Novel nucleosides as potent influenza viral inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.07.017

Influenza virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified ((2R,3S,4R,5R)-3- acetoxy-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3,4-dimethyl- tetrahydrofuran-2-yl) methyl benzo

Full text of DOI:10.1016/j.bmc.2010.07.017

Bioorganic & Medicinal Chemistry 18 (2010) 6329–6339
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel nucleosides as potent influenza viral inhibitors
*
Manohar Sharma Vedula , Sreenu Jennepalli, Ratnakar Aryasomayajula, Subhash Reddy Rondla,
Madanmohan Reddy Musku, Rathnakar Reddy Kura, Parthasaradhi Reddy Bandi
New Drug Discovery, Hetero Research Foundation, Hetero Drugs Limited, Plot No. 80 & 81, APIE, Balanagar, Hyderabad, Andhra Pradesh 500 037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Influenza virus infection constitutes a significant health problem in need of more effective therapies. We
have recently identified ((2R,3S,4R,5R)-3-acetoxy-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-
3,4-dimethyl-tetrahydrofuran-2-yl) methyl benzoate (18c) as a potent influenza virus inhibitor. We
now here report the synthesis and evaluation of a series of C-30 modified ribose nucleosides. These novel
compounds were prepared, primarily by taking known ((2R,3R,4R)-3-benzoyloxy-4-fluoro-4-methyl-5-
oxo-tetrahydrofuran-2-yl)methyl benzoate (1) and converting it in to C-3 keto sugar (7), reacting C-3
keto group with methyl magnesium bromide, followed by coupling these sugars with purine and pyrim-
idine bases. Anti influenza viral activity was determined by screening against both A and B viral strains.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 25 May 2010
Revised 7 July 2010
Accepted 8 July 2010
Available online 13 July 2010
Keywords:
Nucleosides
Influenza virus
SAR
1. Introduction
times ‘NA’ appears. Influenza B has not undergone antigenic shift,
perhaps because it is not found in birds or animals. Since 1977,
The outbreak of 2009 influenza A virus that is believed to have
originated from Mexico is rapidly spreading across the globe. World
wide more than 209 countries and overseas territories or commu-
nities have reported laboratory confirmed cases of pandemic influ-
enza H1N1 2009, including at least 14711 deaths.1 Thus impact of
influenza infection is felt globally each year when the disease devel-
ops in approximately 20% of the world’s population. Influenza A
virus, in particular, represents a significant health risk to the public.
This is due to both its ability to spread quickly within human pop-
ulations and the high degree of mortality associated with infection.
Influenza viruses are enveloped RNA viruses that are classified
into three types: A–C. Two major surface glycoproteins, hemaglu-
tinin (HA) and neuraminidase (NA) are responsible for the anti-
genic properties of the viruses. For influenza A, there are at least
9 subtypes of NA and 16 subtypes of HA in contrast to only one
subtype of influenza B, NA and HA. Influenza C contains both HA
and a NA activity in a single surface glycoprotein and only one sub-
type is known. Influenza C is not considered a serious disease and
most adults have protective antibodies immunity from influenza A
and B, however, does not last long since virus is constantly under-
going variations.
there have been three influenza A viruses circulating in humans.
These are influenza A subtypes H3N2, H1N1 and H5N1. H5N1 avian
influenza A viruses which have high human mortality rate,2 since
1997 are prime candidates for the next pandemic influenza A virus.
InApril2009anovelflustrainevolvedinitially, dubbed‘swineflu’
and also known as influenza A/H1N1 emerged in Mexico, the United
States, and several other nations. The world health organization
(WHO) officially declared the outbreak to be a pandemic. The WHO’s
declaration of a pandemic level 6 was an indication of spread.
Only two classes of influenza virus antivirals are currently avail-
able, inhibitors of the viral M2 ion channel protein (Amantadine
and Rimantadine) and inhibitors of the viral neuraminidase
(Zanamivir and Oseltamivir) (see Fig. 1).
The emergence of influenza viruses resistant to the M2 inhib-
itors occurs at high frequency in treated patients and thus they
are not recommended for a general and uncontrolled use.3 Many
of the human isolated of H5N1 viruses are already resistant to
these inhibitors.4 For addition, a recent study has shown influ-
enza A virus is resistant to Oseltamivir occurred in 20% of the
children treated with this drug.5 In fact, H5N1 viruses that are
partially resistant to Oseltamivir have recently been reported.6
Similar information of influenza B virus is limited. Recently a
study assessed the prevalence and transmissibility of influenza
B viruses with reduced sensitivity to neuraminidase inhibitors.6
The emergence of influenza virus resistant to existing classes of
antiviral drugs highlights the need for additional antiviral drugs
against influenza. Therefore, a need exists for novel antiviral
agents that address one or more locations in the viral replication
cycle.
Influenza A undergoes a progressive antigenic drift and in addi-
tion undergoes an antigenic shift in which a ‘new’ HA and some-
Abbreviations: HA, hemaglutinin; NA, neuraminidase; WHO, world health
organization; NIs, nucleoside inhibitors; MDCK cells, Madin-Darby canine kidney
cells; AACF, Antimicrobial Acquisition and Coordinating facility.
* Corresponding author. Tel.: +91 40 2377 4009; fax: +91 40 2377 8786.
E-mail address: sharmavm@heterodrugs.com (M.S. Vedula).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.017

6330
M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
OH
HO
H
O
NH2
Rimantadine
H
O
COOH
NH2
HO
HN
O
O
HN
HN
NH2
NH2
O
O
HN
Zanamivir
Oseltamivir
Amantadine
O
O
NH
HN
N
H
N
OH
OH
N
HN
O
O
BzO
AcO
H3C
NH2
CH3
O
F
Peramivir
18c
Figure 1. Structures of Amantadine, Rimantidine, Zanamivir, Oseltemivir, Peramivir and ((2R,3S,4R,5R)-3-acetoxy-5-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-fluoro-3,4-
dimethyltetrahydrofuran-2-yl)methyl benzoate (18c).
The design and synthesis of nucleoside analogues7,8 as potential
antiviral and anticancer agents is a very active field of research,
within this area, systems of particular interest include 20-deoxy
nucleosides.9 These nucleosides are compounds in which hydroxyl
group at 20-position has been replaced with different groups. In
parallel fluorinated sugars have received a great interest for their
biological medicinal applications10 indeed the introduction of a
C–F bond in a molecule is of great importance for its physicochem-
ical properties. For example, fluorine substituted nucleoside, Gem-
citabine is used in cancer and herpes therapies. Introduction of
fluorine atom can improve the bioavailability of drug owing to
increasing lipophilicity. Considering all these aspects and necessity
to find novel nucleosides having antiviral activity, we decided to
synthesize 30 modified 20-deoxy nucleoside analogues. Nucleoside
inhibitors (NIs) described in literature, have involved some modifi-
cations at any of the 20, 30, 40 or 50-position, the present modifica-
tion leading to di substitutions at each carbon of both 20 and 30
nucleosides are less prevalent. Most likely due to the added syn-
thetic challenges associated with their synthesis.
2. Chemistry
Recently, there has been an intensive effort seeking influenza
viral drugs with novel chemical structures,11 or that hits new
Table 1
In vitro anti-influenza activity of 13, 13a–i, 22 and 24 against Flu A, Flu B and inhibitory activity of influenza virus infection of MDCK cell
R
R1
Flu A(H5N1)
Flu B
MDCK
Vietnam/1203/2004H
Florida/4/2006
EC50
(
lg/ml)
EC50
(
lg/ml)
IC50
9.2
>100
>100
(lg/ml)
13
13a
13b
H
H
H
Cl
OH
OC2H5
7.1
33
48
3.7
65
>100
13c
13d
H
H
50
23
20
27
>100
>100
HN
HN
13e
H
31
32
>100
HN
O
13f
H
H
32
24
17
25
>100
>100
HN
N
13g
O
N
13h
13i
H
H
>100
32
42
30
>100
36
N
F
HN
Cl
NH2
22
24
H
H
>100
>100
>100
>100
>100
>100
EC50, virus inhibitory concentration, 50% endpoint; IC50, cell inhibitory concentration 50% endpoint.

M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
6331
targets or work in new ways to inhibit influenza virus and to over-
come resistance. As part of our effort to synthesize novel antivirals,
we report here the synthesis and evaluation of nucleoside ana-
logues against influenza virus. Our initial structure–activity rela-
tionship (SAR) was focused on the evaluation of modifications at
C-3 position of sugar. A limited SAR was also performed on
heterobases.
2-keto ribofuranose intermediates is known to proceed stereo
selectively with addition of the alkyl groups to the b face, leading
to the formation of the corresponding ribo derivatives. However,
in our case the resultant 3-ketosugar 7 when treated with freshly
prepared methyl magnesium bromide produced a mixture of
a
and b isomers 8 and 8A in the ratio of 1.2:1.
We studied the solvent effect in this reaction by taking THF in-
stead of diethyl ether. Formation of C-3 b-hydroxy isomer, approx-
imately four times more was observed in this solvent. The resulting
tertiary alcohols were then converted 11 and 11A by subsequent
removal of silyl protecting group with Bu4NF, followed by benzoyl-
ation gave compounds 10 and 10A. Demethylation of anomeric
methoxy group and acetylation of both the hydroxyls was achieved
under AC2O/CH3COOH/H2SO4 conditions to give key intermediates
11 and 11A.
The effect of modification of C-3 position of sugar on SAR was
investigated by first coupling sugar with 6-chloropurine. Many gly-
cosylation protocols for coupling are known in the literature. These
include the silver salt, the chloromercury, the fusion, the sodium
salt, the phase transfer, and the boron trifluoride etherate methods.
We employed Vorbrüggen conditions (N,O-bis(trimethylsilyl)acet-
For the initial studies, a series of nucleosides modified at C-30
position (Tables 1 and 2) were synthesized from novel sugar inter-
mediates (11) and (11A). These intermediates were synthesized
using a route described in Scheme 1.
The starting material 1 required for the synthesis of 11 and 11A
was prepared according to literature procedure.12 Di benzoyl fluoro
lactone 1 was reduced with lithium tri tertiary butoxy aluminum
hydride (Li(OtBu)3AlH) in THF followed by methylation of anomer-
ic hydroxyl group under boran trifluoride ethereate (BF3ꢀOEt2) con-
ditions. Some quantity (20%) of mono debenzoylated product 4
was obtained under these conditions. In the next step 3 and 4 were
together deprotected to afford compound 5. C-5 hydroxy com-
pound was then protected with TBDPS in subsequent step to fur-
nish the compound 6. As a next step to introduce methyl group
at C-3 carbon, oxidation reaction was performed by oxidizing the
hydroxyl group at this position to keto group with Dess–Martin
periodinane, in quantitative yield. Addition of organometallics to
amide, ACN, SnCl4 or ACN, DBU, TMSOTf) to yield a mixture of a/b
diastereoisomers. Separation of individual isomers was accom-
plished by column chromatography. Coupling of sugar 11 with 6-
chloropurine under the above conditions afforded nucleosides 12
with a configuration of
a at anomeric carbon and 13 with b config-
Table 2
uration. Similarly 11A gave 21 and 22. Under similar conditions 6-
chloro-9H-purin-2-amine yielded only b-anomer 13j, 22a from 11
and 11A, respectively. Nucleophilic displacement of chlorine atom
of 12 and 13 with different amines gave compounds 12a–i and
13a–i.
In an effort to further explore the structural effects on furanose
we de protected 12, 12a–i and 13, 13a–i (Scheme 2) using metha-
nolic ammonia at room temperature to give the desired b anomeric
In vitro anti-influenza activity of 18 and 18a–c, against Flu A, Flu B and inhibitory
activity of influenza virus infection of MDCK cells
B
Flu A(H5N1)
Vietnam/1203/
2004H
Flu B
Florida/
4/2006
MDCK
EC50
(l
g/ml)
EC50
(lg/ml)
IC50, (
lg/ml)
18
Uracil
Thymine
5-Fluoruracil
N-Benzoylcytosine
25
>100
26
34
>100
23
>100
>100
>100
>100
18a
18b
18c
nucleoside compounds 16a–j as well as
a anomeric compounds
18
7.7
14a–j with free hydroxyl groups at C-50 and C-30.
O
O
O
O
O
OH
OCH3
CH3
OCH3
CH3
a
b
BzO
BzO
BzO
BzO
HO
+
CH3
CH3
BzO
BzO
BzO
F
F
F
F
2
1
4
3
O
O
O
OCH3
CH3
OCH3
CH3
OCH3
e
c
d
HO
TBDPSO
TBDPSO
3 + 4
CH3
O
HO
HO
F
F
F
7
5
6
O
O
O
OCH3
O
OCH3
CH3
OCH3
OAc
g
h
BzO
HO
TBDPSO
TBDPSO
i
BzO
HO
H3C
HO
H3C
HO
H3C
AcO
CH3
CH3
CH3
F
8
+
F
10
F
9
F
11
H3C
f
O
O
O
O
OAc
CH3
OCH3
OCH3
CH3
OCH3
CH3
g
BzO
BzO
H3C
HO
HO
i
h
H3C
AcO
H3C
HO
H3C
HO
CH3
F
11A
F
F
F
10A
9A
8A
Scheme 1. Synthesis of crucial sugar intermediates 11 and 11A. Reagents and conditions: (a) lithium tri tert-butoxy aluminum hydride, THF; (b) BF3ꢀOEt2, MeOH; (c) NH3/
MeOH; (d) TBDPSCl, imidazole, dry DCM; (e) Dess–Martin periodinane, dry DCM; (f) MeMgI, dry diethyl ether; (g) dry THF, TBAF; (h) dry DCM, BzCl, DMAP, TEA; (i) H2SO4,
acetic acid, acetic anhydride.

6332
M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
R1
R1
R1
R1
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
N
N
N
N
HO
BzO
AcO
H3C
BzO
AcO
H3C
R
R
HO
R
R
CH3
CH3
HO
H3C
CH3
d
CH3
HO
H3C
d
F
F
F
F
12a=R=H, R1=OH
12b=R=H, R1=OEt
14a=R=H, R1=OH
13a=R=H, R1=OH
16a=R=H, R1=OH
14b=R=H, R1=OEt
13b=R=H, R1=OEt
16b=R=H, R1=OEt
b
b
Cl
Cl
N
N
O
BzO
OAc
CH3
N
N
N
N
O
O
N
N
AcO
a
BzO
AcO
H3C
BzO
R
R
+
F
H3C
CH3
AcO
H3C
CH3
11
F
F
13=R=H, 13j=R=NH2
12=R=H
d
d
c
c
R1
R1
R1
R1
N
N
N
N
N
N
N
N
N
N
N
O
N
O
O
F
O
N
N
N
N
HO
R
BzO
AcO
H3C
R
HO
BzO
AcO
H3C
R
R
HO
H3C
14c-j
14j=R=H,R1=NH2
CH3
CH3
d
HO
H3C
CH3
CH3
d
F
F
F
12c=R=H, R1=cyclopropylamino
12d=R=H, R1=cyclobutylamino
12e=R=H, R1=furfurylamino
13c=R=H, R1=cyclopropylamino
13d=R=H, R1=cyclobutylamino
13e=R=H, R1=furfurylamino
16c-j
16j=R=H,R1=NH2
12f=R=H, R1=aminomethylcyclopropyl
12g=R=H, R1=morpholino
13f=R=H, R1=aminomethylcyclopropyl
13g=R=H, R1=morpholino
12h=R=H, R1=N-methylpiperizino
12i=R=H, R1=4-fluorobenzylamino
13h=R=H, R1=N-methylpiperizino
13i=R=H, R1=4-fluorobenzylamino
e
e
15, 15a
14a
17, 17a
16a
Scheme 2. Synthesis of nucleosides 12, 12a–i, 13j, 13, 13a–i, 13j, 14a–j, 16a–j, 15, 15a, 17 and 17a. Reagents and conditions: (a) 6-chloropurine/6-chloro-9H-purin-2-amine,
ACN, DBU, TMSOTf, 65 °C overnight/N,O-bis(trimethylsilyl)acetamide, ACN, SnCl4, 70 °C, 2 h; (b) ethanol reflux, 16 h; (c) R1NH/R1NH2, ethanol, reflux, 1 h; (d) NH3/MeOH;
overnight; (e) DCM, RCOCl, at 0 °C.
In order to evaluate the effect of the modification of heterobase,
analogues 18, 18a–c, and 25 were prepared by coupling 11 and 11A
with commercially available pyrimidine bases. Coupling reaction
was performed in the presence of N,O-bis(trimethylsilyl)acetamide,
3. Results and Discussions
The modified C-30-hydroxy nucleosides were evaluated for their
antiviral effects on two flu viral strains, FluA (H5N1) Vietnam/
1203/2004H and Flu B florida/4/2006 in MDCK cell line at the
NIAID Antimicrobial Acquisition and Coordinating facility (AACF).
In addition, cytotoxicity was evaluated in parallel in MDCK cell
line. Selectivity index (SI) was determined by taking the ratio of
cell inhibitory concentration, 50% endpoint (IC50) to virus inhibi-
tory concentration, 50% endpoint (EC50): SI = (IC50/EC50).
ACN, SnCl4 by the methodology of Vorbrüggen. No
a anomer was
observed in these conditions. Removal of ester groups under meth-
anolic ammonia conditions furnished compounds 19, 19a–c and 26.
To study the effect of substitutions at the C50–OH position on the
above analogues, a series of ester prodrugs 15–15a, 17–17a and 20–
20e were prepared. The synthesis of these derivatives was accom-
plished by reacting C50–OH with acid chlorides (see Scheme 3).
Configuration at newly created stereo center, that is, at C-3 car-
bon in 11 and 11A was determined by observing cross peaks in
NOESY between methyl at C-3 and hydrogen at C-4 in compound
11. Similar observation was made between C-2-methyl and C-3-
3.1. Effect of purine/pyrimidine bases
As said earlier coupling of 11 and 11A with different hetero
bases yielded
14a–i, 21 and 23), as well as b nucleosides. However, all
sides were found to be inactive even up to 100 M concentration.
a
nucleosides and their derivatives (12, 12a–i, 14,
methyl for compound 11A, inferring that methyl is
a in compound
a
nucleo-
11 and b in 11A. Likewise in nucleosides cross peak between a dou-
blet at d 1.9 (30-CH3) and multiplet at d 4.45–4.52 (C40-CH) in 2D
NOESY confirmed that these protons are in proximity, indicating
l
Hence these compounds were omitted from the present discussion.
For the initial SAR, to begin with, we tested the purine nucleosides
obtained from 11 for their antiviral activity. Furanosides 13, 13a–i
with b configuration at anomeric carbon were active with an IC50
values ranging from 7.1–50
Many of the b nucleoside compounds (13, 13a–i) were less toxic
as shown (Table 1) with CC50 values of >100 g/ml. Most active
compound in this series is ((2R,3S,4R,5R)-3-acetoxy-5-(6-chloro-
9H-purin-9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl
benzoate 13. However, we were not interested to evaluate this
that they are in
a plane. In the case of a-furanosides (12) stereo-
chemistry at anomeric position was confirmed by observation of
cross peak between C-20-Me at d 1.46 and anomeric proton doublet
at d 6.62. Such a cross peak was absent in b-furanosides 13, 13j,
18b and 18c (see Fig. 2).
The solid-state structure of representative compound in this
series (12) (Fig. 3) was determined by single crystal X-ray crystal-
lography, stereochemistry at different centers were found to be
C(9)-R, C(10)-S, C(11)-R and C(12)-S.
lg/ml in flu-A and 3.7–65 lg/ml flu-B.
l

M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
6333
R1
R1
N
N
N
N
N
N
O
O
N
N
BzO
H3C
AcO
BzO
H3C
AcO
R
R
CH3
CH3
R1
R1
+
N
N
F
F
b
b
N
21
R=H, R1=Cl
22
N
N
N
O
22=R=H, R1=Cl
O
N
N
HO
H3C
HO
a
HO
R
22a=R=NH2, R1=Cl
R
CH3
H3C
HO
CH3
F
F
O
OAc
CH3
24
23
BzO
H3C
AcO
11 A
R=H, R1=NH2
R=H, R1=NH2
F
c
NH2
NHBz
N
N
N
O
O
O
O
b
N
HO
H3C
HO
BzO
CH3
H3C
AcO
CH3
F
F
26
25
Scheme 3. Synthesis of nucleosides 21, 22, 22a, 23, 24, 25 and 26 from the sugar intermediate 11A. Reagents and conditions: (a) 6-chloropurine/6-chloro-9H-purin-2-amine,
ACN, DBU, TMSOTf, 65 °C overnight/N,O-bis(trimethylsilyl)acetamide, ACN, SnCl4, 70 °C, 2 h; (b) NH3/MeOH; room temperature, 16 h; (c) N-benzoylcytosine, N,O-
bis(trimethylsilyl)acetamide, ACN, SnCl4, 70 °C, 2 h.
compound further as it contained a reactive chloro group at the 6th
H
position of purine nucleus. In an effort to find a compound with
more activity, we screened the corresponding amino compounds
by replacing chloro group with various amines. Cycloalkyl amino
derivatives 13d, 13f heterocyclic alkyl derivative 13h were slightly
better active than their corresponding aromatic alkyl amine com-
pounds 13e and 13i. NIs with guanine derivatives 13j and 22a were
not active.
O
O
1
1
OAc
OAc
CH3
BzO
BzO
5
5
4
4
3 2
2
3
H3C
AcO
H3C
CH3
F
AcO
F
11
11A
To evaluate the effect of other bases, we shifted our focus from
purine to pyrimidine bases (Scheme 4; Table 3) uracil 18, thymine
18a, 5-fluorouracil 18b and N-benzoylcytosine 18c. The activity of
these NIs were declined in the order of 18c>18b>18>18a. The most
active compound was N-benzoylcytosine analogue 18c.
Cl
Cl
N
N
N
N
N
H
N
H
O
N
X
N
O
1'
3' 2'
H
1'
H
BzO
5'
BzO
5'
4'
4'
3'2'
AcO
H3C
CH3
AcO
H3C
CH3
3.2. Effect of modification of the sugar
F
F
13
12
As we introduced a new stereo center at C-30, two diastereo-
meric analogues were obtained. We separated both the isomers
Figure 2. 1H NMR NOE correlations of compound 11 and 11A, 12, and 13.
Figure 3. ORTEP drawing of (3-acetoxy-5-(6-chloro-9H-purin-9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl benzoate (12).

6334
M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
B
B
B
O
O
O
O
OAc
CH3
HO
c
R
a
BzO
AcO
H3C
BzO
b
HO
H3C
CH3
HO
H3C
CH3
CH3
cO
A
F
F
F
H3C
F
20=B=uracil, R=n-butyroyl
11
18=B=uracil
18a=B=thyamine
18b=B=5-fluorouracil
18c=B=N-benzoylcytosine
19=B=uracil
20a=B=uracil, R=isobutyroyl
20b=B=uracil, R=velaryl
19a=B=thyamine
19b=B=5-fluorouracil
19c=B=cytosine
20c=B=5-fluorouracil, R=butyroyl
20d=B=5-fluorouracil, R=isobutyroyl
20e=B=5-fluorouracil , R=velaryl
Scheme 4. Synthesis of compounds 18, 18a–c, 19, 19a–c, 20 and 20a–e. Reagents and conditions: (a) pyrimidine base (uracil/thymine/6-fluorouracil/N-benzyoylcytosine),
N,O-bis(trimethylsilyl)acetamide, ACN, SnCl4, 70 °C, 2 h; (b) NH3/MeOH; overnight; (c) acid chlorides DCM, at 0 °C.
Table 3
In vitro anti-influenza activity of 18c and 13, against Flu A, Flu B in various strains and inhibitory activity of influenza virus infection of MDCK cells
Compound
Assay
Virus
Cell line
EC50
(l
g/ml)
IC50
SI
FluA(H5N1)
Vietnam/1203/2004H
FluB
Florida/4/2006
FluB
Florida/4/2006
FluB
O
Neutral red
Neutral red
MDCK
MDCK
18
>100
>100
>5.6
>91
1.1
NH
Visual
MDCK
MDCK
MDCK
0.591
>100
>100
>100
>170
>53
N
Virus yield
Visual-CONF
EC90:1.9
0.59
N
O
O
Florida/4/2006
FluB
BzO
AcO
>170
CH3
Florida/4/2006
F
H3C
18c
FluA(H5N1)
Vietnam/1203/2004H
FluA(H5N1)
Vietnam/1203/2004H
FluA(H5N1)
Vietnam/1203/2004H
FluB
Florida/4/2006
FluB
Cl
Neutral red
Virus yield
MDCK
MDCK
1.76
19
19
11
N
EC90:4.4
4.1
N
N
O
Visual-CONF
Neutral red
Virus yield
Visual-CONF
MDCK
MDCK
MDCK
MDCK
1.26
1.2
18
18
18
18
15
15
2.9
15
N
BzO
AcO
H3C
CH3
F
EC90:6.3
1.2
13
Florida/4/2006
FluB
Florida/4/2006
Note: SI = (IC50)/(EC50).
and tested them individually. Surprisingly all the isomers obtained
from 11A, irrespective of its configuration either at anomeric cen-
ter or at C-30 were not active, whereas compounds obtained from
11 are active.
Inhibitory activity of inflenza virus infection of MDCK cells
120
100
80
60
40
20
0
fluA( H5N1)Vietnam/1203/2004H
FluB Florida/4/2006
To probe the role of C-50–OH and C-30–OH, we prepared the
deprotected analogues of 13, 13a–i, 18, 18a, 18b, and 18c. The cor-
responding nucleosides having sugar free hydroxyls of these com-
pounds 16, 16a–j and 19, 19a–c (Schemes 2 and 4) failed to show
activity.
To gain a better understanding of the reason for lacking of activ-
ity for these compounds, we synthesized and evaluated monoes-
ters of C-50–OH purine and pyrimidine NIs (17, 17a and 20, 20a–
e) (Schemes 2 and 4). Surprisingly all these compounds were found
to be incapable of inhibiting virus (EC50 >100 lg/ml).
Based on these results we hypothesized that, since we used the
cell based assay to screen the compounds, the ability of a com-
pound to inhibit the influenza virus would be some extent depen-
dent on the cellular uptake. That is why compounds with C-50-
benzoyl–C-20-acetyl esters were active as they acted as prodrugs.
Thus the results suggest that N-benzoylcytosine analogue 18c
was the most active compound (Table 2). This activity was also
confirmed in other standard assays like, increase in neutral red
(NR) dye uptake, and decrease in virus yield assay (Table 3).
It is note worthy to mention that this novel analogue displayed
significant activity with specificity towards Flu B strain.
13 13a 13b 13c 13d 13e 13f 13g 13h 13i 22 24 18 18a 18b 18c
Compound number
4. Conclusion
In conclusion we demonstrated in the present study, the syn-
thesis of new series of nucleoside analogues with significant bio-

M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
6335
logical activity and it is confidently expected that further SAR of
compound 18c will be found more potent and interesting com-
pounds, having useful properties.
After that reaction quenched with satd NaHCO3 solution, metha-
nol was removed under reduced pressure, extracted with ethyl
acetate. The combined organic extracts were washed with water,
brine and dried over Na2SO4. Organic layer was removed under
reduced pressure and the residue was purified by silica gel col-
umn chromatography using 8% ethyl acetate: hexane as eluent
furnished compound 3 (17 g), and 15% ethyl acetate eluded com-
pound 4 (18 g) as a colorless liquid. The overall yield was 56%.
Compound 3: 1H NMR (300 MHz, CDCl3): d 8.01–7.93 (m, 4H),
7.56–7.48 (m, 6H), 5.53 (dd, 1H, J = 24.3 Hz, 7.80 Hz), 4.97 (d,
1H, J = 9.6 Hz), 4.58–4.53 (m, 2H), 4.41–4.38 (m, 1H), 3.30 (s,
5. Experimental section
5.1. General experimental procedures
All reagents and solvents were used without further purification
or drying. All reagents were purchased from Sigma–Aldrich, unless
otherwise specified. Commercial grade anhydrous solvents were
purchased from Rankem chemicals or Merck. All reactions were per-
formed under nitrogen, unless otherwise specified. The NMR exper-
iments were accomplished on a Bruker AVX300 (ultra shield™) at
300 K in CDCl3 (calibrated to the residual nondeuterated solvent sig-
nals). MS analysis was performed on Shimadzu LCMS 2010, EV and
Alliance Waters 2695 micromass QUATTRO MICRO API. Esquire
3000 plus ion-trap mass spectrometer equipped with electrospray
ionization (ESI) in positive and negative mode: spray voltage,
3.5 kV; dissolvation gas 600 l/h. Dry gas, N2, 1.5 l/min; scanning
range, m/z 50–1000. HRMS obtained on a QSTAR XL instrument. Col-
umn chromatography was performed using Merck silica gel 60, 40–
3H), 1.44 (d, 3H, J = 22.8). MS (ESI) m/z 413 [M+Na]+. IR cmꢁ1
:
3439, 2961, 2939, 1728, 1602, 1452, 1270, 1117, 710. Compound
4: 1H NMR (300 MHz, CDCl3): d 8.11–8.08 (m, 2H), 7.61–7.59
(m, 1H), 7.50–7.45 (m, 2H), 5.41 (dd, 1H, J = 23.7 Hz, 7.50 Hz),
4.85 (d, 1H, J = 10.5 Hz), 4.34–4.32 (m, 2H), 3.76–3.69 (m, 1H),
3.33 (s, 3H), 1.49 (d, 3H, J = 22.8). MS (ESI) m/z: 307 [M+NH4]+,
302 [M+H]+.
5.1.3. (2R,3R,4R)-4-Fluoro-2-(hydroxymethyl)-5-methoxy-4-
methyl-tetrahydrofuran-3-ol (5)
Compounds 3 and 4 (17 g, 43.8 mmol, 18 g, 63.3 mmol) in
methanolic ammonia (25% w/w 500 ml) was stirred at rt for
36 h. Completion of the reaction monitored by TLC, methanol
was removed under reduced pressure. Water added to the reac-
tion mixture and extracted with ethyl acetate, the organic layer
was dried over Na2SO4, concentrated under reduced pressure.
Purified by silica gel column chromatography using 20% ethyl
acetate in Hexane to give 5 (16 g, 99%) as an off-white solid. 1H
NMR (300 MHz, CDCl3): d 4.79 (d, 1H, J = 10.8 Hz), 4.01–3.97 (m,
2H), 3.85 (d, 1H,), 3.71–3.66 (m, 1H), 3.44 (s, 3H), 2.05 (br s,
1H, –OH), 2.02 (br s, 1H, –OH), 1.47 (d, 3H, J = 23.4 Hz). MS
(ESI) m/z 203 [M+Na]+. IR cmꢁ1: 3437, 3372, 3294, 2924, 1459,
1102, 1049, 1006, 821, 731.
63 lm and Pharmacia Sephadex LH-20, 20–100 lm. The obtained
fractions from all chromatographic steps were analyzed by TLC (mo-
bile phase: DCM/Methanol [9:1]; stationary phase: Merck silica gel
60 PF254, detected with staining reagents anisaldehyde at VIS,
UV254, UV366). HPLC was performed on a Shimadzu 2010 auto sam-
pler with a photodiode array detector (DAD). LC-parameters: sta-
tionary phase: Zorbax SB-C18, rapid resolution 3.5 lm (4.6–
150 mm), Agilent, USA; temperature: 40 °C; mobile phase: water
(A); methanol (B); flow rate 1.0 ml/min; UV detection wavelength:
205, 280, 360 nm; injection volume: 10 ll; gradient. A summary of
the key crystallographic information for the compound 12, intensity
data were collected on a Bruker SMART CCD area detector diffrac-
tometer with graphite-monochromated radiation (k = 0.71073 Å).
Biological assays were performed at AACF as per their protocols. Vis-
it: http://niaid-aacf.org/protocols/Respiratory.
5.1.4. (2R,3R,4R)-2-((tert-Butyldiphenylsilyloxy)methyl)-4-
fluoro-5-methoxy-4-methyl-tetrahydrofuran-3-ol (6)
To a magnetically stirred solution of 5 (16 g, 88.8 mmol) and
imidazole (9.07 g, 133.2 mmol) in CH2Cl2 (200 ml) cooled to 0 °C,
TBDPSCl (23.1 ml, 88.8 mmol) was added and stirred for 2 h at rt.
Completion of the reaction monitored by TLC, water added to the
reaction mixture and extracted with CH2Cl2. The combined organic
extracts were washed with water, brine and dried over Na2SO4.
Concentrated under reduced pressure, purification of the residue
by silica gel column chromatography using 7% ethyl acetate: hex-
ane as eluent to give 6 (16 g, 43%) as colorless liquid. 1H NMR
(300 MHz, CDCl3): d 7.72–7.69 (m, 4H), 7.42–7.41 (m, 6H), 4.79
(d, 1H, J = 11.1.0 Hz), 3.97–3.87 (m, 4H), 3.35 (s, 3H), 1.86 (br s,
1H), 1.50 (d, 3H). MS (ESI) m/z 441 [M+Na]+.
5.1.1. ((2R,3R,4R)-3-Benzoyloxy-4-fluoro-5-hydroxy-4-methyl-
tetrahydrofuran-2-yl)methyl benzoate (2)
To a stirred solution of compound ((2R,3R,4R)-3-benzoyloxy-4-
fluoro-4-methyl-5-oxo-tetrahydrofuran-2-yl)methyl benzoate (60 g,
161 mmol) in THF (300 ml) cooled to ꢁ10 °C, then Li(tBuO)3AlH
(241.9 ml, 241 mmol) was added and stirred at 0 °C for 1 h. Comple-
tion of the reaction monitored by TLC, quenched with saturated
ammonium chloride solution. THF was removed under reduced
pressure and the aqueous layer was extracted with ethyl acetate.
The combined organic extracts were washed with water, brine and
dried over Na2SO4. Organic layer was removed under reduced pres-
sure and the residue was purified by silica gel column chromatogra-
phy using 16% ethyl acetate: hexane to give 2 (60 g, 99%) as a
colorless liquid. 1H NMR (300 MHz, CDCl3): d 8.11–8.00 (m, 4H),
7.61–7.37 (m, 6H), 5.65 (dd, 1H, J = 23.4 Hz, 7.8 Hz, 7.5 Hz), 5.34
(dd, 1H, J = 9.6 Hz, 2.7 Hz), 4.69–4.66 (m, 1H), 4.57–4.53 (m, 2H),
3.40 (br s, 1H, –OH), 1.60–1.56 (m, 3H, J = 22.5). MS (ESI) m/z 397
[M+Na]+. IR cmꢁ1: 3439, 3070, 2940, 2868, 1733, 1729, 1453, 1274,
1115, 710.
5.1.5. (2R,4S)-2-((tert-Butyldiphenylsilyloxy)methyl)-4-fluoro-
5-methoxy-4-methyl-dihydrofuran-3(2H)-one (7)
A solution of compound 6 (16 g, 38.2 mmol) and Dess–Martin
periodinane (24.3 g, 57.4 mmol) in CH2Cl2 (150 ml) stirred at 0 °C
to room temperature under N2 atmosphere. Completion of the
reaction monitored by TLC and quenched by the saturated solution
of Na2S2O3 and NaHCO3 in equal volumes (100 ml + 100 ml). The
aqueous layers were extracted with CH2Cl2 and the combined or-
ganic extracts were washed with water, brine and dried over
Na2SO4 and concentrated under reduced pressure to give 7 (24 g
as a crude). 1H NMR (300 MHz, CDCl3): d 7.69–7.67 (m, 4H),
7.42–7.39 (m, 6H), 5.07 (d, 1H, J = 12.3 Hz), 4.38 (dd, 1H,
J = 5.1 Hz, 3.3 Hz), 3.889–3.83 (m, 2H), 3.46 (s, 3H), 1.42 (d, 3H,
5.1.2. ((2R,3R,4R)-3-Benzoyloxy-4-fluoro-5-methoxy-4-methyl-
tetrahydrofuran-2-yl)methyl benzoate (3) and (2R,3R,4R)-4-
fluoro-2-(hydroxymethyl)-5-methoxy-4-methyltetrahydrofuran-
3-yl benzoate (4)
To a stirred solution of compound 2 (60 g, 160 mmol) in meth-
anol (300 ml), BF3ꢀOEt2 (60.02 ml, 481 mmol) was added at room
temperature then reaction mixture was heated to 80 °C for 5 h.
J = 24 Hz), 1.03 (s, 9H). MS (ESI) m/z 439 [M+Na]+ IR cmꢁ1: 2934,
.
2859, 1783, 1382, 1114, 998, 703.

6336
M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
5.1.6. (2R,3S,4R)-2-((tert-Butyldiphenylsilyloxy)methyl)-4-
fluoro-5-methoxy-3,4-dimethyltetrahydrofuran-3-ol (8): and
(2R,3R,4R)-2-((tert-butyldiphenylsilyloxy)methyl)-4-fluoro-5-
methoxy-3,4-dimethyltetrahydrofuran-3-ol (8A)
1H, J = 20.4 Hz), 4.73–4.67 (m, 1H), 4.67–4.44 (m, 2H), 2.06 (s,
3H), 1.80 (s, 3H), 1.51 (d, 3H, J = 22.8 Hz), 1.43 (s, 3H). MS (ESI)
m/z: 391 [M+Na]+.
To a solution of 7 (23 g, 55.2 mmol) in dry ether at 0 °C was
added methyl magnesium bromide (60.8 ml, 121 mmol) drop wise
for 30 min and stirred at rt for 4 h. Completion of the reaction mon-
itored by TLC and quenched with saturated ammonium chloride
solution. Aqueous layer was extracted with ethyl acetate and the
combined organic layers were washed with water, brine and dried
over Na2SO4. Organic layers were concentrated under reduced
pressure and purified by silica gel column chromatography using
3% and 3.5% ethyl acetate in hexane gave 8 and 8A (9.1 g and
1.5 g, overall yield 64.2%) as a colorless liquid. Compound 7: 1H
NMR (300 MHz, CDCl3): d 7.74–7.72 (m, 4H), 7.43–7.39 (m, 6H),
4.83 (d, 1H, J = 15.3 Hz), 4.08 (m, 1H), 3.84–3.80 (m, 2H), 3.39 (s,
3H), 1.34 (d, 3H, J = 24.6 Hz), 1.16 (s, 3H), 1.05 (s, 9H). MS (ESI)
m/z 455 [M+Na]+. Compound 8: 1H NMR (300 MHz, CDCl3): d
7.74–7.72 (m, 4H), 7.42–7.39 (m, 6H), 4.81 (d, 1H, J = 11.4 Hz),
4.16–4.14 (m, 1H), 4.00–3.81 (m, 2H), 3.37 (s, 3H), 1.42 (d, 3H,
J = 23.4 Hz), 1.27 (s, 3H), 1.06 (s, 9H). MS (ESI) m/z 455 [M+Na]+.
5.1.10. (2R,3R,4R)-4-Fluoro-2-(hydroxymethyl)-5-methoxy-3,4-
dimethyltetrahydrofuran-3-ol (9A)
To a solution of compound 8A (1.5 g, 3.4 mmol) in THF (25 ml)
at 0 °C was added 1 M TBAF (3.47 ml, 3.4 mmol) drop wise for
10 min and stirred at rt for 1.5 h. Completion of the reaction mon-
itored by TLC and neutralized with satd NaHCO3. Aqueous layer
was extracted with ethyl acetate and the combined organic ex-
tracts were washed with water, brine and dried over Na2SO4 and
concentrated under reduced pressure, purified by silica gel column
chromatography using 50% ethyl acetate: hexane as eluent fur-
nished compound 9A (0.5 g, 75%) as a colorless liquid. 1H NMR
(300 MHz, CDCl3): d 4.84 (d, 1H, J = 15.3 Hz), 4.00–3.98 (m, 3H),
3.45 (s, 3H), 1.34 (d, 3H, J = 24.3 Hz), 1.17 (s, 3H). MS (ESI) m/z:
217 [M+Na]+.
5.1.11. ((2R,3R,4R)-4-Fluoro-3-hydroxy-5-methoxy-3,4-
dimethyltetrahydrofuran-2-yl)methyl benzoate (10A)
To a solution of compound 9A (0.5 g, 2.5 mmol) in dry DCM
(10 ml) at 0 °C was added DMAP (0.47 g, 3.8 mmol), TEA (1.42 ml,
10.3 mmol), stirred for 15 min then drop wise addition of BzCl
(1.7 ml, 15.4 mmol) for 10 min and stirred at 45 °C for 48 h. Com-
pletion of the reaction monitored by TLC and quenched with water.
Aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with satd NaHCO3 solution, brine and
dried over Na2SO4. Concentrated under reduced pressure to yield
compound 10A (0.5 g, 65%). MS (ESI) m/z: 299 [M+Na]+.
5.1.7. (2R,3S,4R)-4-Fluoro-2-(hydroxymethyl)-5-methoxy-3,4-
dimethyltetrahydrofuran-3-ol (9)
To a solution of 7 (6.3 g, 14.5 mmol) in THF (100 ml) at 0 °C was
added 1 N TBAF (14.5 ml, 14.5 mmol) drop wise for 10 min and
stirred at rt for 1.5 h. Completion of the reaction monitored by
TLC and neutralized with satd NaHCO3. Aqueous layer was ex-
tracted with ethyl acetate and the combined organic extracts were
washed with water, brine and dried over Na2SO4 and concentrated
under reduced pressure, purified by silica gel column chromatogra-
phy using 50% ethyl acetate: hexane as eluent furnished 9 (2.8 g,
99%) as a colorless liquid. 1H NMR (300 MHz, CDCl3): d 4.84 (d,
1H, J = 11.1 Hz), 4.11 (m, 1H), 3.88–3.87 (m, 2H), 3.46 (s, 3H),
3.35 (s, 1H), 2.05 (m, 1H), 1.40 (d, 3H, J = 23.4 Hz), 1.27 (d, 3H,
J = 2.1 Hz). MS (ESI) m/z 217 [M+Na]+.
5.1.12. (3R,4R,5R)-5-(Benzoyloxymethyl)-3-fluoro-3,4-
dimethyltetrahydrofuran-2,4-diyl diacetate (11A)
To a solution of compound 10A (0.5 g, 1.67 mmol) in acetic acid
(10 ml) at 0 °C was added H2SO4 (0.36 ml, 3.69 mmol) and acetic
anhydride (0.66 ml, 7.04 mmol), then stirred the reaction at rt for
16 h. Completion of the reaction monitored by TLC, reaction mix-
ture was slowly added to ice cold water, and extracted with ethyl
acetate; organic layer was washed with satd NaHCO3 and dried
over Na2SO4 and concentrated under reduced pressure to yield
5.1.8. ((2R,3S,4R)-4-Fluoro-3-hydroxy-5-methoxy-3,4-
dimethyltetrahydrofuran-2-yl)methyl benzoate (10)
To a solution of 9 (2 g, 10.3 mmol) in dry DCM (40 ml) at 0 °C
was added DMAP (2.5 g, 20.6 mmol), TEA (4.2 ml, 30.9 mmol), stir-
red for 15 min then drop wise addition of BzCl (5.9 ml, 51.5 mmol)
for 10 min and stirred at rt for 18 h. Completion of the reaction
monitored by TLC and quenched with water. Aqueous layer was
extracted with ethyl acetate and the combined organic layers were
washed with satd NaHCO3 solution, brine and dried over Na2SO4.
Concentrated under reduced pressure and purified by silica gel col-
umn chromatography using 3% ethyl acetate: hexane as eluent fur-
nished compound 10 (2.1 g, 68.4%) as a colorless liquid. 1H NMR
(300 MHz, CDCl3): d 8.09–8.06 (m, 4H), 7.57–7.42 (m, 6H), 4.85
(d, 1H, J = 10.2 Hz), 4.60–4.59 (m, 2H), 4.50–4.44 (m, 1H), 3.46 (s,
3H), 1.46 (d, 3H, J = 23.4 Hz), 1.26 (s, 3H). MS (ESI) m/z: 425
[M+Na]+. IR cmꢁ1: 3425, 2927, 1723, 1679, 1602, 1451, 1281,
1124, 710.
compound 11A (0.5 g, 81%) as
a
colorless liquid. 1H NMR
(300 MHz, CDCl3): d 8.08–8.04 (m, 2H), 7.56–7.41 (m, 3H), 6.17
(d, 1H, J = 12.6 Hz), 4.94 (dd, 1H, J = 11.4 Hz, 3 Hz), 4.66–4.65 (m,
1H), 4.55–4.49 (m, 1H), 2.09 (s, 3H), 2.06 (s, 3H), 1.58 (m, 6H).
MS (ESI) m/z: 391 [M+Na]+, 386 [M+NH4]+.
5.1.13. ((2R,3S,4R,5S)-3-Acetoxy-5-(6-chloro-9H-purin-9-yl)-4-
fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl benzoate (12)
and ((2R,3S,4R,5R)-3-acetoxy-5-(6-chloro-9H-purin-9-yl)-4-
fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl benzoate (13)
To a stirred solution of 6-chloropurine (0.87 g, 5.7 mmol) in ace-
tonitrile (20 ml) was added DBU (1.7 ml, 11.4 mmol) and TMSOTf
(2.76 ml, 15.2 mmol) at 0 °C, stirred for 15 min then compound
11 (1.4 g, 3.8 mmol) dissolved in acetonitrile (20 ml) was added
and reaction continued stirring at 65 °C for 8 h. Completion of reac-
tion monitored by TLC and quenched with saturated NaHCO3.
Aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with satd NaHCO3 solution, brine and
dried over Na2SO4. Concentrated under reduced pressure and puri-
fied by silica gel column chromatography using 20% (12) and 25%
(13) ethyl acetate: hexane as eluent furnished compounds 12
(0.7 g) and 13 (0.3 g) as colorless solid, overall yield 57%. Compound
12: 1H NMR (300 MHz, CDCl3): d 8.78 (s, 1H), 8.36 (d, 1H,
J = 3.6 Hz), 8.08–8.04 (m, 2H), 7.60 (m, 1H), 7.49–7.44 (m, 2H),
6.63 (d, 1H, J = 22.5 Hz), 4.90–4.80 (m, 3H), 2.21 (s, 3H), 1.92 (s,
3H), 1.48 (d, 3H, J = 23.1 Hz). 13C NMR: 13.72, 14.35, 29.58, 62.90,
5.1.9. (3R,4S,5R)-5-(Benzoyloxymethyl)-3-fluoro-3,4-
dimethyltetrahydrofuran-2,4-diyl diacetate (11)
To a solution of 10 (2.1 g, 7.04 mmol) in acetic acid (15 ml) at
0 °C was added H2SO4 (1.5 ml, 15.5 mmol) and acetic anhydride
(2.79 ml, 29.5 mmol), and stirred the reaction at rt for 16 h. Com-
pletion of the reaction monitored by TLC, reaction mixture was
slowly added to ice cold water, and extracted with ethyl acetate;
organic layer was washed with satd NaHCO3 and dried over Na2SO4
and concentrated under reduced pressure to yield 11 (1.9 g,
73.27%) as a colorless gummy mass. 1H NMR (300 MHz, CDCl3): d
8.06–8.04 (m, 2H), 7.57–7.55 (m, 1H), 7.47–7.42 (m, 2H), 6.19 (d,

M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
6337
84.39, 88.12, 88.32, 101.67, 121.86, 128.44 (2), 129.59 (3), 133.35,
5.2.2. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-5-(6-(furan-2-
141.64, 148.82, 152.55, 161.98, 166.07, 168.70. MS (ESI) m/z: 485
[M+Na]+, 463 [M+1]+. Compound 13: 1H NMR (300 MHz, CDCl3): d
8.94 (s, 1H), 8.54 (d, 1H, J = 4.2 Hz), 8.06–8.03 (m, 2H), 7.59 (m,
1H), 7.49–7.43 (m, 2H), 6.97 (d, 1H, J = 21.3 Hz), 4.88–4.80 (m,
2H), 4.53–4.47 (m, 1H), 2.19 (s, 3H), 1.91 (s, 3H), 1.49 (d, 3H,
J = 23.1 Hz), 13C NMR: 13.61, 14.15, 29.54, 62.91, 84.42, 86.95,
88.21, 102.38, 128.37 (2), 129.35 (3), 131.24, 133.24, 144.41,
151.13, 151.71, 152.09, 166.04, 168.81. MS (ESI) m/z: 485
[M+Na]+, 463 [M+1]+.
ylmethylamino)-9H-purin-9-yl)-3,4-dimethyltetrahydrofuran-
2-yl)methyl benzoate (13e)
1H NMR (300 MHz, CDCl3): d 8.60 (s, 1H), 8.12–8.00 (m, 3H),
7.48–7.43 (m, 3H), 7.30 (d, 1H, J = 0.9 Hz), 6.46–6.43 (m, 1H),
6.27–6.21 (m, 2H), 6.11 (d, 1H, J = 22.5 Hz), 4.97–4.90 (dd, 1H,
J1 = 15 Hz, J2 = 15 Hz), 4.76–4.71 (dd, 1H, J1 = 12 Hz, J2 = 12 Hz),
4.60–4.41 (m, 3H), 4.22–4.20 (m, 1H), 2.14 (s, 3H), 1.82 (d, 3H,
J = 2.4 Hz), 1.34 (d, 3H, J = 22.2 Hz). MS (ESI) m/z: 546 [M+Na]+.
5.2.3. ((2R,3S,4R,5R)-3-Acetoxy-5-(6-(cyclopropylmethylamino)-
9H-purin-9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-
yl)methyl benzoate (13f)
5.1.14. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-5-(6-hydroxy-9H-
purin-9-yl)-3,4-dimethyltetrahydrofuran-2-yl)methyl benzoate
(13a) and ((2R,3S,4R,5R)-3-acetoxy-5-(6-ethoxy-9H-purin-9-yl)-
4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl benzoate
(13b)
1H NMR (300 MHz, CDCl3): d 8.52 (s, 1H), 8.06–8.01 (m, 3H),
7.60–7.57 (m, 1H), 7.49–7.44 (m, 2H), 6.20 (s, 1H), 6.15 (d, 1H,
J = 22.8 Hz0, 4.90–4.86 (m, 2H), 4.59–4.53 (m, 1H), 3.40–3.34 (m,
2H), 2.19 (s, 3H), 1.91 (d, 3H, J = 2.4 Hz), 1.63 (s, 1H), 1.37 (d, 3H,
J = 22.5 Hz), 0.89–0.88 (m, 1H), 0.49–0.44 (m, 2H), 0.25–0.23 (m,
2H). MS (ESI) m/z: 498 [M+1]+, 499 [M+Na]+.
A stirred solution of 13 (1.0 g, 2.16 mmol) in ethanol (20 ml)
was refluxed for 36 h. Completion of the reaction monitored by
TLC, water added to the reaction mixture and the aqueous layer
was extracted with ethyl acetate and the combined organic layers
were washed with brine and dried over Na2SO4. Concentrated un-
der reduced pressure and purified by silica gel column chromatog-
raphy using 3% methanol in dichloromethane as eluent furnished
compound 13b (0.05 g) as a colorless solid and 5% methanol in
dichloromethane as eluent to give compound 13a (0.45 g). The
overall yield was 52%. Compound 13a: 1H NMR (300 MHz, CDCl3):
d 10.30 (bs, 1H), 8.22 (d, 1H, J = 3.9 Hz), 8.09–8.04 (m, 3H), 7.61–
7.48 (m, 3H), 6.93 (d, 1H, J = 21.6 Hz), 4.84–4.77 (m, 2H), 4.49–
4.47 (m, 1H), 2.17 (s, 3H), 1.89 (d, 3 h, J = 2.4 Hz), 1.46 (d, 3H,
J = 23.1 Hz). MS (ESI) m/z: 445 [M+1]+, 467 [M+Na]+. Compound
13b: 1H NMR (300 MHz, CDCl3): d 8.67 (s, 1H), 8.32 (d, 1H,
J = 3.9 Hz), 8.07–8.03 (m, 2H), 7.60–7.59 (m, 1H), 7.48–7.43 (m,
2H), 6.71 (d, 1H, J = 21.9 Hz), 4.86–4.81 (m, 2H), 4.77–4.76 (m,
2H), 4.68–4.50 (m, 2H), 2.16 (s, 3H), 1.90 (d, 3H, J = 2.11 Hz),
1.50–1.25 (m, 6H). MS (ESI) m/z: 473 [M+1]+, 495 [M+Na]+.
5.2.4. (2R,3S,4R,5R)-3-Acetoxy-4-fluoro-3,4-dimethyl-5-(6-
morpholino-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl
benzoate (13g)
1H NMR (300 MHz, CDCl3): d 8.80 (s, 1H), 8.47 (d, 1H, J = 4.1 Hz),
8.06–8.03 (m, 2H), 7.59–7.56 (m, 1H), 7.48–7.43 (m, 2H), 6.58 (d,
1H, J = 23.4 Hz), 4.86–4.73 (m, 2H), 4.53–4.47 (m, 1H), 3.99–3.83
(m, 4H), 3.54–3.28 (m, 2H), 3.27–3.23 (m, 2H), 2.22 (s, 3H), 1.89
(d, 3H, J = 2.1 Hz), 1.26 (d, 3H, J = 22.8 Hz). MS (ESI) m/z: 536
[M+Na]+, 514 [M+1]+.
5.2.5. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-3,4-dimethyl-5-(6-(4-
methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-2-
yl)methyl benzoate (13h)
1H NMR (300 MHz, CDCl3): d 8.80 (s, 1H), 8.45 (d, 1H, J = 5.1 Hz),
8.07–8.04 (m, 2H), 7.59–7.57 (m, 1H), 7.49–7.44 (m, 2H), 6.54 (d,
1H, J = 23.1 Hz), 4.87–4.76 (m, 2H), 4.57–4.53 (m, 1H), 3.50–3.48
(m, 2H), 3.31 (s, 2H), 2.67–2.60 (m, 3H), 2.38 (s, 1H), 2.24 (s, 1H),
1.88 (d, 3H, J = 2.11 Hz), 1.23 (d, 3H, J = 22.8 Hz). MS (ESI) m/z:
527 [M+1]+, 549 [M+Na]+.
5.1.15. ((2R,3S,4R,5R)-3-Acetoxy-5-(6-(cyclopropylamino)-9H-
purin-9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl
benzoate (13c)
To a stirred solution of compound 13 (1.0 g, 2.16 mmol) in eth-
anol (20 ml) was added compound cyclopropylamine (1.79 ml,
25.90 mmol) and refluxed for 30 min completion of the reaction
monitored by TLC, water added to the reaction mixture and the
aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine and dried over Na2SO4.
Concentrated under reduced pressure, purified by silica gel column
chromatography using 40% ethyl acetate in hexane as eluent fur-
nished compound 13c (0.45 g, 44%) as a colorless solid. 1H NMR
(300 MHz, CDCl3): d 8.64 (s, 1H), 8.08–8.04 (m, 3H), 7.60–7.58
(m, 1H), 7.49–7.44 (m, 2H), 6.37 (s, 1H), 6.11 (d, 1H, J = 22.8 Hz),
4.89–4.84 (m, 1H), 4.75–4.71 (m, 1H), 4.56–4.49 (m, 1H), 2.92–
2.88 (m, 1H), 2.19 (s, 3H), 1.88 (d, 3H, J = 2.1 Hz), 1.35 (d, 3H,
J = 22.5 Hz), 0.88–0.86 (m, 2H), 0.53–0.46 (m, 2H). MS (ESI) m/z:
484 [M+1]+.
5.2.6. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-5-(6-(4-fluoroben-
zylamino)-9H-purin-9-yl)-3,4-dimethyltetrahydrofuran-2-
yl)methyl benzoate (13i)
1H NMR (300 MHz, CDCl3): d 8.56 (s, 1H), 8.06 (s, 1H), 7.96–7.93
(m, 2H), 7.62–7.60 (m, 1H), 7.61–7.58 (m, 2H), 7.25–7.20 (m, 2H),
6.94–6.88 (m, 2H), 6.38 (bs, 1H), 6.10 (d, 1H, J = 22.8 Hz), 4.98–4.91
(m, 1H), 4.64–4.61 (m, 1H), 4.48–4.42 (m, 1H), 4.34–4.30 (m, 2H),
2.12 (s, 3H), 1.76 (s, 3H), 1.34 (d, 3H, J = 22.5 Hz). MS (ESI) m/z: 552
[M+1]+.
5.2.7. ((2R,3S,4R,5R)-3-Acetoxy-5-(2-amino-6-chloro-9H-purin-
9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl
benzoate (13j)
To a stirred solution of 6-chloro-9H-purin-2-amine (0.18 g,
1.08 mmol) in acetonitrile (5 ml) was added N,O-bis(trimethyl-
silyl)acetamide (1.6 ml, 6.52 mmol) at rt and refluxed for 2 h. To
this added compound 11 (0.2 g, 0.54 mmol) in acetonitrile (5 ml)
drop wise at 0 °C, and added SnCl4 (0.09 ml, 0.81 mmol) and re-
fluxed for 15 h, Completion of the reaction monitored by TLC, neu-
tralized the reaction mixture and with saturated NaHCO3 solution,
aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine and dried over Na2SO4.
Concentrated under reduced pressure, purified by silica gel column
chromatography using 5% methanol in dichloromethane as eluent
to give compound 13j (0.08 g, 30%) as a colorless solid. 1H NMR
(300 MHz, CDCl3): d 8.29 (d, 1H, J = 4.2 Hz), 8.06–8.03 (m, 2H),
5.2. Synthesis of compounds 13d–i will follow the above
conditions
5.2.1. ((2R,3S,4R,5R)-3-Acetoxy-5-(6-(cyclobutylamino)-9H-
purin-9-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)methyl
benzoate (13d)
1H NMR (300 MHz, CDCl3): d 8.52 (s, 1H), 8.08–8.05 (m, 3H),
7.71–7.60 (m, 1H), 7.48–7.43 (m, 2H), 6.29 (d, 1H, J = 7.5 Hz),
6.14 (d, 1H, J = 22.8 Hz), 4.94–4.82 (m, 1H), 4.68–4.65 (m, 1H),
4.59–4.55 (m, 1H), 4.23–4.20 (m, 1H), 2.36–1.36 (m, 15H). MS
(ESI) m/z: 498 [M+1]+, 520 [M+Na]+.

6338
M. S. Vedula et al. / Bioorg. Med. Chem. 18 (2010) 6329–6339
7.59–7.56 (m, 1H), 7.49–7.44 (m, 2H), 6.80 (d, 1H, J = 21.6 Hz), 5.09
(s, 2H), 4.83–4.78 (m, 2H), 4.51–4.44 (m, 1H), 2.17 (s, 3H), 1.89 (d,
3H, J = 2.4 Hz), 1.48 (d, 3H, J = 23.1 Hz). MS (ESI) m/z: 478 [M+1],
500 [M+Na].
129.07 (3), 129.68 (2), 132.94 (2), 133.31, 145.94, 155.22, 162.48,
166.18, 169.08. MS (ESI) m/z: 524 [M+1]+. IR cmꢁ1: 3409, 3070,
2924, 1744, 1718, 1665, 1617, 1485, 1393, 1234, 1061, 708.
5.4. Antiviral activity
5.2.8. ((2R,3S,4R,5R)-3-Acetoxy-5-(2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-
yl)methyl benzoate (18)
5.4.1. Increase in neutral red (NR) dye uptake assay
Neutral red is added to the medium; cells not damaged by virus
take up a greater amount of dye, which is read on a computerized
micro plate auto reader. The method as described by McManus
(Appl. Environ. Microbiol. 1976, 31, 35–38) is used. An EC50 is deter-
mined from this dye uptake.
To a stirred solution of uracil (0.30 g, 2.71 mmol) in acetonitrile
(7 ml) was added N,O-bis(trimethylsilyl)acetamide (4 ml,
16.3 mmol) at rt and refluxed for 2 h. To this added compound
11 (0.5gm, 1.35 mmol) in acetonitrile (8 ml) drop wise at 0 °C,
and added SnCl4 (0.2 ml, 2.03 mmol) and refluxed for 15 h, Com-
pletion of the reaction monitored by TLC, neutralized the reaction
mixture and with saturated NaHCO3 solution, aqueous layer was
extracted with ethyl acetate and the combined organic layers were
washed with brine and dried over Na2SO4. Concentrated under re-
duced pressure, purified by silica gel column chromatography
using 35% ethyl acetate in hexane as eluent to give compound 18
(0.35 g, 61%) as a colorless solid. 1H NMR: 1H NMR (300 MHz,
CDCl3): d 8.67 (s, 1H), 8.06–8.03 (m, 2H), 7.59–7.33 (m, 4H), 6.39
(d, 1H, J = 22.8 Hz), 5.76, 5.55 (dd, 1H, J = 2.1 Hz, J = 2.4 Hz), 4.80–
4.43 (m, 3H), 2.13 (s, 3H), 1.81 (d, 3H, J = 2.4 Hz), 1.49 (d, 3H,
J = 23.4 Hz). 13C NMR: 13.50, 14.61, 22.02, 63.08, 84.33, 87.18,
88.26, 102.21, 103.36, 128.48 (2), 129.50 (2), 129.67, 133.35,
140.94, 150.73, 163.01, 166.16, 168.98. MS (ESI) m/z: 443
[M+Na]+. IR cmꢁ1: 3433, 2931, 2346, 1713, 1385, 1274, 952, 712.
5.4.2. Decrease in virus yield assay
Compounds considered active by NR dye uptake was re-tested
using both CPE inhibition and, using the same plate, effect on
reduction of virus yield by assaying frozen and thawed eluates
from each cup for virus titer by serial dilution onto monolayers
of susceptible cells. Development of CPE in these cells is the indica-
tion of presence of infectious virus. As in the initial tests, a known
active drug is run in parallel as a positive control. The 90% effective
concentration (EC90), which is that test drug concentration that
inhibits virus yield by 1 log10, is determined from these data.
5.5. Assay of cytotoxicity
5.5.1. Neutral red uptake
In the neutral red dye uptake phase of the antiviral test de-
scribed above, the two toxicity control wells also receive neutral
red and the degree of color intensity is determined spectrophoto-
metrically. A neutral red IC50 (NR IC50) is subsequently determined.
5.3. Synthesis of compound 18a–c will follow the above
conditions
5.3.1. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-3,4-dimethyl-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydro-
furan-2-yl)methyl benzoate (18a)
5.5.2. Visual observation
In the CPE inhibition tests, two wells of uninfected cells
treated with each concentration of test compound will be run
in parallel with the infected, treated wells. At the time CPE is
determined microscopically, the toxicity control cells will also
be examined microscopically for any changes in cell appearance
compared to normal control cells run in the same plate. These
changes may be enlargement, granularity, cells with ragged
edges, a filmy appearance, rounding, detachment from the surface
of the well, or other changes. A 50% cell inhibitory (cytotoxic)
concentration (IC50) is determined by regression analysis of these
data.
1H NMR (300 MHz, CDCl3): d 8.07 (s, 1H), 8.05–8.04 (m, 2H),
7.60–7.58 (m, 1H), 7.49–7.44 (m, 2H), 7.15–7.13 (m, 1H), 6.38 (d,
1H, J = 23.1 Hz), 4.74–4.65 (m, 2H), 4.49–4.41 (m, 1H), 2.12 (s,
3H), 1.94 (d, 3H, J = 0.9 Hz), 1.83 (d, 3H, J = 2.4 Hz), 1.48 (d, 3H,
J = 23.4 Hz). 13C NMR: 12.55, 13.60, 14.62, 22.05, 29.68, 63.04,
84.17, 86.97, 88.27, 110.50, 128.51 (2), 129.56 (2), 129.69,
133.37, 136.57, 150.72, 163.39, 166.20, 169.90. MS (ESI) m/z: 435
[M+1], 457 [M+Na]. IR cmꢁ1: 3432, 2926, 1753, 1692, 1602,
1547, 1384, 1276, 1115, 954, 833.
5.3.2. ((2R,3S,4R,5R)-3-Acetoxy-4-fluoro-5-(5-fluoro-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)-3,4-dimethyltetrahydrofuran-
2-yl)methyl benzoate (18b)
Acknowledgment
1H NMR: 1H NMR (300 MHz, CDCl3): d 8.56 (bs, 1H), 8.06–8.04
(m, 2H), 7.63–7.58 (m, 1H), 7.50–7.45 (m, 2H), 7.42–7.39 (dd, 1H,
J1 = 6.3 Hz, J2 = 6.0 Hz), 6.39–6.32 (dd, 1H, J1 = 22.5 Hz,
J2 = 22.5 Hz), 4.79–4.47 (m, 1H), 4.68–4.63 (m, 1H), 4.47–4.41
(m, 1H), 2.13 (s, 3H), 1.82 (d, 3H, J = 2.4 Hz), 1.50 (d, 3H,
J = 23.4 Hz). 13C NMR: 13.51, 14.56, 22.01, 63.01, 84.50, 87.43,
88.15, 125.14, 125.60 (2), 128.53 (2), 129.44, 129.69, 133.41,
138.61, 149.21, 156.48, 166.18, 168.95. MS (ESI) m/z: 461
[M+Na]. IR cmꢁ1: 3550, 3479, 3414, 3227, 2283, 1723, 1275,
1113, 780.
We thank NIAID’s Antimicrobial Acquisition and Coordinating
facility (AACF) for screening the compounds and providing the
data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.017. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
5.3.3. ((2R,3S,4R,5R)-3-Acetoxy-5-(4-benzamido-2-oxopyrimi-
din-1(2H)-yl)-4-fluoro-3,4-dimethyltetrahydrofuran-2-yl)
methyl benzoate (18c)
References and notes
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A56/23.
http://who.int/csr/don/2010_01_22/in/
1H NMR (300 MHz, CDCl3): d 8.06 (d, 2H, J = 7.2 Hz), 7.90 (d, 2H,
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Votruba, I.; Dzubák, P.; Hajdúch, M.; Ameral, R.; Birkuš, G.; Wang, T.; Ray, A. S.;