 for
docetaxel and stauroporine, respectively). On the other hand,
compound 9b showed noticeable inhibitory properties toward
chronic myeloid leukemia (K-562) (IC50 =28.8 μM), however,
still lower than docetaxel (IC50 =1.4 μM) and stauroporine
(IC50 =8.0 μM). All tested compounds were not toxic for nor-
mal, non-cancerous retina cells (hTERT RPE-1) at concentra-
tions up to 100 μM.
as internal standard. 13C NMR spectra were recorder for
CD3OD or CDCl3 solutions on the Bruker Avance III at
151.0 MHz. 31P NMR spectra were performed on the Var-
ian Gemini 2000BB at 81.0 MHz or on Bruker Avance
III at 243.0 MHz. IR spectral data were measured on a
Bruker Alpha-T FT-IR spectrometer. Melting points were
determined on a Boetius apparatus. Elemental analyses
were performed by the Microanalytical Laboratory of this
Faculty on a Perkin Elmer PE 2400 CHNS analyzer and
their results were found to be in good agreement ( 0.3%)
with the calculated values.
The following absorbents were used: column chroma-
tography, Merck silica gel 60 (70–230 mesh); analytical
TLC, Merck TLC plastic sheets silica gel 60 F254. TLC
plates were developed in chloroform–methanol solvent
systems. Visualization of spots was efected with iodine
vapors. All solvents were purifed by methods described
in the literature.
Conclusion
In further studies on a concept of the replacement of the
P–CH2–O–CHR– fragment in acyclic nucleoside analogs
(e.g., adefovir) for P–X–HN–C(O)– moieties in addition to
previously obtained series (X=CH2, CH2CH2, CH2CH2CH2,
CH2OCH2CH2), nucleotide analogs containing hydroxyalkyl
linkers (X=CH(OH)CH2, CH(OH)CH2CH2, CH2CH(OH)
CH2) were synthesized. The coupling of the respective
ω-aminophosphonates and nucleobase-derived acetic acids
was accomplished in good yields by application of EDC.
Phosphonate–nucleobase linkers in the synthesized ana-
logs contain two fragments of restricted rotation, namely
the amide bonds and P–CH(OH)–CH2–N (compounds
7a–7h), P–CH(OH)–CH2–C (compounds 8a–8h), and
P–CH2–CH(OH)–C (compounds 9a–9h) residues in which,
as judged from 1H and 13C NMR spectral data, antiperipla-
nar disposition of P and N/C atoms results both from the
steric bulkiness of O,O-diethyl phosphoryl group and intra-
molecular H-bonding. The phosphonates 7a–7h, 8a–8f, 8h,
8j, 9a–9f, 9h, and 9j were subjected to antiviral assays on a
wide variety of DNA and RNA viruses but appeared inactive
at concentrations up to 100 μM. Their cytostatic properties
were evaluated on 9 cancerous cell lines and phosphonates
9b and 9d showed moderate activity towards myeloid leu-
kemia (K-562) (IC50 =28.8 μM) and multiple myeloma cells
(MM.1S) (IC50 =40.7 μM), respectively.
Diethyl 1,2-epoxyethane- and 2,3-epoxypropanephos-
phonates 14 [36] and 15 [15] as well as the protected alde-
hyde 16 [20] and diethyl 3-amino-2-hydroxypropanephos-
phonate 12 [18] were prepared according to the literature
procedures.
Diethyl 2‑amino‑1‑hydroxyethanephosphonate 10 [16] A
mixture of 3.60 g of the epoxide 14 (19.9 mmol) and
3.99 cm3 dibenzylamine (20.8 mmol) was heated at 60 °C
for 20 h. After cooling, the crude product was purifed on
a silica gel column with a chloroform to give pure diethyl
2-(N,N-dibenzylamino)-1-hydroxyethanephosphonate
(6.77 g) in 68% yield. In the next step a solution of 1.53 g
of the diethyl 2-(N,N-dibenzylamino)-1-hydroxyethanephos-
phonate (4.05 mmol) in 10 cm3 ethanol was hydrogenated
over 80 mg Pd–C (10%) at room temperature for 72 h. The
catalyst was removed on a layer of Celite; the solution
was concentrated in vacuo to aford pure phosphonate 10
(0.860 g, 100%) as a yellowish oil.
Diethyl 3‑amino‑1‑hydroxypropanephosphonate 11 [19] A
mixture of 2.02 g of the protected aldehyde 16 (9.76 mmol),
1.13 cm3 diethyl phosphite (8.78 mmol), and 0.136 cm3 tri-
ethylamine (0.976 mmol) was stirred at room temperature
for 24 h. After the solution was concentrated in vacuo, the
residue was chromatographed on a silica gel column with
a chloroform–methanol mixture (200:1, 100:1 v/v) to give
the phosphonate 17 (2.27 g, 67%) as a white powder. In
the next step, a solution of 0.750 g of the phosphonate 17
(2.17 mmol) in 6 cm3 ethanol was hydrogenated over 30 mg
Pd–C (10%) at room temperature for 72 h. The suspension
was fltered through a pad of Celite and washed with etha-
nol. The solution was concentrated in vacuo to aford pure
3-amino-1-hydroxypropanephosphonate 11 (0.460 g, 100%)
as a yellowish oil.
Studies on the analogous phosphonates containing func-
tionalized amino groups within linkers are currently under
way in this laboratory.
Experimental
1H NMR spectra were recorded in CD3OD or CDCl3 on
the following spectrometers: Varian Gemini 2000BB
(200 MHz) and Bruker Avance III (600 MHz) with TMS
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
104.81, 68.25 (d, J = 167.3 Hz, PC), 66.83 and 66.49
(2 × d, J = 7.7 Hz, 2 × POC), 53.90, 39.61 (d, J = 16.4 Hz,
PCCC), 34.73 (d, J=3.4 Hz, PCC), 19.36 and 19.32 (2×d,
J=5.2 Hz, 2×POCC) ppm; 31P NMR (81 MHz, CD3OD):
δ=26.03 ppm.
General procedure
To a solution of aminophosphonates 10–12 (1.00 mmol)
in 2 cm3 DMF or chloroform, the respective acetic acids
13a–13h (1.00 mmol), EDC×HCl (1.00 mmol), and TEA
(1.00 mmol) were added. The reaction mixture was stirred at
room temperature for 48 h and then concentrated in vacuo.
The residue was chromatographed on a silica gel column
with chloroform–methanol mixtures and crystallized from
the appropriate solvents.
Diethyl 3‑[2‑(3,4‑dihydro‑2,4‑dioxopyrimidin‑1(2H)‑yl)‑
acetamido]‑2‑hydrox ypropylphosphonate (9a,
C13H22N3O7P) The crude product obtained from 0.139 g
diethyl 3-amino-2-hydroxypropylphosphonate (12,
0.658 mmol) and 0.112 g (uracil-1-yl)acetic acid (13a,
0.658 mmol) according to the general procedure was chro-
matographed with chloroform–methanol mixtures (50:1,
20:1 v/v) and crystallized from a methanol–diethyl ether
mixture to give compound 19a (0.112 g, 47% yield) as a
white powder. M.p.: 124–125 °C; IR (KBr): V =3313, 3095,
3045, 2982, 2929, 1691, 1650, 1239, 1053, 1023 cm−1; 1H
NMR (600 MHz, CD3OD): δ = 7.53 (d, J = 7.9 Hz, 1H,
HC = CH), 5.69 (d, J = 7.9 Hz, 1H, HC = CH), 4.68 and
4.45 (AB, J = 16.6 Hz, 2H, C(O)CHaHb), 4.19–4.11 (m,
4H, 2 × POCH2CH3), 4.08 (ddt, J = 8.2 Hz, J = 6.6 Hz,
J = 4.8 Hz, J = 4.6 Hz, 1H, PCCH), 3.42 (dd, J = 13.6 Hz,
J = 4.8 Hz, 1H, PCCCHaHb), 3.29 (ddd, J = 13.6 Hz,
J = 6.6 Hz, J = 0.6 Hz, 1H, PCCCHaHb), 2.07 (ddd,
J = 18.9 Hz, J = 15.5 Hz, J = 4.6 Hz, 1H, PCHaHb), 2.01
(ddd, J = 17.9 Hz, J = 15.5 Hz, J = 8.2 Hz, 1H, PCHaHb),
1.36 and 1.35 (2 × t, J = 7.1 Hz, 2 × 3H, 2 × POCH2CH3)
ppm; 13C NMR (151 MHz, CD3OD): δ = 168.32, 165.40,
151.51, 146.46, 100.88, 65.14 (d, J=3.9 Hz, PCC), 62.06
and 61.84 (2 × d, J = 6.5 Hz, 2 × POC), 49.92, 45.83 (d,
J=15.4 Hz, PCCC), 30.56 (d, J=140.7 Hz, PC), 15.26 (d,
J=6.2 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CD3OD):
δ=29.97 ppm.
Diethyl 2‑[2‑(3,4‑dihydro‑2,4‑dioxopyrimidin‑1(2H)‑yl)‑
a c e t a m i d o ] ‑ 1 ‑ hy d r ox y e t hy l p h o s p h o n a t e ( 7 a ,
C12H20N3O7P) According to the general procedure from
0.156 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.791 mmol) and 0.135 g (uracil-1-yl)acetic acid (13a,
0.791 mmol), the amide 7a (0.128 g, 46% yield) was obtained
as a white solid after purifcation on a silica gel column with
chloroform–methanol mixtures (50:1, 20:1 v/v) and crys-
tallization from methanol. M.p.: 174–176 °C; IR (KBr):
V =3340, 3229, 2997, 2940, 2824, 1687, 1046, 1015 cm−1;
1H NMR (200 MHz, CD3OD): δ = 7.55 (d, J = 7.9 Hz,
1H, HC = CH), 5.72 (d, J = 7.9 Hz, 1H, HC = CH), 4.50
and 4.48 (AB, J = 16.7 Hz, 2H, C(O)CHaHb), 4.31–4.13
(m, 4H, 2 × POCH2CH3), 4.08 (ddd, JH1–H2b = 9.2 Hz,
JH1-P = 8.1 Hz, JH1–H2a = 3.7 Hz, 1H, PCHCH2), 3.73
(ddd, JH2a–H2b =13.9 Hz, JH2a-P =7.2 Hz, JH2a–H1 =3.7 Hz,
1H, PCCHaHb), 3.39 (ddd ~ dt, JH2a–H2b = 13.9 Hz,
JH2b–H1 =9.2 Hz, JH2b-P =8.5 Hz, 1H, PCCHaHb), 1.39 and
1.38 (2 × t, J = 7.0 Hz, 2 × 3H, 2 × POCH2CH3) ppm; 13C
NMR (151 MHz, CD3OD): δ = 168.26, 165.33, 151.55,
146.38, 100.97, 65.88 (d, J=164.4 Hz, PC), 63.05 and 62.75
(2 × d, J = 7.3 Hz, 2 × POC), 49.72, 41.12 (d, J = 8.6 Hz,
PCC), 15.40 and 15.38 (2×d, J=5.1 Hz, 2×POCC) ppm;
31P NMR (81 MHz, CD3OD): δ=23.73 ppm.
Diethyl 2‑[2‑(5‑fluoro‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxyethylphosphonate (7b,
C12H19FN3O7P) According to the general procedure from
0.107 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.543 mmol) and 0.102 g (5-fuorouracil-1-yl)acetic acid
(13b, 0.543 mmol), the amide 7b (0.81 g, 41% yield) was
obtained as a white powder after purifcation on a silica gel
column with chloroform–methanol mixtures (50:1, 20:1
v/v). M.p.: 181–183 °C; IR (KBr): V = 3381, 3196, 3049,
Diethyl 3‑[2‑(3,4‑dihydro‑2,4‑dioxopyrimidin‑1(2H)‑yl)‑
acetamido]‑1‑hydrox ypropylphosphonate (8a,
C13H22N3O7P) According to the general procedure from
0.050 g diethyl 3-amino-1-hydroxypropylphosphonate
(11, 0.240 mmol) and 0.040 g (uracil-1-yl)acetic acid
(13a, 0.240 mmol), the amide 8a (0.040 g, 47% yield) was
obtained as a white solid after purifcation on a silica gel
column with chloroform–methanol mixtures (50:1, 20:1
v/v) and crystallization from methanol–diethyl ether. M.p.:
190–191 °C; IR (KBr): V =3333, 3231, 2995, 1698, 1673,
1244, 1028 cm−1; 1H NMR (200 MHz, CD3OD): δ=7.56 (d,
J=8.0 Hz, 1H, HC=CH), 5.70 (d, J=8.0 Hz, 1H, HC=CH),
4.45 (s, 2H, C(O)CH2), 4.29–4.12 (m, 4H, 2×POCH2CH3),
3.98 (ddd, J = 10.6 Hz, J = 7.3 Hz, J = 3.4 Hz, 1H, PCH),
3.51–3.41 (m, 2H, PCCCH2), 2.11–1.73 (m, 2H, PCCH2),
1.38 (t, J = 7.1 Hz, 6H, 2 × POCH2CH3) ppm; 13C NMR
(151 MHz, CD3OD): δ = 172.13, 169.35, 155.41, 151.31,
1
2995, 2829, 1736, 1700, 1661, 1217, 1025, 749 cm−1; H
NMR (200 MHz, CD3OD): δ = 7.82 (d, J = 6.2 Hz, 1H,
HC=CF), 4.47 and 4.44 (AB, J=16.6 Hz, 2H, C(O)CHaHb),
4.31–4.15 (m, 4H, 2×POCH2CH3), 4.08 (ddd, J=9.2 Hz,
J=8.2 Hz, J=3.7 Hz, 1H, PCHCH2), 3.73 (ddd, J=14.0 Hz,
J=6.9 Hz, J=3.7 Hz, 1H, PCCHaHb), 3.45–3.33 (m, 1H,
PCCHaHb), 1.39 (t, J= 7.0 Hz, 6H, 2 × POCH2CH3) ppm;
13C NMR (151 MHz, CD3OD): δ = 168.10, 158.51 (d,
J=25.6 Hz), 150.26, 140.23 (d, J =232.3 Hz), 130.31 (d,
J=34.0 Hz), 65.84 (d, J=164.8 Hz, PC), 63.05 and 62.76
1 3

I. E. Głowacka et al.
(2 × d, J = 6.9 Hz, 2 × POC), 49.70, 41.11 (d, J = 8.6 Hz,
PCC), 15.40 and 15.37 (2×d, J=5.0 Hz, 2×POCC) ppm;
31P NMR (81 MHz, CD3OD): δ=23.71 ppm.
15.26 (d, J=6.0 Hz, 2×POCC) ppm; 31P NMR (243 MHz,
CD3OD): δ=29.95 ppm.
Diethyl 2‑[2‑(5‑bromo‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxyethylphosphonate (7c,
C12H19BrN3O7P) According to the general procedure from
0.177 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.898 mmol) and 0.224 g (5-bromouracil-1-yl)acetic acid
(13c, 0.898 mmol), the amide 7c (0.169 g, 44% yield) was
obtained as a white solid after purifcation on a silica gel
column with chloroform–methanol mixtures (50:1, 20:1
v/v) and crystallization from a methanol–diethyl ether mix-
ture. M.p.: 183–185 °C; IR (KBr): V = 3317, 3234, 3042,
Diethyl 3‑[2‑(5‑fluoro‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxypropylphosphonate (8b,
C13H21FN3O7P) According to the general procedure from
0.160 g diethyl 3-amino-1-hydroxypropylphosphonate (11,
0.758 mmol) and 0.143 g (5-fuorouracil-1-yl)acetic acid
(13b, 0.758 mmol), the amide 8b (0.125 g, 43% yield) was
obtained as a white powder after purifcation on a silica gel
column with chloroform–methanol mixtures (50:1, 20:1
v/v) and crystallization from a methanol–diethyl ether mix-
ture. M.p.: 211–212 °C; IR (KBr): V = 3336, 3217, 3061,
1
2997, 2833, 1692, 1626, 1225, 1021, 622 cm−1; H NMR
1
2929, 1721, 1698, 1659, 1245, 1019, 975 cm−1; H NMR
(600 MHz, CD3OD): δ=8.00 (s, 1H, HC=CBr), 4.50 and
4.47 (AB, J = 16.4 Hz, 2H, C(O)CHaHb), 4.24–4.18 (m,
4H, 2 × POCH2CH3), 4.07 (dt, J = 8.8 Hz, J = 3.7 Hz, 1H,
PCHCH2), 3.71 (ddd, J=13.9 Hz, J=7.4 Hz, J=3.7 Hz, 1H,
PCCHaHb), 3.38 (dt, J=13.9 Hz, J=8.8 Hz, 1H, PCCHaHb),
1.37 (t, J = 7.0 Hz, 6H, 2 × POCH2CH3) ppm; 13C NMR
(151 MHz, CD3OD): δ = 168.03, 160.71, 150.96, 145.81,
95.18, 65.83 (d, J=164.8 Hz, PC), 63.04 and 62.76 (2×d,
J = 7.1 Hz, 2 × POC), 49.73, 41.12 (d, J = 8.6 Hz, PCC),
15.40 and 15.38 (2 × d, J = 5.1 Hz, 2 × POCC) ppm; 31P
NMR (81 MHz, CD3OD): δ=23.71 ppm.
(600 MHz, CD3OD): δ=7.80 (d, J=6.1 Hz, 1H, HC=CF),
4.42 and 4.38 (AB, J = 16.6 Hz, 2H, C(O)CHaHb), 4.23–
4.16 (m, 4H, 2 × POCH2CH3), 3.96 (ddd, J = 10.7 Hz,
J = 7.4 Hz, J = 3.1 Hz 1H, PCHCH2), 3.53–3.40 (m, 2H,
PCCCH2), 2.03–1.96 (m, 1H, PCCHaCHb), 1.90–1.82 (m,
1H, PCCHaCHb), 1.36 (t, J=7.1 Hz, 6H, 2×POCH2CH3)
ppm; 13C NMR (151 MHz, CD3OD): δ=168.02, 158.57 (d,
J=25.6 Hz), 150.20, 140.22 (d, J =232.0 Hz), 130.37 (d,
J=34.0 Hz), 64.37 (d, J=166.1 Hz, PC), 62.90 and 62.55
(2 × d, J = 7.1 Hz, 2 × POC), 49.95, 35.69 (d, J = 16.1 Hz,
PCCC), 30.78 (d, J=3.0 Hz, PCC), 15.42 and 15.39 (2×d,
J=5.3 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CD3OD):
δ=25.14 ppm.
Diethyl 3‑[2‑(5‑bromo‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxypropylphosphonate
(8c, C13H21BrN3O7P) According to the general procedure
from 0.160 g diethyl 3-amino-1-hydroxypropylphospho-
nate (11, 0.758 mmol) and 0.160 g (5-bromouracil-1-yl)
acetic acid (13c, 0.758 mmol), the amide 8c (0.127 g, 44%
yield) was obtained as a white powder after purifcation on
a silica gel column with chloroform–methanol mixtures
Diethyl 3‑[2‑(5‑fluoro‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑2‑hydroxypropylphosphonate (9b,
C13H21FN3O7P) According to the general procedure from
0.128 g diethyl 3-amino-2-hydroxypropylphosphonate (12,
0.606 mmol) and 0.144 g (5-fuorouracil-1-yl)acetic acid
(13b, 0.606 mmol), the amide 9b (0.074 g, 32% yield) was
obtained as a white powder after purifcation on a silica
gel column with chloroform–methanol mixtures (50:1,
20:1 v/v) and HPLC (C18 column, 25:70 methanol:water).
̄
(50:1, 20:1 v/v). M.p.: 203–205 °C; IR (KBr): V = 3326,
1
3189, 3059, 2997, 1725, 1674, 1247, 1021, 586 cm−1; H
NMR (600 MHz, CD3OD): δ = 8.01 (s, 1H, HC = CBr),
4.45 (s, 2H, C(O)CH2), 4.23–4.16 (m, 4H, 2×POCH2CH3,),
3.96 (ddd, JH1–H2b = 10.7 Hz, JH1-P = 7.4 Hz,
JH1–H2a =3.1 Hz, 1H, PCH) 3.48 (ddd, JH3a–H3b =13.5 Hz,
JH3a–H2a = 8.2 Hz, JH2a-P = 7.0 Hz, 1H, PCCCHaHb),
3.41 (ddd, JH3a–H3b = 13.5 Hz, JH3b–H2a = 7.3 Hz,
JH3b–H2b = 4.9 Hz, 1H, PCCCHaHb), 1.99 (ddddd,
JH2a–H2b = 14.0 Hz, JH2a–H3a = 8.2 Hz, JH2a–H3b = 7.3 Hz,
JH2a-P = 6.7 Hz, JH2a–H1 = 3.1 Hz, 1H, PCCHaHb),
1.86 (ddddd, JH2a–H2b = 14.0 Hz, JH2b–H1 = 10.7 Hz,
JH2b-P = 9.5 Hz, JH2b–H3a = 7.0 Hz, JH2b–H3b = 4.9 Hz, 1H,
PCCHaHb) 1.36 (t, J = 7.1 Hz, 6H, 2 × POCH2CH3) ppm;
13C NMR (151 MHz, CD3OD): δ=167.95, 160.77, 150.89,
145.86, 95.13, 64.35 (d, J=167.4 Hz, PC), 62.90 and 62.56
(2 × d, J = 7.1 Hz, 2 × POC), 49.99, 35.68 (d, J = 16.1 Hz,
PCCC), 30.78 (d, J=3.0 Hz, PCC), 15.42 and 15.38 (2×d,
̄
M.p.: 126–128 °C; IR (KBr): V =3358, 3167, 3057, 2995,
1
2931, 2819, 1706, 1661, 1220, 1027, 858 cm−1; H NMR
(600 MHz, CD3OD): δ=7.80 (d, J=6.2 Hz, 1H, HC=CF),
4.44 and 4.42 (AB, J = 16.8 Hz, 2H, C(O)CHaHb), 4.18–
4.11 (m, 4H, 2×POCH2CH3), 4.10–4.05 (m, 1H, PCCH),
3.43 (dd, J = 13.6 Hz, J = 4.7 Hz, 1H, PCCCHaHb), 3.28
(ddd, J=13.6 Hz, J=6.5 Hz, J=0.5 Hz, 1H, PCCCHaHb),
2.08 (ddd, J = 19.0 Hz, J = 15.5 Hz, J = 4.7 Hz, 1H,
PCHaHb), 2.01 (ddd, J = 17.8 Hz, J = 15.5 Hz, J = 8.2 Hz,
1H, PCHaHb), 1.36 and 1.35 (2 × t, J = 7.1 Hz, 2 × 3H,
2 × POCH2CH3) ppm; 13C NMR (151 MHz, CD3OD):
δ = 168.17, 158.67 (d, J = 26.0 Hz), 150.33, 140.23 (d,
J=232.2 Hz), 130.35 (d, J=34.0 Hz), 65.13 (d, J=3.5 Hz,
PCC), 62.07 and 61.84 (2×d, J=6.5 Hz, 2×POC), 49.91,
45.83 (d, J=15.3 Hz, PCCC), 30.59 (d, J=140.9 Hz, PC),
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
J=4.8 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CD3OD):
δ=25.15 ppm.
Diethyl 3‑[2‑(3,4‑dihydro‑5‑methyl‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxypropylphosphonate
(8d, C14H24N3O7P) According to the general procedure
from 0.180 g diethyl 3-amino-1-hydroxypropylphospho-
nate (11, 0.852 mmol) and 0.157 g (thymine-1-yl)acetic
acid (13d, 0.852 mmol), the amide 8d (0.166 g, 52% yield)
was obtained as a white solid after purifcation on a silica
gel column with chloroform–methanol mixtures (50:1,
20:1 v/v) and crystallization from a methanol–diethyl ether
Diethyl 3‑[2‑(5‑bromo‑3,4‑dihydro‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑2‑hydroxypropylphosphonate
(9c, C13H21BrN3O7P) According to the general procedure
from 0.545 g diethyl 3-amino-2-hydroxypropylphospho-
nate (12, 2.58 mmol) and 0.642 g (5-bromouracil-1-yl)
acetic acid (13c, 2.58 mmol), the amide 9c (0.552 g, 48%
yield) was obtained as a white powder after purifcation
on a silica gel column with chloroform–methanol mix-
tures (50:1, 20:1 v/v) and HPLC (C18 column, 25:70
methanol:water). M.p.: 108–109 °C; IR (KBr): V = 3488,
3350, 3159, 3034, 2993, 2928, 2838, 1714, 1668, 1240,
̄
mixture. M.p.: 183–184 °C; IR (KBr): = 3333, 3238,
V
2987, 2833, 1665, 1236, 1024 cm−1; 1H NMR (600 MHz,
CD3OD): δ=7.38 (brq, J=0.8 Hz, 1H, HC=CCH3), 4.41
and 4.39 (AB, J=16.6 Hz, 2H, C(O)CHaHb), 4.22–4.16 (m,
4H, 2 × POCH2CH3), 3.96 (ddd, J = 10.6 Hz, J = 7.4 Hz,
J = 3.1 Hz, 1H, PCHCH2), 3.49–3.39 (m, 2H, PCCC
HaHb), 2.02–1.95 (m, 1H, PCCHaHb), 1.90–1.81 (m, 1H,
PCCHaHb), 1.90 (d, J= 0.8 Hz, 3H, HC= CCH3), 1.36 (t,
J=7.1 Hz, 6H, 2×POCH2CH3) ppm; 13C NMR (151 MHz,
CD3OD): δ=168.37, 165.60, 151.64, 142.23, 109.68, 64.38
(d, J = 167.3 Hz, PC), 62.88 and 62.54 (2× d, J = 7.2 Hz,
2 × POC), 49.79, 35.66 (d, J = 15.9 Hz, PCCC), 30.78
(d, J = 2.7 Hz, PCC),15.41 and 15.36 (2 × d, J = 4.8 Hz,
2 × POCC), 10.78 ppm; 31P NMR (243 MHz, CD3OD):
δ=25.14 ppm.
1
1021, 632 cm−1; H NMR (600 MHz, CDCl3): δ = 7.73
(brs, 2H, HC = CBr, NH), 4.47 (s, 2H, C(O)CH2), 4.22–
4.11 (m, 5H, 2 × POCH2CH3, PCCH), 3.56–3.53 (m,
1H, PCCCHaHb), 3.30 (dt, J = 13.1 Hz, J = 6.4 Hz, 1H,
PCCCHaHb), 2.09–1.97 (m, 2H, PCH2), 1.35 and 1.34
(2×t, J=7.0 Hz, 2×3H, 2×POCH2CH3) ppm; 13C NMR
(151 MHz, CDCl3): δ = 167.20, 160.14, 151.06, 144.87,
96.72, 65.73 (d, J=3.1 Hz, PCC), 62.33 and 62.27 (2×d,
J=6.6 Hz, 2×POC), 50.67, 46.13 (d, J=17.3 Hz, PCCC),
30.93 (d, J = 140.4 Hz, PC), 16.39 and 16.36 (2 × d,
J=6.1 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CDCl3):
δ=29.23 ppm.
Diethyl 3‑[2‑(3,4‑dihydro‑5‑methyl‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑2‑hydroxypropylphosphonate
(9d, C14H24N3O7P) According to the general procedure
from 0.127 g diethyl 3-amino-2-hydroxypropylphospho-
nate (12, 0.601 mmol) and 0.111 g (thymine-1-yl)acetic
acid (13d, 0.601 mmol), the amide 9d (0.113 g, 47% yield)
was obtained as a white solid after purifcation on a silica
gel column with chloroform–methanol mixtures (50:1,
20:1 v/v) and crystallization from a methanol–ethyl acetate
mixture. M.p.: 157–158 °C; IR (KBr): V = 3428, 3186,
3057, 2991, 2936, 2828, 1697, 1651, 1225, 1030 cm−1;
1H NMR (600 MHz, CD3OD): δ = 7.38 (q, J = 1.0 Hz,
1H, HC = CCH3), 4.43 (s, 2H, C(O)CH2), 4.17–4.13 (m,
4H, 2 × POCH2CH3), 4.12–4.08 (m, 1H, PCCH), 3.42
(dd, J = 13.6 Hz, J = 4.7 Hz, 1H, PCCCHaHb), 3.28 (dd,
J = 13.6 Hz, J = 6.7 Hz, 1H, PCCCHaHb), 2.08 (ddd,
J = 18.9 Hz, J = 15.5 Hz, J = 4.7 Hz, 1H, PCHaHb), 2.01
(ddd, J = 17.8 Hz, J = 15.5 Hz, J = 7.4 Hz, 1H, PCHaHb),
1.90 (d, J = 1.0 Hz, 3H, HC = CCH3), 1.36 and 1.35
(2 × t, J = 7.1 Hz, 2 × 3H, 2 × POCH2CH3) ppm;13C NMR
(151 MHz, CD3OD): δ = 168.51, 165.57, 151.68, 142.26,
109.67, 65.13 (d, J=3.6 Hz, PCC), 62.06 and 61.83 (2×d,
J=6.4 Hz, 2×POC), 49.76, 45.83 (d, J=15.4 Hz, PCCC),
30.57 (d, J=140.7 Hz, PC), 15.26 (d, J=6.3 Hz, 2×POCC)
ppm; 31P NMR (243 MHz, CD3OD): δ=29.97 ppm.
Diethyl 2‑[2‑(3,4‑dihydro‑5‑methyl‑2,4‑dioxopyrimi‑
din‑1(2H)‑yl)acetamido]‑1‑hydroxyethylphosphonate (7d,
C13H22N3O7P) According to the general procedure from
0.158 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.801 mmol) and 0.148 g (thymine-1-yl)acetic acid (13d,
0.801 mmol), the amide 7d (0.145 g, 50% yield) was
obtained as a white solid after purifcation on a silica gel
column with chloroform–methanol mixtures (50:1, 20:1
v/v) and crystallization from a methanol–ethyl acetate
mixture. M.p.: 186–187 °C; IR (KBr): V = 3318, 3261,
3043, 2943, 2910, 2830, 1697, 1653, 1245, 1018 cm−1;
1H NMR (200 MHz, CD3OD): δ=7.39 (q, J=1.1 Hz, 1H,
HC = CCH3), 4.47 and 4.44 (AB, J = 16.6 Hz, 2H, C(O)
CHaHb), 4.31–4.14 (m, 4H, 2 × POCH2CH3), 4.08 (ddd,
JH1–H2b = 9.2 Hz, JH1-P = 8.2 Hz, JH1–H2a = 3.6 Hz, 1H,
PCHCH2), 3.73 (ddd, JH2a–H2b = 14.0 Hz, JH2a-P = 7.2 Hz,
JH2a–H1 = 3.6 Hz, 1H, PCCHaHb), 3.38 (ddd ~ dt,
JH2b–H2a = 14.0 Hz, JH2b–H1 = 9.2 Hz, JH2b-P = 8.5 Hz, 1H,
PCCHaHb), 1.92 (d, J= 1.1 Hz, 3H, HC= CCH3), 1.39 (t,
J=7.0 Hz, 6H, 2×POCH2CH3) ppm; 13C NMR (151 MHz,
CD3OD): δ=168.45, 165.54, 151.72, 142.17, 109.78, 65.84
(d, J = 164.3 Hz, PC), 63.04 and 62.73 (2× d, J = 6.9 Hz,
2 × POC), 49.56, 41.11 (d, J = 8.6 Hz, PCC), 15.40 and
15.36 (2×d, J=5.1 Hz, 2×POCC), 10.78 ppm; 31P NMR
(81 MHz, CD3OD): δ=23.74 ppm.
1 3

I. E. Głowacka et al.
Diethyl 2‑[2‑[4‑[(benzyloxycarbonyl)amino]‑2‑oxopyrimi‑
din‑1(2H)‑yl]acetamido]‑1‑hydroxyethylphosphonate (7e,
C20H27N4O8P) According to the general procedure from
0.090 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.456 mmol) and 0.138 g [N4-(benzyloxycarbonyl)cytosine-
1-yl]acetic acid (13e, 0.456 mmol), the amide 7e (0.074 g,
34% yield) was obtained as a colorless oil after purifca-
tion on a silica gel column with chloroform–methanol
mixtures (50:1, 20:1 v/v). IR (flm): V =3279, 3088, 2984,
2931, 1749, 1661, 1215, 1023 cm−1; 1H NMR (200 MHz,
CDCl3): δ=10.39 (brs, 1H, NH), 8.75 (brt, 1H, NH), 7.72
(d, J = 7.4 Hz, 1H, HC = CCH), 7.38–7.28 (m, 6H, C6H5,
HC = CCH), 5.85 (brs, 1H, NH), 5.21 (s, 2H, CH2C6H5),
4.59 and 4.48 (AB, J = 14.9 Hz, 2H, C(O)CHaHb), 4.20–
3.82 (m, 6H, 2×POCH2CH3, PCHCHaHb), 3.58–3.38 (m,
1H, PCCHaHb), 1.31 and 1.20 (2 × t, J = 7.0 Hz, 2 × 3H,
2 × POCH2CH3) ppm;13C NMR (151 MHz, CDCl3):
δ=166.81, 163.67, 156.06, 153.04, 149.30, 135.44, 128.53,
128.37, 128.01, 95.99, 67.54, 66.89 (d, J=161.6 Hz, PC),
63.47 and 62.55 (2×d, J=6.8 Hz, 2×POC), 52.45, 40.79
(d, J=10.1 Hz, PCC), 16.41 (2×d, J=5.4 Hz, 2×POCC)
ppm; 31P NMR (81 MHz, CDCl3): δ=24.42 ppm.
cytosine-1-yl]acetic acid (13e, 0.563 mmol), the amide 9e
(0.100 g, 36% yield) was obtained as a white solid after puri-
fcation on a silica gel column with chloroform–methanol
mixtures (50:1, 20:1 v/v) and crystallization from ethanol.
M.p.: 123–125 °C; IR (KBr): V =3268, 3088, 2981, 2931,
1746, 1666, 1661, 1218, 1027 cm−1; 1H NMR (200 MHz,
CD3OD): δ=7.96 (d, J=7.4 Hz, 1H, HC=CCH), 7.52–7.33
(m, 6H, C6H5, HC=CCH), 5.27 (s, 2H, CH2C6H5), 4.62 (s,
2H, C(O)CH2), 4.25–4.02 (m, 5H, 2×POCH2CH3, PCCH),
3.46 (dd, J=13.9 Hz, J=4.7 Hz, 1H, PCCCHaHb), 3.25 (dd,
J=13.9 Hz, J=7.3 Hz, 1H, PCCCHaHb), 2.21–1.91 (m, 2H,
PCH2), 1.36 (t, J = 7.1 Hz, 6H, 2 × POCH2CH3) ppm; 13C
NMR (151 MHz, CD3OD): δ = 168.05, 164.03, 157.12,
153.16, 150.26, 135.80, 128.21, 128.21, 128.05, 127.89,
127.89, 90.37, 67.21, 65.14 (d, J = 3.6 Hz, PCC), 62.05
and 61.83 (2 × d, J = 6.4 Hz, 2 × POC), 52.02, 45.93 (d,
J=15.4 Hz, PCCC), 30.58 (d, J=140.7 Hz, PC), 15.29 (d,
J=6.0 Hz, 2×POCC) ppm; 31P NMR (81 MHz, CD3OD):
δ=30.88 ppm.
Diethyl 2‑[2‑(3,4‑dihydro‑2,4‑dioxoquinazolin‑1(2H)‑yl)‑
a c e t a m i d o ] ‑ 1 ‑ h y d r ox y e t h y l p h o s p h o n a t e ( 7 f ,
C
16H22N3O7P) According to the general procedure from
Diethyl 3‑[2‑[4‑[(benzyloxycarbonyl)amino]‑2‑oxopyrimi‑
din‑1(2H)‑yl]acetamido]‑1‑hydroxypropylphosphonate (8e,
C21H29N4O8P × 0.5H2O) According to the general procedure
from 0.166 g diethyl 3-amino-1-hydroxypropylphosphonate
(11, 0.787 mmol) and 0.239 g [N4-(benzyloxycarbonyl)
cytosine-1-yl]acetic acid (13e, 0.787 mmol), the amide
8e (0.248 g, 64% yield) was obtained as a white solid
after crystallization from a methanol–diethyl ether mix-
0.158 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.801 mmol) and 0.176 g (3,4-dihydro-2,4-dioxoquinazo-
lin-1-yl)acetic acid (13f, 0.801 mmol), the pure amide 7f
(0.211 g, 66% yield) was obtained as a white powder after a
mere fltration of the reaction mixture. M.p.: 225–226 °C; IR
(KBr): V =3248, 3070, 2986, 2935, 1735, 1637, 1040 cm−1;
1H NMR (600 MHz, CD3OD): δ = 8.06 (dd, J = 7.9 Hz,
J=1.0 Hz, 1H, Haromat.), 7.69 (dt, J=8.4 Hz, J=1.0 Hz, 1H,
Haromat.), 7.27 (dt, J=7.9 Hz, J=0.5 Hz, 1H, Haromat.), 7.22
(d, J=8.4 Hz, 1H, Haromat.), 4.76 and 4.70 (AB, J=16.0 Hz,
2H, C(O)CHaHb), 4.24–4.18 (m, 4H, 2 × POCH2CH3),
4.08 (dt, J = 9.2 Hz, J = 3.7 Hz, 1H, PCHCH2), 3.73
(ddd, J = 13.9 Hz, J = 6.2 Hz, J = 3.7 Hz, 1H, PCCHaHb),
3.37–3.31 (m, 1H, PCCHaHb), 1.37 (t, J = 7.0 Hz, 6H,
2 × POCH2CH3) ppm; 13C NMR (151 MHz, CD3OD):
δ=166.87, 162.78, 150.70, 139.59, 135.06, 127.52, 122.75,
114.90, 114.02, 65.90 (d, J=164.6 Hz, PC), 63.00 and 62.73
(2 × d, J = 6.6 Hz, 2 × POC), 42.44, 41.16 (d, J = 8.8 Hz,
PCC), 15.38 and 15.37 (2×d, J=5.2 Hz, 2×POCC) ppm;
31P NMR (81 MHz, CD3OD): δ=23.05 ppm.
̄
ture. M.p.: 164–165 °C; IR (KBr): V = 3291, 3093, 2977,
1753, 1689, 1657, 1226, 1027 cm−1; 1H NMR (600 MHz,
CD3OD): δ = 7.93 (d, J = 7.3 Hz, 1H, HC = CCH), 7.45–
7.31 (m, 6H, C6H5, HC = CCH), 5.25 (s, 2H, CH2C6H5),
4.59 and 4.54 (AB, J = 15.7 Hz, 2H, C(O)CHaHb), 4.22–
4.15 (m, 4H, 2 × POCH2CH3), 3.97 (ddd, J = 10.7 Hz,
J = 7.4 Hz, J = 3.1 Hz, 1H, PCHCH2), 3.50–3.40 (m, 2H,
PCCCH2), 2.09–1.96 (m, 1H, PCCHaHb), 1.92–1.82 (m,
1H, PCCHaHb), 1.36 (t, J = 7.1 Hz, 6H, 2 × POCH2CH3)
ppm; 13C NMR (151 MHz, CD3OD): δ = 167.91, 164.05,
150.18, 135.81, 128.20, 128.20, 128.04, 127.89, 127.89,
95.35, 67.20, 64.35 (d, J=167.4 Hz, PC), 62.88 and 61.54
(2 × d, J = 7.3 Hz, 2 × POC), 52.01, 35.71 (d, J = 16.2 Hz,
PCCC), 30.75 (d, J=2.7 Hz, PCC), 15.42 and 15.38 (2×d,
J=4.8 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CD3OD):
δ=25.15 ppm.
Diethyl 3‑[2‑(3,4‑dihydro‑2,4‑dioxoquinazolin‑1(2H)‑yl)‑
a ce t a m i d o] ‑ 1 ‑ hyd rox y p ro py l p h o s p h o n ate ( 8 f ,
C17H24N3O7P) According to the general procedure from
0.180 g diethyl 3-amino-1-hydroxypropylphosphonate (11,
0.852 mmol) and 0.187 g (3,4-dihydro-2,4-dioxoquinazolin-
1-yl)acetic acid (13f, 0.852 mmol), the amide 8f (0.168 g,
48% yield) was obtained as a white powder after purifcation
on a silica gel column with chloroform–methanol mixtures
(50:1, 20:1 v/v) and crystallization from an ethanol–diethyl
Diethyl 3‑[2‑[4‑[(benzyloxycarbonyl)amino]‑2‑oxopyrimi‑
din‑1(2H)‑yl]acetamido]‑2‑hydroxypropylphosphonate (9e,
C21H29N4O8P × 0.5H2O) According to the general procedure
from 0.119 g diethyl 3-amino-2-hydroxypropylphosphonate
(12, 0.563 mmol) and 0.171 g [N4-(benzyloxycarbonyl)
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
chloroform–methanol mixtures (50:1, 20:1 v/v). IR (flm):
V =3297, 3090, 2982, 2934, 2873, 1788, 1756, 1693, 1250,
1024 cm−1; 1H NMR (600 MHz, CDCl3): δ=8.87 (s, 1H),
8.29 (s, 1H), 7.32 (t, J = 5.3 Hz, 1H, NH), 5.02 and 4.99
(AB, J = 16.3 Hz, 2H, C(O)CHaHb), 4.60 (t, J = 6.6 Hz,
1H, OH), 4.21–4.17 (m, 4H, 2 × POCH2CH3), 4.10–4.05
(m, 1H, PCHCH2), 3.91 (ddt, J = 14.1 Hz, J = 6.6 Hz,
J=3.7 Hz, 1H, PCCHaHb), 3.44–3.33 (m, 1H, PCCHaHb),
1.48 (s, 18 H, 6 × CH3), 1.36 and 1.35 (2 × t, J = 7.1 Hz,
2×3H, 2×POCH2CH3) ppm; 13C NMR (151 MHz, CDCl3):
δ=166.60, 153.52, 152.08, 150.46, 150.25, 146.08, 128.39,
83.95, 66.61 (d, J=163.0 Hz, PC), 63.23 and 63.08 (2×d,
J = 7.3 Hz, 2 × POC), 49.98, 41.60 (d, J = 5.8 Hz, PCC),
27.79, 16.43 (d, J = 5.5 Hz, 2 × POCC) ppm; 31P NMR
(81 MHz, CDCl3): δ=21.90 ppm.
ether mixture. M.p.: 205–208 °C; IR (KBr): V = 3256,
3205, 3075, 2984, 2954, 1736, 1654, 1635, 1024 cm−1;
1H NMR (600 MHz, CD3OD): δ=8.01(d, J=7.8 Hz, 1H,
Haromat.), 7.69 (dt, J = 8.4 Hz, J = 1.2 Hz, 1H, Haromat.),
7.27 (t, J = 7.8 Hz, 1H, Haromat.), 7.22 (d, J = 8.3 Hz, 1H,
Haromat.), 4.71 and 4.68 (AB, J=16.0 Hz, 2H, C(O)CHaHb),
4.23–4.16 (m, 4H, 2×POCH2CH3), 3.99 (ddd, J=10.7 Hz,
J = 7.7 Hz, J = 3.0 Hz, 1H, PCHCH2), 3.52–3.47 (m, 1H,
PCCCHaHb), 3.41–3.37 (m, 1H, PCCCHaHb), 2.03–1.96
(m, 1H, PCCHaHb), 1.89–1.81 (m, 1H, PCCHaHb), 1.35 (t,
J=7.0 Hz, 6H, 2×POCH2CH3) ppm; 13C NMR (151 MHz,
CD3OD): δ = 168.91, 162.84, 150.86, 139.61, 135.03,
127.51, 122.51, 114.88, 114.06, 64.21 (d, J=167.3 Hz, PC),
62.86 and 62.53 (2×d, J=7.1 Hz, 2×POC), 42.56, 35.49
(d, J=16.0 Hz, PCCC), 30.84 (d, J=3.0 Hz, PCC), 15.41
and 15.38 (2 × d, J = 5.2 Hz, 2 × POCC) ppm; 31P NMR
(243 MHz, CD3OD): δ=25.29 ppm.
Diethyl 3‑[2‑[6‑(tert‑butoxycarbonyl)amino]‑9H‑pu‑
rin‑9‑yl]acetamido]‑1‑hydroxypropylphosphonate (8j,
C19H31N6O7P × 2H2O) According to the general procedure
from 0.180 g diethyl 3-amino-1-hydroxypropylphosphonate
(11, 0.852 mmol) and 0.335 g [6-[bis(tert-butoxycarbonyl)
amino]-9H-purin-9-yl]acetic acid (13g, 0.852 mmol), the
crude product 8g contaminated with triethylamine hydro-
chloride (0.246 g, 49%) was obtained as a yellowish oil
after purifcation on a silica gel column with chloroform–
methanol mixtures (50:1, 20:1 v/v). Further purifcation
with HPLC (water–methanol) gave pure 8j (0.030 g, 7%)
and a 7:3 mixture of 8j and 8g (0.125 g). Yellowish oil;
IR (flm): V = 3289, 3091, 2982, 2935, 1787, 1748, 1247,
1026 cm−1; 1H NMR (600 MHz, CDCl3): δ=8.70 (s, 1H),
8.21 (brs, 1H), 8.12 (s, 1H), 7.64 (brt, J=5.4 Hz, 1H), 4.95
(s, 2H, C(O)CH2), 4.61 (brs, 1H, OH), 4.21–4.12 (m, 4H,
2 × POCH2CH3), 4.03–3.98 (m, 1H), 3.65–3.60 (m, 1H),
3.46–3.41 (m, 1H), 2.09–1.95 (m, 1H), 1.93–1.84 (m,
1H), 1.58 (s, 9H, 3×CH3), 1.33 and 1.32 (2×t, J=7.0 Hz,
2×3H, 2×POCH2CH3) ppm;13C NMR (151 MHz, CDCl3):
δ=166.19, 153.41, 152.19, 150.57, 150.46, 145.55, 128.57,
84.00, 65.99 (d, J=165.2 Hz, PC), 62.93 and 62.89 (2×d,
J=6.3 Hz, 2×POC), 46.40, 36.84 (d, J=15.1 Hz, PCCC),
30.66 (PCC), 27.82, 16.49 (d, J=5.5 Hz, 2×POCC) ppm;
31P NMR (243 MHz, CDCl3): δ=24.32 ppm.
Diethyl 3‑[2‑(3,4‑dihydro‑2,4‑dioxoquinazolin‑1(2H)‑yl)‑
a ce t a m i d o] ‑ 2 ‑ hyd rox y p ro py l p h o s p h o n ate ( 9 f ,
C17H24N3O7P) According to the general procedure from
0.127 g diethyl 3-amino-2-hydroxypropylphosphonate (12,
0.601 mmol) and 0.132 g (3,4-dihydro-2,4-dioxoquinazolin-
1-yl)acetic acid (13f, 0.601 mmol), the amide 9f (0.131 g,
53% yield) was obtained as a white solid after crystallization
from an ethanol–diethyl ether mixture. M.p.: 168–169 °C;
̄
IR (KBr): V =3378, 3249, 3201, 2985, 2946, 1734, 1656,
1
1638, 1020 cm−1; H NMR (200 MHz, CD3OD): δ = 8.08
(ddd, J=8.0 Hz, J=1.0 Hz, J=0.5 Hz, 1H, Haromat.), 7.71
(ddd, J=8.2 Hz, J=7.3 Hz, J=1.5 Hz, 1H, Haromat.), 7.32–
7.20 (m, 2H, Haromat.), 4.74 (s, 2H, C(O)CH2), 4.24–4.03
(m, 5H, 2 × POCH2CH3, PCCH), 3.44 (dd, J = 13.5 Hz,
J = 5.1 Hz, 1H, PCCCHaHb), 3.31 (ddd, J = 13.5 Hz,
J = 6.5 Hz, J = 1.6 Hz, 1H, PCCCHaHb), 2.13 (ddd,
J = 18.8 Hz, J = 15.5 Hz, J = 4.6 Hz, 1H, PCHaHb), 1.99
(ddd, J = 17.7 Hz, J = 15.5 Hz, J = 8.2 Hz, 1H, PCHaHb),
1.36 (t, J = 7.1 Hz, 6H, 2 × POCH2CH3) ppm; 13C NMR
(151 MHz, CD3OD): δ = 168.98, 162.83, 150.87, 139.60,
135.04, 127.51, 122.71, 114.89, 114.05, 65.24 (d, J=4.2 Hz,
PCC), 62.04 and 61.83 (2×d, J=6.2 Hz, 2×POC), 45.82
(d, J=15.6 Hz, PCCC), 42.55, 30.44 (d, J=140.7 Hz, PC),
15.27 (d, J=6.2 Hz, 2×POCC) ppm; 31P NMR (81 MHz,
CD3OD): δ=31.11 ppm.
Diethyl 3‑[2‑[6‑(tert‑butoxycarbonyl)amino]‑9H‑pu‑
rin‑9‑yl]acetamido]‑2‑hydroxypropylphosphonate (9j,
C19H31N6O7P × H2O) According to the general procedure
from 0.123 g diethyl 3-amino-2-hydroxypropylphosphonate
(12, 0.582 mmol) and 0.229 g [6-[bis(tert-butoxycarbonyl)
amino]-9H-purin-9-yl]acetic acid (13g, 0.582 mmol), the
crude product 9g contaminated with triethylamine hydro-
chloride (0.197 g, 57%) was obtained as a colorless oil
after purifcation on a silica gel column with chloroform–
methanol mixtures (100:1, 50:1 v/v). Further purifcation
with HPLC (water–methanol) gave pure 9j (0.019 g, 7%)
Diethyl 2‑[2‑[6‑[bis(tert‑butoxycarbonyl)amino]‑9H‑pu‑
rin‑9‑yl]acetamido]‑1‑hydroxyethylphosphonate (7g,
C23H37N6O9P) According to the general procedure from
0.097 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
0.492 mmol) and 0.290 g [6-[bis(tert-butoxycarbonyl)
amino]-9H-purin-9-yl]acetic acid (13g, 0.492 mmol), the
amide 7g (0.142 g, 50% yield) was obtained as a color-
less oil after purification on a silica gel column with
1 3

I. E. Głowacka et al.
and a 8:2 mixture of 9j and 9g (0.094 g). Colorless oil; IR
(flm): V = 3271, 30,881, 2992, 2955, 1782, 1760, 1234,
1023 cm−1; 1H NMR (200 MHz, CDCl3): δ=8.70 (s, 1H),
8.12 (brs, 1H), 8.09 (s, 1H), 7.48 (brt, J=6.2 Hz, 1H), 4.95
(s, 2H, C(O)CH2), 4.63 (brs, 1H, OH), 4.22–3.99 (m, 5H,
2 × POCH2CH3, PCCH), 3.62–3.46 (m, 1H), 3.37–3.24
(m, 1H), 2.10–1.88 (m, 2H), 1.56 (s, 9H, 3×CH3), 1.30 (t,
J=7.0 Hz, 6H, 2×POCH2CH3) ppm; 13C NMR (151 MHz,
CDCl3): δ=166.39, 152.92, 151.16, 149.98, 149.75, 143.30,
121.37, 82.30, 65.59 (d, J=4.3 Hz, PCC), 62.18 and 62.15
(2 × d, J = 5.5 Hz, 2 × POC), 46.28, 46.04 (d, J = 18.4 Hz,
PCCC), 30.90 (d, J=140.2 Hz, PC), 28.14, 16.37 and 16.33
(2 × d, J = 4.6 Hz, 2 × POCC) ppm; 31P NMR (81 MHz,
CDCl3): δ=30.03 ppm.
ppm; 13C NMR (151 MHz, DMSO-d6): δ=166.56, 160.28,
154.88, 149.69, 144.56, 123.57, 64.46 (d, J=164.6 Hz, PC),
62.28 and 61.94 (2×d, J=6.8 Hz, 2×POC), 45.505, 36.08
(d, J=17.1 Hz, PCCC), 31.60 (d, J=2.6 Hz, PCC), 16.86
and 16.83 (2 × d, J = 5.0 Hz, 2 × POCC) ppm; 31P NMR
(243 MHz, DMSO-d6): δ=24.74 ppm.
Diethyl 3‑[2‑(2‑amino ‑6‑ chloro ‑9H‑purin‑9‑yl)‑
acetamido]‑2‑hydrox ypropylphosphonate (9h,
C14H22ClN6O5P × 0.25H2O) According to the general proce-
dure from 0.485 g diethyl 3-amino-2-hydroxypropylphos-
phonate (12, 2.30 mmol) and 0.523 g (2-amino-6-chloropu-
rin-9-yl)acetic acid (13h, 2.30 mmol), the amide 9h (0.210 g,
22%) was obtained as a white powder after purifcation on a
silica gel column with chloroform–methanol mixtures (50:1,
20:1, 10:1 v/v) and crystallization from an ethanol–diethyl
ether mixture. M.p.: 163–164 °C; IR (KBr): V =3399, 3280,
3245, 3091, 2981, 2936, 2907, 1690, 1614, 1224, 1031,
868 cm−1; 1H NMR (600 MHz, CD3OD): δ=8.07 (s, 1H),
4.92 and 4.88 (AB, J=16.6 Hz, 2H, C(O)CH2), 4.18–4.06
(m, 5H, 2 × POCH2CH3, PCCH), 3.42 (dd, J = 13.7 Hz,
J = 4.8 Hz, 1H, PCCCHaHb), 3.31 (ddd, J = 13.7 Hz,
J=6.5 Hz, J=1.0 Hz, 1H, PCCCHaHb), 2.11–1.98 (m, 2H,
PCH2), 1.34 (t, J = 7.0 Hz, 6H, 2 × POCH2CH3) ppm;13C
NMR (151 MHz, CD3OD): δ = 167.58, 160.31, 154.18,
150.20, 143.88, 123.28, 65.13 (d, J=3.5 Hz, PCC), 62.08
and 61.83 (2×d, J=6.6 Hz, 2×POC), 45.85 (d, J=15.3 Hz,
PCCC), 45.12, 30.62 (d, J = 140.9 Hz, PC), 15.25 (2 × d,
J=5.9 Hz, 2×POCC) ppm; 31P NMR (243 MHz, CD3OD):
δ=29.94 ppm.
Diethyl 2‑[2‑(2‑amino ‑6‑ chloro ‑9H‑purin‑9‑yl)‑
a c e t a m i d o ] ‑ 1 ‑ hyd r ox ye t hy l p h o s p h o n a t e ( 7 h ,
C13H20ClN6O5P) According to the general procedure from
0. 322 g diethyl 2-amino-1-hydroxyethylphosphonate (10,
1.63 mmol) and 0.372 g (2-amino-6-chloropurin-9-yl)acetic
acid (13h, 1.63 mmol), the amide 7h (0.278 g, 27%) was
obtained as a white powder after purifcation on a silica
gel column with chloroform–methanol mixtures (50:1,
20:1, 10:1 v/v). M.p.: 206–208 °C; IR (KBr): V = 3457,
3347, 3212, 3044, 2991, 2930, 1672, 1632, 1565, 1249,
1
1027, 705 cm−1; H NMR (200 MHz, CD3OD): δ = 8.10
(s, 1H), 4.94–4.88 (m, 2H, C(O)CHaHb), 4.30–4.13 (m,
4H, 2 × POCH2CH3), 4.08 (ddd, J = 9.1 Hz, J = 7.9 Hz,
J=3.7 Hz,1H, PCHCH2), 3.72 (ddd, J=14.0 Hz, J=7.3 Hz,
J=3.7 Hz, 1H, PCCHaHb), 3.48–3.37 (m, 1H, PCCHaHb),
1.38 (t, J = 7.0 Hz, 6H, 2 × POCH2CH3) ppm; 13C NMR
(151 MHz, CD3OD): δ = 167.55, 160.31, 154.17, 150.18,
143.87, 123.23, 65.80 (d, J=164.6 Hz, PC), 63.05 and 62.75
(2 × d, J = 7.3 Hz, 2 × POC), 41.17, 41.13 (d, J = 8.5 Hz,
PCC), 15.40 and 15.73 (2×d, J=5.2 Hz, 2×POCC) ppm;
31P NMR (81 MHz, CD3OD): δ=23.67 ppm.
Antiviral activity assays
The compounds were evaluated against diferent herpes
viruses, including herpes simplex virus type 1 (HSV-1)
strain KOS, thymidine kinase-defcient (TK−) HSV-1 KOS
strain resistant to ACV (ACVr), herpes simplex virus type 2
(HSV-2) strain G, varicella-zoster virus (VZV) strain Oka,
TK− VZV strain 07-1, human cytomegalovirus (HCMV)
strains AD-169 and Davis as well as vaccinia virus, adeno
virus-2, human coronavirus, para-infuenza-3 virus, reo-
virus-1, Sindbis virus, Coxsackie virus B4, Punta Toro
virus, respiratory syncytial virus (RSV) and infuenza A
virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and
infuenza B virus (B/HK/5/72), were based on inhibition of
virus-induced cytopathicity or plaque formation in human
embryonic lung (HEL) fbroblasts, African green monkey
kidney cells (Vero), human epithelial cervix carcinoma cells
(HeLa) or Madin Darby canine kidney cells (MDCK). Con-
fuent cell cultures in microtiter 96-well plates were inocu-
lated 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) and the cell cultures were incubated
Diethyl 3‑[2‑(2‑amino ‑6‑ chloro ‑9H‑purin‑9‑yl)‑
acetamido]‑1‑hydrox ypropylphosphonate (8h,
C14H22ClN6O5P × 0.75H2O) According to the general proce-
dure from 0. 366 g diethyl 3-amino-1-hydroxypropylphos-
phonate (11, 1.71 mmol) and 0.389 g (2-amino-6-chlo-
ropurin-9-yl)acetic acid (13h, 1.71 mmol), the amide 8h
(0.099 g, 14%) was obtained as a white powder after puri-
fcation on a silica gel column with chloroform–methanol
mixtures (50:1, 20:1, 10:1 v/v) and crystallization from
methanol. M.p.: 183–184 °C; IR (KBr): V = 3401, 3278,
3213, 3089, 2980, 2935, 2864, 1690, 1631, 1223, 1031,
971 cm−1; 1H NMR (600 MHz, DMSO-d6): δ=8.05 (s, 1H),
6.90 (brs, 2H, NH2) 5.50 (t, J=7.0 Hz, 1H, OH), 4.73 (s, 2H,
C(O)CH2), 4.11–3.94 (m, 4H, 2×POCH2CH3), 3.87–3.71
(m, 1H, PCH), 3.45–3.25 (m, 2H, PCCCHaHb), 1.85–1.50
(m, 2H, PCCH2), 1.23 (t, J=7.0 Hz, 6H, 2×POCH2CH3)
1 3

Acyclic nucleoside phosphonates containing the amide bond: hydroxy derivatives
4. Magee WC, Evans DH (2012) Antiviral Res 96:169
5. Głowacka IE, Piotrowska DG, Andrei G, Schols D, Snoeck R,
Wróblewski AE (2016) Monatsh Chem 147:2163
6. Holm L, Bockermann R, Wellner E, Baecklund J, Holmdahl R,
Kihlberg J (2006) Bioorg Med Chem 14:5921
in the presence of varying concentrations of the test com-
pounds. Viral cytopathicity 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 EC50
or compound concentration required reducing virus-induced
cytopathicity or viral plaque formation by 50%. Cytotoxicity
of the test compounds was expressed as the minimum cyto-
toxic concentration (MCC) or the compound concentration
that caused a microscopically detectable alteration of cell
morphology.
7. Blomberg D, Kreye P, Fowler CS, Brickmann K, Kihlberg J
(2006) Org Biomol Chem 4:416
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Chem 70:703
Cytostatic activity assays
All assays were performed in 96-well microtiter plates. To
each well were added (5–7.5)×104 tumor cells and a given
amount of the test compound. The cells were allowed to pro-
liferate at 37 °C in a humidifed CO2-controlled atmosphere.
At the end of the incubation period, the cells were counted
in a Coulter counter. The IC50 (50% inhibitory concentra-
tion) was defned as the concentration of the compound that
inhibited cell proliferation by 50%.
15. Grifn CE, Kundu SK (1969) J Org Chem 34:1532
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Acknowledgements Financial supports from the National Science
Centre (Grant UMO-2013/09/B/NZ7/00729—synthetic part of the
project) and the KU Leuven (GOA 15/19 TBA—biological screening)
are gratefully acknowledged. The Faculty of Pharmacy authors wish
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Pluskota for excellent technical assistance. Special thanks are for-
warded to the Rega Institute collaborators: Mrs. Leentje Persoons, Mrs.
Ellen De Waegenaere, Mrs. Bianca Stals, Mrs. Kirsten Lepaige, Mr.
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