Novel acyclic phosphonylated 1,2,3-triazolonucleosides with an acetamidomethyl linker: Synthesis and biological activity

Article abstract of DOI:10.1002/ardp.201300468

A new series of 4-substituted [(1,2,3-triazol-1-yl)acetamido] methylphosphonates as acyclic nucleotide analogs were synthesized from diethyl (2-chloroacetamido)methylphosphonate via azidation followed by 1,3-dipolar cycloaddition with selected alkynes derived from natural nucleobases or their mimetics. All compounds were tested for their antiviral activities against DNA and RNA viruses as well as for cytostatic activity or cytotoxicity. Among all tested compounds, [(1,2,3-triazol-1-yl)acetamido]methylphosphonate 6e substituted with the N³-Bz-benzuracil moiety showed activity against the vesicular stomatitis virus (EC₅₀ = 45 μM) in HeLa cell cultures. New 4-substituted [(1,2,3-triazol-1-yl)acetamido]methylphosphonates were synthesized as acyclic nucleotide analogs and tested for their antiviral activities against DNA and RNA viruses. [(1,2,3-Triazol-1-yl)-acetamido] methylphosphonate 6e (n = 1; R = N³-Bz-benzuracil) was found to be active against the vesicular stomatitis virus (EC₅₀ = 45 μM) in HeLa cells.

Full text of DOI:10.1002/ardp.201300468

506
Arch. Pharm. Chem. Life Sci. 2014, 347, 506–514
Full Paper
Novel Acyclic Phosphonylated 1,2,3-Triazolonucleosides with
an Acetamidomethyl Linker: Synthesis and Biological Activity
Iwona E. Głowacka1, Jan Balzarini2, and Andrzej E. Wróblewski1
1
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Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Łódz, Łódz, Poland
Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
2
A new series of 4-substituted [(1,2,3-triazol-1-yl)acetamido]methylphosphonates as acyclic nucleotide
analogs were synthesized from diethyl (2-chloroacetamido)methylphosphonate via azidation followed
by 1,3-dipolar cycloaddition with selected alkynes derived from natural nucleobases or their mimetics.
All compounds were tested for their antiviral activities against DNA and RNA viruses as well as for
cytostatic activity or cytotoxicity. Among all tested compounds, [(1,2,3-triazol-1-yl)acetamido]-
methylphosphonate 6e substituted with the N3-Bz-benzuracil moiety showed activity against the
vesicular stomatitis virus (EC50 ¼ 45 mM) in HeLa cell cultures.
Keywords: Antiviral / Cycloaddition / Synthesis / Triazoles
Received: December 17, 2013; Revised: January 28, 2014; Accepted: January 31, 2014
DOI 10.1002/ardp.201300468
Introduction
and is considered as the first biologically active ANP [3–5].
Small modifications within a carbon linker led to the
discovery of other analogs including adefovir [PMEA] {9-[2-
(phosphonomethoxy)ethyl]adenine}, which is active against
herpesviruses, retroviruses, and HBV [6, 7], as well as tenofovir
{(R)-9-[2-(phosphonomethoxy)propyl]adenine} [(R)-PMPA],
which is used as an anti-HIV and anti-HBV agent [8]. On the
other hand, cidofovir {(S)-9-[3-hydroxy-2-(phosphonomethoxy)-
propyl]cytosine} [(S)-HPMPC] shows activity against herpes-
viruses (such as cytomegalovirus) and adenoviruses [9] (Fig. 1).
A significant body of information on the structure–activity
relationship of ANPs has been collected in the literature
[10–14]. However, there is still a great interest in search for
new modified aliphatic spacers to achieve a correct balance
between potency and selectivity, which resulted in elabora-
tion of various ANP derivatives with modified carbon linkers
(Fig. 2) [15–18].
Moreover, several analogs of ANPs containing an 1,2,3-
triazole ring between the phosphonoalkyl and the nucleobase
moieties have been reported and some of them showed
interesting activity (Fig. 3) [19–21].
On the other hand, some known modified nucleosides are
equipped with nucleobase moieties functionalized with, e.g.,
carbamoyl, carbamate, or ureidyl groups or they may serve as
linkers as well (Fig. 4) [22–31]. However, the primary reason to
incorporate the acetamido group next to the phosphate or
diphosphate is a replacement of the ionic phosphate by the
Effective treatment of viral infections as well as cancer is still
an unsolved problem of contemporary medicine, which has
stimulated an intensive search for new drugs in the last
decades.
Among available therapeutics, nucleosides and nucleotides
belong to the most important classes of antiviral and
cytostatic compounds. However, their clinical applications
are hampered by toxic side effects [1] and drug resistance [2].
For this reason a search for new nucleoside mimetics with
improved activity and lower toxicity is conducted by research
groups around the world. So far, various structural analogs
have been obtained including modification of both the
nucleobase and sugar moieties. Among them, numerous
acyclic nucleoside phosphonates (ANPs) with promising
activity have been synthesized by connecting a phosphonate
group with functionalized purine or pyrimidine bases via an
acyclic linker.
(S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-
HPMPA], which shows a broad spectrum of antiviral (anti-
DNA and RNA viruses) activity, was obtained as early as in 1986
Correspondence: Dr. Iwona E. Głowacka, Bioorganic Chemistry
Laboratory, Faculty of Pharmacy, Medical University of Łódz, Muszyn-
skiego 1, 90-151 Łódz, Poland.
E-mail: iwona.glowacka@umed.lodz.pl
Fax: þ48 42 678 83 98
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[(1,2,3-Triazol-1-yl)acetamido]methylphosphonates
507
Figure 1. Acyclic nucleoside phosphonates (ANPs) active against viral infections.
Figure 2. Acyclic nucleoside phosphonates (ANPs) with modified carbon linkers.
Figure 3. Acyclic nucleoside phosphonates (ANPs) having natural nucleobases (or their analogs) connected to the 1,2,3-triazole ring by
a methylene linker.
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Figure 4. Modified nucleosides containing carbamoyl, carbamate, or ureidyl linker.
neutral amide bond, which may be considered as a non-
charged phosphate bioisostere [32, 33]. Furthermore, the
replacement of the phosphate internucleotide bonds with an
acetamido linkage has also been investigated [34]. Although
the phosphate and carbamoyl groups differ in length,
flexibility, polarity, lipophilicity, and hydrogen bonding
capabilities, increased stability toward enzymatic hydrolysis
phosphorus atom and a 1,2,3-triazole ring substituted at C4
with selected nucleobases are presented (Scheme 1). In
addition to the canonical nucleobases (in 4a–4d) and their
close mimetics (in 4e and 4f), several nucleobase analogs
containing substituted heterocyclic (in 3e, 4g, and 4h) and
benzene (in 3a–3d) rings [37, 38] were used.
in comparison to phosphates makes this replacement Results and discussion
valuable and thus it deserves further studies.
In continuation of our involvement in the search for new
biologically active nucleotide analogs [20, 21, 35, 36], the
synthesis and biological evaluation of a new series of acyclic
phosphonylated 1,2,3-triazolonucleosides 5a–e and 6a–h
containing an acetamidomethyl linker located between a
The starting diethyl (2-chloroacetamido)methylphosphonate
1 was obtained from diethyl aminomethylphosphonate and
chloroacetyl chloride following the literature procedure
described for the preparation of analogous diethyl (2-bromo-
acetamido)methylphosphonate [39].
Scheme 1. Retrosynthesis of phosphonylated 1,2,3-triazoloacyclonucleosides.
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[(1,2,3-Triazol-1-yl)acetamido]methylphosphonates
509
Antiviral activity and cytotoxicity evaluation
All the 1,2,3-triazoloacyclonucleotides 5 and 6 were evaluated
for their antiviral activities against a wide variety of DNA and
RNA viruses, using the following cell-based assays: (a) human
embryonic lung (HEL) cells: herpes simplex virus-1 (KOS),
herpes simplex virus-2 (G), herpes simplex virus-1 (TKꢀ ACVr
KOS), vaccinia virus, and vesicular stomatitis virus; (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, and Punta Toro virus; (d)
HeLa cell cultures: vesicular stomatitis virus, Coxsackie virus
B4, and respiratory syncytial virus; (e) Crandell-Rees Feline
Kidney (CRFK) cell cultures: 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),
influenza A virus H3N2 subtype (A/HK/7/87), and influenza B
virus (B/HK/5/72). Ganciclovir, cidofovir, acyclovir, brivudin,
(S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], Hippeastrum hy-
brid agglutinin (HHA), Urtica dioica agglutinin (UDA), dextran
sulfate (molecular weight 10,000 (DS-10000)), ribavirin,
zanamivir, amantadine, and rimantadine were used as the
reference compounds. The antiviral activity was expressed as
the EC50: the compound concentration required to reduce
virus-induced cytopathogenicity by 50% (other viruses). In
these series of tested compounds, only 6e (R ¼ N3-Bz-
benzuracil) was moderately active against vesicular stomatitis
virus (EC50 ¼ 45 mM) in HeLa, but not HEL cells.
Scheme 2. Reagents and conditions: (a) NaN3, Bu4NBr, toluene,
90°C, 4 h.
The conversion of phosphonate 1 into diethyl (2-azidoacet-
amido)methylphosphonate 2 was achieved with tetrabutyl-
ammonium azide, which was generated in situ from sodium
azide and tetrabutylammonium bromide in toluene at 90°C
(Scheme 2). This procedure is simple, safe, and efficient
leading to the formation of 2 in 82% yield.
The 1,2,3-triazoloacyclonucleotides 5 and 6 were synthe-
sized by the Hüisgen 1,3-dipolar cycloaddition of the diethyl
(2-azidoacetamido)methylphosphonate 2 and the selected
terminal alkynes 3 (phenylacetylene 3a, 1-ethynyl-2-fluoro-
benzene 3b, 1-ethynyl-3-fluorobenzene 3c, 1-ethynyl-2,4-
difluorobenzene 3d, 5-ethynyl-1-methyl-1H-imidazole 3e),
natural propargylated nucleobases (N9-propargyladenine 4a
[40], N1-propargylthymine 4b [40], N1-propargyluracil 4c [40,
41], and N4-acetyl-N1-propargylcytosine 4d [41]) and several
modified nucleobases (N3-benzoyl-N1-propargyluracil 4e [21,
42], N3-benzoyl-N1-propargylquinazoline-2,4-dione 4f [21], 5,6-
dimethyl-N1-propargylbenzimidazole 4g [43], and 3-acetyl-N-
propargylindole 4h [44]) (Scheme 3).
According to our previous experience, cycloadditions were
carried out in a microwave oven at 40–45°C [20]. A complete
conversion of azidophosphonate 2 into the respective 1,2,3-
triazoloacyclonucleotides 5 or 6 was achieved in less than 10min.
Structure and purity of all 1,2,3-triazoloacyclonucleotides
were established by 1H, 31P, and 13C NMR and IR techniques as
well as by elemental analysis.
The cytotoxicity of the tested compounds toward the
uninfected host cells was defined as the minimum cytotoxic
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-
Scheme 3. Reagents and conditions: (a) CuSO4 · 5H2O (0.05 equiv.), sodium ascorbate (0.1 equiv.), H2O–EtOH (1:1), MW, 40–45°C,
10 min.
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yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli-
um (MTS) assay system. None of the tested compounds
appeared cytotoxic toward screened cell lines at 250 mM.
effected with iodine vapors. All solvents were purified by methods
described earlier in the literature.
All microwave irradiation experiments were carried out in a
microwave reactor Plazmatronika RM 800. The reaction was
carried out in 50 mL glass vials.
Evaluation of cytostatic activity
The cytostatic activity of the tested compounds was defined
as the 50% inhibitory concentration (IC50), causing a 50%
decrease in cell proliferation. It was determined against
murine leukemia L1210, human lymphocyte CEM, and
human cervix carcinoma HeLa cells. None of the compounds
were inhibitory (cytostatic) at 250 mM.
Synthesis of diethyl (2-chloroacetamido)-
methylphosphonate 1
To a solution of chloroacetyl chloride (0.524 mL, 6.58 mmol) in
toluene (10 mL) cooled to 0°C containing anhydrous potassium
carbonate (1.161 g, 8.40 mmol), a solution of diethyl amino-
methylphosphonate (1.100 g, 6.58 mmol) in toluene (2 mL) was
added dropwise and the reaction mixture was stirred at 0°C for
40 min. Then, it was allowed to warm up to room temperature
and it was further stirred for 20 h. The suspension was diluted in
chloroform (15 mL) and filtered through a layer of Celite. The
solution was concentrated in vacuo, and the crude product was
purified on a silica gel column with a chloroform–methanol
mixture (50:1 or 20:1 v/v) to give diethyl (2-chloroacetamido)-
methylphosphonate 1 (1.283 g, 80%) as a white solid. m.p. ¼ 62–
63°C; IR (KBr): n ¼ 3263, 3078, 2984, 2929, 1680, 1235, 1023,
969 cmꢀ1; 1H NMR (300 MHz, CDCl3): d ¼ 6.83 (brs, 1H, NH), 4.21–
4.12 (m, 4H, 2 ꢁ POCH2CH3), 4.09 (d, J ¼ 1.1 Hz, 2H, CH2Cl), 3.74
(dd, J ¼ 12.4 Hz, J ¼ 5.9 Hz, 2H, PCH2), 1.35 (2t, J ¼ 7.1 Hz, 6H,
2 ꢁ POCH2CH3);13C NMR (75.5 MHz, CDCl3): d ¼ 166.2 (d, J ¼ 5.1
Conclusion
Transformation of diethyl (2-chloroacetamido)methylphos-
phonate 1 into the unknown diethyl (2-azidoacetamido)-
methylphosphonate 2 was efficiently accomplished employing
in situ generated tetrabutylammonium azide.
The 1,2,3-triazoloacyclonucleotides 5a–e and 6a–h were
obtained in good yields from phosphonate 2 by the copper(I)-
catalyzed 1,3-dipolar cycloaddition under microwave irradia-
tion with several aromatic and heteroaromatic alkynes 3a–e
and propargylated natural nucleobases as well as their
selected mimetics 4a–h.
All synthesized compounds were examined for their
antiviral activities against a variety of DNA and RNA viruses,
and for their cytostatic activity or cytotoxicity.
Compound 6e (R ¼ N3-Bz-benzuracil) showed activity
against vesicular stomatitis virus (EC50 ¼ 45 mM) in HeLa cell
cultures.
–
Hz, C O), 62.4 (d, J ¼ 6.6 Hz, POC), 42.0 (s, CH2Cl), 34.8
–
(d, J ¼ 157.5 Hz, PC), 16.1 (d, J ¼ 5.7 Hz, POCC); 31P NMR
(121.5 MHz, CDCl3): d ¼ 22.65 ppm. Anal. calcd. for C7H15ClN4O4P:
C, 34.51; H, 6.21; N, 5.75. Found: C, 34.28; H, 6.40; N, 5.93.
Synthesis of diethyl (2-azidoacetamido)-
methylphosphonate 2
A
suspension of tetrabutylammonium bromide (0.096 g,
0.297 mmol) and sodium azide (0.245 g, 3.77 mmol) in toluene
(8 mL) containing diethyl (2-chloroacetamido)methylphospho-
nate 1 (0.483 g, 1.98 mmol) was heated to 90°C for 4 h. After
cooling, the suspension was filtered through a layer of Celite. The
solution was evaporated in vacuo, and the crude product was
purified on a silica gel column with a chloroform–methanol
mixture (100:1 v/v) to give diethyl (2-azidoacetamido)methyl-
phosphonate 2 (0.382 g, 77%) as a colorless oil; IR (film): n ¼ 3240,
The lack of any biological activity might be due to the poor
uptake by the (virus-infected) cells and/or by a lack of efficient
interaction with any potential target protein important for
the integrity of the tumor cells or replication of the viruses.
3066, 2985, 2931, 2107, 1688, 1220, 1052, 1028 cmꢀ1 1H NMR
;
Experimental
(300 MHz, CDCl3): d ¼ 6.78 (brs, 1H, NH), 4.21–4.11 (m, 4H,
2 ꢁ POCH2CH3), 4.03 (d, J ¼ 1.1 Hz, 2H, CH2N3), 3.73 (dd, J ¼ 12.4
13
1H NMR spectra were taken in CDCl3 or CD3OD on the following
spectrometers: 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, CD3OD, or DMSO-d6 solutions on
a Varian Mercury-300 and Bruker Avance III (600 MHz) spectro-
meter at 75.5 and 151 MHz, respectively. 31P NMR spectra were
taken in CDCl3 or CD3OD on Varian Mercury-300 at 121.5 MHz.
IR spectral data were measured on an Infinity MI-60 FT-IR
spectrometer. Melting points were determined on a Boetius
apparatus and are uncorrected. Elemental analyses were
performed by the microanalytical laboratory of this faculty on
a Perkin Elmer PE 2400 CHNS analyzer.
Hz, J ¼ 6.0 Hz, 2H, PCH2), 1.35 (2t, J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3);
–
–
C NMR (75.5 MHz, CDCl ): d ¼ 167.0 (d, J ¼ 5.4 Hz, C O), 62.9
3
(d, J ¼ 6.6 Hz, POC), 52.3 (s, CH2N3), 34.8 (d, J ¼ 157.3 Hz, PC), 16.5
(d, J ¼ 5.9 Hz, POCC); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.89 ppm.
Anal. calcd. for C7H15N4O4P: C, 33.60; H, 6.04; N, 22.39. Found: C,
36.78; H, 6.12; N, 22.60.
General procedure for the preparation of 1,2,3-triazoles
To a solution of azidoalkylphosphonates 2 (1.00 mmol) in EtOH
(1 mL) and H2O (1 mL), CuSO4 · 5H2O (0.05 mmol), sodium
ascorbate (0.10 mmol), and alkynes (1.00 mmol) were added
sequentially. The suspension was irradiated in the microwave
reactor at 40–45°C for 10 min. After cooling, the solvent was
removed by vacuum evaporation. The residue was suspended in
chloroform (5 mL) and filtered through a layer of Celite. The
solution was concentrated in vacuo, and the crude product was
The following adsorbents were used: column chromatography,
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
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Arch. Pharm. Chem. Life Sci. 2014, 347, 506–514
[(1,2,3-Triazol-1-yl)acetamido]methylphosphonates
511
purified on a silica gel column with a chloroform–methanol
mixture (50:1, 20:1, or 10:1 v/v) to give the appropriate 1,2,3-
triazoles 5a–e and 6a–h.
(d, J ¼ 6.9 Hz, POC), 52.4 (s, CH2N), 35.0 (d, J ¼ 157.7 Hz, PC), 16.3 (d,
J ¼ 5.7 Hz, POCC); 31P NMR (243 MHz, CDCl3): d ¼ 21.73 ppm. Anal.
calcd. for C15H20FN4O4P: C, 48.65; H, 5.44; N, 15.13. Found: C,
48.51; H, 5.24; N, 14.95.
Diethyl [2-(4-phenyl-1,2,3-triazol-1-yl)acetamido]-
methylphosphonate 5a
Diethyl {2-[4-(2,4-difluorophenyl)-1,2,3-triazol-1-yl]-
acetamido}methylphosphonate 5d
Yield: 90%; after chromatography on a silica gel column with
chloroform–methanol (100:1 v/v) appropriate fractions were
crystallized from an ethyl acetate–hexane mixture to give white
needles. m.p.: 131–132°C; IR (KBr): n ¼ 3244, 3066, 2985, 2930,
1687, 1221, 1046, 1025 cmꢀ1; 1H NMR (300 MHz, CDCl3): d ¼ 8.04
(s, 1H, HC50), 7.85 (brs, 1H, NH), 7.82–7.80 (m, 2H, Haromat.), 7.45–
7.31 (m, 3H, Haromat.), 5.21 (s, 2H, CH2N), 4.16–4.07 (m, 4H,
2 ꢁ POCH2CH3), 3.76 (dd, J ¼ 12.3 Hz, J ¼ 6.0 Hz, 2H, PCH2), 1.27 (2t,
J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR (151 MHz, CDCl3):
Yield: 82%; after chromatography on a silica gel column with
chloroform–methanol (100:1, 50:1 v/v) appropriate fractions were
crystallized from an ethyl acetate–hexane mixture to give white
needles. m.p.: 105–106°C; IR (KBr): n ¼ 3242, 3073, 2989, 2935,
1688, 1231, 1049, 1028, 756 cmꢀ1 1H NMR (600 MHz, CDCl3):
;
d ¼ 8.29 (dt, J ¼ 8.6 Hz, J ¼ 6.5 Hz, 1Haromat.), 8.12 (d, J ¼ 3.5 Hz, 1H,
HC50), 7.59 (ddt, J ¼ 8.6 Hz, J ¼ 2.6 Hz, J ¼ 0.8 Hz, 1H, Haromat.), 7.33
(brs, 1H, NH), 6.94 (ddd, J ¼ 11.0 Hz, J ¼ 8.6 Hz, J ¼ 2.6 Hz, 1H,
–
d ¼ 165.5 (d, J ¼ 5.4 Hz, C O), 148.2, 130.4, 128.9, 128.3, 125.8,
H
aromat.), 5.22 (s, 2H, CH2N), 4.17–4.12 (m, 4H, 2 ꢁ POCH2CH3), 3.77
–
121.3, 62.9 (d, J ¼ 6.6 Hz, POC), 52.6 (s, CH2N3), 35.0 (d, J ¼ 157.6 Hz,
PC), 16.3 (d, J ¼ 5.6 Hz, POCC); 31P NMR (121.5 MHz, CDCl3):
d ¼ 21.96 ppm. Anal. calcd. for C15H21N4O4P: C, 51.13; H, 6.01;
N, 15.90. Found: C, 51.44; H, 5.99; N, 15.66.
(dd, J ¼ 12.4 Hz, J ¼ 5.9 Hz, 2H, PCH2), 1.34 (t, J ¼ 7.1 Hz, 3H,
POCH2CH3), 1.32 (t, J ¼ 7.1 Hz, 3H, POCH2CH3); 13C NMR (151 MHz,
–
CDCl ): d ¼ 165.4 (d, J ¼ 5.5 Hz, C O), 162.6 (dd, J ¼ 251.0 Hz,
–
3
J ¼ 12.0 Hz, CF), 159.3 (dd, J ¼ 251.0 Hz, J ¼ 12.0 Hz, CF), 141.0
(d, J ¼ 1.7 Hz), 128.8 (dd, J ¼ 9.7 Hz, J ¼ 5.2 Hz, Caromat), 123.9 (d,
J ¼ 12.2 Hz), 114.8 (dd, J ¼ 13.3 Hz, J ¼ 3.8 Hz, Caromat), 112.0
(dd, J ¼ 21.2 Hz, J ¼ 3.2 Hz, Caromat), 104.1 (d, J ¼ 26.0 Hz, Caromat),
63.0 (d, J ¼ 6.6 Hz, POC), 52.6 (s, CH2N), 35.0 (d, J ¼ 157.8 Hz, PC),
16.3 (d, J ¼ 6.0 Hz, POCC); 31P NMR (243 MHz, CDCl3): d ¼ 21.29
ppm. Anal. calcd. for C15H19F2N4O4P: C, 46.40; H, 4.93; N, 14.43.
Found: C, 46.48; H, 4.90; N, 14.38.
Diethyl {2-[4-(2-fluorophenyl)-1,2,3-triazol-1-yl]acetamido}-
methylphosphonate 5b
Yield: 86%; after chromatography on a silica gel column with
chloroform–methanol (100:1 v/v) appropriate fractions were
crystallized from an ethyl acetate–hexane mixture to give white
needles. m.p.: 127–129°C; IR (KBr): n ¼ 3215, 3149, 3058, 2995,
2938, 1680, 1204, 1023, 755 cmꢀ1 1H NMR (600 MHz, CDCl3):
;
Diethyl {2-[4-(1-methylimidazolo-5-yl)-1,2,3-triazol-1-yl]-
acetamido}methylphosphonate 5e
d ¼ 8.30 (brt, J ¼ 4.9 Hz, 1H), 8.24 (dt, J ¼ 7.6 Hz, J ¼ 1.8 Hz, 1H,
H
aromat.), 8.18 (d, J ¼ 3.5 Hz, 1H, HC50), 7.30–7.26 (m, 1H, Haromat.),
7.22 (dt, J ¼ 7.6 Hz, J ¼ 1.1 Hz, 1H, Haromat.), 7.11 (ddd, J ¼ 10.9 Hz,
J ¼ 8.2 Hz, J ¼ 1.1 Hz, 1H, Haromat.), 5.25 (s, 2H, CH2N), 4.15–4.06 (m,
4H, 2 ꢁ POCH2CH3), 3.77 (dd, J ¼ 12.2 Hz, J ¼ 5.9 Hz, 2H, PCH2),
1.27 (2t, J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR (151 MHz, CDCl3):
Yield: 76%; after chromatography on a silica gel column
with chloroform–methanol (100:1, 50:1 v/v) appropriate frac-
tions solidified as a white powder. m.p.: 111–112°C; IR (KBr):
n ¼ 3564, 3279, 3136, 2932, 1661, 1219, 1052, 1021 cmꢀ1
;
d ¼ 165.9 (d, J ¼ 5.5 Hz, C O), 159.2 (d, J ¼ 249.2 Hz, CF), 141.4 (d,
1H NMR (300 MHz, CDCl3): d ¼ 7.96 (s, 1H, HC50), 7.70 (brt,
J ¼ 5.8 Hz, 1H), 7.59 (s, 1H), 7.28 (s, 1H), 5.20 (s, 2H, CH2N),
4.18–4.09 (m, 4H, 2 ꢁ POCH2CH3), 3.92 (s, 3H, CH3), 3.75
(dd, J ¼ 12.2 Hz, J ¼ 5.8 Hz, 2H, PCH2), 1.31 (2t, J ¼ 7.0 Hz,
6H, 2 ꢁ POCH2CH3); 13C NMR (151 MHz, CDCl3): d ¼ 166.3
–
–
J ¼ 2.5 Hz, Caromat), 129.3 (d, J ¼ 8.5 Hz, Caromat), 127.8 (d, J ¼ 3.5 Hz,
C
aromat), 124.5 (d, J ¼ 3.1 Hz, Caromat), 124.4 (d, J ¼ 12.8 Hz, Caromat),
122.0, 115.6 (d, J ¼ 21.5 Hz, Caromat), 63.0 (d, J ¼ 6.7 Hz, POC),
52.4 (s, CH2N), 34.9 (d, J ¼ 157.6 Hz, PC), 16.2 (d, J ¼ 6.0 Hz, POCC);
31P NMR (243 MHz, CDCl3): d ¼ 21.67 ppm. Anal. calcd. for
C15H20FN4O4P: C, 48.65; H, 5.44; N, 15.13. Found: C, 48.51; H,
5.24; N, 15.19.
–
(d, J ¼ 4.2 Hz, C O), 139.7, 137.4, 127.2, 123.8, 123.8, 62.8
–
(d, J ¼ 6.6 Hz, POC), 51.6 (s, CH2N), 34.4 (d, J ¼ 158.4 Hz, PC),
32.8, 15.3 (d, J ¼ 5.7 Hz, POCC); 31P NMR (121 MHz, CDCl3):
d ¼ 22.10 ppm. Anal. calcd. for C13H21N6O4P: C, 43.82; H, 5.94;
N, 23.59. Found: C, 43.61; H, 5.74; N, 23.40.
Diethyl {2-[4-(3-fluorophenyl)-1,2,3-triazol-1-yl]acetamido}-
methylphosphonate 5c
Diethyl {2-[4-((6-aminopurin-9-yl)methyl)-1,2,3-triazol-1-yl]-
acetamido}methylphosphonate 6a
Yield: 76%; after chromatography on a silica gel column with
chloroform–methanol (100:1, 50:1 v/v) appropriate fractions were
crystallized from an ethyl acetate–hexane mixture to give white
needles. m.p.: 101–102°C; IR (KBr): n ¼ 3242, 3058, 2989, 2935,
Yield: 81%; after chromatography on a silica gel column with
chloroform–methanol (100:1, 50:1 v/v) appropriate fractions were
crystallized from a methanol–diethyl ether mixture to give white
needles. m.p. ¼ 176–178°C; IR (KBr): n ¼ 3310, 3232, 3072, 2996,
2919, 1692, 1661, 1596, 1572, 1229, 1048, 1021 cmꢀ1; solubility of
6a in methanol, water, and DMSO was insufficient to measure the
13C NMR spectrum; 1H NMR (600 MHz, CDCl3): d ¼ 8.41 (s, 1H),
8.02 (s, 1H), 7.87 (s, 1H), 6.43 (brs, 1H), 5.57 (brs, 2H), 5.54 (s, 2H,
CH2N), 5.07 (s, 2H), 4.15–4.09 (m, 4H, 2 ꢁ POCH2CH3), 3.70
(dd, J ¼ 12.2 Hz, J ¼ 5.8 Hz, 2H, PCH2), 1.32 (t, J ¼ 7.0 Hz, 6H,
2 ꢁ POCH2CH3); 31P NMR (243 MHz, CDCl3): d ¼ 21.21 ppm. Anal.
calcd. for C15H22N9O4P · 2H2O: C, 39.22; H, 5.70; N, 27.44. Found: C,
39.00; H, 5.97; N, 27.67.
1688, 1231, 1049, 1028, 756 cmꢀ1 1H NMR (600 MHz, CDCl3):
;
d ¼ 8.31 (brt, J ¼ 5.1 Hz, 1H), 8.11 (s, 1H, HC50), 7.59 (dt, J ¼ 7.6 Hz,
J ¼ 1.4 Hz, 1H, Haromat.), 7.56 (ddd, J ¼ 9.9 Hz, J ¼ 2.6 Hz, J ¼ 1.4 Hz,
1H, Haromat.), 7.37 (dt, J ¼ 8.4 Hz, J ¼ 5.9 Hz, 1H, Haromat.), 7.02 (ddt,
J ¼ 8.4 Hz, J ¼ 2.4 Hz, J ¼ 1.4 Hz, 1H, Haromat.), 5.23 (s, 2H, CH2N),
4.18–4.09 (m, 4H, 2 ꢁ POCH2CH3), 3.78 (dd, J ¼ 12.2 Hz, J ¼ 5.9 Hz,
2H, PCH2), 1.30 (2t, J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR
–
–
(151 MHz, CDCl ): d ¼ 165.6 (d, J ¼ 5.4 Hz, C O), 163.2 (d, J ¼ 245.5
3
Hz, CF), 146.9 (d, J ¼ 3.0 Hz, Caromat), 132.6 (d, J ¼ 8.6 Hz, Caromat),
130.4 (d, J ¼ 8.6 Hz, Caromat), 121.9, 121.3 (d, J ¼ 2.8 Hz, Caromat),
115.0 (d, J ¼ 21.4 Hz, Caromat), 112.6 (d, J ¼ 23.1 Hz, Caromat), 63.1
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I. E. Głowacka et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 506–514
Diethyl {2-[4-((5-methyl-2,4-dioxopyrimidin-1-yl)methyl)-
1,2,3-triazol-1-yl]acetamido}methylphosphonate 6b
8.07 (brt, J ¼ 5.9 Hz, 1H, PCH2NH), 7.98–7.94 (m, 2H, Haromat.), 7.92
(s, 1H, HC50), 7.87 (d, J ¼ 8.3 Hz, 1H, Haromat.), 7.72 (ddd, J ¼ 8.3 Hz,
J ¼ 7.4 Hz, J ¼ 1.4 Hz, 1H, Haromat.), 7.67–7.62 (m, 1H, Haromat.),
7.52–7.46 (m, 2H, Haromat.), 7.28 (dt, J ¼ 7.4 Hz, J ¼ 0.9 Hz, 1H,
Yield: 86%; after chromatography on a silica gel column with
chloroform–methanol (50:1, 20:1 v/v) appropriate fractions were
crystallized from a methanol–diethyl ether mixture to give white
needles. m.p. ¼ 175–176°C; IR (KBr): n ¼ 3305, 3147, 3085, 2990,
H
aromat.), 5.40 (s, 2H), 5.10 (s, 2H), 4.12–4.02 (m, 4H, 2 ꢁ
POCH2CH3), 3.70 (dd, J ¼ 12.2 Hz, J ¼ 5.9 Hz, 2H, PCH2), 1.27 (2t,
J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR (75.5 MHz, CDCl3):
2934, 2882, 1677, 1048, 1026 cmꢀ1 1H NMR (600 MHz, CDCl3):
;
–
d ¼ 9.74 (s, 1H, NH), 7.94 (s, 1H, HC50), 7.83 (brt, J ¼ 5.8 Hz, 1H,
d ¼ 168.6, 165.3 (d, J ¼ 5.4 Hz, C O), 161.0, 149.4, 142.3, 140.2,
–
136.2, 135.1, 131.5, 130.4, 129.2, 129.2, 128.8, 125.5, 123.7, 115.4,
115.3, 63.1 (d, J ¼ 4.9 Hz, POC), 52.3, 38.9, 34.9 (d, J ¼ 157.5 Hz, PC),
16.5 (d, J ¼ 5.8 Hz, POCC), 16.4 (d, J ¼ 5.8 Hz, POCC); 31P NMR
(121.5 MHz, CDCl3): d ¼ 22.72 ppm. Anal. calcd. for C25H27N6O7P:
C, 54.15; H, 4.91; N, 15.16. Found: C, 54.40; H, 4.71; N, 15.36.
–
PCH NH), 7.35 (br q, J ¼ 1.1 Hz, 1H, HC CCH ), 5.06 (s, 2H), 4.99 (s,
–
2
3
2H), 4.18–4.13 (m, 4H, 2 ꢁ POCH2CH3), 3.73 (dd, J ¼ 12.5 Hz,
–
–
J ¼ 5.8 Hz, 2H, PCH ), 1.92 (d, J ¼ 1.1 Hz, 3H, HC CCH ), 1.33 (t,
2
3.
J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR (75.5 MHz, CD3OD):
–
–
d ¼ 166.9 (d, J ¼ 4.0 Hz, C O), 166.2, 152.2, 143.3, 142.0, 126.5,
111.4, 63.9 (d, J ¼ 6.6 Hz, POC), 52.7, 43.5, 35.6 (d, J ¼ 158.0 Hz, PC),
16.8 (d, J ¼ 6.0 Hz, POCC),12.5 (s, CH3); 31P NMR (243 MHz, CDCl3):
d ¼ 21.98 ppm. Anal. calcd. for C15H23N6O6P ꢁ H2O: C, 41.67; H,
5.83, N, 19.44. Found: C, 41.66; H, 5.56; N, 19.70.
Diethyl {2-[4-((3-benzoyl-2,4-dioxopyrimidin-1-yl)methyl)-
1,2,3-triazol-1-yl]acetamido}methylphosphonate 6f
Yield: 93%; after chromatography on a silica gel column with
chloroform–methanol (50:1, 20:1 v/v) appropriate fractions were
collected as a colorless oil. IR (film): n ¼ 3246, 3079, 2989, 1747,
1698, 1667, 1237, 1050, 734, 699 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d ¼ 8.20 (brt, J ¼ 5.9 Hz, 1H, PCH2NH), 7.95 (s, 1H, HC50), 7.93–7.89
Diethyl {2-[4-((2,4-dioxopyrimidin-1-yl)methyl)-1,2,3-
triazol-1-yl]acetamido}methylphosphonate 6c
Yield: 75%; after chromatography on a silica gel column with
chloroform–methanol (50:1, 20:1 v/v) appropriate fractions
–
(m, 2H, Haromat.), 7.67 (d, J ¼ 8.0 Hz, 1H, HC CH), 7.67–7.62 (m, 1H,
–
Haromat.), 7.51–7.46 (m, 2H, Haromat.), 5.78 (d, J ¼ 8.0 Hz, 1H,
solidified as
a
white powder. m.p. ¼ 156–157°C; IR (KBr):
–
HC CH), 5.13 (s, 2H), 5.01 (s, 2H), 4.15–4.06 (m, 4H, 2 ꢁ POCH CH ),
n ¼ 3305, 3147, 3085, 2990, 2934, 2882, 1677, 1048, 1026 cmꢀ1
;
–
2
3
3.72 (dd, J ¼ 12.1 Hz, J ¼ 5.9 Hz, 2H, PCH2), 1.27 (t, J ¼ 7.1 Hz, 6H,
2 ꢁ POCH2CH3); 13C NMR (75.5 MHz, CDCl3): d ¼ 168.9, 165.5 (d,
1H NMR (300 MHz, CD3OD): d ¼ 8.03 (s, 1H, HC50), 7.70 (d,
–
–
–
–
J ¼ 7.9 Hz, 1H, HC CH), 5.66 (d, J ¼ 7.9 Hz, 1H, HC CH), 5.20 (d,
–
–
J ¼ 5.4 Hz, C O), 166.5, 162.5, 149.9, 144.6, 135.4, 131.3, 130.6,
J ¼ 1.1 Hz, 2H), 5.03 (s, 2H), 4.15–4.10 (m, 4H, 2 ꢁ POCH2CH3), 3.75
13
129.3, 126.0, 102.5, 63.3 (d, J ¼ 6.5 Hz, POC), 52.4, 43.6, 35.6 (d,
J ¼ 157.2 Hz, PC), 16.6 (d, J ¼ 4.1 Hz, POCC); 31P NMR (121.5 MHz,
CDCl3): d ¼ 22.94 ppm. Anal. calcd. for C21H25N6O7P: C, 50.00; H,
5.00; N, 16.66. Found: C, 50.22; H, 4.88; N, 16.40.
(d, J ¼ 11.9 Hz, 2H, PCH2), 1.31 (t, J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3);
–
–
C NMR (75.5 MHz, CD OD): d ¼ 166.9 (d, J ¼ 5.4 Hz, C O), 166.1,
3
152.1, 146.2, 143.1, 126.6, 102.7, 64.0 (d, J ¼ 6.6 Hz, POC), 52.8,
43.8, 35.6 (d, J ¼ 158.0 Hz, PC), 16.8 (d, J ¼ 5.9 Hz, POCC); 31P NMR
(121.5 MHz, CD3OD): d ¼ 22.22 ppm. Anal. calcd. for C14H21N6O6P:
C, 42.00; H, 5.29; N, 20.99. Found: C, 42.24; H, 5.14; N, 21.20.
Diethyl {2-[4-((5,6-dimethylbenzimidazol-1-yl)methyl)-
1,2,3-triazol-1-yl]acetamido}-methylphosphonate 6g
Yield: 74%; after chromatography on a silica gel column with
chloroform–methanol (50:1, 20:1 v/v) appropriate fractions
Diethyl {2-[4-((N4-acetylamino-2-oxopyrimidin-1-yl)-
methyl)-1,2,3-triazol-1-yl]acetamido}methylphosphonate
6d
solidified as
a
white powder. m.p. ¼ 172–174°C; IR (KBr):
n ¼ 3416, 3217, 3142, 3058, 2974, 2942, 1693, 1216, 1044,
979 cmꢀ1; 1H NMR (300 MHz, CDCl3): d ¼ 8.18 (brt, J ¼ 5.9 Hz, 1H,
PCH2NH), 7.95 (s, 1H), 7.63 (s, 1H), 7.53 (s, 1H), 7.21 (s, 1H), 5.42 (s,
2H), 5.05 (s, 2H), 4.13–4.02 (m, 4H, 2 ꢁ POCH2CH3), 3.70 (dd,
J ¼ 12.3 Hz, J ¼ 5.9 Hz, 2H, PCH2), 2.35 (s, 3H, CH3.), 2.34 (s, 3H,
CH3.), 1.25 (2 ꢁ t, J ¼ 7.1 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR
Yield: 83%; after chromatography on a silica gel column with
chloroform–methanol (10:1, 5:1 v/v) appropriate fractions solidi-
fied as a white powder. m.p. ¼ 175–177°C; IR (KBr): n ¼ 3426,
3281, 3145, 3042, 3009, 2934, 1710, 1666, 1050, 1025, 975 cmꢀ1
;
1H NMR (300 MHz, CD3OD): d ¼ 8.03 (s, 1H, HC50), 7.97 (d,
–
–
–
J ¼ 7.3 Hz, 1H, HC CH),7.41 (d, J ¼ 7.3 Hz, 1H, HC CH), 5.13 (s, 2H),
–
–
–
(75.5 MHz, CDCl ): d ¼ 165.2 (d, J ¼ 5.0 Hz, C O), 142.7, 141.9,
3
5.11 (s, 2H), 4.17–4.07 (m, 4H, 2 ꢁ POCH2CH3), 3.69 (d, J ¼ 12.1 Hz,
2H, PCH2), 2.20 (s, 3H, C(O)CH3), 1.31 (t, J ¼ 6.9 Hz, 6H, 2 ꢁ
POCH2CH3); 13C NMR (75.5 MHz, CD3OD): d ¼ 171.2, 165.4 (d,
141.3, 132.6, 131.6, 124.2, 120.5, 119.5, 110.1, 62.9 (d, J ¼ 6.6 Hz,
POC), 52.0, 40.2, 34.8 (d, J ¼ 157.5 Hz, PC), 20.6, 20.2, 16.3 (d,
J ¼ 5.4 Hz, POCC), 16.2 (d, J ¼ 5.4 Hz, POCC); 31P NMR (121.5 MHz,
CDCl3): d ¼ 22.73 ppm. Anal. calcd. for C19H27N6O4P: C, 52.53; H,
6.26; N, 19.34. Found: C, 52.31; H, 6.11; N, 19.67.
–
–
J ¼ 4.5 Hz, C O), 166.7, 156.5, 148.8, 141.4, 125.9, 97.4, 63.0 (d,
J ¼ 6.6 Hz, POC), 51.9, 45.1, 34.7 (d, J ¼ 158.0 Hz, PC), 24.4, 16.2 (d,
J ¼ 5.7 Hz, POCC); 31P NMR (121.5 MHz, CD3OD): d ¼ 23.23 ppm.
Anal. calcd. for C16H24N7O6P · H2O: C, 41.83; H, 5.70; N, 21.34.
Found: C, 41.80; H, 5.78; N, 21.52.
Diethyl {2-[4-((3-acetylindol-1-yl)methyl)-1,2,3-triazol-1-yl]-
acetamido}methylphosphonate 6h
Yield: 95%; after chromatography on a silica gel column with
chloroform–methanol (50:1, 20:1 v/v) appropriate fractions were
crystallized from a methanol–diethyl ether mixture to give a
white solid. m.p. ¼ 128–130°C; IR (KBr): n ¼ 3395, 3235, 3134,
3067, 2986, 2930, 1692, 1639, 1026, 754 cmꢀ1; 1H NMR (300 MHz,
CDCl3): d ¼ 8.37–8.33 (m, 1H), 7.87 (s, 1H), 7.70 (brt, J ¼ 5.8 Hz, 1H,
PCH2NH), 7.62 (s, 1H), 7.43–7.40 (m, 1H), 7.31–7.25 (m, 2H), 5.46 (s,
2H), 5.06 (s, 2H), 4.12–4.02 (m, 4H, 2 ꢁ POCH2CH3), 3.68 (dd,
Diethyl {2-[4-((3-benzoyl-2,4-dioxoquinazolin-1-yl)methyl)-
1,2,3-triazol-1-yl]acetamido}methylphosphonate 6e
Yield: 84%; after chromatography on a silica gel column with
chloroform–methanol (20:1, 10:1 v/v) appropriate fractions
solidified as a white powder. m.p. ¼ 87–88°C; IR (KBr): n ¼ 3446,
3067, 2985, 1748, 1699, 1663, 1233, 1023, 811, 754, 679 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d ¼ 8.17 (dd, J ¼ 7.9 Hz, J ¼ 1.4 Hz, 1H),
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Arch. Pharm. Chem. Life Sci. 2014, 347, 506–514
[(1,2,3-Triazol-1-yl)acetamido]methylphosphonates
513
J ¼ 12.3 Hz, J ¼ 5.8 Hz, 2H, PCH2), 2.51 (s, 3H, C(O)CH3), 1.25 (t,
J ¼ 6.8 Hz, 6H, 2 ꢁ POCH2CH3); 13C NMR (75.5 MHz, CDCl3):
[3] E. De Clercq, A. Holý, I. Rosenberg, T. Sakuma, J. Balzarini,
P. C. Maudgal, Nature 1986, 323, 464–467.
–
–
–
d ¼ 193.3 (s, C O), 165.4 (d, J ¼ 5.2 Hz, C O), 142.8, 136.4,
–
[4] E. De Clercq, T. Sakuma, M. Baba, R. Pauwels, J. Balzarini,
135.3, 126.2, 124.2, 123.4, 122.6, 122.4, 117.2, 110.0, 63.1 (d,
J ¼ 6.6 Hz, POC), 52.1, 42.1, 34.9 (d, J ¼ 157.7 Hz, PC), 27.6, 16.4 (d,
J ¼ 5.7 Hz, POCC); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.62 ppm.
Anal. calcd. for C20H26N5O5P · H2O: C, 51.61; H, 6.06; N, 15.05.
Found: C, 51.51; H, 5.76; N, 15.15.
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The antiviral assays were based on inhibition of virus-induced
cytopathicity in HEL [herpes simplex virus type 1 (HSV-1), HSV-2
(G), vaccinia virus and vesicular stomatitis virus], Vero (para-
influenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro
virus), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and
respiratory syncytial virus), MDCK (influenza A (H1N1 and H3N1)
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the cell cultures) in the presence of varying concentrations (250,
50, 10, … mM) of the test compounds. Viral cytopathicity was
recorded as soon as it reached completion in the control virus-
infected cell cultures that were not treated with the test
compounds. The antiviral concentration was expressed as the
EC50 or 50%-effective compound concentration required to
inhibit virus-induced cytopathicity by 50%.
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ꢀ
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Cytotoxicity and cytostatic assay
The cytotoxicity of the test compounds was monitored as a
microscopically visible alteration of cell morphology, and expressed
as the MCC or compound concentration required to afford a
microscopically detectable alteration of cell culture morphology.
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as the 50% cytostatic concentration (IC50
) or compound
concentration required to inhibit cell proliferation by 50%. For
this purpose, cells were seeded in 200-mL wells of 96-well
microtiter plates and allowed to proliferate for 2 (L1210) to 3
(CEM) or 4 (HeLa) days in the absence or presence of different
serial concentrations of the test compounds. At the end of the
exponential proliferation phase, the cells were counted by an
automated Coulter ZI particle counter (Analis, Ghent, Belgium).
[20] I. E. Głowacka, J. Balzarini, A. E. Wróblewski, Arch. Pharm.
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The authors wish to express their gratitude to Mrs. Małgorzata
Pluskota, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, and Mrs. Lizette
van Berckelaer for excellent technical assistance. The synthetic part of
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ꢀ
this work was supported by the Medical University of Łódz internal
[24] P. G. Baraldi, B. Cacciari, G. Spalluto, X. Ji, M. E. Olah,
G. Stiles, S. Dionisotti, C. Zocchi, E. Ongini, K. A. Jacobson, J.
Med. Chem. 1996, 39, 802–806.
funds (503-3014-01). The virological/cytostatic part of this work was
supported by the KU Leuven (GOA No. 10/014).
[25] P. G. Baraldi, B. Cacciari, M. J. P. de Las Infantas, R. Romagnoli,
G. Spalluto, R. Volpini, S. Costanzi, S. Vittori, G. Cristalli,
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41, 3174–3185.
The authors have declared no conflict of interest.
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