Synthesis, antiviral, cytotoxic and cytostatic evaluation of N1-(Phosphonoalkyl)uracil derivatives

Article abstract of DOI:10.1007/s00706-016-1701-2

A series of N¹-(phosphonoalkyl)uracils was prepared in a two-step reaction sequence from x- aminoalkylphosphonates and (E)-3-ethoxyacryloyl isocyanate followed by the uracil ring closure. Under standard conditions (NCS; NBS; I₂/CAN) all N¹-(phosphonoalkyl)uracils were transformed into the respective 5-halogeno derivatives to be later benzoylated at N3. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses. One compound was slightly active against human cytomegalovirus in HEL cell cultures (EC₅₀ = 45 μM) while another showed weak activity against varicella-zoster virus (TK⁺ VZV strain OKA and TK^- VZV strain 07-1) with EC₅₀ = 43 and 53 μM, respectively. In addition, several compounds exhibited noticeable inhibitory effects on the proliferation of human cervical carcinoma cells (HeLa) at a concentration lower than 200 μM.

Full text of DOI:10.1007/s00706-016-1701-2

Monatsh Chem (2016) 147:1081–1090
DOI 10.1007/s00706-016-1701-2
ORIGINAL PAPER
Synthesis, antiviral, cytotoxic and cytostatic evaluation
of N1-(phosphonoalkyl)uracil derivatives
Dorota Rygielska-Tokarska1 Graciela Andrei2 Dominique Schols2
•
•
•
Robert Snoeck2 Iwona E. Głowacka1
•
Received: 17 December 2015 / Accepted: 9 February 2016 / Published online: 1 March 2016
Ó Springer-Verlag Wien 2016
Abstract A series of N1-(phosphonoalkyl)uracils was
Introduction
prepared in a two-step reaction sequence from x-
Acyclic nucleoside and nucleotide analogues have been
aminoalkylphosphonates and (E)-3-ethoxyacryloyl iso-
cyanate followed by the uracil ring closure. Under standard
conditions (NCS; NBS; I2/CAN) all N1-(phospho-
noalkyl)uracils were transformed into the respective
5-halogeno derivatives to be later benzoylated at N3. All
compounds were evaluated in vitro for activity against a
broad variety of DNA and RNA viruses. One compound
was slightly active against human cytomegalovirus in HEL
cell cultures (EC50 = 45 lM) while another showed weak
activity against varicella-zoster virus (TK? VZV strain
OKA and TK- VZV strain 07-1) with EC50 = 43 and
53 lM, respectively. In addition, several compounds
exhibited noticeable inhibitory effects on the proliferation
of human cervical carcinoma cells (HeLa) at a concentra-
tion lower than 200 lM.
used in clinical practice for almost 50 years and have
become cornerstones in the treatment of patients with viral
infections [1–3]. Nucleoside analogues such as acyclovir,
ganciclovir, and penciclovir as well as acyclic nucleoside
phosphonates (ANPs), namely adefovir, cidofovir, and
tenofovir, belong to the most widely used antiviral drugs
(Fig. 1) [4–6]. The ribose moiety in natural nucleosides/
nucleotides was replaced by linear or branched aliphatic
units to obtain analogues with specific activity following
activation by viral and/or cellular kinases. The antiviral
activity of these compounds relies on enzyme inhibition or
on incorporation as active metabolites into the viral nucleic
acids resulting in inhibition of viral genome replication.
In the last decade, several new drugs, including nucle-
oside and nucleotide analogues, have been approved for the
treatment of viral infections and in cancer chemotherapy.
Many nucleoside/nucleotide analogues and their derivatives
are currently under preclinical and clinical trials [7] which
demonstrates how rapidly this area of research grows.
However, some of these analogues have poor oral
bioavailability and are associated with toxicity, limiting their
application for treatment of cancer and viral infections.
Moreover, a long-term treatment with antiviral drugs may
result in lower sensitivity of the viruses to chemotherapeu-
tics (drug resistance) [8, 9]. For this reason, a search for new
compounds with higher antiviral and antitumor activities,
good bioavailability, better solubility, and proper balance
between efficacy and long-term toxicity still continues.
The known synthetic strategies to ANPs commonly
apply alkylation of heterocyclic bases [10] while the con-
struction of nucleobase skeletons from the appropriate
terminal primary amines has been less frequently used [11–
Graphical abstract
Keywords Nucleotides Á Halogenation Á
Antiviral activity Á NMR spectroscopy Á Cyclizations
& Iwona E. Głowacka
iwona.glowacka@umed.lodz.pl
1
Bioorganic Chemistry Laboratory, Faculty of Pharmacy,
´
Medical University of Lodz, Muszynskiego 1, 90-151 Lodz,
Poland
2
Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, 3000 Louvain, Belgium
123

1082
D. Rygielska-Tokarska et al.
Fig. 1 Acyclic nucleoside and
nucleotide analogues
aminoalkylphosphonates 2b–2d were synthesized from the
corresponding x-azidoalkylphosphonates 1b–1d [28–32] by
catalytic hydrogenation [23, 26]. However, several literature
reports [33, 34] noticed the formation of significant amounts
of symmetrical secondary amines as by-products during
hydrogenolysis of azides depending on the azide concentra-
tion and the azide to catalyst ratio. When x-
azidophosphonates1b–1d weresubjected to hydrogenationin
the presence of 10 % Pd–C, symmetrical secondary amines
3b–3d contaminated (11–33 %) the major phosphonates 2b–
2d as judged from the 31P NMR spectra (Scheme 2). The
mixtures of x-aminoalkylphosphonates 2b–2d containing
various amounts of symmetrical secondary amines 3b–3d
were separated on silica gel columns.
Scheme 1
Alkylation of nucleobases with substituted alkylphos-
phonates is generally recognized as a capricious process
since dealkylation of phosphonate esters accompany the
formation of a carbon-nucleobase bond and this was the
primary reason to replace O,O-dimethyl and O,O-diethyl
esters with O,O-diisopropyl phosphonates [35, 36]. Based on
this experience and that of other groups, we concluded that
the alternative approach to independently construct the
pyrimidine ring through functionalization of x-
aminoalkylphosphonates is more feasible than the alkylation.
The N1-(phosphonoalkyl)uracils 5a–5d were synthe-
sized from x-aminoalkylphosphonates 2b–2d in a two-step
procedure which involved reaction with (E)-3-ethoxy-
acryloyl isocyanate [37] in situ generated from (E)-3-
ethoxyacryloyl chloride [38–40] and silver cyanate fol-
lowed by the uracil ring closure in the presence of 2 M
H2SO4 (Scheme 3) [18, 39, 40].
15]. Herein, we report on the efficient synthesis and bio-
logical evaluation of a new series of acyclic nucleotide
analogues by application of the latter approach (Scheme 1).
Results and discussion
x-Aminoalkylphosphonates can be prepared by several
methods. Arbuzov reaction [16] of N-(x-bromoalkyl)ph-
thalimides with trialkyl phosphites seems to be most
frequently applied. Alternatively, x-aminoalkylphospho-
nates can be synthesized from nitriles [17] or from x-
azidophosphonates employing hydrogenolysis [18].
Diethyl aminoalkylphosphonates 2a–2d (aminomethyl-,
2-aminoethyl-, 3-aminopropyl-, and 4-aminobutylphospho-
nates), used in this study, are known and have already been
described in the literature [19–27]. Thus, diethyl
aminomethylphosphonate (2a) was prepared in total 90 %
yield from N-(bromomethyl)phthalimide followed by the
treatment with hydrazine [19–21] whereas x-
The conversion of uracil phosphonates 5a–5d into
5-chlorouracils 6a–6d and 5-bromouracils 8a–8d was
achieved by treatment with N-chlorosuccinimide (NCS)
[41] and N-bromosuccinimide (NBS) [41] respectively,
Scheme 2
Reagents and conditions: a. H2, 10% Pd-C, EtOH, r.t. 20 h
123

Synthesis, antiviral, cytotoxic and cytostatic evaluation of N1-(phosphonoalkyl)uracil…
1083
based assays: (a) human embryonic lung (HEL) cells:
herpes simplex virus-1 (KOS), herpes simplex virus-2 (G),
herpes simplex virus-1 (TK- ACVr KOS), vaccinia virus
and vesicular stomatitis virus, human cytomegalovirus
(AD-169 strain and Davis strain), varicella-zoster virus
(TK? VZV strain OKA and TK- VZV strain 07-1);
(b) CEM cell cultures: human immunodeficiency virus
(HIV-1 and HIV-2); (c) Vero cell cultures: para-influenza-3
virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta
Toro virus; (d) HeLa cell cultures: vesicular stomatitis
virus, Coxsackie virus B4 and respiratory syncytial virus
(RSV); (e) Crandell-Rees Feline Kidney (CRFK) cell cul-
tures: feline corona virus (FIPV) and feline herpes virus
(FHV); (f) Madin Darby Canine Kidney (MDCK) cell
cultures: influenza A virus H1N1 subtype (A/PR/8), influ-
enza A virus H3N2 subtype (A/HK/7/87) and influenza B
virus (B/HK/5/72). Ganciclovir, cidofovir, acyclovir, bri-
vudin, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA],
Hippeastrum hybrid agglutinin (HHA), Urtica dioica
agglutinin (UDA), dextran sulfate (molecular weight 5000,
DS-5000), ribavirin, oseltamivir carboxylate, amantadine,
and rimantadine were used as the reference compounds.
The antiviral activity was expressed as the EC50: the
compound concentration required to reduce virus-plaque
formation (VZV) or virus-induced cytopathogenicity by
50 % (other viruses). It was found that the acyclic phos-
phonate analogues 10a and 10d containing the N3-
benzoyluracil group as a modified nucleobase exhibited
noticeable activity. Thus, compound 10a showed activity
against cytomegalovirus (AD-169 strain) in human
embryonic lung (HEL) cells (EC50 = 45 lM), whereas
compound 10d proved to be active against VZV (TK?
strain OKA and TK- strain 07-1) with EC50 = 43 and
53 lM, respectively. The antiviral activity of compound
10d against VZV TK- strain 07-1 appeared to be slightly
better than the activity of acyclovir and brivudin used as
reference compounds (EC50 = 160 and 103 lM, respec-
tively). To rationalize the observed activity of the N3-
benzoyl derivatives 10a and 10d one can refer to their
easier transport through cell membranes in comparison to
the precursors 5a and 5d which appeared inactive. After
entering into the cell debenzoylation would provide
nucleoside analogues capable of Watson-Crick bas pairing.
Our previous results [44] support this conclusion.
Scheme 3
Reagents and conditions: a. (E)-3-ethoxyacryloyl isocyanate, DMF,
0 °C → r.t, 12 h b. 2M H2SO4, dioxane, reflux, 20 h
Scheme 4
Reagents and conditions: a. benzoyl chloride, NEt3, CH2Cl2, 0 °C to
r.t., 72 h. b. NCS, pyridine, 100 °C, 0.5 h. c. NBS, pyridine, 100 °C, 0.5 h. d. I2,
CAN, CH3CN, reflux, 2 h
whereas 5-iodouracils 9a–9d were synthesized using iodine
and cerium(IV) ammonium nitrate (CAN) [42]
(Scheme 4). Although iodination and bromination of ura-
cils 5a–5d proceeded regioselectively at C5, chlorination
of uracil phosphonates 5c, 5d under the applied conditions
provided mixtures of C5 and C6 regioisomers 7c, 7d in an
approximately 8:2 ratio.
Moreover, based on the literature report [43] and on our
previous observations regarding the effect of the benzoyl
substituent at N-3 of a nucleobase on the antiviral activity [29,
44], the N3-benzoyluracil analogues 10a–10d were synthe-
sized from phosphonates 5a–5d as shown on Scheme 4.
Structures and purity of all synthesized compounds were
The cytotoxicity of the test compounds toward unin-
fected host cells was defined as the minimum cytotoxic
established by H, 31P and 13C NMR and IR techniques as
1
well as by elemental analysis.
concentration (MCC) that causes
a microscopically
detectable alteration of normal cell morphology. The 50 %
cytotoxic concentration (CC50), causing a 50 % decrease in
cell viability was determined using a colorimetric 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) assay system. None of
the tested compounds affected cell morphology of HEL,
Antiviral activity and cytotoxic evaluation
All the synthesized compounds 5a–5d, 6a–6d, and 8a–10d
were evaluated for their antiviral activities against a wide
variety of DNA and RNA viruses, using the following cell-
123

1084
D. Rygielska-Tokarska et al.
HeLa, Vero, MDCK, and CRFK cells (MCC or CC50) at
well as N3-benzoyluracils. All synthesized compounds were
tested in vitro for their antiviral activities against a broad
variety of DNA and RNA viruses and compound 10a was
slightly active against human cytomegalovirus in HEL cell
cultures (EC50 = 45 lM) while 10d showed activity against
both TK? and TK-VZV strains with EC50 values of 43 lM
(Oka strain) and of 53 lM (07-1 strain), which was two to
threefold better than the potency of acyclovir and brivudin
against 07-1 (EC50 = 160 and 103 lM, respectively).
Among them, several compounds exhibited a very poor
inhibitory effect on the proliferation of human cervical
carcinoma cells (HeLa) at a concentration lower than
200 lM. In contrast, none of the compounds affected the
proliferation of CEM cells and L1210 cells up to a con-
centration of 250 lM.
compound concentrations up to 100 lM.
Evaluation of cytostatic activity
The cytostatic activity of the tested compounds was defined
as the 50 % inhibitory concentration (IC50), or compound
concentration causing a 50 % decrease in cell proliferation.
IC50 values were determined against murine leukemia L1210
cells, human lymphocyte CEM cells and human cervical
carcinoma HeLa cells. Several synthesized compounds
showed marginal inhibitory effect on the proliferation of
HeLa cells at concentrations lower than 200 lM (Table 1).
Conclusion
A series of N1-(phosphonoalkyl)uracils 5a–5d has been
efficiently obtained from x-aminoalkylphosphonates 2a–2d
in a two-step process which involved a reaction with (E)-3-
ethoxyacryloyl isocyanate followed by the uracil ring clo-
sure. The uracil phosphonates 5a–5d were successfully
transformed into 5-chloro-, 5-bromo-, and 5-iodouracils as
Experimental
1H NMR was taken in CDCl3 on the following spectrom-
eters: Varian Mercury-300 and Bruker Avance III
(600 MHz) with TMS as an internal standard; chemical
shifts d in ppm with respect to TMS; coupling constants
J in Hz. 13C NMR spectra were recorded for CDCl3 solu-
tion on the Varian Mercury-300 and Bruker Avance III
(600 MHz) spectrometer at 75.5 and 151 MHz, respec-
tively. 31P NMR spectra were taken in CDCl3 on the
Varian Mercury-300 and Bruker Avance III (600 MHz)
spectrometer at 121.5 and 243 MHz, respectively. IR
spectra were measured on an Infinity MI-60 FT-IR spec-
trometer as KBr pellets or as films, and absorptions are
given in cm-1. Melting points were determined on a
Boetius apparatus. Elemental analyses were performed by
Microanalytical Laboratory of this Faculty on Perkin Elmer
PE 2400 CHNS analyzer and their results were found to be
in good agreement ( 0.3 %) with the calculated values.
The following adsorbents were used: column chromatog-
raphy, Merck silica gel 60 (70–230 mesh); analytical TLC,
Table 1 Inhibitory effect of the tested compounds against the pro-
liferation of murine leukemia (L1210), human T-lymphocyte (CEM),
and human cervix carcinoma cells (HeLa)
Compounds
ICa50 (lM)
L1210
CEM
HeLa
5a
[250
[250
[250
[250
C250
[250
[250
[250
[250
[250
[250
[250
[250
[250
C250
[250
[250
[250
C250
[250
0.33 0.17
[250
[250
[250
[250
C250
[250
[250
[250
[250
[250
[250
[250
C250
[250
[250
[250
200 66
[250
C250
[250
213 52
C250
6a
8a
C250
9a
208 59
10a
130
158
8
1
5b
6b
195 78
195 78
233 23
117 81
99 51
8b
9b
Merck TLC plastic sheets silica gel 60 F254
.
10b
Applied reagents such as silver cyanate (Alfa Aesar, cata-
logue number: 45411), CAN (TokyoChemical Industry(TCI)
catalogue number: C1806) and inorganic reagents are com-
mercially available and were used as received. Solvents were
dried according to the literature methods. (E)-3-Ethoxyacry-
loyl chloride was obtained according to the known protocol
[18]. Diethyl aminomethylphosphonate (2a) was prepared
according to literature (colorless oil, yield 90 %) [19–24].
5c
6c
100 55
90 56
8c
9c
129 97
117 61
126 87
97 69
10c
5d
6d
8d
C250
9d
199 73
166 118
0.54 0.12
General procedure for the preparation
of aminoalkylphosphonates 2b–2d
10d
5-Fluorouracil
18
5
a
The x-azidoalkylphosphonates 1b–1d (1.0 mmol) were
dissolved in 10 cm3 ethanol and 10 % Pd–C (16 mg) was
50 % Inhibitory concentration or compound concentration required
to inhibit tumor cell proliferation by 50 %
123

Synthesis, antiviral, cytotoxic and cytostatic evaluation of N1-(phosphonoalkyl)uracil…
1085
added. The suspension was stirred under hydrogen atmo-
sphere (balloon) at room temperature for 20 h. The catalyst
was filtered off through a layer of Celite and the solvent
was evaporated to give a mixture of x-aminoalkylphos-
phonates 2b–2d and 3b–3d (ca. 8:2). The crude products
were chromatographed on silica gel columns with chloro-
form–methanol mixtures (50:1, 20:1, 10:1, v/v) to give
pure x-aminoalkylphosphonates 2b–2d and pure com-
pounds 3b–3d.
previously dried in vacuo for 2 h with protection from light.
After 45 min, the mixture was allowed to cool and then was
transferred via cannula to a solution of the respective x-
aminoalkylphosphonate 2 (2.50 mmol) in 10 cm3 dry DMF
at 0 °C. Solid AgCl was washed with dry toluene
(2 9 8 cm3) to remove the residual 3-ethoxyacryloyl iso-
cyanate which was added to the reaction flask. The reaction
mixture was stirred at room temperature overnight. The solid
residue was filtered through a layer of Celite and the solvent
was evaporated. The crude product was chromatographed on
a silica gel column with chloroform–methanol mixtures
(100:1, 20:1 v/v) to afford phosphonates 4a–4d.
Diethyl 2-aminoethylphosphonate (2b) [22–24]
Colorless oil; yield 80 %.
Bis[2-(O,O-diethylphosphoryl)ethyl]amine (3b) [45]
Pale yellow oil; yield 11 %.
Diethyl (E)-[3-(3-ethoxyacryloyl)ureido]methylphospho-
nate (4a, C11H21N2O6P)
Creamy solid; yield 79 %; m.p.: 56–58 °C; IR (KBr):
Diethyl 3-aminopropylphosphonate (2c) [19, 24–26]
Yellowish oil; yield 80 %.
ꢀ
m = 3428, 3239, 3133, 2984, 2936, 1684, 1617, 1563,
1
1304, 1229, 1168, 1120, 1054, 1023, 979, 807 cm-1; H
Bis[3-(O,O-diethylphosphoryl)propyl]amine
NMR (300 MHz, CDCl3): d = 10.00 (br s, 1H, NH), 9.01
(dt, J = 5.9, 1.9 Hz, 1H, HNCH2), 7.66 (d, J = 12.3 Hz,
1H, EtOCH=CH), 5.39 (d, J = 12.3 Hz, 1H, EtOCH=CH),
4.22–4.12 (m, 4H, 2 9 POCH2CH3), 3.97 (q, J = 6.9 Hz,
2H, OCH2CH3), 3.77 (dd, J = 11.9, 5.9 Hz, 2H, CH2P),
1.37 (t, J = 6.9 Hz, 3H, OCH2CH3), 1.34 (t, J = 7.2 Hz,
6H, 2 9 POCH2CH3) ppm; 13C NMR (75.5 MHz, CDCl3):
d = 168.3 (s, C=O), 162.9 (s, EtOCH=CH), 155.5 (d,
J = 6.6 Hz, C=O), 98.0 (s, EtOCH=CH), 67.7 (s, OCH2-
CH3), 62.7 (d, J = 6.3 Hz, POC), 35.3 (d, J = 157.2 Hz,
CP), 16.6 (d, J = 5.7 Hz, POCC), 14.7 (s, OCH2CH3)
ppm; 31P NMR (121.5 MHz, CDCl3): d = 23.25 ppm.
(3c, C14H33NO6P2)
ꢀ
Pale yellow oil; yield 13 %; IR (film): m = 3436, 2984,
2936, 2910, 1648, 1227, 1053, 1027, 964 cm-1; H NMR
1
(600 MHz, CDCl3): d = 4.14–4.04 (m, 8H, 4 9 POCH2-
CH3), 2.68 (t, J = 6.4 Hz, 4H, 2 9 CH2CH2CH2P), 2.03
(br s, 1H, NH), 1.82–1.73 (m, 8H, 2 9 CH2CH2P), 1.32 (t,
J = 7.1 Hz, 12H, 4 9 POCH2CH3) ppm; 13C NMR
(151 MHz, CDCl3): d = 61.5 (d, J = 6.4 Hz, POC), 49.7
(d, J = 16.7 Hz, CCCP), 23.4 (d, J = 142.1 Hz, CP), 22.8
(d, J = 4.7 Hz, CCP), 16.4 (d, J = 5.7 Hz, POCC) ppm;
31P NMR (243 MHz, CDCl3): d = 32.20 ppm.
Diethyl 4-aminobutylphosphonate (2d) [19, 27]
Yellowish oil; yield 70 %.
Diethyl (E)-2-[3-(3-ethoxyacryloyl)ureido]ethylphospho-
nate (4b, C12H23N2O6P)
Bis[4-(O,O-diethylphosphoryl)butyl]amine
Colorless solid; yield 65 %; m.p.: 61–63 °C; IR (KBr):
(3d, C16H36NO6P2)
ꢀ
m = 3297, 3231, 3149, 3102, 2984, 2938, 1705, 1680, 1618,
1536, 1227, 1164, 1032, 965, 846 cm-1 1H NMR
;
ꢀ
Pale yellow oil; yield 23 %; IR (film): m = 3441, 2982, 2936,
1
1642, 1230, 1052, 1026, 962 cm-1; H NMR (600 MHz,
(300 MHz, CDCl3): d = 9.81 (br s, 1H, NH), 8.92 (t,
J = 5.7 Hz, 1H, HNCH2), 7.64 (d, J = 12.3 Hz, 1H,
EtOCH=CH), 5.38 (d, J = 12.3 Hz, 1H, EtOCH=CH),
4.21–4.00 (m, 4H, 2 9 POCH2CH3), 3.97 (q, J = 7.2 Hz,
2H, OCH2CH3), 3.58 (dt, J = 14.7, 7.2 Hz, 2H, CH2CH2P),
2.08 (dt, J = 18.0, 7.2 Hz, 2H, CH2P), 1.36 (t, J = 7.2 Hz,
3H, OCH2CH3), 1.34 (t, J = 7.2 Hz, 6H, 2 9 POCH2CH3)
ppm; 13C NMR (75.5 MHz, CDCl3): d = 168.1 (s, C=O),
162.7 (s, EtOCH=CH), 155.4 (s, C=O), 98.2 (s,
EtOCH=CH), 67.6 (s, OCH2CH3), 62.0 (d, J = 6.6 Hz,
POC), 34.2 (d, J = 3.1 Hz, CCP), 26.5 (d, J = 139.1 Hz,
CP), 16.7 (d, J = 6.0 Hz, POCC), 14.8 (s, OCH2CH3) ppm;
31P NMR (121.5 MHz, CDCl3): d = 29.76 ppm.
CDCl3): d = 4.13–4.03 (m, 8H, 4 9 POCH2CH3), 2.59 (t,
J = 7.1 Hz, 4H, 2 9 CH2CH2CH2CH2P), 2.00 (br s, 1H,
NH), 1.76–1.70 (m, 4H, 2 9 CH2P), 1.66–1.54 (m, 8H,
2 9 CH2CH2CH2P), 1.31 (t, J = 7.1 Hz, 12H, 4 9 POCH2-
CH3) ppm; 13C NMR (151 MHz, CDCl3): d = 61.4 (d,
J = 6.5 Hz, POC), 49.3 (s, CCCCP), 30.9 (d, J = 16.0 Hz,
CCCP), 25.6 (d, J = 140.9 Hz, CP), 20.3 (d, J = 5.2 Hz,
CCP), 16.4 (d, J = 6.2 Hz, POCC) ppm; 31P NMR
(243 MHz, CDCl3): d = 32.07 ppm.
General procedure for the preparation
of acryloylureas 4a–4d
Diethyl (E)-3-[3-(3-ethoxyacryloyl)ureido]propylphospho-
A
solution of 1.009 g 3-ethoxyacryloyl chloride
nate (4c, C13H25N2O6P)
(7.50 mmol) in 20 cm3 dry toluene was refluxed under an
ꢀ
Yellow oil; yield 73 %; IR (film): m = 3285, 3140, 2981,
1681, 1613, 1548, 1242, 1167, 1105, 1027, 966 cm-1; H
1
argon atmosphere with 2.173 g silver cyanate (14.50 mmol)
123

1086
D. Rygielska-Tokarska et al.
1
NMR (300 MHz, CDCl3): d = 9.67 (br s, 1H, NH), 8.76 (t,
J = 6.0 Hz, 1H, HNCH2), 7.64 (d, J = 12.3 Hz, 1H,
EtOCH=CH), 5.38 (d, J = 12.3 Hz, 1H, EtOCH=CH),
4.16–4.04 (m, 4H, 2 9 POCH2CH3), 3.98 (q, J = 7.0 Hz,
2H, OCH2CH3), 3.38 (dt, J = 12.6, 6.0 Hz, 2H, CH2CH2-
CH2P), 1.94–1.72 (m, 4H, CH2CH2P), 1.36 (t, J = 7.0 Hz,
3H, OCH2CH3), 1.32 (t, J = 7.0 Hz, 6H, 2 9 POCH2CH3)
ppm; 13C NMR (75.5 MHz, CDCl3): d = 168.4 (s, C=O),
162.5 (s, EtOCH=CH), 155.5 (s, C=O), 98.1 (s,
EtOCH=CH), 67.4 (s, OCH2CH3), 61.7 (d, J = 6.3 Hz,
POC), 39.9 (d, J = 18.0 Hz, CCCP), 23.2 (d,
J = 142.6 Hz, CP), 23.0 (d, J = 4.9 Hz, CCP), 16.6 (d,
J = 6.0 Hz, POCC), 14.6 (s, OCH2CH3) ppm; 31P NMR
(121.5 MHz, CDCl3): d = 32.56 ppm.
1452, 1382, 1343, 1239, 1049, 977, 732 cm-1; H NMR
(300 MHz, CDCl3): d = 9.29 (br s, 1H, NH), 7.35 (d,
J = 7.9 Hz, 1H, HC-6), 5.76 (dd, J = 7.9, 1.4 Hz, 1H,
HC-5), 4.19 (dq, J = 7.9, 6.9 Hz, 4H, 2 9 POCH2CH3),
4.17 (d, J = 11.5 Hz, 2H, CH2P), 1.34 (t, J = 6.9 Hz, 6H,
2 9 POCH2CH3) ppm; 13C NMR (75.5 MHz, CDCl3):
d = 163.8 (s, C=O), 150.6 (d, J = 2.0 Hz, C=O), 144.2 (s,
C-6), 102.9 (s, C-5), 63.5 (d, J = 6.6 Hz, POC), 42.0 (d,
J = 156.3 Hz, CP), 16.6 (d, J = 6.0 Hz, POCC) ppm; 31P
NMR (121.5 MHz, CDCl3): d = 19.35 ppm.
Diethyl 2-[3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl]-
ethylphosphonate (5b) [46, 47]
Colorless oil; yield: 83 %.
Diethyl 3-[3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl]-
propylphosphonate (5c) [46, 47]
Colorless oil; yield: 84 %.
Diethyl (E)-4-[3-(3-ethoxyacryloyl)ureido]butylphospho-
nate (4d, C14H27N2O6P)
ꢀ
Yellowish oil; yield 72 %; IR (film): m = 3421, 3283,
3144, 3103, 2982, 2941, 1704, 1679, 1617, 1549, 1234,
Diethyl 4-[3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl]-
butylphosphonate (5d) [46, 47]
Colorless oil; yield: 85 %.
1168, 1105, 1057, 1027, 965, 819 cm-1
;
1H NMR
(300 MHz, CDCl3): d = 9.52 (br s, 1H, NH), 8.68 (t,
J = 5.8 Hz, 1H, HNCH2), 7.63 (d, J = 12.2 Hz, 1H,
EtOCH=CH), 5.36 (d, J = 12.2 Hz, 1H, EtOCH=CH),
4.15–4.03 (m, 4H, 2 9 POCH2CH3), 3.97 (q, J = 7.0 Hz,
2H, OCH2CH3), 3.31 (dt, J = 6.4, 5.8 Hz, 2H, CH2CH2-
CH2CH2P), 1.81–1.62 (m, 6H, CH2CH2CH2P), 1.36 and
1.32 (2t, J = 6.9 Hz, 6H, 2 9 POCH2CH3) ppm; 13C
NMR (151 MHz, CDCl3): d = 168.4 (s, C=O), 162.4 (s,
EtOCH=CH), 155.5 (s, C=O), 98.2 (s, EtOCH=CH), 67.2
(s, OCH2CH3), 61.5 (d, J = 6.6 Hz, POC), 38.9 (s,
CCCCP), 30.4 (d, J = 15.5 Hz, CCCP), 25.2 (d,
J = 141.8 Hz, CP), 19.8 (d, J = 5.5 Hz, CCP), 16.3 (d,
J = 6.3 Hz, POCC), 14.4 (s, OCH2CH3) ppm; 31P NMR
(121.5 MHz, CDCl3): d = 32.19 ppm.
General procedure for the chlorination
and bromination reactions
To the solution of the respective phosphonate 5a–5d
(0.30 mmol) in 5 cm3 pyridine, N-halogenosuccinimide
(NCS or NBS, 0.45 mmol) was added and the mixture was
stirred for 30 min at 100 °C. Saturated aqueous NaHCO3
solution (10 cm3) was added and the mixture was extracted
with chloroform (3 9 20 cm3). The combined organic
phases were dried over MgSO4, filtered, and the solvent
was removed. The crude product was purified by column
chromatography or crystallization.
Diethyl [5-chloro-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]methylphosphonate (6a, C9H14ClN2O5P)
General procedure for the preparation of uracil
derivatives 5a–5d
Colorless solid; yield 72 % [after column chromatography
(ethyl acetate) and crystallization from ethyl acetate-
petroleum ether mixture]; m.p.: 136–138 °C; IR (KBr):
To a solution of the respective acryloylurea 4a–4d
(1.0 mmol) in 8 cm3 dioxane, 8 cm3 H2SO4 (2 M) was
added and the mixture was refluxed for 20 h. NaOH (2 M)
was added to reach pH 7 and the reaction mixture was
concentrated to dryness. To the solid residue, chloroform
was added and the mixture was stirred for 15 min, then
filtered through a layer of Celite and concentrated. The
product was purified by column chromatography with
chloroform–methanol mixtures (50:1, 20:1 v/v) to give
pure uracil derivatives 5a–5d.
ꢀ
m = 3406, 3172, 3043, 2984, 2945, 2831, 1712, 1690,
1633, 1338, 1224, 1061, 1012, 971 cm-1 1H NMR
;
(300 MHz, CDCl3): d = 9.92 (br s, 1H, NH), 7.61 (s,
1H, HC-6), 4.21 (dq, J = 7.9, 6.9 Hz, 4H, 2 9 POCH2-
CH3), 4.19 (d, J = 10.7 Hz, 2H, CH2P), 1.35 (t,
J = 6.9 Hz, 6H, 2 9 POCH2CH3) ppm; 13C NMR
(75.5 MHz, CDCl3): d = 159.4 (s, C=O), 149.9 (d,
J = 2.0 Hz, C=O), 141.0 (s, C-6), 109.4 (s, C-5), 63.6 (d,
J = 6.3 Hz, POC), 42.2 (d, J = 156.0 Hz, CP), 16.6 (d,
J = 6.0 Hz, POCC) ppm; 31P NMR (121.5 MHz, CDCl3):
d = 18.80 ppm.
Diethyl [3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]methylphosphonate (5a, C9H15N2O5P)
White solid; yield 58 %; m.p.: 122–124 °C; IR (KBr):
m = 3428, 3172, 3057, 2991, 2870, 2822, 1689, 1632,
Diethyl 2-[5-chloro-3,4-dihydro-2,4-dioxopyrimidin-
ꢀ
1(2H)-yl]ethylphosphonate (6b, C10H16ClN2O5P)
123

Synthesis, antiviral, cytotoxic and cytostatic evaluation of N1-(phosphonoalkyl)uracil…
1087
Creamy solid; yield 69 % [after column chromatography
ethyl acetate-hexane mixture); m.p.: 139–142 °C; IR
ꢀ
(chloroform/methanol 100:1 v/v)]; m.p.: 149–151 °C; IR
(KBr): m = 3150, 2982, 2947, 2818, 1688, 1627, 1451,
1
(KBr): m = 3171, 3029, 2994, 2926, 2839, 1701, 1665,
1429, 1355, 1245, 1210, 1187, 1045, 1020, 971 cm-1; H
ꢀ
1
1631, 1368, 1250, 1069, 1018, 963, 793 cm-1; H NMR
NMR (300 MHz, CDCl3): d = 9.38 (br s, 1H, NH), 7.45
(s, 1H, HC-6), 4.17–4.04 (m, 4H, 2 9 POCH2CH3), 3.76
(t, J = 7.3 Hz, 2H, CH2CH2CH2CH2P), 1.88–1.61 (m, 6H,
CH2CH2CH2P), 1.33 (t, J = 7.1 Hz, 6H, 2 9 POCH2CH3)
ppm; 13C NMR (75.5 MHz, CDCl3): d = 159.6 (s, C=O),
150.1 (s, C=O), 141.2 (s, C-6), 108.8 (s, C-5), 61.9 (d,
J = 6.6 Hz, POC), 48.8 (s, CCCCP), 29.7 (d, J = 14.3 Hz,
CCCP), 25.2 (d, J = 141.7 Hz, CP), 19.7 (d, J = 4.9 Hz,
(600 MHz, CDCl3): d = 9.18 (br s, 1H, NH), 7.61 (s, 1H,
HC-6), 4.19–4.10 (m, 4H, 2 9 POCH2CH3), 4.05 (dt,
J = 16.1, 6.8 Hz, 2H, CH2CH2P), 2.26 (dt, J = 18.2,
6.8 Hz, 2H, CH2P), 1.35 (t, J = 7.1 Hz, 6H, 2 9 POCH2-
CH3) ppm; 13C NMR (151 MHz, CDCl3): d = 159.6 (s,
C=O), 149.9 (s, C=O), 142.2 (s, C-6), 108.4 (s, C-5), 62.3
(d, J = 6.4 Hz, POC), 44.6 (d, J = 3.0 Hz, CCP), 25.0 (d,
J = 140.9 Hz, CP), 16.4 (d, J = 5.7 Hz, POCC) ppm; 31P
NMR (243 MHz, CDCl3): d = 26.78 ppm.
CCP), 16.7 and 16.6 (2 9 d, J = 5.9 Hz, POCC) ppm; 31
P
NMR (121.5 MHz, CDCl3): d = 31.46 ppm.
Diethyl 3-[5-chloro-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]propylphosphonate (6c, C11H18ClN2O5P)
Colorless solid; yield 53 % (after column chromatography
(ethyl acetate) and crystallization from ethyl acetate-
petroleum ether mixture); m.p.: 64–66 °C; IR (KBr):
Diethyl 4-[6-chloro-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]butylphosphonate (7d, C12H20ClN2O5P)
White solid; yield 12 % [after column chromatography
(hexane/ethyl acetate 2:1, 1:2 v/v)]; m.p.: 138–141 °C; IR
ꢀ
(KBr): m = 3412, 3231, 2961, 2924, 2854, 1745, 1698,
1482, 1439, 1254, 1027 cm-1 1H NMR (300 MHz,
;
ꢀ
m = 3545, 3044, 2988, 1701, 1654, 1624, 1458, 1272,
1242, 1227, 1015, 962 cm-1; 1H NMR (300 MHz, CDCl3):
d = 9.95 (br s, 1H, NH), 7.63 (s, 1H, HC-6), 4.24–4.07 (m,
4H, 2 9 POCH2CH3), 3.90 (t, J = 7.2 Hz, 2H, CH2CH2-
CH2P), 2.06 (dqu, J = 14.6, 7.2 Hz, 2H, CH2CH2P), 1.81
(dt, J = 18.2, 7.2 Hz, 2H, CH2P), 1.36 (t, J = 6.9 Hz, 6H,
2 9 POCH2CH3) ppm; 13C NMR (75.5 MHz, CDCl3):
d = 159.7 (s, C=O), 150.2 (s, C=O), 141.6 (s, C-6), 108.8
(s, C-5), 62.2 (d, J = 6.6 Hz, POC), 49.1 (d, J = 14.6 Hz,
CCCP), 22.4 (d, J = 142.8 Hz, CP), 22.3 (d, J = 4.9 Hz,
CCP), 16.7 (d, J = 5.7 Hz, POCC) ppm; 31P NMR
(121.5 MHz, CDCl3): d = 31.35 ppm.
CDCl3): d = 8.60 (br s, 1H, NH), 5.11 (s, 1H, HC-5),
4.16–3.98 (m, 4H, 2 9 POCH2CH3), 3.67 (dt, J = 13.7,
7.0 Hz, 1H, CHaHbCH2CH2CH2P), 3.49 (dt, J = 13.7,
7.1 Hz, 1H, CHaHbCH2CH2CH2P), 1.94–1.65 (m, 6H,
CH2CH2CH2P), 1.32 and 1.31 (2 9 t, J = 7.0 Hz, 6H,
2 9 POCH2CH3) ppm; 13C NMR (151 MHz, CDCl3):
d = 161.6 (s, C=O), 150.6 (s, C=O), 85.6 (s, C-6), 79.5 (s,
C-5), 62.2 (d, J = 6.5 Hz, POC), 62.0 (d, J = 7.1 Hz,
POC), 46.3 (s, CCCCP), 28.2 (d, J = 11.7 Hz, CCCP),
24.3 (d, J = 141.3 Hz, CP), 19.1 (d, J = 4.8 Hz, CCP),
16.4 (d, J = 6.6 Hz, POCC), 16.3 (d, J = 6.6 Hz, POCC)
ppm; 31P NMR (121.5 MHz, CDCl3): d = 33.19 ppm.
Diethyl 3-[6-chloro-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]propylphosphonate (7c, C11H18ClN2O5P)
Colorless oil; yield 6 % [after column chromatography
Diethyl [5-bromo-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]methylphosphonate (8a, C9H14BrN2O5P)
ꢀ
(ethyl acetate)]; IR (film): m = 3229, 3077, 2925, 2852,
White solid; yield 69 % (after column chromatography
(hexane/ethyl acetate 1:2 and ethyl acetate v/v) and
crystallization from ethyl acetate-petroleum ether mixture);
1
1745, 1696, 1471, 1240, 1098, 1024, 970 cm-1; H NMR
(300 MHz, CDCl3): d = 8.18 (br s, 1H, NH), 5.14 (s, 1H,
HC-5), 4.19–4.02 (m, 4H, 2 9 POCH2CH3), 3.86 (dt,
J = 13.9, 7.0 Hz, 1H, CHaHbCH2CH2CH2P), 3.47 (dt,
J = 13.9, 6.8 Hz, 1H, CHaHbCH2CH2CH2P), 2.18–1.84
(m, 4H, CH2CH2P), 1.34 and 1.32 (2 9 t, J = 7.0 Hz, 6H,
2 9 POCH2CH3) ppm; 13C NMR (151 MHz, CDCl3):
d = 161.5 (s, C=O), 150.6 (s, C=O), 86.3 (s, C-6), 79.5 (s,
C-5), 62.5 (d, J = 6.4 Hz, POC), 62.2 (d, J = 6.8 Hz,
POC), 47.8 (d, J = 13.5 Hz, CCCP), 22.1 (d,
J = 142.4 Hz, CP), 21.1 (d, J = 4.3 Hz, CCP), 16.4 (d,
ꢀ
m.p.: 120–122 °C; IR (KBr): m = 3415, 3164, 3040, 2943,
1
2851, 1707, 1626, 1422, 1338, 1224, 1012, 985 cm-1; H
NMR (300 MHz, CDCl3): d = 10.06 (br s, 1H, NH), 7.72
(s, 1H, HC-6), 4.21 (dq, J = 8.1, 7.0 Hz, 4H, 2 9 POCH2-
CH3), 4.20 (d, J = 11.1 Hz, 2H, CH2P), 1.35 (t,
J = 7.0 Hz, 6H, 2 9 POCH2CH3) ppm; 13C NMR
(75.5 MHz, CDCl3): d = 159.5 (s, C=O), 150.1 (d,
J = 2.0 Hz, C=O), 143.5 (s, C-6), 97.2 (s, C-5), 63.6 (d,
J = 6.6 Hz, POC), 42.2 (d, J = 156.0 Hz, CP), 16.6 (d,
J = 6.0 Hz, POCC) ppm; 31P NMR (121.5 MHz, CDCl3):
d = 18.82 ppm.
J = 6.6 Hz, POCC), 16.3 (d, J = 5.3 Hz, POCC) ppm; 31
P
NMR (121.5 MHz, CDCl3): d = 33.88 ppm.
Diethyl 4-[5-chloro-3,4-dihydro-2,4-dioxopyrimidin-
Diethyl 2-[5-bromo-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]ethylphosphonate (8b, C10H16BrN2O5P)
1(2H)-yl]butylphosphonate (6d, C12H20ClN2O5P)
White solid; yield 65 % (after column chromatography
(hexane/ethyl acetate 2:1, 1:2 v/v) and crystallization from
White solid; yield 75 % (after column chromatography
(chloroform/methanol 200:1 v/v) and crystallization from
123

1088
D. Rygielska-Tokarska et al.
(0.15 mmol) in 10 cm3 anhydrous acetonitrile was refluxed
for 2 h. After the solvent was removed, the residue was
washed with diethyl ether (3 9 15 cm3) and purified by
column chromatography with 2-propanol-hexane mixture
(1:3, v/v) to give phosphonates 9a–9d.
ethyl acetate-petroleum ether mixture); m.p.: 134–135 °C;
IR (KBr): m = 3162, 3038, 2990, 2834, 1704, 1660, 1623,
ꢀ
1251, 1054, 1016, 962 cm-1; 1H NMR (300 MHz, CDCl3):
d = 10.06 (br s, 1H, NH), 7.73 (s, 1H, HC-6), 4.19–4.03
(m, 4H, 2 9 POCH2CH3), 4.02 (dt, J = 16.2, 6.9 Hz, 2H,
CH2CH2P), 2.23 (dt, J = 18.0, 6.9 Hz, 2H, CH2P), 1.30 (t,
J = 6.8 Hz, 6H, 2 9 POCH2CH3) ppm; 13C NMR
(75.5 MHz, CDCl3): d = 159.9 (s, C=O), 150.3 (s, C=O),
144.8 (s, C-6), 96.1 (s, C-5), 62.5 (d, J = 6.6 Hz, POC),
44.6 (d, J = 3.4 Hz, CCP), 25.2 (d, J = 140.3 Hz, CP),
16.6 (d, J = 6.0 Hz, POCC) ppm; 31P NMR (121.5 MHz,
CDCl3): d = 27.46 ppm.
Diethyl
[5-iodo-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]methylphosphonate (9a, C9H14IN2O5P)
Colorless solid; yield 72 % (after crystallization from ethyl
alcohol-petroleum ether); m.p.: 116–118 °C; IR (KBr):
ꢀ
m = 3156, 3008, 2984, 2929, 2827, 1694, 1613, 1418, 1352,
1226, 1055, 1017, 967 cm-1; 1H NMR (300 MHz, CDCl3):
d = 9.24(brs, 1H, NH),7.78(s, 1H, HC-6), 4.20(dq,J = 8.1,
7.0 Hz, 4H, 2 9 POCH2CH3), 4.17 (d, J = 11.3 Hz, 2H,
Diethyl 3-[5-bromo-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]propylphosphonate (8c, C11H18BrN2O5P)
Creamy solid; yield 85 % [after column chromatography
(chloroform/methanol 200:1 v/v)]; m.p.: 48–50 °C; IR
CH2P), 1.35 (t, J = 7.0 Hz, 6H, 2 9 POCH2CH3) ppm; 13
C
NMR (75.5 MHz, CDCl3): d = 160.4 (s, C=O), 150.3 (d,
J = 2.0 Hz, C=O), 148.5 (s, C-6), 68.8 (s, C-5), 63.6 (d,
J = 6.6 Hz, POC), 42.2 (d, J = 155.7 Hz, CP), 16.6 and 16.5
(2 9 d, J = 6.0 Hz, POCC) ppm; 31P NMR (121.5 MHz,
CDCl3): d = 18.02 ppm.
ꢀ
(KBr): m = 3549, 3504, 3162, 3052, 2986, 2824, 1702,
1
1618, 1452, 1346, 1272, 1242, 1180, 1014, 962 cm-1; H
NMR (300 MHz, CDCl3): d = 9.84 (br s, 1H, NH), 7.72
(s, 1H, HC-6), 4.22–4.04 (m, 4H, 2 9 POCH2CH3), 3.88
(t, J = 7.2 Hz, 2H, CH2CH2CH2P), 2.04 (dqu, J = 15.0,
7.2 Hz, 2H, CH2CH2P), 1.78 (dt, J = 18.0, 7.2 Hz, 2H,
CH2P), 1.34 (t, J = 6.8 Hz, 6H, 2 9 POCH2CH3) ppm;
13C NMR (75.5 MHz, CDCl3): d = 159.9 (s, C=O), 150.5
(s, C=O), 144.2 (s, C-6), 96.5 (s, C-5), 62.2 (d, J = 6.6 Hz,
POC), 49.0 (d, J = 15.2 Hz, CCCP), 22.4 (d,
J = 142.6 Hz, CP), 22.3 (d, J = 4.9 Hz, CCP), 16.6 (d,
J = 6.0 Hz, POCC) ppm; 31P NMR (121.5 MHz, CDCl3):
d = 31.36 ppm.
Diethyl 2-[5-iodo-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]ethylphosphonate (9b, C10H16IN2O5P)
ꢀ
Colorless oil; yield 77 %; IR (film): m = 3473, 3167, 3051,
2984, 2817, 1691, 1613, 1443, 1420, 1359, 1284, 1226,
1
1097, 1051, 1024, 973, 794 cm-1; H NMR (300 MHz,
CDCl3): d = 10.04 (br s, 1H, NH), 7.84 (s, 1H, HC-6),
4.21–4.04 (m, 4H, 2 9 POCH2CH3), 4.04 (dt, J = 15.7,
6.9 Hz, 2H, CH2CH2P), 2.25 (dt, J = 18.2, 6.9 Hz, 2H,
CH2P), 1.32 (t, J = 7.0 Hz, 6H, 2 9 POCH2CH3) ppm;
13C NMR (75.5 MHz, CDCl3): d = 161.0 (s, C=O), 150.6
(s, C=O), 149.7 (s, C-6), 67.7 (s, C-5), 62.5 (d, J = 6.3 Hz,
POC), 44.5 (d, J = 2.6 Hz, CCP), 25.2 (d, J = 140.3 Hz,
CP), 16.6 (d, J = 5.7 Hz, POCC) ppm; 31P NMR
(121.5 MHz, CDCl3): d = 27.49 ppm.
Diethyl 4-[5-bromo-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]butylphosphonate (8d, C12H20BrN2O5P)
White solid; yield 85 % (after column chromatography
(chloroform/methanol 200:1 v/v) and crystallization from
ethyl acetate-petroleum ether mixture); m.p.: 112–114 °C;
Diethyl 3-[5-iodo-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]propylphosphonate (9c) [42]
ꢀ
IR (KBr): m = 3141, 3074, 2980, 2928, 2817, 1705, 1686,
1
1621, 1424, 1353, 1247, 1211, 1040, 959 cm-1; H NMR
White solid; yield 71 % (after crystallization from chloro-
form–diethyl ether mixture); m.p.: 82–84 °C.
(300 MHz, CDCl3): d = 9.36 (br s, 1H, NH), 7.56 (s, 1H,
HC-6), 4.17–4.02 (m, 4H, 2 9 POCH2CH3), 3.77 (t,
J = 7.2 Hz, 2H, CH2CH2CH2CH2P), 1.88–1.61 (m, 6H,
CH2CH2CH2P), 1.33 (t, J = 7.0 Hz, 6H, 2 9 POCH2CH3)
ppm; 13C NMR (75.5 MHz, CDCl3): d = 159.6 (s, C=O),
150.3 (s, C=O), 143.8 (s, C-6), 96.6 (s, C-5), 61.9 (d,
J = 6.6 Hz, POC), 48.8 (s, CCCCP), 29.8 (d, J = 14.3 Hz,
CCCP), 25.2 (d, J = 141.7 Hz, CP), 19.7 (d, J = 4.9 Hz,
Diethyl 4-[5-iodo-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
yl]butylphosphonate (9d, C12H20IN2O5P)
ꢀ
Colorless oil; yield 91 %; IR (film): m = 3445, 3165, 3050,
2984, 2816, 1690, 1612, 1441, 1420, 1228, 1051, 1026,
1
966 cm-1; H NMR (300 MHz, CDCl3): d = 9.63 (br s,
1H, NH), 7.67 (s, 1H, HC-6), 4.18–4.02 (m, 4H,
2 9 POCH2CH3), 3.77 (t, J = 7.1 Hz, 2H, CH2CH2CH2-
CH2P), 1.88–1.61 (m, 6H, CH2CH2CH2P), 1.33 (t,
J = 7.1 Hz, 6H, 2 9 POCH2CH3) ppm; 13C NMR
(75.5 MHz, CDCl3): d = 160.9 (s, C=O), 150.7 (s, C=O),
148.8 (s, C-6), 68.1 (s, C-5), 61.9 (d, J = 6.6 Hz, POC),
48.6 (s, CCCCP), 29.8 (d, J = 14.9 Hz, CCCP), 25.1 (d,
J = 142.0 Hz, CP), 19.6 (d, J = 3.7 Hz, CCP), 16.8 and
CCP), 16.7 and 16.6 (2 9 d, J = 6.0 Hz, POCC) ppm; 31
P
NMR (121.5 MHz, CDCl3): d = 31.46 ppm.
General procedure for the iodination reactions
A solution of the respective phosphonate 5a–5d
(0.30 mmol), iodine (0.18 mmol) and (NH4)2Ce(NO3)6
123

Synthesis, antiviral, cytotoxic and cytostatic evaluation of N1-(phosphonoalkyl)uracil…
1089
16.7 (2 9 d, J = 5.9 Hz, POCC) ppm; 31P NMR
(121.5 MHz, CDCl3): d = 31.55 ppm.
7.68–7.66 (m, 1H), 7.53–7.51 (m, 2H), 7.43 (d,
J = 8.0 Hz, 1H, HC-6), 5.83 (d, J = 8.0 Hz, 1H, HC-5),
4.18–4.08 (m, 4H, 2 9 POCH2CH3), 3.91 (t, J = 7.5 Hz,
2H, CH2CH2CH2P), 2.07 (dqu, J = 14.9, 7.5 Hz, 2H,
CH2CH2P), 1.79 (dt, J = 18.5, 7.5 Hz, 2H, CH2P), 1.35 (t,
J = 7.1 Hz, 6H, 2 9 POCH2CH3) ppm; 13C NMR
(150 MHz, CDCl3): d = 168.7 (s, C=O), 162.4 (s, C=O),
149.8 (s, C=O), 144.4 (s, C-6), 135.1, 131.5, 130.4, 129.2,
102.2 (s, C-5), 61.9 (d, J = 6.6 Hz, POC), 49.0 (d,
J = 13.7 Hz, CCCP), 22.3 (d, J = 143.0 Hz, CP), 22.2
(d, J = 4.6 Hz, CCP), 16.5 (d, J = 5.8 Hz, POCC) ppm;
31P NMR (243 MHz, CDCl3): d = 29.96 ppm.
General procedure for the benzoyl derivatives
10a–10d
To a solution of the respective phosphonate 5a–5d
(0.20 mmol) in 6 cm3 dichloromethane, 0.022 g triethy-
lamine (0.22 mmol) and 0.030 g benzoyl chloride
(0.21 mmol) were added at 0 °C and the mixture was
stirred at room temperature for 72 h. The solution was
washed with water (3 9 15 cm3), dried over MgSO4, and
purified by column chromatography with chloroform–
methanol mixtures (200:1, 20:1, v/v) to give pure phos-
phonates 10a–10d.
Diethyl 4-[3-benzoyl-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]butylphosphonate (10d, C19H25N2O6P)
ꢀ
Colorless oil; yield 73 %; IR (film): m = 3451, 3083, 2983,
2939, 2872, 1747, 1704, 1663, 1439, 1241, 1027,
Diethyl [3-benzoyl-3,4-dihydro-2,4-dioxopyrimidin-1(2H)-
1
yl]methylphosphonate (10a, C16H19N2O6P)
ꢀ
963 cm-1; H NMR (600 MHz, CDCl3): d = 7.94–7.93
Colorless oil; yield 63 %; IR (film): m = 3482, 3090, 2988,
2939, 1750, 1706, 1668, 1598, 1437, 1381, 1341, 1241,
(m, 2H), 7.67–7.65 (m, 1H), 7.52–7.50 (m, 2H), 7.30 (d,
J = 8.0 Hz, 1H, HC-6), 5.81 (d, J = 8.0 Hz, 1H, HC-5),
4.14–4.04 (m, 4H, 2 9 POCH2CH3), 3.78 (t, J = 7.3 Hz,
2H, CH2CH2CH2CH2P), 1.88–1.83 (m, 2H, CH2CH2CH2-
P), 1.81–1.76 (m, 2H, CH2P), 1.72–1.64 (m, 2H,
CH2CH2P), 1.32 (t, J = 7.0 Hz, 6H, 2 9 POCH2CH3)
ppm; 13C NMR (150 MHz, CDCl3): d = 168.8 (s, C=O),
162.4 (s, C=O), 149.8 (s, C=O), 144.1 (s, C-6), 135.0,
131.6, 130.4, 129.2, 102.2 (s, C-5), 61.6 (d, J = 6.6 Hz,
POC), 48.5 (s, CCCCP), 29.5 (d, J = 14.6 Hz, CCCP),
25.0 (d, J = 141.9 Hz, CP), 19.5 (d, J = 4.7 Hz, CCP),
16.4 (d, J = 5.7 Hz, POCC) ppm; 31P NMR (243 MHz,
CDCl3): d = 30.88 ppm.
1047, 1023, 976 cm-1 1H NMR (600 MHz, CDCl3):
;
d = 7.95–7.94 (m, 2H), 7.68–7.66 (m, 1H), 7.53–7.50 (m,
2H), 7.44 (d, J = 8.0 Hz, 1H, HC-6), 5.87 (d, J = 8.0 Hz,
1H, HC-5), 4.16–4.09 (m, 4H, 2 9 POCH2CH3), 4.18 (d,
J = 11.3 Hz, 2H, CH2P), 1.34 (t, J = 7.1 Hz, 6H,
2 9 POCH2CH3) ppm; 13C NMR (151 MHz, CDCl3):
d = 168.2 (s, C=O), 162.0 (s, C=O), 149.5 (d, J = 2.3 Hz,
C=O), 143.8 (s, C-6), 135.2, 131.3, 130.5, 129.2, 102.7 (s,
C-5), 63.4 (d, J = 6.5 Hz, POC), 42.2 (d, J = 155.7 Hz,
CP), 16.3 (d, J = 5.7 Hz, POCC) ppm; 31P NMR
(243 MHz, CDCl3): d = 17.86 ppm.
Diethyl 2-[3-benzoyl-3,4-dihydro-2,4-dioxopyrimidin-
Antiviral activity assays
1(2H)-yl]ethylphosphonate (10b, C17H21N2O6P)
ꢀ
Colorless oil; yield 64 %; IR (film): m = 3494, 3088, 2984,
2931, 2910, 1746, 1705, 1664, 1441, 1391, 1353, 1242,
The compounds were evaluated against the following viru-
ses: herpes simplex virus type 1 (HSV-1) strain KOS,
thymidine kinase-deficient (TK-) HSV-1 KOS strain resis-
tant to ACV (ACVr), herpes simplex virus type 2 (HSV-2)
strains Lyons and G, varicella-zoster virus (VZV) strain Oka,
TK- VZV strain 07-1, human cytomegalovirus (HCMV)
strains AD-169 and Davis, vaccinia virus Lederle strain,
respiratory syncytial virus (RSV) strain Long, vesicular
stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3,
Influenza virus A (subtypes H1N1, H3N2), influenza virus B,
Reovirus-1, Sindbis, Reovirus-1, Punta Toro, human
immunodeficiency virus type 1 strain IIIB, and human
immunodeficiency virus type 2 strain ROD. The antiviral,
other than anti-HIV, assays were based on inhibition of
virus-induced cytopathicity or plaque formation in human
embryonic lung (HEL) fibroblasts, African green monkey
cells (Vero), human epithelial cells (HeLa), or Madin-Darby
canine kidney cells (MDCK). Confluent cell cultures in
microtiter 96-well plates were inoculated with 100 CCID50
1051, 1026, 987 cm-1 1H NMR (600 MHz, CDCl3):
;
d = 7.96–7.95 (m, 2H), 7.68–7.66 (m, 1H), 7.53–7.50 (m,
2H), 7.41 (d, J = 8.0 Hz, 1H, HC-6), 5.81 (d, J = 8.0 Hz,
1H, HC-5), 4.18–4.09 (m, 4H, 2 9 POCH2CH3), 4.05 (dt,
J = 15.9, 7.0 Hz, 2H, CH2CH2P), 2.26 (dt, J = 18.2,
7.0 Hz, 2H, CH2P), 1.36 (t, J = 7.0 Hz, 6H, 2 9 POCH2-
CH3) ppm; 13C NMR (151 MHz, CDCl3): d = 168.6 (s,
C=O), 162.4 (s, C=O), 149.6 (s, C=O), 144.9 (s, C-6),
135.1, 131.5, 130.5, 129.2, 101.9 (s, C-5), 62.2 (d,
J = 6.5 Hz, POC), 44.8 (d, J = 4.0 Hz, CCP), 24.9 (d,
J = 140.7 Hz, CP), 16.4 (d, J = 6.0 Hz, POCC) ppm; 31P
NMR (243 MHz, CDCl3): d = 26.76 ppm.
Diethyl 3-[3-benzoyl-3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl]propylphosphonate (10c, C18H23N2O6P)
ꢀ
Colorless oil; yield 56 %; IR (film): m = 3448, 3083, 2984,
1
2932, 1747, 1704, 1663, 1439, 1242, 1028, 967 cm-1; H
NMR (600 MHz, CDCl3): d = 7.95–7.94 (m, 2H),
123

1090
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