Anti-flavivirus Activity of Different Tritylated Pyrimidine and Purine Nucleoside Analogues

DOI: 10.1002/open.201500216

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ChemistryOpen p. 227 - 235 (2016)

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Authors:

McGuigan, Christopher

Serpi, Michaela

Slusarczyk, Magdalena

Ferrari, Valentina

Pertusati, Fabrizio

Meneghesso, Silvia

Derudas, Marco

Farleigh, Laura

Zanetta, Paola

Bugert, Joachim

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Article abstract of DOI:10.1002/open.201500216

A series of tritylated and dimethoxytritylated analogues of selected pyrimidine and purine nucleosides were synthesized and evaluated for their in vitro inhibitory activity against two important members of the genus Flavivirus in the Flaviviridae family, the yellow fever (YFV) and dengue viruses (DENV). Among all compounds tested, the 5′-O-tritylated and the 5′-O-dimethoxytritylated 5-fluorouridine derivatives exerted potency against YFV. Interestingly in the series of purine analogues, the 5′O, N-bis-tritylated fludarabine derivative revealed strong inhibitory activity against DENV at μm concentrations, however significantly weaker potency against YFV.

Full text of DOI:10.1002/open.201500216

DOI: 10.1002/open.201500216

Anti-flavivirus Activity of Different Tritylated Pyrimidine

and Purine Nucleoside Analogues

Christopher McGuigan,*^[a]Michaela Serpi,^[a]Magdalena Slusarczyk,^[a]Valentina Ferrari,^[a]

Fabrizio Pertusati,^[a]Silvia Meneghesso,^[a]Marco Derudas,^[a]Laura Farleigh,^[b]Paola Zanetta,^[b]

and Joachim Bugert^[b]

A series of tritylated and dimethoxytritylated analogues of se-

lected pyrimidine and purine nucleosides were synthesized

and evaluated for their in vitro inhibitory activity against two

important members of the genus Flavivirus in the Flaviviridae

family, the yellow fever (YFV) and dengue viruses (DENV).

Among all compounds tested, the 5’-O-tritylated and the 5’-O-

dimethoxytritylated 5-fluorouridine derivatives exerted potency

against YFV. Interestingly in the series of purine analogues, the

5’O, N-bis-tritylated fludarabine derivative revealed strong in-

hibitory activity against DENV at mm concentrations, however

significantly weaker potency against YFV.

Introduction

Medicinal chemists have directed significant efforts to the syn-

thesis of nucleoside and nucleotide analogues endowed with

antiviral and anticancer activities.^[1]Their synthesis is not

always straightforward and often it is complicated by several

issues, such us their poor solubility in organic solvents, the

presence of several reactive functional groups, and the sus-

ceptibility of the glycosidic bond to undergo hydrolytic cleav-

age. To overcome all these problems, different chemical strat-

egies have been elaborated. Among them, chemical manipula-

tion by means of protecting groups remains a fundamental

synthetic approach to prepare nucleoside analogues. Different

protecting groups are commonly employed in nucleoside and

nucleotide chemistry including both base and acid-labile. The

triphenyl methyl group, most commonly indicated as the trityl

group is typically used to protect selectively primary, over sec-

ondary hydroxyl functions.^[2]The positive charge on the alpha

carbon, stabilized by the resonance effect of three aromatic

rings makes trityl ethers acid-labile. Since its discovery one

hundred years ago, the trityl group has become a key protec-

tive group, widely used in nucleoside, oligonucleotide, peptide,

carbohydrate chemistry, and indeed in almost all other fields of

organic and bioorganic chemistry.^[3]Typically, the trityl group is

introduced prior to manipulation of the structure and then re-

moved when appropriate during the synthesis. However some

recent studies indicated that trityl-containing compounds

themselves possess a certain biological activity. To some

degree, such discoveries may have been serendipitous. To the

best of our knowledge the first account reporting on a trityl

compound biologically active was in 2002 from Hernandez

et al.^[4]In their studies, 5’-O-tritylthymidine (1, Figure 1) emerg-

es as a novel inhibitor of human mitochondrial thymidine

kinase. After that, several other studies described the biological

potential of nucleoside analogues bearing the trityl group.^[5,6]

[a] Prof. C. McGuigan, Dr. M. Serpi, Dr. M. Slusarczyk, V. Ferrari, Dr. F. Pertusati,

Dr. S. Meneghesso, Dr. M. Derudas

School of Pharmacy and Pharmaceutical Sciences, Cardiff University

King Edward VII Avenue, Cardiff CF10 3NB (United Kingdom)

E-mail: cmcguigan@cardiff.ac.uk

[b] L. Farleigh, P. Zanetta, Dr. J. Bugert

Medical Microbiology and Infectious Diseases, School of Medicine

Cardiff University, Heath Park, Cardiff CF14 4XN (United Kingdom)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/open.201500216.

ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

This is an open access article under the terms of the Creative Commons

Attribution-NonCommercial-NoDerivs License, which permits use and

distribution in any medium, provided the original work is properly cited,

the use is non-commercial and no modifications or adaptations are

made.

Figure 1. Examples of tritylated nucleosides reported as flavivirus inhibitors.

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In particular in 2013, De Burghgraeve et al.^[7]during the

screening of a large compound library aimed at recognizing

hit molecules, identified 2’,5’-bis-O-tritylated uridine (2) as a fla-

vivirus inhibitor. As a consequence, several other trityl uridine

nucleosides, including different regioisomers, were prepared

and evaluated in vitro against two important members of Fla-

viviridae family, yellow fever (YFV) and dengue (DENV) virus-

es.^[8]Among the different compounds 3’,5’-bis-O-tritylated uri-

dine (3) was endowed with the best inhibitory properties (YFV

IC₅₀=1.0 mm; DENV IC₅₀=1.75 mm).^[9]The finding of this lipo-

philic structure being endowed with high antiviral activity for

flaviviruses, encouraged the same authors to undertake further

research. However, modifications of the trityl groups, com-

bined with minor variations of the heterocyclic base generally

led only to weak in vitro flavivirus inhibition and in some cases

to marked in vitro cytotoxicity. From these studies, only 3’,5’-

bis-O-tritylated 5-fluoro-2’-deoxyuridine (4) showed no cellular

toxicity up to a concentration of 25 mm, while having EC₅₀

values for YFV and DENV respectively of 1.05 mgmL^À1and

Results and Discussion

Chemistry

Tritylation of nucleoside analogues has always been a relatively

straightforward procedure. Generally, it is sufficient to stir the

selected nucleosides with an excess of trityl chloride at an ele-

vated temperature (1108C).^[13]However some reports have

shown that the temperature can be as low as 80–908C. For

our synthesis, we followed the milder approach reported by

Van Aerschot.^[8]Briefly, the selected nucleoside was treated in

anhydrous pyridine, with an excess of trityl chloride (2.8 equiv-

alents), and the resulting mixture heated at 808C for 18 h.

After aqueous work-up, the desired tritylated compounds were

isolated by column chromatography on silica gel. In order to

avoid decomposition of the trityl moiety, 0.5% of triethylamine

was added to the chromatographic eluent in order to neutral-

ize the silica gel acidity. Although this was not strictly necessa-

ry for trityl-bearing derivatives A–D (provided that their purifi-

cation was performed in a reasonable time), we found its use

imperative during the purification of 4,4’-dimethoxytrityl deriv-

atives (E–H). The reaction always returned compounds with

a diverse degree of tritylation (5’-, 3’-, and 2’-) depending upon

the reactivity of the nucleoside. The different regioisomers

were separated by column chromatography. The general syn-

thetic procedure for the preparation of the trityl ethers of

these compounds is summarized in Scheme 1, and complete

compound information can be found in Table 1.

1.2 mgmL^{À1 [6a]}

In the attempt to explain the mode of action of this class of

compounds, De Burghgraeve et al. undertook further investiga-

tions.^[7]Their preliminary data suggested that the antiviral ac-

tivity of this class of compounds might be due to an inhibition

of the viral RNA-dependent RNA polymerase rather than to

a mechanism interfering during an early or a late stage of the

viral life cycle such as virus entry, assembly, or release.

Despite being under partial control, yellow fever case num-

bers are now increasing globally, posing a serious risk of local

epidemic outbreaks. The global incidence of dengue has also

grown dramatically in recent decades. Both viruses are now

considered global threats for the entire word population. Al-

though a safe and effective vaccine against YFV exists, there is

currently no vaccine available for dengue virus and its devel-

opment has proven to be very difficult because of the exis-

tence of multiple serotypes. With no therapeutic agent avail-

able to treat and/or prevent severe epidemics of either yellow

fever or dengue virus infections, prevention is currently limited

to vector (mosquites) control measures.^[10]Therefore, there is

an obvious and urgent need for the development of effective

anti-flavivirus drugs.^[11]

Scheme 1. General synthetic methods to trityl compounds 5–24A–D. Re-

agents and conditions: a) trityl chloride (2.8 equiv), pyridine (4.5 mLmmol^À1),

808C, 18 h; b) trityl chloride, (2.8 equiv), DMAP (2.8 equiv), pyridine

(4.5 mLmmol^À1), 808C, 18 h.

Based on these considerations and the interesting data re-

ported in the literature for the tritylated nucleosides, we

became interested in assessing the impact of the trityl group

on several pyrimidine nucleosides not included in the previous

studies against both YFV and DENV.

In order to expand the structure–activity relationship (SAR),

and considering that flavivirus inhibition profile has been

linked to the critical requirement of bulkiness in the region be-

tween the 3’ and 5’ positions^[14]we decided to synthesize also

the 4,4’-dimethoxytrityl derivatives (5–20E–H, Scheme 2,

Table 2) of nucleosides 5–20.

5’-2’-O-Bis tritylated regioisomers were distinguished from

those 5’-3’-O-bis tritylated analogues using 2D NMR experi-

ments. Correlation spectroscopy (COSY) analysis allowed the

correct assignment of each proton for all the compounds. Cou-

pling of the OH signal to either the signal of 2’ or 3’ indicated

which position is unalkylated.

Taking into account that in different studies, hindered nu-

cleoside analogues (NAs) including 3’,5’-O-trityl derivatives of

adenine nucleoside analogues were found to elicit anti-flavivi-

rus activity to some extent,^{[6 g,12]}we decided to include also

a few selected purine analogues in our investigations. The

structures of the pyrimidine and purine nucleosides used in

our studies are shown in Figure 2.

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Figure 2. Nucleoside analogues (NAs) considered for this study. RBV: ribavirin, 5-FUR: 5-fluorouridine, 5-BrU: 5-bromouridine, 4’-AzidoU: 4’-azidouridine,

AraU: 2’-b-d-arabinouridine, 3TC: lamivudine, d4T: stavudine, 5-IdU: 5-iodo-2’-deoxyuridine, 5-FUDR: 5-fluoro-2’-deoxyuridine, 2’-FdU: 2’-fluoro-2’-deoxyuri-

dine, 5-AzaC: 5-azacytidine, AZT: 4’-azidothymidine, 5-MeU: 5-methyluridine, PCV: penciclovir, 3’-dA: 3’-deoxyadenosine, BCNA: bicyclic nucleoside analogue.

was assessed using a cell viability assay in which Vero cells

were infected with either yellow fever (YFV) or dengue viruses

(DENV, serotype D2) and further treated with the tested com-

pounds at three different concentrations (5, 10, and 20 mm).

Cell viability was determined by measurement of the level of

ATP present seven days post infection when compared to vehi-

cle only (DMSO) controls.^[15]The results are reported as per-

centage of viable cells left after the assay. Tables 3 and 4 gath-

ered only compounds exhibiting an antiviral activity and in-

clude also their CC₅₀values, defined as the concentration re-

quired to decrease cell growth by 50%. The toxicity of all the

Scheme 2. General synthetic method to 4,4’-dimethoxytrityl compounds 5–

tested compounds on uninfected cells was also determined in

parallel.

20E–H. Reagents and conditions: a) 4,4’-dimethoxytrityl chloride, (2.8 equiv),

pyridine (4.5 mLmmol^À1), 808C, 18 h; b) 4,4’-dimethoxytrityl chloride,

(2.8 equiv), DMAP (2.8 equiv), pyridine (4.5 mLmmol^À1), 808C, 18 h.

As expected, the parent nucleosides did not show any anti-

viral activity versus both the flaviviruses, considered in this

study.

Biological evaluation

In the series of the tritylated nucleoside analogues two com-

pounds: the 5’-O-tritylated 5-FUR derivative (6D) and the 5’-O-

tritylated thymidine derivative (1) showed a similar protective

The antiviral effect of all the parent nucleosides and their cor-

responding tritylated and 4,4’-dimethoxytritylated analogues

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Table 1. Summary of all synthesized tritylated compounds.

Table 2. Summary of all synthesized 4,4’-dimethoxytritylated compounds.

Cmpd

Nucleoside

5’

3’

2’

Cmpd

Nucleoside

5’

3’

2’

RBV (5)

5-FUR (6)

5-BrU (7)

4’-AzidoU (8)

Uridine (9)

AraU (10)

3TC (11)

N-acetyl-3TC (12)

d4T (13)

5-IdU (14)

5-FUDR (15)

2’-FdU (16)

5-AzaC (17)

AZT (18)

5-MeU (19)

Fludarabine (20)

Fludarabine(20)

Thymidine (21)

PCV (22)

OTr

–

OTr

–

OTr

–

OTr

–

10E

10F

12H

15F

15H

16F

16H

20H

RBV (5)

5-FUR (6)

ODMTr

–

5-FUR (6)

4’-AzidoU (8)

Uridine (9)

AraU (10)

ODMTr

–

ODMTr

AraU (10)

N-acetyl-3TC (12)

5-FUDR (15)

2’-FdU (16)

Fludarabine (20)

10B

10C

10D

11D-NHTr

12D

13D

14D

15D

16B

16D

17D

18D

19B

19C

19D

20B

20D-NHTr

21B

RBV: ribavirin, 5-FUR: 5-fluorouridine, 4’-AzidoU: 4’-azidouridine, AraU:

2’-b-d-arabinouridine, 3TC: lamivudine, 5-FUDR: 5-fluoro-2’-deoxyuridine,

2’-FdU: 2’-fluoro-2’-deoxyuridine, ODMTr: O-4,4’-dimethoxytrityl.

–

OTr

–

OTr

–

effect on the target cells, infected with YFV, when tested at

20 mm, with respectively 31% and 26% of the cells maintaining

full metabolic activity. Only a low percentage (8.6%) of YFV in-

hibition was achieved by the 5’-O-tritylated-5-Me-uridine deriv-

ative 19D, at the highest concentration tested. On the other

hand, no inhibitory properties were observed for all these

compounds (6D, 1, and 19D) in the DENV (D2) inhibition

assay at any of the concentrations tested.

22B-NHTr

22B

23C

24B

24D

The 5’,3’-bis-O-tritylated ribavirin (RBV) analogue (5B) and

the 5’-O-tritylated 2’-FdU analogue (16D) displayed some inter-

esting inhibitory activity against DENV with respectively 17%

and only 11% viable cells counted at 20 mm.

PCV (22)

3’-dA (23)

BCNA (24)

OTr

–

However, among all the trityl analogues, the 5’-bis-O,N-trity-

lated derivative (20D-NHTr) of the anticancer agent fludara-

bine (20) displayed the strongest anti-DENV inhibitory activity

with 72% of viable cells counted at 5 mm and 100% at both

10 mm and 20 mm. Interestingly, this compound, devoid of any

significant anti-YFV inhibitory effect, seems to selectively target

RBV: ribavirin, 5-FUR: 5-fluorouridine, 5-BrU: 5-bromouridine, 4’-AzidoU:

4’-azidouridine, AraU: 2’-b-d-arabinouridine, 3TC: lamivudine, d4T: stavu-

dine, 5-IdU: 5-iodo-2’-deoxyuridine, 5-FUDR: 5-fluoro-2’-deoxyuridine,

2’-FdU: 2’-fluoro-2’-deoxyuridine, 5-AzaC: 5-azacytidine, AZT: 4’-azidothy-

midine, 5-MeU: 5-methyluridine, PCV: penciclovir, 3’-dA: 3’-deoxyadeno-

sine, BCNA: bicyclic nucleoside analogue, OTr: O-trityl.

Table 3. Antiviral activity and toxicity of selected tritylated compounds.^[a]

YFV inhibition [% Cells alive]

D2 inhibition [% Cells alive]

Toxicity CC₅₀

Cmpd

14D

16D

19D

Nucleoside

5’

3’

2’

5 mm

10 mm

20 mm

5 mm

10 mm

20 mm

[mm]

RBV (5)

5-FUR (6)

5-IdU (14)

OTr

–

16.85Æ1.51

>20

31.24Æ2.34

2.47Æ0.06

10.88Æ1.44

2’-FdU (16)

5-MeU (19)

Fludarabine (20)

Thymidine (21)

PCV (22)

–

8.59Æ0.85

0.84

0.95Æ0.17

25.77Æ1.03

8.09Æ0.04

20D-NHTr

21B

0.28

71.98Æ5.75

100Æ1.48

100Æ7.83

1.25Æ0.03

22B-NHTr

[a] Determined as an inhibition percentage of yellow fever (YFV, 17D strain of ASIBI) and dengue viruses (DENV, serotype D2). Values are given as

meansÆSD for n=3. RBV: ribavirin, 5-FUR: 5-fluorouridine, 5-IdU: 5-iodo-2’-deoxyuridine, 2’-FdU: 2’-fluoro-2’-deoxyuridine, 5-MeU: 5-methyluridine,

PCV: penciclovir, OTr: O-trityl.

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Table 4. Antiviral activity and toxicity of selected 4,4’-dimethoxytritylated compounds.^[a]

YFV inhibition [% Cells alive]

D2 inhibition [% Cells alive]

Toxicity CC₅₀

Cmpd

Nucleoside

5’

3’

2’

5 mm

10 mm

20 mm

5 mm

10 mm

20 mm

[mm]

12H

15H

16H

5-FUR (6)

Uridine (9)

N-acetyl-3TC (12)

5-FUDR (15)

2’-FdU (16)

ODMTr

–

9.22Æ0.46

30.89Æ1.85

100Æ8.68

>20

1.06Æ0.27

7.00Æ0.98

100Æ5.23

70.45Æ18.31

100Æ5.4

77.99Æ14.81

95.17Æ10.4

[a] Determined as an inhibition percentage of yellow fever (YFV, 17D strain of ASIBI) and dengue viruses (DENV, serotype D2). Values are given as

meansÆSD for n=3. 5-FUR: 5-fluorouridine, 3TC: lamivudine, 5-FUDR: 5-fluoro-2’-deoxyuridine, 2’-FdU: 2’-fluoro-2’-deoxyuridine.

DENV. Despite being the analogue of an anticancer agent,

Conclusion

compound 20D showed no cytotoxicity in our assays, up to

a 20 mm.

This noncytotoxic profile was observed also for all the other

We herein report the synthesis of tritylated and 4,4’-dime-

thoxytritylated pyrimidine and purine nucleoside analogues

and the evaluation of their inhibitory activity against YF and

DEN viruses. The activity was reported as % of viable cells re-

sulting from treatment with our compounds after exposure to

the viruses. In the series of 38 tritylated analogues, two com-

pounds, 6D and 1, showed selective and moderate YFV inhibi-

tion (31% and 26%, respectively) at 20 mm concentration. In

the DENV inhibition assay, the tritylated fludarabine derivative

20D-NHTr displayed pronounced anti-DENV inhibitory activity,

reaching 100% of virus inhibition at a concentration of 10 mm.

Within the panel of 16 4,4’-dimethoxytritylated compounds,

five of the monosubstituted derivatives (6H, 9H, 12H, 15H,

and 16H) revealed selective anti-YFV inhibitory activity ranging

from 7 to 95% at 10 mm and from 70 to 100% at 20 mm. All

the tested compounds proved to be noncytotoxic at concen-

trations up to 20 mm.

In conclusion, our preliminary biological data support an

anti-flavivirus activity for tritylated/4,4’-dimethoxytritylated nu-

cleoside analogues. Previous studies have suggested that

these drugs might act as potential inhibitors of the DENV viral

RNA-dependent RNA polymerase (RdRp).^[7]However, evidence

to unravel their mechanism of action have not been provided

yet. In fact, their structure–activity relationship remains unclear

and requires additional studies and therefore it is not possible

for us here to speculate on a possible mechanism. It might be

however that the prior identified lead impacted on cellular

metabolic processes rather than a viral target.

analogues considered in this work with no evidence of toxicity

at concentration up to 20 mm.

Next, the antiviral effect of dimethoxytritylation of selected

nucleoside analogues was evaluated similarly to the tritylated

congeners against YF and DEN viruses. Table 4 collects the

result for the compounds of this series showing some antiviral

activity. In particular the 5’-O-DMTr-FUR derivative (6H), and,

the 5’-O-DMTr-5-FUDR derivative (15H) emerged as the most

active compounds against YFV with 100% inhibition at 20 mm.

Compound 15H was found to retain high inhibition also at

10 mm with 95% of viable cells counted. Differently from the

5’-O-tritylated-2’-deoxy-2’-fluorouridine analogue 16D, its 5’-O-

dimethoxytrityl congener 16H had pronounced inhibitory ac-

tivity but surprisingly only against YFV (78% at 20 mm).

Intriguing results were also found for the 5’-O-dimethoxytri-

tylated-uridine analogue 9H, which showed inhibitory activity

only against YFV with an inhibition of 1% at 5 mm; 7% at

10 mm; and 100% at 20 mm.

All 4,4’-dimethoxytritylated compounds tested were devoid

of anti-DENV activity and were found to be not toxic at con-

centrations up to 20 mm.

As mentioned already, compounds 3 and 2 have been previ-

ously reported as relatively potent anti-YFV and anti DENV

agents.^[6]In contrast, our assays revealed no potency either as

anti-YFV or anti-DENV agents. Explanation of the differing re-

sults from those reported could be found in the differing de-

tection methods used.^[7]We assayed the metabolic activity of

cells, quantitating ATP, which is rapidly degraded in dying cells.

Using such an assay indicating active intracellular processes, it

is possible to dissect toxic and viral effects that lead to cells

with intact membranes but no metabolic activity; that latter

method (methylene blue dye exclusion) was used in the prior

reports. The ATP assay used here is more sensitive, indicates

both toxic and viral effects leading to the loss of metabolic ac-

tivity, and is the assay of choice to identify compounds, which

lead to increased cell survival.^[14,15]

Experimental Section

General experimental

All solvents and reagents were used as obtained from commercial

sources unless otherwise indicated. All reactions were performed

under an argon atmosphere. The H and ¹³C NMR spectra were re-

corded on a Bruker spectrometer (Billerica, MA, USA) operating at

500 MHz for H and 125 MHz for ¹³C. CDCl₃was used as the solvent

for NMR experiments, unless otherwise stated. ¹H chemical shift

values (d) are referenced to the residual nondeuterated compo-

nents of the NMR solvents (d=7.26 ppm for CHCl₃, etc.). The ¹³C

chemical shifts (d) are referenced to CDCl₃(central peak, d=

77.0 ppm). Fluorine chemical shifts are referenced to CFCl₃. Mass

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spectra (MS) were measured in positive-mode electrospray ioniza-

tion (ESI). Thin-layer chromatography (TLC) was performed on silica

gel 60 F254 plastic sheets. Column chromatography was per-

formed using silica gel (35–75 mesh) or on an Isolera Biotage

system (Uppsala, Sweden). Purity of prepared compounds was de-

termined to be>95% by high-performance liquid chromatography

(HPLC)–UV analysis (Thermo HPLC connected with UV detector;

Varian Pursuit XS, 4.6 mmꢁ150 mm, 5.0 mm, Palo Alto, CA, USA).

C-6), 90.1 (C-1’), 87.5 (C(Ph)₃), 84.4 (C-4’), 75.8 (C-2’), 71.0 (C-3’),

63.2 ppm (C-5’); MS (ES+) found: m/z 527.10 [M+Na]⁺, calcd for

[C₂₈H₂₅FN₂O₆]: m/z 504.50 [M]; Reverse-phase HPLC (H₂O/CH₃CN

from 90:10 to 0:50 in 30 min), flow=1 mLmin^À1, l=245 nm, t_R=

17.52 min.

Dimethoxytritylated derivatives of 5-fluorouridine (6) were pre-

pared according to the general procedure 1 from 6 (0.30 g,

1.14 mmol) and 4,4’-dimethoxytrityl chloride (1.24 g, 3.66 mmol,

3.2 equiv/mol) in pyridine (6 mL). Column chromatography purifica-

tion using a gradient of MeOH/triethylamine (1:0.5% to 2:0.5%) in

CH₂Cl₂as an eluent yielded three products, which were further re-

purified by preparative TLC using MeOH/triethylamine (2:0.5%) in

CH₂Cl₂to yield: 5’,3’-bis-O-dimethoxytrityl-5fluorouridine (6F), 5’,2’-

bis-O-dimethoxytrityl-5-fluorouridine (6G), and 5’-O-dimethoxytri-

tyl-5-fluorouridine (6H).

Chemistry

General Procedure 1. A mixture of a nucleoside (1.0 eqmol^À1), an

appropriate trityl chloride (2.2 eqmol^À1), and 4-dimethylaminopyri-

dine (DMAP, 2.8 moleq^À1, unless stated otherwise) in anhydrous

pyridine (4.5 mLmmol^À1) was heated at 808C under an argon at-

mosphere for 18 h. The reaction was quenched by addition of

MeOH (2 mLmmol^À1) at rt, and kept stirring at rt for 30 min. The

solution was then concentrated and diluted in CH₂Cl₂. The organic

solution was washed with saturated solution of NaHCO₃(3ꢁ

20 mL), and the combined aqueous layers were extracted with

CH₂Cl₂. Combined organic layers were dried over Na₂SO₄, filtered,

and concentrated under vacuum. The residue was purified by

column chromatography on silica gel (eluent system gradient

MeOH in CH₂Cl₂=1–3% containing 0.5% triethylamine).

5’-O-Dimethoxytrityl-5-fluorouridine (6H) was obtained as a yel-

lowish solid (0.17 g, 26%); ¹H NMR (500 MHz, 258C, [D₆]DMSO,):

d=7.91 (d, J_HÀF=7.0 Hz, 1H, H-6), 7.40 (dd, J=8.5, 2.5 Hz, 2H, H-

Ph), 7.33–7.23 (m, 7H, H-Ph), 6.90 (apparent d, J=8.5 Hz, 4H, H-

Ph), 5.73 (dd, J=4.0, 1.5 Hz, 1H, H-1’), 5.48 (d, J=5.0 Hz, 1H, C-2’-

OH), 5.14 (d, J=5.0 Hz, 1H, C-3’-OH), 4.14 (apparent q, J=5.0 Hz,

1H, H-2’), 4.08 (apparent q, J=5.0 Hz, 1H, H-3’), 3.98–3.96 (m, 1H,

H-4’), 3.75 (s, 6H, 2 x OCH₃), 3.29 (dd, J=11.0, 3.5 Hz, 1H, H-5’),

3.19 ppm (dd, J=11.0, 3.5 Hz, 1H, H-5’); ¹³C NMR (125 MHz, 258C,

[D₆]DMSO,): d=158.2, 158.1 (CH₃O-C-Ph), 156.9 (d, ²J_CÀF=26.0 Hz,

Experimental data for all active compounds are reported below

and for inactive compounds in the Supporting Information.

C-4), 149.8 (C-2), 144.7 (C-Ph), 139.9 (d, J_CÀF=230.5 Hz, C-5), 135.4,

135.2 (C-Ph), 129.7, 128.9, 128.2, 127.9, 127.6, 126.7, 125.3 (CH-Ph),

124.6 (d, J_CÀF=33.8 Hz, C-6), 113.3, 113.2 (CH-Ph), 89.0 (C-1’), 85.8

(C(Ph)₃), 82.6 (C-4’), 73.2 (C-2’), 69.5 (C-3’), 63.1 (C-5’), 55.0 ppm

(OCH₃); ¹⁹F NMR (470 MHz, 258C, [D₆]DMSO,): d=À167.25 ppm; MS

(ES+) found: m/z 587 [M+Na]⁺, calcd for [C₃₀H₂₉FN₂O₈]: m/z

564.55 [M]; Reverse-phase HPLC (H₂O/CH₃CN 90:10 to 0:50 in

30 min), flow=1 mLmin^À1, l=245 nm, t_R=14.85 min.

Tritylated derivatives of ribavirin (5) were prepared according to

the general procedure 1 from 5 (0.500 g, 2.05 mmol) and trityl

chloride (1.6 g, 5.74 mmol). Column purification with a gradient of

MeOH/triethylamine (1:0.5% to 2:0.5%) in CH₂Cl₂as eluent yielded

5’,3’,2’-tri-O-tritylribavirin (5A), 5’,3’-bis-O-tritylribavirin (5B) and

5’,2’-bis-O-tritylribavirin (5C).

5’,3’-Bis-O-tritylribavirin (5B) was obtained as a white solid

Dimethoxytritylated derivatives of uridine (9) were prepared ac-

cording to the general procedure 1 from 9 (0.150 g, 0.61 mmol),

4,4’-dimethoxytrityl chloride (0.578 g, 1.71 mmol), and DMAP

(0.209 g, 1.71 mmol) in anhydrous pyridine (10 mL). Column chro-

(0.268 g, 18%); H NMR (500 MHz, 258C, CDCl₃): d=8.21 (s, 1H, H-

3), 7.43–7.38 (m, 6H, H-Ph), 7.34–7.18 (m, 24H, H-Ph), 6.64 (br s,

1H, NH₂), 5.89 (d, J=3.08 Hz, 1H, H-1’), 5.66 (br s, 1H, NH₂), 4.43–

4.39 (m, 1H, H-3’), 4.19–4.14 (m, 1H, H-4’), 3.61–3.56 (m, 1H, H-2’),

3.47–3.43 (m, 1H, H-5’), 3.00 (dd, J=10.9, 5.5 Hz, 1H, H-5’),

2.85 ppm (d, J=4.5 Hz, 1H, OH-2’); ¹³C NMR (125 MHz, 258C,

CDCl₃): d=160.4 (C=O), 156.9 (C-5), 144.1 (C-3), 143.5, 143.3 (C-Ph),

128.7, 128.6, 128.3, 127.8, 127.1 (CH-Ph), 92.4 (C-1’), 88.1, 87.1

(C(Ph)₃), 83.5 (C-4’), 74.3 (C-2’), 74.1 (C-3’), 64.0 ppm (C-5’); MS

(ES+) found: m/z 751.3 [M+Na]⁺, calcd for [C₄₆H₄₀N₄O₅]: m/z

728.83 [M]; Reverse-phase HPLC (H₂O/CH₃CN from 90:10 to 0:100

in 30 min), flow=1 mLmin^À1, l=254 nm, t_R=28.10 min.

matography eluting with

a gradient of MeOH/triethylamine

(1:0.5% to 3.5:0.5%) in CH₂Cl₂as an eluent gave 5’,2’-bis-O-

dimethoxytrityluridine (9G) and 5’-dimethoxytrityluridine (9H).

5’-O-Dimethoxytrityluridine (9H) was obtained as a yellowish

solid (0.16 g, 48%); H NMR (500 MHz, 258C, CDCl₃): d=8.01 (d, J=

8.0 Hz, H-6), 7.43–7.38 (m, 2H, H-Ph), 7.33–7.28 (m, 6H, H-Ph), 7.26–

7.22 (m, 1H, H-Ph), 6.89–6.82 (m, 4H, H-Ph), 5.92 (d, J=2.5 Hz, H-

1’), 5.38 (d, J=8.0 Hz, H-5), 4.44 (dd, J=6.0, 5.5 Hz, H-3’), 4.36 (dd,

J=5.0, 2.5 Hz, H-2’), 4.22–4.18 (m, 1H, H-4’), 3.80 (s, 3H, OCH₃), 3.79

(s, 3H, OCH₃), 3.55 (dd, J=11.0, 2.5 Hz, 1H, H-5’), 3.50 ppm (dd, J=

11.0, 2.5 Hz, 1H, H-5’); ¹³C NMR (125 MHz, 258C, CDCl₃): d=163.7

(C-2), 158.7, 158.6 (CH₃O-C-Ph) 151.1 (C-4), 144.4 (C-Ph), 140.3 (C-6),

135.5 (C-Ph), 135.2 (C-Ph), 130.3, 130.2, 130.1, 129.2, 128.1, 127.9,

127.8, 127.1, 113.4, 113.3, 113.2 (CH-Ph), 102.3 (C-5), 90.6 (C-1’), 83.8

(C-4’), 87.0 (C(Ph)₃), 75.5 (C-2’), 69.7 (C-3’), 61.9 (C-5’), 55.2 ppm

(OCH₃); MS (ES+) found: m/z 569.2 [M+Na]⁺, calcd for

[C₃₀H₃₀N₂O₈]: m/z 546.57 [M]; Reverse-phase HPLC (H₂O/CH₃CN

from 80:20 to 10:90 in 15 min, then to CH₃CN 100% in 25 min),

flow=1 mLmin^À1, l=245 nm, t_R=10.56 min.

Tritylated derivatives of 5-fluorouridine (6) were prepared accord-

ing to the general procedure 1 from 6 (0.30 g, 1.14 mmol) and

trityl chloride (1.02 g, 3.66 mmol) in pyridine (6 mL). Column chro-

matography purification using a gradient of MeOH/triethylamine

(1:0.5% to 2:0.5%) in CH₂Cl₂as an eluent yielded 5’,3’-bis-O-trityl-5-

fluorouridine (6B), 5’,2’-bis-O-trityl-5-fluorouridine (6C), and 5’-O-

trityl-5-fluorouridine (6D).

5’-O-Trityl-5-fluorouridine (6D) was obtained as a white solid

(0.183 g, 32%); H NMR (500 MHz, 258C, CDCl₃,): d=7.42 (d, J_HÀF

6.0 Hz, 1H, H-6), 7.22–7.20 (m, 6H, H-Ph), 7.05–7.02 (m, 6H, H-Ph),

6.98–6.95 (m, 6H, H-Ph), 5.72 (dd, J=4.5, 1.5 Hz, 1H, H-1’), 4.04 (t,

J=5.0 Hz, 1H, H-3’), 3.96 (t, J=5.0 Hz, 1H, H-2’), 3.90–3.87 (m, 1H,

H-4’), 3.30 (dd, J=11.0, 2.5 Hz, 1H, H-5’), 3.25 ppm (dd, J=11.0,

2.5 Hz, 1H, H-5’); ¹³C NMR (125 MHz, 258C, [D₆]DMSO,): d=161.0

(d, J_CÀF=22.5 Hz, C-2), 152.7 (C-4), 144.4 (d, J_CÀF=241.5 Hz, C-5),

143.3 (C-Ph), 128.6, 128.3, 127.3 (CH-Ph), 123.4 (d, J_CÀF=33.75 Hz,

5’-O-Dimetoxytrityl-N-acetyl-lamivudine (12H) was prepared ac-

cording to the general procedure 1 from N-acetyl-lamivudine (12)

(0.80 g, 2.95 mmol), 4,4’-dimethoxytrityl chloride (1.2 g, 2.95 mmol),

and DMAP (0.036 g, 0.295 mmol) in anhydrous pyridine (5 mL).

Column chromatography with a gradient of MeOH/triethylamine

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(1:0.5% to 2:0.5%) in CH₂Cl₂as an eluent yielded 12H as a white

solid (0.55 g, 33%); H NMR (500 MHz, 258C, CDCl₃): d= 9.73 (bs,

(1:0.5% to 2.5:0.5%) in CH₂Cl₂to give 5’,3’-bis-O-trityl-2’-fluoro-2’-

deoxyuridine 16B and 5’-O-trityl-2’-fluoro-2’-deoxyuridine 16D.

1H, NH), 8.43 (d, J=7.5 Hz, 1H, H-6), 7.49–7.47 (m, 2H, H-Ph), 7.38–

7.29 (m, 7H, H-Ph), 7.21 (d, J=7.5 Hz, H-5), 6.90–6.88 (m, 4H, H-Ph),

6.37 (dd, J=5.5, 2.0 Hz, 1H, H-1’), 5.34 (t, J=5.0 Hz, 1H, H-4’), 3.84

(s, 6H, OCH₃), 3.68 (dd, J=12.5, 2.5 Hz, 1H, H-5’a), 3.64–3.60 (m,

2H, H-5’b and H-2’a), 3.29 (d, J=12.5 Hz, H-2’b), 2.27 ppm (s, 3H,

CH₃); ¹³C NMR (125 MHz, 258C, CDCl_,): d=170.5 (COCH₃), 163.0

(C-2), 158.7 (CH₃O-C-Ph), 154.9 (C-4), 145.7 (C-5), 145.3 (C-Ph), 135.3,

135.1 (C-Ph), 128.4, 128.1, 113.7, (CH-Ph), 87.9 (C-5), 87.6 (CH-1’),

87.3 (C(Ph)₃), 63.5 (CH₂-5’), 55.3 (OCH₃), 39.7 (CH₂-2’), 24.9 ppm

(COCH₃); MS (ES+) found: m/z 596.2 [M+Na]⁺, calcd for

[C₃₁H₃₁N₃O₆S]: m/z 573.1974 [M]; Reverse-phase HPLC (H₂O/CH₃CN

from 80:20 to 0:100 in 30 min), flow=1 mLmin^À1, l=254 nm, t_R=

21.59 min.

5’-O-Trityl-2’-fluoro-2’-deoxyuridine (16D) was obtained as

a white solid (0.79 g, 80%); ¹H NMR (500 MHz, 258C, [D₆]DMSO):

d=11.43 (bs, 1H, NH), 7.77 (d, J=8.1 Hz, 1H, H-6), 7.41–7.29 (m,

15H, H-Ph), 5.91–5.87 (m, 1H, H-1’), 5.70–5.69 (m, 1H, OH-3’), 5.31

(d, J=8.1 Hz, 1H, H-5), 5.18–5.07 (m, 1H, H-2’), 4.41–4.32 (m, 1H,

H-3’), 4.03–4.00 (m, 1H, H-4’), 3.34–3.28 ppm (m, 2H, H-5’); ¹³C NMR

(125 MHz, 258C, [D₆]DMSO): d=163.1 (C-4), 150.0 (C-2), 143.3

(‘ipso’ C-Ph), 140.8 (C-6), 128.3, 127.9, 127.2 (CH-Ph), 101.4 (C-5),

93.4 (d, J_C-F=184.5 Hz, C-2’), 88.6 (d, J_C-F=35.7 Hz, C-1’), 86.3

(C(Ph)₃), 80.8 (C-4’), 67.9 (d, J_C-F=16.6 Hz, C-3’), 62.2 ppm (C-5’);

¹⁹F NMR ([D₆]DMSO, 470 MHz): d=À199.47 ppm; MS (ES+) found:

m/z 511.14 [M+Na]⁺, calcd for [C₂₈H₂₅FN₂O₅]: m/z 488.51 [M]; Re-

verse-phase HPLC (H₂O/CH₃CN from 50:50 to 0:100 in 30 min),

flow=1 mLmin^À1, l=254 nm, t_R=6.35 min.

5’-O-Trityl-5-iodo-2’-deoxyuridine (14D) was prepared according

to the general procedure 1 from 5-iodo-2’-deoxyuridine (14) (0.5 g,

1.41 mmol), trityl chloride (1.26 g, 4.52 mmol), and DMAP (0.172 g,

1.41 mmol) in anhydrous pyridine (10 mL). Column chromatogra-

phy eluting with a gradient of MeOH (1% to 4%) in CH₂Cl₂gave

5’-O-trityl-5-iodo-2’-deoxyuridine (14D) as a white solid (0.46 g,

Dimethoxytritylated derivatives of 2’-fluoro-2’-deoxyuridine (16)

were prepared according to the general procedure 1 from 16

(0.200 g, 0.81 mmol), 4,4’-dimethoxytrityl chloride (0.769 g,

2.27 mmol), and DMAP (0.278 g, 2.27 mmol) in anhydrous pyridine

(10 mL). After work-up, the crude sample was purified by column

chromatography using gradient of MeOH/triethylamine (1:0.5% to

2.5:0.5%) in CH₂Cl₂to give 5’,3’-bis-O-dimethoxytrityl-2’-fluoro-2’-

deoxyuridine (16F) and 5’-O-dimethoxytrityl-2’-fluoro-2’-deoxyuri-

dine (16H).

55%); H NMR (500 MHz, 258C, CDCl₃): d=9.34 (bs, 1H, NH), 8.16

(s, 1H, H-6), 7.47–7.46 (m, 6H, H-Ph), 7.36–7.33 (m, 6H, H-Ph), 7.29–

7.26 (m, 3H, H-Ph), 6.34 (dd, J=6.0, 8.0 Hz, 1H, H-1’), 4.58–4.57 (m,

1H, H-3’), 4.15–4.13 (m, 1H, H-4’), 3.45–3.39 (m, 2H, H-5’), 2.56–2.52

(m, 1H, H-2’a), 2.32–2.28 ppm (m, 1H, H-2’b); ¹³C NMR (125 MHz,

258C, CDCl₃): d=160.0 (C-2), 149.7 (C-4), 144.2 (C-6), 143.3 (C-Ph),

128.6, 128.1, 127.4 (CH-Ph), 88.3 (C-5), 87.7 (C(Ph)₃), 86.3 (C-4’), 85.5

(C-1’), 72.3 (C-3’), 63.6 (C-5’), 49.5 ppm (C-2’); MS (ES+) found: m/z

619.2 [M+Na]⁺, calcd for [C₂₈H₂₅IN₂O₅] m/z 596.4130 [M]; Reverse-

phase HPLC (H₂O/CH₃CN from 80:20 to 0:100 in 30 min), flow=

1 mLmin^À1, l=254 nm, t_R=20.71 min.

5’-O-Dimethoxytrityl-2’-fluoro-2’-deoxyuridine (16H) was ob-

tained as a white solid (0.053 g, 12%); ¹H NMR (500 MHz, 258C,

CDCl₃): d=7.91 (d, J=8.5 Hz, 1H, H-6), 7.41–7.38 (m, 2H, Ar), 7.35–

7.25 (m, 8H, H-Ph), 6.89–6.85 (m, 3H, H-Ph), 6.09 (dd, J=16.5,

1.5 Hz, 1H, H-1’), 5.36 (d, J=8.5 Hz, 1H, H-5), 5.05 (dddd, J=52.5,

4.5, 1.5 Hz, 1H, H-2’), 4.55 (dddd, J=20.0, 8.0, 4.0, Hz, 1H, H-3’),

4.13–4.08 (m, 1H, H-4’), 3.83 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.66

(dd, J=11.5, 2.5 Hz, 1H, H-5’), 3.55 ppm (dd, J=11.5, 2.5 Hz, 1H, H-

5’); ¹³C NMR (125 MHz, 258C, CDCl₃): d=162.4 (C-2), 158.8, 158.7

(CH₃O-C-Ph), 149.7 (C-4), 144.2 (C-Ph), 139.8 (C-6), 135.1, 135.0 (C-

Ph), 130.2, 130.1, 128.2, 128.1, 127.2, 113.4 (CH-Ph), 102.5 (C-5), 93.9

Dimethoxytritylated derivatives of 5-fluoro-2’deoxyuridine (15)

were prepared according to the general procedure 1 from 15

(0.30 g, 1.22 mmol) and 4,4’-dimethoxytrityl chloride (0.90 g,

2.68 mmol) in pyridine (6 mL). Column chromatography purifica-

tion using gradient of MeOH/triethylamine (1:0.5% to 2:0.5%) in

CH₂Cl₂as an eluent followed by preparative TLC purification with

MeOH/triethylamine (2:0.5%) in CH₂Cl₂yielded 5’,3’-bis-O-dime-

thoxytrityl-5-fluoro-2’-deoxyuridine (15F) and 5’-O-dimethoxytrityl-

5-fluoro-2’-deoxyuridine (15H).

(d, J_CF=185.7 Hz, C-2’), 87.6 (d, J_CF=33.6 Hz, C-1’), 82.3 (C-4’), 87.0

(C(Ph)₃), 69.1 (d, ²J_CF=17.0 Hz, C-3’), 61.1 (C-5’), 55.3 ppm (OCH₃);

MS (ES+) found: m/z: 571.2 [M+Na]⁺, calcd for [C₃₀H₂₉FN₂O₇]: m/z

548.56 [M]; Reverse-phase HPLC (H₂O/CH₃CN from 80:20 to 10:90

in 15 min, then to CH₃CN 100% in 25 min, flow=1 mLmin^À1, l=

245 nm, t_R=11.64 min.

5’-O-Dimethoxytrityl-5-fluoro-2’-deoxyuridine (15H) was ob-

tained as a yellowish solid (0.135 g, 20%); H NMR (500 MHz, 258C,

Tritylated derivatives of 5-methyluridine (19) were prepared ac-

cording to general procedure 1 from 19 (0.50 g, 1.94 mmol) and

trityl chloride (1.50 g, 5.42 mmol) in anhydrous pyridine (8.0 mL).

Column chromatography using a gradient of MeOH/triethylamine

(1:0.5% to 2:0.5%) in CH₂Cl₂as eluent yielded 5’,3’-bis-O-trityl-5-

methyluridine (19B), 5’,2’-bis-O-trityl-5-methyluridine (19C) and 5’-

O-trityl-5-methyluridine (19D).

MeOD): d=7.92 (d, J_HÀF=6.5 Hz, 1H, H-6), 7.45 (dd, J=9.0, 1.5 Hz,

2H, H-Ph), 7.35–7.29 (m, 6H, H-Ph), 7.25–7.23 (m, 1H, H-Ph), 6.89–

6.87 (m, 4H, H-Ph), 6.25–6.22 (m, 1H, H-1’), 4.52–4.49 (m, 1H, H-3’),

4.05–4.03 (m, 1H, H-4’), 3.80 (s, 6H, 2 x OCH₃), 3.42 (dd, J=10.5,

4.0 Hz, 1H, H-5’), 3.35 (dd, J=10.5, 4.0 Hz, 1H, H-5’), 2.43–2.38 (m,

1H, H-2’), 2.35–2.29 ppm (m, 1H, H-2’); ¹³C NMR (125 MHz, 258C,

CDCl₃): d=160.6 (C-2), 158.7 (CH₃O-C-Ph), 156.7 (C-4), 144.8 (C-Ph),

143.4 (d, ¹J_CÀF=239.5 Hz, C-5), 129.8, 128.8, (CH-Ph), 124.3 (d,

²J_CÀF=32.8 Hz, C-6), 113.7 (CH-Ph), 88.7 (C(Ph)₃), 87.8 (C-1’), 87.0 (C-

4’), 72.3 (C-3’), 63.9 (C-5’), 55.3 (OCH₃), 40.7 ppm (C-2’); MS (ES+)

found: m/z 571.6 [M+Na]⁺, calcd for [C₃₀H₂₉FN₂O₇]: m/z 548.55

[M]; Reverse-phase HPLC (H₂O/CH₃CN from 90:10 to 0:50 in

30 min), flow=1 mLmin^À1, l=245 nm, t_R=18.44 min.

5’-O-Trityl-5-methyl-uridine (19D) was obtained as a white solid

(0.36 g, 37%); ¹H NMR (500 MHz, 258C, [D₆]DMSO): d=11.38 (bs,

1H, NH), 7.50–7.27 (m, 16H, H-Ph, H-6), 5.81 (d, J=5.2 Hz, 1H, H-

1’), 5.49 (d, J=5.7 Hz, 1H, OH-2’), 5.19 (d, J=5.6 Hz, 1H, OH-3’),

4.21–4.17 (m, 1H, H-2’), 4.14–4.11 (m, 1H, H-3’), 3.98–3.96 (m, 1H,

H-4’), 3.28–3.16 (m, 2H, H-5’), 1.44 ppm (d, J=1.1 Hz, 3H, CH₃);

¹³C NMR (125 MHz, 258C, [D₆]DMSO): d=163.6 (C-4), 150.6 (C-2),

143.4 (‘ipso’ C-Ph), 135.8 (C-6), 128.2, 128.0, 127.1 (CH-Ph), 109.4 (C-

5), 88.0 (C-1’), 86.4 (C(Ph)₃), 82.7 (C-4’), 73.0 (C-2’), 70.0 (C-3’), 63.7

(C-5’), 11.6 ppm (CH₃); MS (ES+) found: m/z 523.16 [M+Na]⁺,

calcd for [C₂₉H₂₈N₂O₆]: m/z 500.54 [M]; Reverse-phase HPLC (H₂O/

Tritylated derivatives of 2’-fluoro-2’-deoxyuridine (16) were pre-

pared according to the general procedure 1 from 16 (0.50 g,

2.03 mmol) and trityl chloride (1.60 g, 5.69 mmol) in anhydrous pyr-

idine (10 mL). After work-up, the crude sample was purified by

column chromatography using gradient of MeOH/triethylamine

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CH₃CN from 95:5 to 0:100 in 30 min), flow=1 mLmin^À1, l=

254 nm, t_R=20.36 min.

Tritylated derivatives of penciclovir (22) were prepared according

to general procedure 1 from 22 (0.57 g, 2.25 mmol) and trityl chlo-

ride (1.86 g, 6.75 mmol) in anhydrous pyridine (10 mL). Column

chromatography eluting using a gradient of MeOH/triethylamine

(1:0.5% to 3:0.5%) in CH₂Cl₂yielded 5’,3’-bis-O-trityl-penciclovir

(22B) and 2-N,5’,3’-O-tri-trityl penciclovir (22B-NHTr).

Tritylated derivatives of fludarabine (20) were prepared according

to the standard procedure 1 from 20 (0.30 g, 1.05 mmol) and trityl

chloride (0.64 g, 2.31 mmol) in pyridine (6 mL). Column chromatog-

raphy purification using a gradient of MeOH/triethylamine (1:0.5%

to 2:0.5%) in CH₂Cl₂as eluent gave two products which were fur-

ther repurified by preparative TLC with MeOH/triethylamine

(2:0.5%) in CH₂Cl₂to yield 5’, 3’-bis-O-trityl-fludarabine (20B) and

5’-O,N-trityl-fludarabine (20D-NHTr).

2-N-5’,3’-O-Tri-trityl penciclovir (22B-NHTr) was obtained as

a white solid (0.47 g, 21%); ¹H NMR (500 MHz, 258C, [D₆]DMSO):

d=10.51 (bs, 1H, NH), 7.26 (s, 1H, H-8), 7.31–7.28 (m, 31H, H-Ph,

NH), 7.19–7.17 (m, 6H, H-Ph), 7.07–7.04 (m, 6H, H-Ph), 6.97–6.96

(m, 3H, H-Ph), 3.24–3.21 (m, 2H, H-1’), 2.91–2.85 (m, 4H, H-4’, H-5’),

1.62–1.58 (m, 1H, H-3’), 1.13–1.09 ppm (m, 2H, H-2’); ¹³C NMR

(125 MHz, 258C, [D₆]DMSO): d=156.5 (C-6), 150.3 (C-2), 149.3 (C-4),

144.6, 143.8 (‘ipso’ C-Ph), 137.2 (C-8), 128.7, 128.4, 128.1, 127.8,

127.4, 126.9, 126.3 (CH-Ph), 117.0 (C-5), 85.8 (C(Ph)₃), 62.7 (C-4’, C-

5’), 41.3 (C-1’), 37.3 (C-3’), 28.4 ppm (C-2’); MS (ES+) found: m/z

1003.39 [M+Na]⁺, calcd for [C₆₇H₅₇N₅O₃] m/z: 980.20 [M]; Reverse-

phase HPLC (H₂O/CH₃CN from 50:50 to 0:100 in 40 min), flow=

1 mLmin^À1, l=254 nm, t_R=36.64 min.

5’-Bis-O,N-trityl-fludarabine (20D-NHTr) was obtained as a white

solid (0.05 g, 6%); H NMR (500 MHz, 258C, CDCl₃): d=8.16 (s, 1H,

H-8), 7.45–7.33 (m, 8H, H-Ph), 7.38–7.36 (m, 8H, H-Ph), 7.38–7.36

(m, 14H, H-Ph), 6.18 (d, J=3.5 Hz, 1H, H-1’), 4.36 (apparent broad

d, J=2.5 Hz, 1H, H-3’), 4.25–4.20 (m, 2H, H-2’, NH), 4.05 (q, J=

3.5 Hz, 1H, H-4’), 3.89 (bs, 1H, 3’-OH), 3.62 (dd, J=11.0, 3.0 Hz, 1H,

H-5’), 3.42 ppm (dd, J=11.0, 3.0 Hz, 1H, H-5’); ¹³C NMR (125 MHz,

258C, CDCl₃): d=159.9 (¹J_CÀF=209.0 Hz, C-2), 155.7 (³J_CÀF=20.8 Hz,

C-6), 149.3 (³J_CÀF=18.0 Hz, C-4), 144.2, 142.9 (C-Ph), 140.1 (CH-8),

129.0, 128.6, 128.1, 127.9, 127.5, 127.1 (CH-Ph), 118.8 (⁴J_CÀF=3.8 Hz,

C-5), 88.2, 88.1 (C(Ph)₃), 85.1 (C-1’), 83.0 (C-4’), 76.7 (C-3’), 76.6 (C-2’),

63.6 ppm (C-5’); ¹⁹F NMR (470 MHz, 258C, CDCl₃): d=À49.84; MS

(ES+) found: m/z 792.3 [M+Na]⁺, calcd for [C₄₈H₄₀FN₅O₄]: m/z

769.86 [M]; Reverse-phase HPLC (H₂O/CH₃CN from 90:10 to 0:50 in

30 min), flow=1 mLmin^À1, l=245 nm, t_R=27.40 min.

Biological assays

Antiviral assay: Trityl compounds were prepared as stock solution

of 100 mm in dimethyl sulfoxide (DMSO) and then were diluted to

a final assay concentration of 20, 10, 5 mm. Dilutions were made by

adding the appropriate amount of DMSO to lyophilized compound

to achieve stock concentrations of 10 to 100 mm. Dissolved com-

pounds were stored at À208C and brought to rt prior to assay. All

compounds were well dissolved in DMSO, and precipitates were

not observed upon thawing.

Tritylated derivatives of thymidine (21) were prepared according to

the general procedure 1 from 21 (0.30 g, 1.24 mmol) and trityl

chloride (0.75 g, 2.72 mmol) in pyridine (6 mL). Column chromatog-

raphy using a gradient of MeOH/triethylamine (1:0.5% to 3:0.5%)

in CH₂Cl₂as an eluent gave 5’,3’-bis-O-trityl-thymidine (21B) and

5’-O-trityl-thymidine (1).

Cell culture: Vero cells were grown in Dulbecco’s modified Eagle’s

medium (DMEM, Sigma-Aldrich) with 10% FBS (Sigma-Aldrich).

Cells were plated into 96-well plates at a density of 10³cells per

well.

5’,3’-Bis-O-trityl-thymidine (21B) was obtained as a white solid

(0.147 g, 16%); ¹H NMR (500 MHz, 258C, CDCl₃): d=7.53 (d, J=

1.0 Hz, 1H, H-6), 7.41–7.39 (m, 6H, H-Ph), 7.27–7.23 (m, 24H, H-Ph),

6.47 (t, J=7.5 Hz, 1H, H-1’), 4.45–4.42 (m, 1H, H-3’), 3.87–3.85 (m,

1H, H-4’), 3.24 (dd, J=11.0, 2.5 Hz, 1H, H-5’), 2.86 (dd, J=11.0,

2.5 Hz, 1H, H-5’), 1.97–1.94 (m, 2H, H-2’), 1.41 ppm (d, J=1.0 Hz,

3H, CH₃); ¹³C NMR (125 MHz, 258C, CDCl₃): d=163.3 (C=O, C-2),

150.1 (C=O, C-4), 144.0, 143.3 (C-Ph), 135.8 (C-6), 129.0, 128.7,

128.5, 128.2, 128.0, 127.9, 127.4, 127.3, 125.3 (CH-Ph), 111.0 (C-5),

87.8, 87.4 (C(Ph)₃), 85.4 (C-4’), 84.9 (C-1’), 75.4 (C-3’), 63.8 (C-5’), 39.7

(C-2’), 11.7 ppm (CH₃); MS (ES+) found: m/z 749.38 [M+Na]⁺,

calcd for [C₄₈H₄₂N₂O₅]: m/z 726.85 [M]; Reverse-phase HPLC (H₂O/

CH₃CN from 90:10 to 0:50 in 30 min), flow=1 mLmin^À1, l=

245 nm, t_R=24.56 min.

Virus culture: Cells were then infected with dengue virus (serotype

D2 (strain New Guinea C), ATCC Number: VR-1584), and yellow

fever vaccine (17D strain Asibi; grown from a dose of Sanofi Pas-

teur STAMARIL (1000IU from embryonated chicken eggs) and

adapted to Vero cells (Farleigh and Bugert, 2014, unpublished re-

sults) at a moi of 1, or left uninfected to determine compound tox-

icity.

Cell viability was determined after seven days using CellTiterGlo

PROMEGA, following the manufacturer’s instructions. All experi-

ments were performed in triplicate.

Acknowledgements

5’-O-Trityl-thymidine (1) was obtained as a white solid (0.19 g,

33%); ¹H NMR (500 MHz, 258C, [D₆]DMSO): d=7.50 (d, J=1.0 Hz,

1H, H-6), 7.41–7.38 (m, 6H, H-Ph), 7.36–7.33 (m, 6H, H-Ph), 7.30–

7.26 (m, 3H, H-Ph), 6.21 (t, J=6.5 Hz, 1H, H-1’), 5.30 (d, J=4.5 Hz,

C-3’-OH), 4.35–4.31 (m, 1H, H-3’), 3.90–3.88 (m, 1H, H-4’), 3.24 (dd,

J=10.5, 3.0 Hz, 1H, H-5’), 3.17 (dd, J=10.5, 3.0 Hz, 1H, H-5’), 2.29-

2.23 (m, 1H, H-2’), 2.18–2.13 (m, 1H, H-2’), 1.47 ppm (d, J=1.0 Hz,

3H, CH₃); ¹³C NMR (125 MHz, 258C, [D₆]DMSO): d=163.6 (C=O,

C-2), 150.3 (C=O, C-4), 143.4, (C-Ph), 135.6 (C-6), 128.2, 127.9, 127.1

(CH-Ph), 109.5 (C-5), 86.3 (C(Ph)₃), 85.3 (C-4’), 83.6 (C-1’), 70.4 (C-3’),

63.9 (C-5’), 40.0 (C-2’), 11.7 ppm (CH₃); MS (ES+) found: m/z 507.54

[M+Na]⁺, calcd for [C₂₉H₂₈N₂O₅]: m/z 484.54 [M]; Reverse-phase

HPLC (H₂O/CH₃CN 90:10 to 0:50 in 30 min), flow=1 mLmin^À1, l=

245 nm, t_R=6.29 min.

The authors would like to thank Andrew Gibbs from the School

of Pharmacy and Pharmaceutical Sciences, Cardiff University,

Cardiff, King Edward VII Avenue, Cardiff CF10 3NB, United King-

dom, for excellent technical assistance.

Keywords: 4,4’-dimetoxytrityl · antiviral · dengue · flavivirus ·

nucleoside analogues · trityl · yellow fever

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Received: November 26, 2015

Published online on && &&, 0000

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FULL PAPERS

C. McGuigan,* M. Serpi, M. Slusarczyk,

V. Ferrari, F. Pertusati, S. Meneghesso,

M. Derudas, L. Farleigh, P. Zanetta,

J. Bugert

Trityl you succeed! Trityl-containing

compounds have been recently shown

to possess a certain biological activity. A

series of tritylated and dimethoxytrity-

lated analogues of selected pyrimidine

and purine nucleosides were prepared.

Interestingly, in the series of purine ana-

logues, the 5’O, N-bis-tritylated fludara-

bine derivative revealed moderate and

strong inhibitory activity respectively

against the yellow fever (YFV) and

dengue viruses (DENV), two important

members of the genus Flavivirus in the

Flaviviridae family.

&& – &&

Anti-flavivirus Activity of Different

Tritylated Pyrimidine and Purine

Nucleoside Analogues

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