Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase

Article abstract of DOI:10.1016/j.ejmech.2014.05.073

8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

Full text of DOI:10.1016/j.ejmech.2014.05.073

Accepted Manuscript
Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination
of their inhibitory activities against Plasmodium falciparum Purine Nucleoside
Phosphorylase
Alba Gigante , Eva-María Priego , Paula Sánchez-Carrasco , Luis Miguel Ruiz-Pérez ,
Johan Vande Voorde , María- José Camarasa , Jan Balzarini , Dolores González-
Pacanowska , María-Jesús Pérez-Pérez
PII:
S0223-5234(14)00505-4
10.1016/j.ejmech.2014.05.073
EJMECH 7031
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 February 2014
Revised Date: 26 May 2014
Accepted Date: 31 May 2014
Please cite this article as: A. Gigante, E.-M. Priego, P. Sánchez-Carrasco, L.M. Ruiz-Pérez, J.V. Voorde,
M.-.J. Camarasa, J. Balzarini, D. González-Pacanowska, M.-J. Pérez-Pérez, Microwave-assisted
synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against
Plasmodium falciparum Purine Nucleoside Phosphorylase, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.05.073.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their
inhibitory activities against Plasmodium falciparum Purine Nucleoside Phosphorylase
Alba Gigante, Eva-María Priego, Paula Sánchez-Carrasco, Luis Miguel Ruiz-Pérez, Johan Vande
Voorde, María- José Camarasa, Jan Balzarini, Dolores González-Pacanowska and María-Jesús Pérez-
Pérez*
Compounds 8 (R=3-OCH3), 10 (R=3-CF3) and 11 (R=4-CF3)
selectively inhibited PfPNP with IC50 =3-8 µM

ACCEPTED MANUSCRIPT
Microwave-assisted synthesis of C-8 aryl and heteroaryl
inosines and determination of their inhibitory activities
against Plasmodium falciparum Purine Nucleoside
Phosphorylase
Alba Gigante,a Eva-María Priego, a Paula Sánchez-Carrasco,b Luis Miguel Ruiz-
Pérez, b Johan Vande Voorde,c María- José Camarasa,a Jan Balzarini,c Dolores
González-Pacanowskab and María-Jesús Pérez-Péreza*
a Instituto de Química Médica (IQM-CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain
b Instituto de Parasitología y Biomedicina “López-Neyra” (IPBLN-CSIC). Parque Tecnológico de
Ciencias de la Salud, Avda. del Conocimiento, s/n.18016 Armilla, Granada, Spain
c Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium.
Abbreviations- MAOS, microwave-assisted organic synthesis ; PfPNP : Plasmodium falciparum
Purine Nucleoside Phosphorylase.
* Corresponding author: Dr. María-Jesús Pérez-Pérez
Instituto de Química Médica (CSIC)
c/ Juan de la Cierva 3
E-28006 Madrid (Spain)
Phone number: 34 91 258 75 79
Fax number: 34 91 5644853
e-mail: mjperez@iqm.csic.es
1

ACCEPTED MANUSCRIPT
Abstract— 8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to
difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine
nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These
compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three
compounds have shown a pronounced specific inhibition of P. falciparum-encoded purine
nucleoside phosphorylase, an important target for antimalarial chemotherapy.
Keywords- Inosine; microwave-assisted synthesis; direct C-Arylation; antiviral; phosphorylase
2

ACCEPTED MANUSCRIPT
1. – Introduction.
C-8 Arylpurine nucleosides are interesting chemical entities due to their potential as therapeutic
agents and as tools for biomolecular applications, as recently reviewed [1]. These compounds are
mostly synthesized by Suzuki or Stille cross-coupling processes starting from the corresponding 8-
bromonucleosides. More recently a direct arylation reaction at position 8 of purine nucleosides has
been described by two independent groups [2, 3] mediated by a Pd-Cu catalyst system with aryl
iodides using an appropriate base. This reaction has been applied to the unprotected nucleosides to
obtain C-8 aryl nucleosides in a single reaction step [2, 3]. Moreover the reaction works well with
either electron-donating or electron-withdrawing aryl iodides allowing the introduction of different
aryl substituents. This interesting synthetic procedure has been applied mostly to adenosine
derivatives [2, 3] and has been scarcely explored for guanosine and inosine [4].
Our research group has been involved in the synthesis and the biological evaluation of inosine
nucleosides, and we have reported their behaviour as inhibitors of nucleoside-processing enzymes
[5] as well as their capacity to inhibit new blood vessel formation [6-8]. Moreover it is well-known
that inosine derivatives are interesting entities for evaluation against purine nucleoside
phosphorylase, a key enzyme in the purine salvage pathway of humans and different pathogens [9,
10]. Among the latest, PNP from Plasmodium falciparum is probably the best studied target [11-
14]. Thus, the recently described direct arylation reaction sounds appealing to our interests to access
C-8 aryl inosine nucleosides (Figure 1) and to explore their biological potential as
chemotherapeutics.
So our initial approach was to apply the reaction conditions set up by Fairlamb for adenosines [4]
and to introduce a variety of differently substituted aryl groups at position 8 of inosine. However,
our attempts to apply this described procedure to inosine did not meet a successful outcome. Only a
very low conversion to the desired 8-aryl compound was detected accompanied by serious
difficulties to isolate a pure compound for biological testing. This different behaviour of inosine
compared to adenosine could be partially ascribed to low solubility, a problem already described in
3

ACCEPTED MANUSCRIPT
other synthetic approaches. Therefore, our working strategy has been to increase the solubility of
inosine by protection of the nucleoside OHs, prior to performing the arylation reaction. Very
recently other authors have also used a protection/deprotection procedure while performing C-C
cross coupling reactions in inosines [15]. The reported examples in adenosine require, in general,
long reaction times, typically overnight [4] or even up to 60 hours [2]. We have found that
application of microwave-assisted synthesis has allowed performing the reaction on protected
inosine in one hour. The synthesized inosines have been evaluated against purine nucleoside
phosphorylases of different origins. In parallel their antiviral activity has also been determined.
2.- Results and discussion.
2.1. Chemistry.
The described procedures for the direct C-8 arylation of adenosines [2-4] require the participation of
an appropriate base (mostly Cs2CO3 and/or piperidine) whose role has been well-studied by
Fairlamb and co. [3, 4]. Therefore the protection of the sugar OHs had to be performed with a basic-
stable protecting group, and this made us to consider the commercially available 2´,3´-O-
isopropylideninosine (1) as a convenient starting material. Reaction of 1 with 4-iodoanisole in the
presence of Cs2CO3 (2.5 eq), CuI (3.0 eq), piperidine (0.4 eq), Pd(OAc)2 (0.05 eq) and 4-
iodoanisole (2.0 eq) in dry and degassed DMF (5 mL/mmol) in a vacuum-dried Schlenk tube at 80
°C overnight afforded the C-8 arylated compound (2) in approximately 22% yield (Scheme 1).
HPLC-MS analysis of the reaction mixture indicated that the only accompanying nucleoside
product was the starting material. Attempts to increase the yield by prolonging the reaction time
and/or by increasing the quantities of the reactives were almost unsuccessful; only increasing the
temperature slightly improved the conversion. At this stage, the use of microwave (MW) heating
was considered. Indeed Alami [16] had previously successfully applied microwave irradiation for
the direct C-8 arylation of non-ribose adenines employing high temperatures (160 °C). However
when Farirlamb´s group applied these conditions to the arylation reaction of 2´-deoxyadenosine [4],
4

ACCEPTED MANUSCRIPT
extensive breaking of the glycosidic bond was detected ascribed to the sensitivity of this bond to
such high temperatures. Based on the higher stability of ribonucleosides compared to their 2´-
deoxynucleoside analogues, we decided to assay microwave-irradiation for the synthesis of 2
applying lower temperatures. We were glad to see that the microwaved-reaction of 1 with 4-
iodoanisole in DMF in the presence of Cs2CO3, CuI, Pd(OAc)2 and piperidine at 120 °C for 1 h
showed a clear conversion to the coupling product 2 with no detection of glycosidic bond cleavage.
However, the work-up procedure was found problematic. Removal of the metal salts was partially
performed by filtration, but attempts to completely eliminate these salts by acid treatment as
described by other authors led to extensive decomposition through breakage of the glycosidic bond.
In our hands, the solid isolated after filtration and subsequent chromatography was deeply coloured,
probably indicating the concomitant presence of some metal salts. We were seriously concerned
about the coexistence of metal containing by-products that might be a source of influencing and
misinterpretation of the biological results. Indeed the use of copper salts, extensively applied in
click chemistry, is being reconsidered when applied to the synthesis of compounds meant for
biological screening, since such salts may have a clear impact on cellular events masking the real
properties of the compounds [17-19]. Therefore, to assure the removal of any metal contaminants,
the compound obtained after chromatography was extensively treated with Quadrasil MP in DMF
overnight. Filtration of the resin afforded the 8-arylnucleoside 2 as a white solid in 52% overall
yield.
The next step involved the removal of the isopropylidene group but the acid conditions required
provoked partial breaking of the glycosidic bond generating a variable proportion of 8-(4-
methoxyphenyl)hypoxanthine. The cleanest deprotection was observed by time-controlled treatment
with a mixture of TFA/dioxane/H2O (4:1:1) at rt, and quenching with NH4HCO3. In this way,
compound 3 was isolated in 98% yield.
The pathway step-up for the synthesis of 3 as a model compound was applied to the reaction of 1
with different aryl iodides. The results obtained are shown in Scheme 2. Thus reaction of 2´,3´-O-
5

ACCEPTED MANUSCRIPT
isopropylideneinosine (1) with a variety of aryl iodides (3-iodoanisole, 4-iodotoluene, 4(3)-
trifluoromethylphenyl iodides) under microwave conditions (120 °C, 1h) using Cs2CO3, CuI,
Pd(OAc)2 and piperidine afforded the 8-arylinosines (4-7) in good yields. No significant differences
were observed when employing electrondonating or electronwithdrawing groups at the aryl moiety,
in agreement with that reported by other authors [4, 20]. The reaction also took place when an aryl
bromide was used (i.e., 4-bromoanisole) but the yield was significantly lower than that obtained
with the corresponding aryl iodide. In all cases, Quadrasil MP treatment was crucial to obtain a pure
compound. Removal of the isopropylidene group in compounds 4-7 afforded the deprotected
ribonucleosides 8-11 in good to excellent yields.
Based on these results, the reaction was extended to the synthesis of 4-pyridyl or 3-pyridyl 8-
substituted inosines (Scheme 3). Thus reaction of 2´,3´-O-isopropylideneinosine (1) with 4-
iodopyridine or 3-iodopyridine afforded the corresponding coupling products at position 8, 12 and
13, in 55 and 54% yields, respectively. Deprotection of the isopropylidene group provided the 8-
pyridylinosines 14 and 15, in 78 and 69% yields, respectively.
2.2 Hydrolitic stability studies.
It has been described that 8-substituted purine nucleosides are more sensitive to acidic conditions
than their non-substituted analogues [1, 21] and indeed we have experienced serious difficulties to
remove the isopropylidene group by acid treatment without affecting the glycosidic bond. In this
scenario, we considered relevant to determine the hydrolytic stability of the synthesised 8-
substituted inosines at different pH values. The compounds were incubated at 37 °C in buffered
solutions at three different pHs (1.2, 5.0 and 7.4) and the evolution was followed by HPLC at
different time points (0, 1h, 4h and 24h). Compounds 3, 8-11, 14 and 15 were found to be
completely stable in the buffer solutions at pH 5.0 and 7.4 up to 24 h (data not shown). Only at pH
1.2, the compounds suffer from the breakage of the glycosidic bond with half-lifes around 5 h
6

ACCEPTED MANUSCRIPT
(Figure S1 in Supplementary data). Interestingly the two pyridyl derivatives 14 and 15 showed a
higher stability with larger half-lifes (between 10 and 15 h).
2.3 Biological evaluation
The synthesized compounds 3, 8-11, 14 and 15 were evaluated for their inhibitory activity against a
wide variety of DNA and RNA viruses (including, among others, herpes simplex virus type 1,
herpes simplex virus type 2, vaccinia virus, vesicular stomatitis virus, cytomegalovirus, human
immunodeficiency virus and influenza virus). Compound 9 could not be tested due to solubility
problems. Unfortunately, no significant antiviral activity was observed at 100 µM. It should be
mentioned that no cytotoxicity was detected against the host cell lines (i.e. CEM, HEL, HeLa, Vero
and MDCK) at concentrations up to 100 µM suggesting that the compounds are free of any toxic
contaminant.
Compounds 3, 8-11, 14 and 15 were also evaluated against purine nucleoside phosphorylase of
Plasmodium falciparum (PfPNP). This enzyme, that preferentially uses inosine as substrate, has a
central role in purine salvage for the parasite and is a promising validated target for antimalarial
intervention [12, 14]. Immucillins have been deeply investigated as transition state analogues
inhibiting PfPNP and compounds such as 5′-methylthio-immucillin-H kills P. falciparum in culture
[22]. More recently, another immucillin, DADMe-Immucillin-G, has been shown to kill the parasite
in a primate animal model [14]. When tested against PfPNP using inosine as the natural substrate,
compounds 8, 10 and 11 showed significant inhibitory activity against this enzyme in the low µM
range (Ki = 8.1, 3.7 and 5.3 µM, respectively), with values quite comparable to the Km for the
natural substrate inosine (Km = 5.9 µM) (Table 1). Compounds 8, 10 and 11 were also investigated
as potential inhibitors or substrates against human and prokaryotic (i.e. E. coli, Mycoplasma
hyorhinis) PNP. When the compounds were evaluated as potential inhibitors of the conversion of
100 µM inosine to hypoxanthine, no signs of direct inhibitory activity were observed for both
enzymes. Also, no substrate activity was found under conditions where 100 µM inosine was
7

ACCEPTED MANUSCRIPT
converted to hypoxanthine by 96-98% within 1 hour. These findings point to a significant degree of
selectivity of these compounds as specific anti-PfPNP agents. Unfortunately the compounds were
devoid of significant inhibitory activity of growth of intraerythrocytic forms of the Plasmodium
falciparum chloroquine-sensitive strain 3D7 when tested at 25 µM, while chloroquine in the same
assay gave an IC50 value of 5 nM. The lack of cellular activity may be due to inefficient uptake by
the erythrocytes and/or the parasites in addition to limited intracellular efficacy. Specific uptake
studies might provide more insights on this issue.
3. Conclusions
A number of 8-aryl and pyridyl-substituted inosines have been synthesized making use of the
recently reported direct arylation reaction mediated by Pd-Cu complexes. Special attention has been
paid to the removal of metal contaminants that has been accomplished including an extensive
Quadrasil MP treatment. The 8-substituted inosines obtained proved to be stable at pH values of 7.4
and 5.0, while at pH=1.2 their half-lives varied between 5 and 10 h. Concerning the biological
evaluation, no significant toxicity was detected in different types of cell cultures supporting the
absence of metal-contaminants. Moreover compounds 8, 10 and 11 were able to inhibit P.
falciparum PNP at low micromolar Ki values similar to that of the Km of inosine. The lack of
inhibitory or substrate properties of these inosine derivatives 8 against human and prokaryotic PNP
let us conclude that these 8-arylinosines are highly selective inhibitors of PfPNP and therefore
represent interesting starting points for further development of specific inhibitors of the enzyme.
4. Experimental
4.1 Synthesis.
Melting points were obtained on a Reichert-Jung Kofler apparatus and are uncorrected. The
elemental analysis was performed with a Heraeus CHN-O-RAPID instrument. The elemental
8

ACCEPTED MANUSCRIPT
compositions of the compounds fell within ± 0.4% of the calculated values. Electrospray mass
spectra were measured on a quadrupole mass spectrometer equipped with an electrospray source
(Hewlett-Packard, LC/MS HP 1100). 1H and 13C NMR spectra were recorded on a Varian INNOVA
300 operating at 299 MHz (1H) and 75 MHz (13C), respectively, and Varian INNOVA-400
operating at 399 MHZ (1H) and 99 MHz (13C), respectively.
Analytical TLC was performed on silica gel 60 F254 (Merck) precoated plates (0.2 mm). Spots were
detected under UV light (254 nm) and/or charring with ninhydrin. Separations on silica gel were
performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a
ChromatotronR (Kiesegel 60 PF254 gipshaltig (Merck)), with layer thickness of 1 and 2 mm and
flow rate of 4 or 8 mL/min, respectively. Flash column chromatography was performed in a Biotage
Horizon instrument.
Microwave reactions were performed using the Biotage Initiator 2.0 single-mode cavity instrument
from Biotage (Uppsala). Experiments were carried out in sealed microwave process vials utilizing
the standard absorbance level (400 W maximum power). The temperature was measured with an IR
sensor on the outside of the reaction vessel.
High-performance liquid chomatography (HPLC) analysis was performed using an Aligent
Technologies 1120 Compact LC and an ACE5 C18-300 column (150x4.6 mm, 300Å). UV
absorption was monitored at diode array.
4.1.1. 2’,3’-O-Isopropylidene-8-(4’’-methoxyphenyl)inosine (2). To a microwave vial containing
oven-dried 2´,3´-O-isopropilideneinosine (1) (200 mg, 0.65 mmol) and Cs2CO3 (528 mg, 1.62
mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol) and 4-iodoanisole (304 mg, 1.30
mmol) in dry DMF (5 mL) were added. The vial was sealed and deoxygenated. Then dried and
deoxygenated piperidine (26 µL, 0.26 mmol) was added. The mixture was microwaved irradiated at
120 ºC for 1h. The reaction mixture was diluted with 150 mL of dichloromethane:methanol (1:1)
and filtered. The filtrate was evaporated to dryness and the residue obtained was purified by flash
chromatography (dichloromethane:methanol, 10:1). The purified solid was solved in DMF, treated
9

ACCEPTED MANUSCRIPT
with Quadrasil MP overnight, filtered and coevaporated with diethyl ether to yield 140 mg (52%) of
2 as a white amorphous solid. EM (ES, positive mode): m/z 415 (M+H)+.1H NMR (400 MHz,
DMSO-d6): δ 1.27, 1.44 (s, 6H, C(CH3)2), 3.57 (m, 2H, H-5´), 3.84 (s, 3H, OCH3), 4.13 (m, 1H, H-
4´), 5.02 (m, 2H, H-3´, OH), 5.51 (dd, J = 5.9, 2.2 Hz, 1H, H-2´), 5.86 (d, J = 2.1 Hz, 1H, H-1´),
7.16 (d, J = 8.6 Hz, 2H, H-3´´), 7.65 (d, J = 8.5 Hz, 2H, H-2´´), 8.11 (s, 1H, H-2), 12.52 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): δ 25.2, 27.1 (C(CH3)2), 55.4 (OCH3), 61.6 (C-5´), 81.7 (C-
3´), 82.4 (C-2´), 87.2 (C-4´), 90.1 (C-1´), 113.2 (C(CH3)2), 114.4 (C-2´´), 121.1 (C-1´´), 124.4 (C-
5), 130.9 (C-3´´), 145.6 (C-2), 148.4 (C-4), 149.6 (C-8), 156.4 (C-6), 160.8 (C-4´´). Anal. Calc. for
C20H22N4O6 (%): C, 57.97; H, 5.35; N, 13.52. Found: C, 57.69; H, 5.64; N, 13.49.
4.1.2. 8-(4’’-Methoxyphenyl)inosine (3). A solution of 2 (100 mg, 0.24 mmol) in a mixture of
TFA/H2O/dioxano (3:1:1) (2.4 ml) was stirred at rt for 15 min. The reaction was quenched by
addition of a saturated solution of NH4HCO3 and extracted with EtOAc (3 x 15 ml). The organic
extracts were dried on anhydrous MgSO4, filtered and evaporated. The residue was purified by
CCTLC using as eluent dichloromethane/methanol (10:1 to 5:1) to yield 88 mg (98%) of 3 as a
1
white solid. Mp: 136-137 ºC. EM (ES, positive mode): m/z 375 (M+H)+. H NMR (400 MHz,
DMSO-d6): δ 3.53, 3.67 (m, 2H, H-5´), 3.84 (s, 3H, OCH3), 3.90 (m, 1H, H-4´), 4.14 (dd, J = 2.1,
4.8 Hz, 1H, H-3´), 5.10 (dd, J = 6.0, 6.0 Hz, 1H, H-2´), 5.11 (br s, 2H, OH), 5.47 (br s, 1H, OH),
5.72 (d, J = 6.8 Hz, 1H, H-1´), 7.12 (d, J = 8.7 Hz, 2H, H-3´´), 7.65 (d, J = 8.7 Hz, 2H, H-2´´), 8.06
(s, 1H, H-2); 13C NMR (100 MHz, DMSO-d6): δ 56.0 (OCH3), 62.8 (C-5´), 71.5 (C-3´), 71.9 (C-2´),
87.0 (C-4´), 89.9 (C-1´), 114.9 (C-3´´), 122.4 (C-1´´), 125.2 (C-5), 131.6 (C-2´´), 147.9 (C-2), 149.9
(C-4), 150.6 (C-8), 158.3 (C-6), 161.2 (C-4´´). Anal. Calc. for C17H18N4O6 (%): C, 54.54; H, 4.85;
N, 14.97. Found: C, 54.36; H, 5.08; N, 14.69.
4.1.3. 2’,3’-O-Isopropylidene-8-(3’’-methoxyphenyl)inosine (4). Following the conditions described
for the synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol) reacted
with 3-iodoanisole (152 µl, 1.30 mmol) in the presence Cs2CO3 (528 mg, 1.62 mmol), CuI (371 mg,
1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol) and piperidine (26 µl, 0.26 mmol) in anhydrous DMF
10

ACCEPTED MANUSCRIPT
(3.2 ml). After work-up, chromatography purification and Quadrasil® MP treatment as described for
2, 148 mg (55%) of 4 were obtained as a white amorphous solid. MS (ES, positive mode): m/z 415
1
(M+H)+. H NMR (400 MHz, DMSO-d6): δ 1.19, 1.41 (s, 6H, C(CH3)2), 3.56 (m, 2H, H-5´), 3.81
(s, 3H, OCH3), 4.09 (m, 1H, H-4´), 4.98 (m, 2H, H-3´, OH), 5.50 (dd, J = 6.2, 2.8 Hz, 1H, H-2´),
5.86 (d, J = 2.6 Hz, 1H, H-1´), 7.22 (m, 3H, H-4´´, H-5´´, H-6´´), 7.49 (t, J = 7.9 Hz, 1H, H-2´´),
8.09 (s, 1H, H-2), 12.53 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 25.2, 27.0 (C(CH3)2), 55.3
(OCH3), 61.5 (C-5´), 81.6 (C-3´), 82.5 (C-2´), 87.2 (C-4´), 90.1 (C-1´), 113.3 (C(CH3)2), 114.8,
116.0, 121.4 (C-4´´, C-5´´, C-6´´), 124.5 (C-5), 130.0 (C-2´´), 130.1 (C-1´´), 145.9 (C-2), 148.5 (C-
4), 149.4 (C-8), 156.4 (C-6), 159.3 (C-3´´). Anal. Calc. for C20H22N4O6 (%): C, 57.97; H, 5.35; N,
13.52. Found: C, 57.71; H, 5.44; N, 13.39.
4.1.4. 2’,3’-O-Isopropylidene-8-(4’’-toly)inosine (5). Following the conditions described for the
synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol) reacted under
microwave conditions in anhydrous DMF (3.2 ml) with 4-iodotoluene (284 mg, 1.30 mmol) in the
presence of Cs2CO3 (528 mg, 1.62 mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol)
and piperidine (26 µl, 0.26 mmol). After work-up, chromatography purification and Quadrasil® MP
treatment as described for 2, 158 mg (61%) of 5 were obtained as a white amorphous solid. MS
1
(ES, positive mode): m/z 399 (M+H)+. H NMR (400 MHz, DMSO-d6): δ 1.27, 1.43 (s, 6H,
C(CH3)2), 2.40 (s, 3H, CH3), 3.57 (m, 2H, H-5´), 4.12 (m, 1H, H-4´), 5.01 (m, 2H, H-3´, OH), 5.51
(dd, J = 6.2, 2.7 Hz, 1H, H-2´), 5.86 (d, J = 2.7 Hz, 1H, H-1´), 7.41 (d, J = 7.9 Hz, 2H, H-3´´), 7.59
(d, J = 8.0 Hz, 2H, H-2´´), 8.12 (s, 1H, H-2), 12.52 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ
21.7 (CH3), 25.9, 27.7 (C(CH3)2), 62.2 (C-5´), 82.3 (C-3´), 83.1 (C-2´), 87.8 (C-4´), 90.8 (C-1´),
113.9 (C(CH3)2), 125.2 (C-5), 126.7 (C-1´´), 130.0 (C-2´´), 130.2 (C-3´´), 140.8 (C-4´´), 146.4 (C-
2), 149.1 (C-4), 150.4 (C-8), 157.0 (C-6). Anal. Calc. for C20H22N4O5 (%): C, 60.29; H, 5.57; N,
14.06. Found: C, 60.05; H, 5.68; N, 14.22.
11

ACCEPTED MANUSCRIPT
4.1.5. 2’,3’-O-Isopropylidene-8-(4’’-trifluoromethylphenyl)inosine (6). Following the conditions
described for the synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol)
in dry DMF (3.2 ml) reacted with 4-trifluoromethylphenyl iodide (191 µl, 1.30 mmol) in the
presence of Cs2CO3 (528 mg, 1.62 mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol)
and piperidine (26 µl, 0.26 mmol). After work-up, chromatography purification and Quadrasil® MP
treatment as described for 2, 135 mg (46%) of 6 were obtained as a white amorphous solid. MS
(ES, positive mode): m/z 453 (M+H)+, 475 (M+Na)+. 1H NMR (400 MHz, DMSO-d6): δ 1.27, 1.43
(s, 6H, C(CH3)2), 3.56 (m, 2H, H-5´), 4.15 (m, 1H, H-4´), 5.30 (m, 2H, H-3´, OH), 5.53 (dd, J = 6.2,
2.3 Hz, 1H, H-2´), 5.87 (d, J = 2.3 Hz, 1H, H-1´), 7.97 (m, 4H, H-2´´, H-3´´), 8.17 (s, 1H, H-2),
12.60 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 25.8, 27.6 (C(CH3)2), 62.2 (C-5´), 82.3
(C-3´), 83.3 (C-2´), 88.2 (C-4´), 90.8 (C-1´), 113.9 (C(CH3)2), 124.6 (q ap, J = 272.2 Hz, CF3),
125.4 (C-5), 126.6 (d, J = 3.1 Hz, C-3´´), 130.9 (d, J = 31.9 Hz, C-4´´), 130.9 (C-2´´), 133.6 (C-1´´),
146.7 (C-2), 148.8, 149.3 (C-8, C-4), 157.0 (C-6). Anal. Calc. for C20H19F3N4O5 (%): C, 53.10; H,
4.23; N, 12.38. Found: C, 53.00; H, 4.35; N, 12.42.
4.1.7. 2’,3’-O-Isopropylidene-8-(3’’-trifluoromethylphenyl)inosine (7). Following the conditions
described for the synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol)
in dry DMF (3.2 ml) reacted with 3-trifluoromethylphenyl iodide (187 µl, 1.30 mmol) in the
presence of Cs2CO3 (528 mg, 1.62 mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol)
and piperidine (26 µl, 0.26 mmol). After work-up, chromatography purification and Quadrasil® MP
treatment as described for 2, 162 mg (55%) of 7 were obtained as a white amorphous solid. MS
(ES, positive mode): m/z 453 (M+1)+, 475 (M+Na)+. 1H NMR (400 MHz, DMSO-d6): δ 1.26, 1.40
(s, 6H, C(CH3)2), 3.52 (m, 2H, H-5´), 4.12 (m, 1H, H-4´), 4.99 (m, 2H, H-3´, OH), 5.53 (dd, J =
6.3, 2.6 Hz, 1H, H-2´), 5.82 (d, J = 2.6 Hz, 1H, H-1´), 7.83 (t, J = 8.0 Hz, 1H, H-5´´), 7.95 (d, J =
7.9 Hz, 1H, H-4´´), 8.00 (m, 2H, H-2´´, H-6´´), 8.14 (s, 1H, H-2), 12.57 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): δ 25.8, 27.6 (C(CH3)2), 62.1 (C-5´), 82.3 (C-3´), 83.1 (C-2´), 88.1 (C-4´),
91.0 (C-1´), 113.9 (C(CH3)2), 124.5 (q ap, J = 272.7 Hz, CF3), 125.3 (C-5), 126.7 (d, J = 4.3 Hz, C-
12

ACCEPTED MANUSCRIPT
2´´), 127.6 (d, J = 4.1 Hz, C-4´´), 130.3 (d, J = 32.4 Hz, C-3´´), 130.7 (C-1´´), 130.9 (C-5´´), 133.9
(C-6´´), 146.8 (C-2), 148.9 (C-4), 149.3 (C-8), 157.0 (C-6). Anal. Calc. for C20H19F3N4O5 (%): C,
53.10; H, 4.23; N, 12.38. Found: C, 52.95; H, 4.41; N, 12.35.
4.1.8. 8-(3’’-Methoxyphenyl)inosine (8). Following the conditions described for the synthesis of
compound 3, compound 4 (100 mg, 0.24 mmol) was treated a mixture of TFA/H2O/dioxane (3:1:1)
(2.4 mL) at rt for 15 min. The reaction was quenched by addition of a saturated solution of
NH4HCO3. After work-up and CCTLC purification, 76 mg (85%) of 8 were obtained as a white
solid. Mp: 205-207 ºC. MS (ES, positive mode): m/z 375 (M+H)+. 1H NMR (400 MHz, DMSO-d6):
δ 3.54, 3.67 (m, 2H, H-5´), 3.83 (s, 3H, OCH3), 3.88 (m, 1H, H-4´), 4.15 (m, 1H, H-3´), 5.03 (s, 1H,
OH), 5.10 (m, 1H, H-2´), 5.15 (s, 1H, OH), 5.47 (s, 1H, OH), 5.76 (d, J = 6.7 Hz, 1H, H-1´), 7.15
(d, J = 8.3 Hz, 1H, H-4´´), 7.29 (m, 2H, H-2´´, H-6´´), 7.49 (t, J = 7.9 Hz, 1H, H-5´´), 8.12 (s, 1H,
H-2), 12.35 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 55.6 (OCH3), 62.4 (C-5´), 71.1 (C-
3´), 71.7 (C-2´), 86.6 (C-4´), 89.7 (C-1´), 115.2 (C-2´´), 116.4 (C-4´´), 122.1 (C-6´´), 125.2 (C-5),
130.5 (C-5´´), 130.8 (C-1´´), 146.0 (C-2), 149.4 (C-4), 150.7 (C-8), 156.8 (C-6), 159.7 (C-3´´).
Anal. Calc. for (C17H18N4O6) (%): C, 54.54; H, 4.85; N, 14.97. Found: C, 54.36; H, 5.08; N, 14.69.
4.1.9. 8-(4’’-Methylphenyl)inosine (9). Following the conditions described for the synthesis of
compound 3, compound 5 (100 mg, 0.25 mmol) was treated a mixture of TFA/H2O/dioxane (3:1:1)
(2.5 mL) at rt for 15 min, and quenched by addition of a saturated solution of NH4HCO3. After
work-up and CCTLC purification, 78 mg (87%) of 9 were obtained as a white solid. Mp: 296-297
1
ºC. MS (ES, positive mode): m/z 359 (M+H)+. H NMR (400 MHz, DMSO-d6): δ 2.39 (s, 3H,
CH3), 3.60 (m, 2H, H-5´), 3.87 (m, 1H, H-4´), 4.13 (m, 1H, H-3´), 5.07 (m, 3H, H-2´, 2 OH), 5.44
(s, 1H, OH), 5.71 (d, J = 6.8 Hz, 1H, H-1´), 7.38 (d, J = 7.4 Hz, 2H, H-3´´), 7.60 (d, J = 7.5 Hz, 2H,
H-2´´), 8.10 (s, 1H, H-2), 12.08 (br s, 1H, NH); (1H NMR values are in agreement with those
partially previously described [23]). 13C NMR (100 MHz, DMSO-d6): δ 21.7 (CH3), 62.7 (C-5´),
71.3 (C-3´), 71.8 (C-2´), 86.8 (C-4´), 90.0 (C-1´), 125.4 (C-5), 127.1 (C-1´´), 130.0, 130.1 (C-2´´,
13

ACCEPTED MANUSCRIPT
C-3´´), 140.6 (C-4´´), 146.2 (C-2), 149.6 (C-4), 151.2 (C-8), 157.3 (C-6). Anal. Calc for
C17H18N4O5 (%): C, 56.98; H, 5.06; N, 15.63. Found: C, 56.75; H, 5.35; N, 15.49.
4.1.10. 8-(4’’-Trifluoromethylphenyl)inosine (10). Following the conditions described for the
synthesis of compound 3, compound 6 (100 mg, 0.22 mmol) was treated a mixture of
TFA/H2O/dioxane (3:1:1) (2.2 mL) at rt for 15 min and quenched by addition of a saturated solution
of NH4HCO3. After work-up and CCTLC purification, 64 mg (70%) of 10 were obtained as a white
1
solid. Mp >170 ºC (decomp). MS (ES, positive mode): m/z 413 (M+H)+. H NMR (500 MHz,
DMSO-d6): δ 3.54, 367 (m, 2H, H-5´), 3.91 (dd, J = 7.4, 4.6 Hz, 1H, H-4´), 4.14 (m, 1H, H-3´),
5.08 (m, 2H, H-2´, OH), 5.19 (s, 1H, OH), 5.46 (s, 1H, OH), 5.70 (d, J = 6.8 Hz, 1H, H-1´), 7.95 (s,
4H, H-2´´, H-3´´), 8.14 (s, 1H, H-2). 13C NMR (125 MHz, DMSO-d6): δ 62.3 (C-5´), 71.0 (C-3´),
71.7 (C-2´), 86.9 (C-4´), 89.8 (C-1´), 124.4 (q ap, J = 272.4 Hz, CF3), 125.4 (C-5), 126.6 (d, J = 4.1
Hz, C-3´´), 130.6 (d, J = 32.1 Hz, C-4´´), 130.8 (C-2´´), 133.7 (C-1´´), 146.7 (C-2), 149.3 (C-4),
149.6 (C-8), 157.2 (C-6). Anal. Calc for C17H15F3N4O5 (%): C, 49.52; H, 3.67; N, 13.59. Found: C,
49.31; H, 3.88; N, 13.70.
4.1.11. 8-(3’’-Trifluoromethylphenyl)inosine (11). Following the conditions described for the
synthesis of compound 3, compound 7 (100 mg, 0.22 mmol) was treated a mixture of
TFA/H2O/dioxane (3:1:1) (2.2 mL) at rt for 15 min and quenched by addition of a saturated solution
of NH4HCO3. After work-up and CCTLC purification, 55 mg (60%) of 11 were obtained as a white
solid. Mp: 194-195 ºC. MS (ES, positive mode): m/z 413 (M+H)+. 1H NMR (500 MHz, DMSO-d6):
δ 3.54, 3.66 (m, 2H, H-5´), 3.90 (dd, J = 7.6, 4.7 Hz, 1H, H-4´), 4.14 (dd, J = 7.6, 4.8 Hz, 1H, H-
3´), 5.06 (m, 2H, H-2´, OH), 5.17 (d, J =4.8 Hz, 1H, OH), 5.48 (d, J = 6.2 Hz, 1H, OH), 5.69 (d, J =
6.7 Hz, 1H, H-1´), 7.84 (t, J = 7.8 Hz, 1H, H-5´´), 7.97 (d, J = 7.8 Hz, 1H, H-4´´), 8.05 (m, 2H, H-
2´´, H-6´´), 8.16 (s, 1H, H-2). 13C NMR (125 MHz, DMSO-d6): δ 61.9 (C-5´), 70.5 (C-3´), 71.2 (C-
2´), 86.3 (C-4´), 89.3 (C-1´), 123.8 (q ap, J = 272.3 Hz, CF3), 125.0 (C-5), 126.5 (d, J = 4.5 Hz, C-
2´´), 126.8 (d, J = 4.5 Hz, C-4´´), 130.1 (d, J = 32.1 Hz, C-3´´), 130.2, 130.3 (C-5´´, C-1´´), 133.3
14

ACCEPTED MANUSCRIPT
(C-6´´), 146.0 (C-2), 148.9 (C-8), 149.1 (C-4), 156.4 (C-6). Anal. Calc for C17H15F3N4O5 (%): C,
49.52; H, 3.67; N, 13.59. Found: C, 49.25; H, 3.90; N, 13.42.
4.1.12 2’,3’-O-Isopropylidene-8-(4’’-pyridyl)inosine (12). Following the conditions described for
the synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol) in dry DMF
(3.2 ml) reacted with 4-iodopyridine (266 mg, 1.30 mmol) in the presence of Cs2CO3 (528 mg, 1.62
mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol) and piperidine (26 µl, 0.26 mmol)
in the microwave at 120 °C for 1 h. The reaction was diluted with CHCl3:MeOH (1:1) (150 mL)
and filtered. The filtrate was evaporated to dryness and the residue obtained was purified by flash
chromatography (dichloromethane:methanol, 10:1). The purified solid was solved in DMF, treated
with Quadrasil MP overnight, filtered and coevaporated with diethyl ether (2x 5mL) to yield 138
1
mg (55%) of 12 as a white amorphous solid. MS (ES, positive mode): m/z 386 (M+H)+. H NMR
(400 MHz, DMSO-d6): δ 1.27, 1.44 (s, 6H, C(CH3)2), 3.55 (m, 2H, H-5´), 4.15 (m, 1H, H-4´), 5.02
(m, 2H, H-3´, OH), 5.53 (dd, J = 6.3, 2.6 Hz, 1H, H-2´), 5.90 (d, J = 2.5 Hz, 1H, H-1´), 7.74 (m,
2H, H-2´´), 8.17 (s, 1H, H-2), 8.85 (m, 2H, H-3´´), 12.68 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): δ 25.9, 27.7 (C(CH3)2), 62.1 (C-5´), 82.3 (C-3´), 83.2 (C-2´), 88.3 (C-4´), 90.8 (C-1´),
113.9 (C(CH3)2), 124.2 (C-2´´), 125.5 (C-5), 137.0 (C-1´´), 147.1 (C-2), 147.9 (C-3´´), 149.5 (C-4),
151.0 (C-8), 157.0 (C-6). Anal. Calc for C18H19N5O5 (%): C, 56.10; H, 4.97; N, 18.17. Found: C,
55.93; H, 5.13; N, 18.25.
4.1.13 2’,3’-O-Isopropylidene-8-(3’’-pyridyl)inosine (13). Following the conditions described for
the synthesis of compound 2, 2’,3’-O-isopropylideninosine (1) (200 mg, 0.65 mmol) in dry DMF
(3.3 ml) reacted under MW conditions with 3-iodopyridine (266 mg, 1.30 mmol) in the presence
Cs2CO3 (528 mg, 1.62 mmol), CuI (371 mg, 1.95 mmol), Pd(OAc)2 (7 mg, 0.03 mmol) and
piperidine (26 µl, 0.26 mmol). Following the work-up described for the synthesis of 12, compound
1
13 was obtained (135 mg, 54%). EM (ES, positive mode): m/z 386 (M+H)+. H NMR (500 MHz,
DMSO-d6): δ 1.27, 1.43 (s, 6H, C(CH3)2), 3.53 (m, 2H, H-5´), 4.13 (dd, J = 9.0, 5.9 Hz, 1H, H-4´),
5.00 (m, 2H, H-3´, OH), 5.53 (dd, J = 6.2, 2.1 Hz, 1H, H-2´), 5.82 (d, J = 2.0 Hz, 1H, H-1´), 7.79
15

ACCEPTED MANUSCRIPT
(m, 1H, H-5´´), 8.15 (s, 1H, H-2), 8.17 (m, 1H, H-6´´), 8.81 (m, 1H, H-4´´), 9.29 (s, 1H, H-2´´),
12.57 (s, 1H, NH). 13C NMR (125 MHz, DMSO-d6): δ 25.6, 27.5 (C(CH3)2), 61.9 (C-5´), 82.1 (C-
3´), 83.0 (C-2´), 87.89 (C-4´), 90.6 (C-1´),113.7 (C(CH3)2), 124.0 (C-5´´), 125.2 (C-5), 126.1 (C-
1´´), 137.0 (C-6´´), 146.6 (C8), 147.9 (C-2), 149.1 (C-4), 150.2 (C-2´´), 151.4 (C-4´´), 156.8 (C-6).
Anal. Calc for C18H19N5O5 (%): C, 56.10; H, 4.97; N, 18.17. Found: C, 55.99; H, 5.10; N, 18.28.
4.1.14 8-(4’’-Pyridyl)inosine (14). Following the conditions described for the synthesis of
compound 3, compound 12 (100 mg, 0.26 mmol) was treated a mixture of TFA/H2O/dioxane
(3:1:1) (2.6 mL) at rt for 15 min and quenched by addition of a saturated solution of NH4HCO3.
After work-up and CCTLC purification, 63 mg (78%) of 14 were obtained as a white solid. Mp >
1
200 ºC (decomp). MS (ES, positive mode): m/z 346 (M+H)+. H NMR (400 MHz, DMSO-d6): δ
3.55, 3.68 (m, 2H, H-5´), 3.92 (dd, J =7.4, 4.4 Hz, 1H, H-4´), 4.15 (m, 1H, H-3´), 4.95 (dd, J =5.1,
5.1 Hz, 1H, OH), 5.05 (dd, J = 6.0, 6.0 Hz, 1H, H-2´), 5.11 (d, J = 4.8 Hz, 1H, OH), 5.40 (d, J = 6.3
Hz, 1H, OH), 5.74 (d, J = 6.6 Hz, 1H, H-1´), 7.73 (d, J = 5.9 Hz, 2H, H-2´´), 8.13 (s 1H, H-2), 8.79
(d, J = 5.8 Hz, 1H, H-3´´), 12.52 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 61.7 (C-5´), 70.3
(C-3´), 71.2 (C-2´), 86.3 (C-4´), 89.2 (C-1´), 123.4 (C-2´´), 125.0 (C-5), 136.5 (C-1´´), 146.0 (C-2),
147.8 (C-8), 149.2 (C-4), 150.1 (C-3´´), 156.2 (C-6). Anal. Calc. for C15H15N5O5 (%): C, 52.17; H,
4.38; N, 20.38. Found: C, 52.03; H, 4.44; N, 20.25.
4.1.15. 8-(3-Pyridyl)inosine (15). Following the conditions described for the synthesis of compound
3, compound 13 (100 mg, 0.26 mmol) was treated a mixture of TFA/H2O/dioxane (3:1:1) (2.6 mL)
at rt for 15 min and it was quenched by addition of a saturated solution of NH4HCO3. After work-up
and CCTLC purification, 55 mg (69%) of 15 were obtained as a white solid. Mp > 200 ºC
(decomp). MS (ES, positive mode): m/z 346 (M+H)+. 1H NMR (500 MHz, DMSO-d6): δ 3.25 (br s,
2H, 2 OH), 3.53 (dd, J = 11.9, 4.2 Hz, 1H, H-5´), 3.66 (dd, J = 11.9, 4.0 Hz, 1H, H-5´), 3.91 (m,
1H, H-4´), 4.13 (m, 1H, H-3´), 5.06 (dd, J = 5.9, 5.9 Hz, 1H, H-2´), 5.36 (br s, 1H, OH), 5.68 (d, J =
6.7 Hz, 1H, H-1´), 7.60 (dd, J = 7.5, 4.9 Hz, 1H, H-5´´), 8.06 (s 1H, H-2), 8.11 (d, J = 7.8 Hz, 1H,
H-6´´), 8.74 (d, J = 3.6 Hz, 1H, H-4´´), 8.89 (s, 1H, H-2´´). 13C NMR (125 MHz, DMSO-d6): δ 62.5
16

ACCEPTED MANUSCRIPT
(C-5´), 71.2 (C-3´), 71.9 (C-2´), 87.0 (C-4´), 89.8 (C-1´), 124.1 (C-5´´), 125.5 (C-5), 126.3 (C-1´´),
137.4 (C-6´´), 147.5, 147.7 (C-8, C-2), 149.7 (C-4), 150.1 (C-2´´), 151.1 (C-4´´), 158.8 (C-6). Anal
Calc for C15H15N5O5 (%): C, 52.17; H, 4.38; N, 20.28. Found: C, 52.10; H, 4.42; N, 20.31.
4.2. Hydrolytic stability studies. Solutions were prepared using 10 µL of a 5 mM stock solution of
each compound in DMSO and 990 µL of phosphate buffer (pH 7.4) or citrate buffer (pH 5.0) or
trifluoroacetate buffer (pH 1.2) to obtain a final concentration of 50 µM. The samples were
incubated at 37 ºC. Aliquots were taken at different time points (t = 0, 0.5, 1, 4 and 24 h) and
analyzed by HPLC. Injection volume: 20 µL. Flow rate: 1.0 mL/min. Bottle A: Acetonitrile; bottle
B. water containing 0.05% trifluoroacetic acid. Gradient employed: 10% of bottle A to 100% bottle
A in 20 min.
4.3. Biological assays
4.3.1. Antiviral assays. The compounds were evaluated against the following viruses: herpes
simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK−) HSV-1 KOS strain
resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strains Lyons and G, 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 and Punta Toro. The antiviral assays were based on inhibition of virus-induced
cytopathicity in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero),
human epithelial cervix carcinoma cells (HeLa) or Madin-Darby canine kidney cells (MDCK).
Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the cell cultures) in the presence of varying
concentrations of the test compounds. Viral cytopathicity was recorded as soon as it reached
completion in the control virus-infected cell cultures that were not treated with the test compounds.
Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-
induced cytopathicity by 50%.
17

ACCEPTED MANUSCRIPT
4.3.2. Human and prokaryotic PNP assays. The separation of inosine from hypoxanthine was
performed by HPLC on a reverse phase column RP-18 (Merck, Darmstadt, Germany) using a linear
gradient [from 100 % buffer A (50 mM NaH2PO4 (Acros Organics, Geel, Belgium) and 5 mM
heptane sulfonic acid; pH 3.2) to 75 % buffer A and 25 % acetonitrile] as follows: 10 min linear
gradient of 100 % buffer A to 98 % buffer A + 2 % acetonitrile; 10 min linear gradient to 90 %
buffer A + 10% acetonitrile; 5 min linear gradient to 75 % buffer A + 25 % acetonitrile; 5 min
linear gradient to 100 % buffer A followed by equilibration at 100 % buffer A for 10 min.
To study the phosphorolysis of the different compounds and inosine by PNPHyor, PNPEcoli and
human PNP (ProSpec, Rehovot, Israel) the compounds (100 µM) were exposed to the enzyme (20
nM PNPHyor and PNPEcoli or 6 nM human PNP) and incubated at 37 °C in PNP buffer (50 mM
MOPS; 0.2 mM EDTA; 200 mM potassium phosphate; pH = 6.5) in a total volume of 500 µL.
After 60 min, a 100 µL-fraction was withdrawn and subjected to HPLC analysis. For analysis of the
inhibitory activity of the test compounds against PNP-catalysed hydrolysis of 100 µM inosine, the
test compounds were added to PNP at 250 µM and hydrolysis of inosine to hypoxanthine was
measured after 60 min incubation by the above-described HPLC analysis.
4.3.3. PfPNP inhibition assays. The coding sequence of PfPNP was previously cloned in pET28a
and expression was performed in E. coli BL21 (DE3) cells, by induction with 1 mM IPTG at 37 ºC
for 4 h. The purification was performed as previously described.[13] To monitor the reaction, the
activity of the enzyme was coupled to the xanthine oxidase, that catalyze the oxidation of
hypoxanthine to uric acid, whose appearance can be followed at 293nm (molar extinction
coefficient for uric acid: 12.9 mM-1 cm-1). The reaction mixture to determine the activity contained
50 mM KH2PO4, pH 7.5, 60 mU xanthine oxidase, 25 nM (0.73 µg) PfPNP, and 25 µM inosine at
25 ºC in a final volume of 1 ml. For inhibition studies, the reaction rates were measured with
variable inhibitor concentrations and inosine as substrate (25 µM) and at least five concentrations
were tested.
Supplementary data.
18

ACCEPTED MANUSCRIPT
Supplementary data associated with this article can be found in the online version. These data
include MOL files, 1H and 13C NMR spectra of the most important compounds described in this
article, and percentage of intact nucleosides in the function of time at pH=1.2.
Acknowledgements
A.G. thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral
fellowship. This project has been supported by the Spanish CICYT (SAF2009-13914-C02-01 and
SAF2012-39760-C02-01 to MJC, MJPP, EMP; and SAF2010-20059, to DGP). The antiviral and
human/bacterial PNP experiments were performed with financial support from the KU Leuven
(GOA 10/014). The technical assistance of Mrs. Ria Van Berwaer is greatly appreciated.
References
[1] N.M. Thomsen, V. Vongsutilers, P.M. Gannett, Crit. Rev. Eukaryot. Gene Expr., 21 (2011) 155-
176.
[2] I. Cerna, R. Pohl, M. Hocek, Chem. Commun., (2007) 4729-4730.
[3] T.E. Storr, A.G. Firth, K. Wilson, K. Darley, C.G. Baumann, I.J.S. Fairlamb, Tetrahedron, 64
(2008) 6125-6137.
[4] T.E. Storr, C.G. Baumann, R.J. Thatcher, S. De Ornellas, A.C. Whitwood, I.J.S. Fairlamb, J.
Org. Chem., 74 (2009) 5810-5821.
[5] E. Casanova, A.I. Hernández, E.M. Priego, S. Liekens, M.J. Camarasa, J. Balzarini, M.J. Pérez-
Pérez, J. Med. Chem., 49 (2006) 5562-5570.
[6] S. Liekens, A.I. Hernández, D. Ribatti, E. De Clercq, M.J. Camarasa, M.J. Pérez-Pérez, J.
Balzarini, J. Biol. Chem., 279 (2004) 29598-29605.
19

ACCEPTED MANUSCRIPT
[7] S. Liekens, A. Bronckaers, A.I. Hernández, E.M. Priego, E. Casanova, M.J. Camarasa, M.J.
Pérez-Pérez, J. Balzarini, Mol. Pharmacol., 70 (2006) 501-509.
[8] S. Liekens, A. Bronckaers, M. Belleri, A. Bugatti, R. Sienaert, D. Ribatti, B. Nico, A. Gigante,
E. Casanova, G. Opdenakker, M.J. Pérez-Pérez, J. Balzarini, M. Presta, Mol. Cancer Ther., 11
(2012) 817-829.
[9] A. Bzowska, E. Kulikowska, D. Shugar, Pharmacol. Ther., 88 (2000) 349-425.
[10] E.A.T. Ringia, V.L. Schramm, Curr. Top. Med. Chem., 5 (2005) 1237-1258.
[11] C. Schnick, M.A. Robien, A.M. Brzozowski, E.J. Dodson, G.N. Murshudov, L. Anderson, J.R.
Luft, C. Mehlin, W.G.J. Hol, J.A. Brannigan, A.J. Wilkinson, Acta Crystallographica Section D-
Biological Crystallography, 61 (2005) 1245-1254.
[12] D.C. Madrid, L.M. Ting, K.L. Waller, V.L. Schramm, K. Kim, J. Biol. Chem., 283 (2008)
35899-35907.
[13] H. Cui, G.F. Ruda, J. Carrero-Lérida, L.M. Ruiz-Pérez, I.H. Gilbert, D. González-Pacanowska,
Eur. J. Med. Chem., 45 (2010) 5140-5149.
[14] M.B. Cassera, K.Z. Hazleton, E.F. Merino, N. Obaldia, III, M.-C. Ho, A.S. Murkin, R.
DePinto, J.A. Gutierrez, S.C. Almo, G.B. Evans, Y.S. Babu, V.L. Schramm, PLoS ONE, 6 (2011)
e26916.
[15] V. Gurram, N. Pottabathini, R. Garlapati, A.B. Chaudhary, B. Patro, M.K. Lakshman, Chem.--
Asian J., 7 (2012) 1853-1861.
[16] S. Sahnoun, S. Messaoudi, J.F. Peyrat, J.D. Brion, M. Alami, Tetrahedron Lett., 49 (2008)
7279-7283.
[17] L.M. Gaetke, C.K. Chow, Toxicology, 189 (2003) 147-163.
[18] D.C. Kennedy, C.S. McKay, M.C.B. Legault, D.C. Danielson, J.A. Blake, A.F. Pegoraro, A.
Stolow, Z. Mester, J.P. Pezacki, J. Am. Chem. Soc., 133 (2011) 17993-18001.
[19] E. Lallana, R. Riguera, E. Fernandez-Megia, Angew. Chem.-Int. Edit., 50 (2011) 8794-8804.
[20] B. Vankova, V. Krchnak, M. Soural, J. Hlavac, ACS Comb. Sci., 13 (2011) 496-500.
20

ACCEPTED MANUSCRIPT
[21] V. Vongsutilers, J. Daft, K. Shaughnessy, P. Gannett, Molecules, 14 (2009) 3339-3352.
[22] L.-M. Ting, W. Shi, A. Lewandowicz, V. Singh, A. Mwakingwe, M.R. Birck, E.A.T. Ringia,
G. Bench, D.C. Madrid, P.C. Tyler, G.B. Evans, R.H. Furneaux, V.L. Schramm, K. Kim, J. Biol.
Chem., 280 (2005) 9547-9554.
[23] M.H. Hung, L.M. Stock, Heterocycles, 18 (1982) 67-75.
21

ACCEPTED MANUSCRIPT
Figure 1. Proposed 8-aryl and heteroarylinosines
Scheme 1. Reagents and conditions: (a) 4-Iodoanisole; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF;
80 ºC, 16 h (22% yield); (b) 4-Iodoanisole; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF; MW 120 ºC,
1 h (52% yield); (c) TFA 60%, H2O:1,4-dioxane, 15 min, and then quenching with NH4HCO3
(98% yield).
Scheme 2. Reagents and conditions: (a) Ar-I; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF; MW 120
ºC, 1h (46-61%); (b) TFA 60%, H2O: 1,4-dioxane, 15 min, and then quenching with NH4HCO3 (60-
87% yield).
Scheme 3. Reagents and conditions: (a) 4-iodo- or 3-iodopyridine, Cs2CO3, Pd(OAc)2, CuI,
piperidine; DMF; MW 120 ºC; 1 h (12: 55%; 13: 54%); (b) TFA 60%, H2O: 1,4-dioxane, 15 min,
and then quenching with NH4HCO3 (14: 78%; 15: 69%yield)
22

ACCEPTED MANUSCRIPT
O
N
N
R
HN
OH
N
O
HO
OH
R= CH3, OCH3, CF3
Figure 1. Proposed 8-arylinosines
23

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: (a) 4-Iodoanisole; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF;
80 ºC, 16 h (22% yield); (b) 4-Iodoanisole; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF; MW 120 ºC,
1 h (52% yield); (c) TFA 60%, H2O:1,4-dioxane, 15 min, and then quenching with NH4HCO3
(98% yield).
24

ACCEPTED MANUSCRIPT
Scheme 2. Reagents and conditions: (a) Ar-I; Cs2CO3, Pd(OAc)2, CuI, piperidine; DMF; MW 120
ºC, 1h (46-61%); (b) TFA 60%, H2O: 1,4-dioxane, 15 min, and then quenching with NH4HCO3 (60-
87% yield)
25

ACCEPTED MANUSCRIPT
Scheme 3. Reagents and conditions: (a) 4-iodo- or 3-iodopyridine, Cs2CO3, Pd(OAc)2, CuI,
piperidine; DMF; MW 120 ºC; 1 h (12: 55%; 13: 54%); (b) TFA 60%, H2O: 1,4-dioxane, 15 min,
and then quenching with NH4HCO3 (14: 78%; 15: 69%yield)
26

ACCEPTED MANUSCRIPT
Table 1
Inhibition of P. falciparum PNP by compounds 3, 8-11, 14 and 15.
Compound
PfPNP Ki (µM)
a
-
3
8
9
8.1
b
-
3.69
5.33
>80
10
11
14
15
a
-
a No inhibition at 200 ꢀM for PfPNP. b Poor solubility
27

ACCEPTED MANUSCRIPT
Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and
determination of their inhibitory activities against Plasmodium falciparum Purine
Nucleoside Phosphorylase
Alba Gigante, Eva-María Priego, Paula Sánchez-Carrasco, Luis Miguel Ruiz-Pérez,
Johan Vande Voorde, María- José Camarasa, Jan Balzarini, Dolores González-
Pacanowska and María-Jesús Pérez-Pérez
Highlights
•
Novel 8-aryl and pyridyl inosines have been synthesized by a direct arylation
reaction mediated by Pd/Cu complexes.
•
•
Extensive Quadrasil MP treatment has allowed removal of metal contaminants.
Several aryl inosines selectively inhibited Plasmodium falcipuarum PNP but not
human or prokaryotic PNPs.