Synthesis and antiviral properties of novel 7-heterocyclic substituted 7-deaza-adenine nucleoside inhibitors of Hepatitis C NS5B polymerase

Article abstract of DOI:10.1016/j.bmc.2012.05.067

Previous investigations in our laboratories resulted in the discovery of a novel series of potent nucleoside inhibitors of Hepatitis C virus (HCV) NS5B polymerase bearing tetracyclic 7-substituted 7-deaza-adenine nucleobases. The planarity of such modified systems was suggested to play a role in the high inhibitory potency observed. This paper describes how we envisaged to maintain the desired planarity of the modified nucleobase by means of an intra-molecular H-bond, engaging a H-bond donor atom on an appropriately substituted 7-heterocyclic residue with the adjacent amino group of the nucleobase. The success of this strategy is reflected by the identification of several novel potent nucleoside inhibitors of HCV NS5B bearing a 7-heterocyclic substituted 7-deaza-adenine nucleobase. Amongst these, the 1,2,4-oxadiazole analog 11 showed high antiviral potency against HCV replication in replicon cells and efficient conversion to the corresponding NTP in vivo, with high and sustained levels of NTP measured in rat liver following intravenous and oral administration.

Full text of DOI:10.1016/j.bmc.2012.05.067

Bioorganic & Medicinal Chemistry 20 (2012) 4801–4811
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral properties of novel 7-heterocyclic substituted
7-deaza-adenine nucleoside inhibitors of Hepatitis C NS5B polymerase
⇑
M. Emilia Di Francesco , Salvatore Avolio, Marco Pompei, Silvia Pesci, Edith Monteagudo, Vincenzo Pucci,
Claudio Giuliano, Fabrizio Fiore, Michael Rowley, Vincenzo Summa
Istituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A., Merck Research Laboratories Rome, Via Pontina Km 30,600, 00040 Pomezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous investigations in our laboratories resulted in the discovery of a novel series of potent nucleoside
inhibitors of Hepatitis C virus (HCV) NS5B polymerase bearing tetracyclic 7-substituted 7-deaza-adenine
nucleobases. The planarity of such modified systems was suggested to play a role in the high inhibitory
potency observed. This paper describes how we envisaged to maintain the desired planarity of the mod-
ified nucleobase by means of an intra-molecular H-bond, engaging a H-bond donor atom on an appropri-
ately substituted 7-heterocyclic residue with the adjacent amino group of the nucleobase. The success of
this strategy is reflected by the identification of several novel potent nucleoside inhibitors of HCV NS5B
bearing a 7-heterocyclic substituted 7-deaza-adenine nucleobase. Amongst these, the 1,2,4-oxadiazole
analog 11 showed high antiviral potency against HCV replication in replicon cells and efficient conversion
to the corresponding NTP in vivo, with high and sustained levels of NTP measured in rat liver following
intravenous and oral administration.
Received 15 February 2012
Revised 15 May 2012
Accepted 29 May 2012
Available online 6 June 2012
Keywords:
HCV polymerase
Nucleoside inhhibitor
Nucleoside triphosphate
20-C-methyl-ribose
7-Deaza-adenine
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
O
N
NH2
N
NH
Hepatitis C virus (HCV) infection is a serious epidemic which af-
fects an estimated 170 million people.1 In the majority of cases, the
infection evolves in cirrhosis and ultimately in hepatocellular car-
cinoma.2 The standard of care (SOC), a combination PEGylated
O
P
OPh
O
O
O
N
O
N
H
R1O
O
O
O
2'
2'
a (PEG-IFNa
) and ribavirin,3 is poorly tolerated, re-
O
interferon-
R2O
R1, R2 =
F
HO
F
quires long treatment periods and has suboptimal efficacy, with
only 50% of genotype 1 infected patients achieving sustained viro-
logic response (SVR).4
mericitabine, RG-7128 (1)
PSI-7977 (2)
To overcome these issues significant efforts have been devoted
to develop direct-acting antiviral (DAA) agents targeting essential
virally-encoded non-structural enzymes (NS). The recent approval
of the two HCV NS3 protease inhibitors telaprevir6 and boceprevir7
in combination with SOC holds great promise for improved efficacy
and reduction of treatment time.5
A further area of intense research is the viral RNA-dependent
RNA polymerase (RdRp) NS5B. HCV is a 9.6 kb positive single
strand RNA virus that replicates mainly in the liver.8 NS5B poly-
merase is essential to the replication process, producing first
strand copy of the RNA genome and secondly catalyzing the syn-
thesis of the positive strand RNA copies of the progeny virus.9 Gi-
ven its pivotal role in the viral life cycle, NS5B has been long
recognized as a key target for antiviral intervention, and numerous
Figure 1. HCV nucleoside inhibitors in clinical trials.
inhibitors of NS5B have progressed to clinical studies, both in the
nucleoside (NI) and non-nucleoside (NNI) classes.10
NIs are at the heart of antiviral treatment for a variety of viral
infections,11 and the success of this class of compounds has pro-
vided a compelling rational for the discovery efforts towards nucle-
oside analogs to treat HCV infection. The significant challenges
associated with the development of a NI for HCV are counterbal-
anced by the advantage offered by these agents.12 In fact NIs target
the highly conserved RdRp active site, being therefore pan-geno-
type inhibitors and offering a higher genetic barrier to resistance
compared with NS3 inhibitors and NS5B non-nucleoside agents, a
key feature for the success of any antiviral therapy.12,13 Several
nucleoside analogs have demonstrated significant efficacy in HCV
infected patients and are currently in late stage clinical trials,
⇑
Corresponding author. Tel.: +1 713 794 5265; fax: +1 713 792 6882.
E-mail address: medifrancesco@mdanderson.org (M.E. Di Francesco).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.067

4802
M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
including mericitabine (RG-7128, Fig. 1)14 and the monophosphate
prodrug PSI-797715 (2, Fig. 1).
starting from 7-deaza-6-chloro-adenine and 30,50-bisbenzoyl-20-C-
methyl-ribose.20 With practical amounts of 6 in hand, the pyrazole
derivatives 7a and 7b were obtained in good yields (55–65%) via
Suzuki coupling with the required boronic acids,22a while Stille
cross coupling was employed to access pyrimidine 7c and oxazole
7d, albeit in more modest yields (11–19%).22b Cross-coupling of
iodide 6 with trimethylsilyl acetylene in Sonogashira conditions
followed by treatment with ammonium hydroxyde in methanol
afforded alkyne 8 in 92%.23 The latter was in turn reacted with
trimethylsilylmethyl azide in the presence of in situ generated
Cu(I) en route to the 1,2,3-triazole analogue 9. Finally, intermediate
6 was reacted with zinc cyanide via a microwave promoted
Pd(0) cross coupling to give the nitrile derivative 10 in 80%. The
latter was converted to the 1,2,4-oxadiazole 11 in a 3-step
sequence involving formation of the corresponding amideoxime,
treatment of the latter with triethylorthoformate and deprotection
of the resulting bis-ethoxy derivative with BBr3. All the above
described 7-heterocyclic analogs were synthesized in an efficient
manner through convergent synthetic routes which completely
avoided the use of protecting group manipulations, despite the
highly functionalized nature of the nucleoside intermediates
To be efficacious as antiviral agents, NIs need to be converted
into the corresponding nucleoside triphosphate (NTP) via a step-
wise process catalyzed by cellular kinases.12 Typically the NTP is
then recognized by the RdRp and incorporated within the growing
RNA chain where it acts as a chain terminator, preventing the addi-
tion of further nucleotides by means of a structural modification
within the ribose unit.16 Nucleoside analogs bearing a methyl
group in the 20-C-position of the ribose have long being recognized
as potent and specific inhibitors of HCV NS5B.17a,b Amongst these,
the 7-deaza-20-C-methyladenosine analogue MK-0608 (3, Fig. 2)18a
showed remarkably high antiviral potency in vitro (inhibition of
RNA replication in a subgenomic replicon assay EC50 = 0.3 lM)
and achieved robust reduction of viral load in HCV infected chim-
panzees (P3 log) with no emergency of resistant viral strain.18b
These findings prompted extensive investigations to identify
novel analogs of MK-060819 and research in our laboratories re-
sulted in the discovery of a novel series of potent 7-deaza-ade-
nine-20-C-methyladenosine analogues bearing
a
tetracyclic
nucleobase (4, Fig. 2),20 wherein the planarity of the modified
nucleobase was suggested to play a key role in achieving high lev-
els of intrinsic potency. This paper describes how we capitalized on
these findings and envisaged an intra-molecular H-bond within a
series of appropriately substituted 7-hetrocyclic-7-deaza-adenine
analogs as an alternative way to achieve a similar planar arrange-
ment (5, Fig. 2). Synthesis and in vitro antiviral properties of
nucleosides belonging to this novel series will be described, as well
as in vivo characterization of the most promising analogs. The high
levels of selectivity and antiviral potency observed in vitro, com-
bined with efficient and sustained conversion to NTP in rat liver,
offer promise for future development of selected analogs within
this series of novel NIs of HCV NS5B.
24
employed.
Guided by the initial biological data on the analogs within this
series, we proceeded to investigate further oxadiazole derivatives
focusing on isomeric analogs of compound 11 (Scheme 2). Acid
hydrolysis of the nitrile derivative 10 followed by acetylation of
the ribose hydroxyl groups gave in 90% yields the carboxylic acid
intermediate 12. The latter was progressed to the 1,3,5-oxadiazole
analog 13 by coupling with methyl amideoxime followed by cycli-
zation and removal of the acetyl group. Notably, the same syn-
thetic approach failed to produce the unsubstituted 1,3,5-
oxadiazole analog. Finally, the 1,3,4-oxadiazole isomer 15 was pre-
pared by coupling of the carboxylic acid intermediate 12 with Boc-
hydrazide, followed by Boc-deprotection and cyclization with tri-
ethylorthoformate.
2. Synthesis
2.2. Synthesis of 20-C-methyl-20-deoxy-20-F-ribose nucleoside
analogs
Investigation of 7-heterocyclic-7-deaza-adenine analogs started
initially in the context of the 20-C-methyl-20-hydroxyribose series,
and was further expanded to another widely represented class of
20-C nucleoside inhibitors, the 20-C-methyl-20-deoxy-20-fluoro ri-
bose analogs.14,15 Every novel nucleoside analog was also con-
verted into the corresponding NTP, according to the efficient
synthesis and purification platform we recently disclosed.20 Se-
lected compounds within the latter series were converted into a
corresponding phosphoramidate monophosphate prodrug.21
Selected 7-heterocyclic substitutions were also investigated in
the context of the 20-C-methyl-20-deoxy-20-fluoro ribose series
(Scheme 3). The required 20-deoxy-20-fluoro 7-iodinated interme-
diate 19 was prepared through a 3-step synthetic sequence start-
ing from the fully protected ribose building block 16.25 Following
selective 1-O-deacetylation with tributyltin methoxide,26 nucleo-
side intermediate 18 was obtained in practical yields as a 1:1 mix-
2.1. Synthesis of 20-C-methyl-ribose nucleoside analogs
ture of
a and b anomers following a glycosidic coupling in
Mitsunobu conditions.27 A slow gradient silica gel chromato-
graphic separation of the two anomers and subsequent removal
of the benzoate protecting groups afforded the required 7-iodo-
20-fluoro building block 19. The latter was in turn converted to
Central to the synthesis of several 7-heterocyclic analogs was
the versatile advanced iodinated intermediate 6 (Scheme 1), avail-
able in multi-gram amounts through a convergent synthetic route
NH2
H
H
N
X
HET
N
7
NH
6
N
7
6
R
7
N
N
N
HO
O
N
N
HO
HO
N
N
O
O
2'
2'
2'
HO
OH
HO
OH
HO
OH
4
5
MK-0608 (3)
NTP IC50 = 0.1 μM
R = Substituted Alkyl,
Aryl, Heteroaryl
Figure 2. 7-Deaza-adenine analogs.

M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
4803
H
N
NH2
H
N
R
R =
N
b
N
a
N
N
(a)
RB(OH)2
or
N
HO
O
N
N
(b) RSnBu3
d
c
HO
7a-d
OH
N
O
NH2
N
I
NH2
N
N
N
N
N
N
HO
N
HO
(c)
N
O
HO
(d)
O
O
NH2
N
N
N
HO
OH
9
8
6
HO
OH
HO
HO
O
N
N
N
NH2
(f)
(e)
N
HO
NH2
O
N
N
HO
N
O
N
N
HO
HO
11
10
HO
OH
Scheme 1. Reaction conditions: (a) Pd(PPh3)4, Na2CO3, H2O/dioxane, 100 °C, 55–65%; (b) PdCl2(PPh3)2, 120 °C, DMF, microwave irradiation, 11–19%; (c) (i) TMS-acetylene,
Pd(PPh3)4, Et3N, CuI, DMF/THF; (ii) NH4OH/MeOH; 92% over 2 steps; (d) (i)TMSCH2N3, CuSO4.5H2O, ascorbate, tBuOH, H2O, RT to 50 °C; (ii) 1 M aq NaOH/MeOH, 50 °C; (e)
Zn(CN)2, Pd(PPh3)4, DMF, 150 °C, microwave irradiation, 80%; (f) (i) NH2OH.HCl, EtOH, 50 °C; (ii) CH(OEt)3, BF3ꢀEt2O, 100 °C; (iii) BBr3, DCM, 0 °C to RT; 25% over 3 steps.
N
O
OH
O
O
NH2
N
N
NH2
N
(c)
N
(d), (e)
HO
AcO
(a), (b)
N
N
O
N
10
H2N
OH
N
Me
HO
OH
AcO
OAc
12
13
H
N
Boc
O
NH2
(f)
(g)
O
N
NH2
N
N
HO
H
(h), (k)
NH2
AcO
O
N
NH2
O
N
N
N
N
N
HO
HO
AcO
OAc
14
15
Scheme 2. Reagents and conditions: (a) 1.3 M aq HCl, 110 °C; (b) AcCl, AcOH; 96% over 2 steps; (c) TBTU, HOBt, iPr2EtN, DMF; (d) DMF, 140 °C; (e) 1 M NaOH, MeOH; 35% over
3 steps; (f) DCI, HOBt, NMM, THF, 0 °C to RT; (g) HCl/dioxane; (h) HC(OEt)3, BF3ꢀEt2O, DMF, 70 °C; (k) 4 M HCl in dioxane, then 6 M aq NH4OH; 13% over 4 steps.
the 1,2,4-oxadiazole 21 and to the oxazole and pyrazole derivatives
22a and 22b with the similar synthetic protocols to those already
described for the corresponding ribose analogs.
representatives of monophosphate prodrugs belonging to the 20-C-
methyl-ribose and to the 20-C-methyl-20-deoxy-20-fluorine
structural classes are reported in Scheme 4a and b, respectively.
Conversion of the pyrazole derivative 7a into the phosphoramidate
prodrug 23 was achieved in a straightforward manner by chemose-
lective O-phosphorylation of the unprotected nucleoside according
to the Uchiyama protocol (Scheme 4a).28 In contrast, synthesis of
the 20-deoxy-20-fluoro analog 28 (Scheme 4b) proved more chal-
lenging. Following extensive optimization, it was found that pro-
tection of the secondary hydroxyl group of 19 was required and
that the tetra-hydropyranyl group (THP) was the optimal choice.
Suzuki coupling with the THP-protected pyrazole boronic acid 25
gave intermediate 26, which was in turn O-phosphorylated with
2.3. Synthesis of 20-C-methyl-ribose and 20-C-methyl-20-deoxy-
20-F-ribose monophosphate produgs
Selected nucleoside analogs which combined very high levels of
intrinsic potency against HCV NS5B with modest level of cellular
antiviral potency, were converted into the corresponding mono-
phosphate prodrugs.21 The aim of this investigation was to assess
if a by-pass of the often rate-determining first phosphorylation
step could improve the efficiency of conversion to the correspond-
ing NTP and hence the cellular antiviral potency. Synthesis of two
L-alanine-N-chlorophenoxyphosphinyl-ethyl ester and progressed

4804
M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
Cl
I
Cl
N
I
N
N
BzO
(b)
BzO
N
N
O
F
(a)
N
O
BzO
H
Anomer separation
OAc
O
F
OH
α:β = 1:1 (SiO2 gel chromatography)
BzO
BzO
F
BzO
18
16
17
O
N
NH2
N
N
NC
NH2
N
N
N
N
HO
N
(e)
(d)
HO
O
NH2
N
O
I
20
21
HO
HO
F
F
(c)
N
N
HO
O
NH2
N
R
β
B(OH)
2
(f)
N
N
HO
H
F
N
HN
N
O
N
HO
O
or
N
N
19
SnBu3
(g)
O
HO
b
a
22a-b
F
Scheme 3. Reagents and conditions: (a) Sn(nBu)3OMe, 70%; (b) DIAD, Ph3P, 45%; (c) NH3/MeOH, 40%; (d) Zn(CN)2, Pd(PPh3)4, DMF, 150 °C, microwave irradiation, 30%; (e) (i)
NH2OH.HCl, EtOH, 50 °C; (ii) CH(OEt)3, BF3ꢀEt2O, 100 °C; (iii) BBr3, DCM, 0 °C to RT; 27% over 3 steps; (f) Pd(PPh3)4, Na2CO3, H2O/dioxane, 100 °C, 68%; (g) PdCl2(PPh3)2, 120 °C,
DMF, microwave irradiation, 20%.
N
NH
O
P
O
H
N
EtO2C
Cl
P
HN
O
NH2
N
O
EtO2C
OPh
OPh
(a)
7a
19
N
N
N
(a)
HO
HO
23
N
N
I
N
THP
THP
NH2
HO
HO
(e)
NH2
N
N
O
O
(b), (c), (d)
B(OH)
2
25
(b)
N
N
N
24
N
THPO
THPO
F
F
26
N
NH
O
O
P
N
N
H
PhO
O
O
P
EtO2C
N
Cl
HN
O
THP
P
(g)
NH2
N
(f)
O
EtO2C
HN
EtO2C
OPh
OPh
NH2
N
O
N
N
N
N
HO
F
THPO
F
27
28
t
Scheme 4. Reaction conditions: Reagents and conditions: (a) BuMgCl, THF; (b) TBDMS-Cl, ImH; (c) 3,4-dihydro-2H-pyran, PTSA; (d) TBAF; 50% over 3 steps; (e) Pd(PPh3)4,
Na2CO3, H2O/dioxane, 100 °C, 70%; (f) tBuMgCl, THF; (g) HCO2H, 0 °C; 18% over 2 steps.
to prodrug 28 in practical yields after removal of the THP protect-
ing groups.
the two inhibitor classes were profiled in rodent species to assess
NTP formation in vivo.
3.1. Nucleoside analogs: in vitro and in vivo studies
3. Results and discussion
Following our previous findings within the tetracyclic series (4,
Fig. 2), which suggested that the overall planarity of the modified
nucleobases could be instrumental to ensure high level of intrinsic
potency,20 we set out to investigate whether alternative modifica-
tions introduced in the 7-position of the 7-deaza-adenine base
could maintain the desired planarity as well as the high intrinsic
All the 7-heterocyclic nucleoside and nucleoside monophos-
phate prodrug analogs described above were evaluated for their
antiviral potency against the replication of HCV RNA in HBI10A
hepatoma cell lines containing a subgenomic HCV replicon.29 The
corresponding NTPs were tested for their inhibitory potency
against NS5B polymerase.30 The most promising candidates from

M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
4805
potency, and potentially offer advantages in terms of improved
antiviral cellular potency. Towards this end, we envisaged that
with a series of appropriately substituted 7-hetrocyclic-7-deaza-
adenine analogs we could engage the 6-amino group into a 7-
membered ring intra-molecular H-bond with the 7-heterocyclic
substituent, thus achieving the required system planarity (5,
Fig. 2).
Indeed a head to head comparison between the phenyl analog
2920 and the pyrimidine 7c (Table 1, entries 12 and 3, respectively)
suggested that the introduction of a H-bond acceptor atom adja-
cent to the carbon linked to the 7-position to the 7-deaza-adenine
base could prove beneficial to the inhibitory potency, with NTP 32
viral activity in the replicon assay, with EC50 = 7.8
zole 7d and a remarkable boost in cellular potency for oxadiazole
l
M for the oxa-
11, with EC50 = 1.9 lM. This value is comparable to that of past
and current HCV NIs in the clinic,14,15 and suggests an efficient con-
version of 11 to the corresponding NTP species in the cellular
media.
Encouraged by these results, we focused our efforts on further
exploring 5-membered ring heterocyclic substituents, and ex-
panded our investigations to the isomeric pyrazole analogs 7a
and 7b (Table 1, entries 1 and 2). Analog 7a, bearing a nitrogen atom
adjacent to the carbon linked to the 7-position of the nucleobase,
showed excellent levels of potency against HCV NS5B, with
showing an IC50 = 12
lM against NS5B while its phenyl analog 41
IC50 = 0.1 lM for its corresponding NTP 30. In contrast, the isomeric
was devoid of activity (NTP IC50 >100
l
M).
pyrazole 7b, bearing the nitrogen atoms two positions away from
the carbon linked to the 7-position and thus unable to engage the
6-amino group in the 7-membered ring intra-molecular H-bond,
A much more significant effect was observed when we investi-
gated the 7-substitution with 5-membered heterocycles bearing a
H-bond acceptor adjacent to carbon linked to the deaza-adenine
base. Indeed, both the oxazole analog 7d and the 1,2,4-oxadiazole
11 showed significantly improved intrinsic potency, both inhibit-
showed a dramatic loss in potency with IC50 = 8.4 lM for NTP 31.
These findings corroborated our initial hypothesis, and
prompted us to find further evidence to support it. Towards this
end, we turned to 1H NMR analysis to gain insight on the possible
involvement of the protons of the 6-amino group in an
ing NS5B polymerase with NTP IC50 = 0.5 lM (Table 1, entries 4
and 6). Both these analogs also displayed interesting levels of anti-
Table 1
H
H
N
HET X
N
N
HO
N
O
HO
R1
Entry
HET
R1
Compound nucleoside/NTP
HCV NSSB polymeraseb IC50
0.1
(
lM)
HCV replicona EC50
>100
(lM)
H
N
N
1
OH
7a/30
N
2
3
4
OH
OH
OH
7b/31
7c/32
7d/33
8.4
12
30
NH
N
N
>100
7.8
N
0.5
O
N
N
N
N
Me
Me
5
OH
9/34
8.4
42
O
6
7
OH
OH
11/35
13/36
0.5
8.4
1.9
42
N
N
O
N
O
N
8
9
OH
F
15/37
21/38
>95
0.4
46
41
N
O
N
N
N
10
11
12
F
22a/39
22b/40
29/41
0.3
37
35
56
O
H
N
N
F
0.07
>100
OH
a
Cellular activity determined in the HCV bicistronic replicon assay, using HBI10A cells stably transfected with genotype 1b HCV replicon RNA, for details see Ref. 30; EC50
are measured in the presence of 10% fetal bovine serum (FBS); EC50 values are the mean of P3 experiments, standard deviation was within 20% of the reported value.
b
Enzyme potency measured in a NS5B polymerization assay, using an heteromeric RNA template, for details see Ref. 31; IC50 values are the mean of P3 experiments,
standard deviation was within 20% of the reported value.

4806
M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
intra-molecular H-bond with an acceptor atom on the 7-substitu-
ent (Fig. 3). In fact, in solvents that do not form strong hydrogen
bonds, significantly de-shielded chemical shift for NH-protons of-
ten indicate their participation in hydrogen bonding.31a,b We
started our investigations from pyrazole 7b, unable to engage in
the intra-molecular H-bond, and set the chemical shift for its two
isochronous NH2 protons at 6.74 ppm in CD3CN as a reference value
(Fig. 3a). When the isomeric pyrazole 7a, bearing a hydrogen bond
acceptor in the position adjacent to the carbon linked to the nucle-
obase, was analyzed in the same NMR conditions a dramatic down-
field shift was observed for the chemical shift of the two
NH2-protons, found at 11.20 and 10.90 ppm, respectively (Fig. 3b).
Similarly to 7a, a significant downfield chemical shift was also
observed for the 1,2,4-oxadiazole 11 (10.12 and 9.53 ppm, Fig. 3c),
suggesting the involvement of 6-NH2 protons in intra-molecular
hydrogen-bond with acceptor atoms on the C-7 substituent.
supported the presence of an intra-molecular hydrogen bond also
for analogs 9, 13 and 15 (data not shown), suggesting that the ob-
served suboptimal potency is unlikely to be derived from a lack of
planarity of the heterocycle-substituted nucleobase.
Our investigations within the series were next extended to
structural variation within the ribose unit. The replacement of
the 20-hydroxyl group with a fluorine atom has been amongst
the most successful ribose modifications reported in the nucleo-
side field, as exemplified by the two furthest advanced nucleoside
clinical candidates in the HCV field (1 and 2, Fig. 1).14,15 We were
pleased to observe that by combining the 20-C-methyl-20-deoxy-
20-fluoro-ribose unit with the most promising 5-membered hetero-
cyclic substituents identified from our previous efforts, we could
maintain and even further improve the inhibitory potency against
NS5B, as exemplified by oxadiazole 21, oxazole 22a and pyrazole
22b, with NTP IC50 = 0.4, 0.3 and, remarkably, 0.07 lM, respectively
The above findings, combined with the compelling in vitro po-
tency observed for compounds 7d, 11 and 7a, prompted us to fur-
ther investigate analogs within the series. Unexpectedly, both the
1,2,3-triazole derivative 9 and the isomeric oxadiazoles 13 and
15 showed a significant loss in inhibitory potency against NS5B
(Table 1, entries 9–11). Despite the excellent levels of enzymatic
potency observed, all these 20-deoxy-20-fluorinated analogs
showed only marginal levels of antiviral potency in the cellular as-
say (EC50 = 35–41 l
M). Noticeably, the 20-fluoro oxadiazole analog
21 had a significantly reduced replicon potency compared to oxa-
(Table 1, entries 5, 7 and 8, NTP IC50 = 8.4, 8.4 and >95
lM, respec-
diazole 11, its direct analog in the 20-C-methyl-ribose series, which
tively). Interestingly, similar NMR studies to those described above
showed high levels of antiviral potency with EC50 = 1.9 lM.
Figure 3. 1H NMR experiments to investigate the presence of an intra-molecular H-bond with compounds 7a, 7b and 11. For all the compounds: NMR experiments showed
the NH protons chemical shift to be independent of concentration in the range 0.5–5 mM. The reported data were acquired at 1 mM concentration, T = 283 K and acquired in
CD3CN (chemical shift are reported in ppm using the residual proton signal of CD3CN at 1.94 ppm as internal reference).

M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
4807
Table 2
Having identified novel and potent nucleoside inhibitors of
NS5B, some of which showing also compelling antiviral cellular po-
tency, we proceeded to further evaluate the most promising ana-
logs, such as oxadiazole 11 and the two pyrazole analogs 7a and
22b, in the 20-hydroxy and 20-deoxy-20-florine series, respectively.
Their in vitro profile was further investigated by assessing the
N
NH
O
P
HN
O
NH2
N
O
EtO2C
OPh
selectivity against the human DNA polymerases
a
, b,
c. Pleasingly,
N
N
in all cases, the corresponding NTPs had IC50 >10
lM.
HO
R1
We next pursued the initial evaluation of the pharmacokinetic
properties of the above key compounds, with particular focus on
the efficiency of their conversion to NTP in vivo, either as nucleo-
side analogs or following their derivatization into the correspond-
ing monophosphate prodrugs as appropriate.
Within the selected nucleoside analogs, oxadiazole 11 appeared
as the candidate of choice for further in vivo evaluation due to the
high potency of the corresponding NTP combined with the remark-
able antiviral activity observed in the cellular assay, suggestive of
efficient intracellular conversion to the corresponding NTP
Entry
R1
Compound
HCV replicona EC50
(lM)
1
2
F
OH
28
23
>50
1.8
a
See note (a) in Table 1.
(IC50 = 0.5 lM, EC50 = 1.9 lM, respectively). Initial evaluation of
Remarkably, when the same approach was applied to the 20-hy-
droxy analog 7a (NTP IC50 = 0.1 M, replicon EC50 = >100 M) the
corresponding NMP prodrug 23 offered a significant boost in cellu-
lar potency, with EC50 = 1.8 M (Table 2, entry 2). This finding is in
line with the remarkable difference in replicon activity observed
for nucleoside analogs belonging to the two series, as exemplified
the pharmacokinetic profile of 11 was obtained following adminis-
tration to Wistar Han (WH) Rats (1 mg/Kg intravenous and orally).
Compound 11 showed high plasmatic clearance of the parent
nucleoside (>hepatic flow), a volume of distribution of 7.0 L and
a moderate bioavailability (F = 14.7%). In a second experiment, 11
was dosed to WH rats intravenously at 20 mg/Kg, and levels of
NTP formed in rat liver where measured up to 12 h from dosing.32
Pleasingly, while the concentration of the parent nucleoside 11 in
plasma was decreasing rapidly, in rat liver the corresponding
NTP was formed efficiently, with NTP levels of 11.9, 3.9 and
0.5 nmol/g at 3, 6 and 12 h from dosing, respectively. The ratio be-
tween the concentration of the NTP in liver and of 11 in plasma
was found in the range of 20–25 fold in favor of liver NTP at all
timepoints, suggesting both a high hepatic extraction of the nucle-
oside 11 into the liver, target organ of HCV replication, and an effi-
cient conversion to the NTP species, required for inhibition of NS5B
polymerase. Following dosing of 11 at 20 mg/Kg orally, NTP liver
levels at 12 h were found to be comparable to those of the intrave-
nous administration (0.4 nmol/g) suggesting that significant levels
of NTP in liver could be achieved also following oral administration
of 11. Further characterization of oxadiazole 11 in preclinical spe-
cies is ongoing and results will be reported in due time.
l
l
l
by the oxadiazole analogs 11 (EC50 = 1.9
lM) and 21
(EC50 = 41
l
M) belonging to the 20-C-methyl-ribose and to the 20-
deoxy-20-fluorine series, respectively. These data suggest that fur-
ther efforts to identify nucleoside or monophosphate prodrug with
satisfactoty levels of antiviral potency within the 7-heterocyclic-
substituted 7-deaza-adenine series should focus primarily on 20-
C-methyl-ribose analogs. Moreover, the high cellular potency ob-
served for the NMP prodrug 23 warrants further investigations,
including in vivo experiments to assess if efficient conversion to
NTP is observed in hamster liver and the full evaluation of other
NMP prodrugs of the most potent 20-C-methyl-ribose analogs iden-
tified within this series.
4. Conclusions
On the basis of previous findings in a related series, we investi-
gated novel 7-heteroaromatic substituted 7-deaza-nucloside ana-
logs bearing a 20-C-methyl substitution within the ribose ring.
We envisaged that the presence of an appropriately situated H-
bond acceptor within the heteroaromatic substituent could engage
the adjacent amino group in an intra-molecular H-bond, thus
imparting to the modified nucleobase a planar arrangement and
maintaining high levels of intrinsic potency. The proposed hypoth-
esis was supported by SAR studies and 1H NMR evidences. We
identified several potent novel and potent nucleoside inhibitors
of HCV NS5B polymerase, such as oxadiazole 11 and pyrazoles 7a
3.2. Nucleoside monophosphate prodrug analogs: in vitro and
in vivo studies
The other two selected analogs, pyrazole 22b (20-deoxy-20-fluo-
rine series) and oxazole 7a (20-C-methyl-ribose series) combining
excellent potency against NS5B (NTP IC50 = 0.07 and 0.1
tively) and modest level of cellular antiviral potency (EC50 = 35 and
>100 M, respectively) were singled out to be evaluated as the cor-
lM, respec-
l
responding monophosphate prodrugs (NMP). It is known that for
many nucleosides the first phosphorylation is the rate limiting step,
and, in such cases, a kinase bypass with a NMP prodrug might restore
the efficient conversion to NTP and therefore the desired cellular
antiviral potency.21 In order to explore this approach, 22b was con-
verted into the corresponding phosphramidate prodrug 28. No
improvement in the antiviral potency was observed in the replicon
and 22b (NTP IC50 = 0.5, 0.1 and 0.07
lar oxadiazole 11 was found to be a potent inhibitor of HCV repli-
cation in vitro (EC50 = 1.9 M) and was efficiently phosphorylated
lM, respectively). In particu-
l
in vivo, resulting in high and substained levels of NTP levels in
rat liver following intravenous and oral administration. These
properties, combined the high levels of antiviral potency of 11,
comparable or better to that of other HCV nucleoside inhibitors
that have been or currently are in clinical studies, offer great prom-
ise for its further development. Moreover, successful application of
the kinase by-pass approach resulted in restored cellular potency
for analog 23, the monophosphate prodrug of nucleoside 7a
assay (Table 2, entry 1, 28 EC50 >50 lM), suggesting that the first
intracellular phosphorylation might not be the only rate determin-
ing step towards the formation of the required NTP. Indeed, when
prodrug 28 was dosed at 10 mg/Kg in hamster, no NTP formation
was detected in liver, with the only species detected being the
NMP.
(EC50 = 1.8 lM). Future work will focus on further evaluation of
the novel potent inhibitors identified, as well as on continued
investigation of further 7-heterocyclic-7-deaza-adenine analogs
within the 20-C-methyl ribose series.
Hamster was chosen instead of rat in view of the significantly higher plasma
stability of prodrug 28 compared to rat.

4808
M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
iodide 19 (vide infra) in DMF, and the resulting mixture was heated
5. Experimental section
at 120 °C for 3 h under microwave irradiation. The reaction mix-
ture was cooled to RT, partitioned between water/hexanes and
the water phase was purified by preparative RP-HPLC eluting with
MeCN/water containing 0.1% TFA to give the title compound as a
solid.
1H NMR spectra were obtained on a Bruker 400 MHz spectrom-
eter. The purity of the nucleoside analogs was determined by ana-
lytical RP-HPLC. Data were obtained by two methods: method (1)
on an Acquity Waters UPLC system equipped with a ZQ micromass
spectrometer, using a flow rate of 0.5 mL/min, a HSS T3, C18,
5.2.1. Oxazole 7d
1.8
phase comprising MeCN +0.1% HCO2H (solvent A) and H2O +0.1%
HCO2H (solvent B); method parameters were 0% solvent
(0.0 min) to 100% solvent A (2.7 min) and then 100% solvent A
(0.2 min); method (2) on a Waters Alliance 2795 HPLC–MS system
equipped with a diode array and a ZQ mass spectrometer, using a
l
m, 2.1 ꢁ 50 mm column as the stationary phase, and a mobile
Solid (11%). 1H NMR (300 MHz, DMSO-d6) d 9.88 (br s, 1H), 8.60
(s, 1H), 8.36 (s, 1H), 8.33 (br s, 1H), 8.23 (s, 1H), 7.45 (s, 1H), 6.21 (s,
1H), 4.06 (d, J = 9.6 Hz, 1H), 3.99–3.87 (m, 2H), 3.73 (m, 1H), 0.77 (s,
3H); MS (ES+) C15H17N5O5 requires: 347. Found: 348 [M+H]+. RP-
HPLC method 1, tR = 1.65 min; purity 99.6%; method 2,
tR = 5.55 min; purity 98.13%.
A
flow rate of 1.0 mL/min, an Atlantis T3, C18, 5
lm, 4.6 ꢁ 50 mm
column as the stationary phase; method parameters were 0% sol-
vent A (0.0 min) to 20% (5.2 min), then 95% (7.0 min) and 100%
(8.5 min). All the nucleoside analogs showed purities higher than
95%. High resolving power accurate mass measurement electro-
spray (ES) mass spectral data were acquired by use of a Bruker Dal-
tonics apex-Qe Fourier transform ion cyclotron resonance mass
spetrometer (FT-ICR MS). External calibration was accomplished
with oligomers of polypropylene.
5.2.2. Pyrimidine 7c
Solid 19%; 1H NMR (300 MHz, DMSO-d6, 300 K) d 10.79 (br s,
1H), 8.89 (d, J = 5.0 Hz, 2H), 8.82 (s, 1H), 8.48 (br s, 1H), 8.40 (s,
1H), 7.43 (t, J = 5.0 Hz, 1H), 6.26 (s, 1H), 4.04 (d, J = 9.1 Hz, 1H),
3.99–3.86 (m, 2H), 3.71 (m, 1H), 0.78 (s, 3H); MS (ES+)
C
16H18N6O4 requires: 358. Found: 359 [M+H]+.
5.2.3. Oxazole 22a
5.1. General procedure for the Suzuki Coupling and selected
examples
20% isolated yield; 1H NMR (300 MHz, CD3CN/D2O) d 8.43 (s,
1H), 8.26 (s, 1H), 7.78 (s, 1H), 7.27 (s, 1H), 6.45 (d, J = 17.1 Hz,
1H), 4.23 (dd, J = 24.0, 9.5 Hz, 1H), 4.06–3.99 (m, 2H), 3.83 (dd,
J = 12.0, 2.4 Hz, 1H), 1.06 (d, J = 22.8 Hz, 3H); 19F NMR (300 MHz,
CD3CN/D2O) d ꢂ163.14; MS (ES+) C15H16FN5O4 requires: 349.
Found: 350 (M+H+). HRMS (ESI) m/z Calcd for C15H17FN5O4
350.1266, measured 350.1261. RP-HPLC method 1, tR = 1.61 min;
purity 95.3%.
Pd(PPh3)4 (0.1 equiv) was added to a 0.1 M solution in dioxane
of hetroaryl-boronic acid (1.5 equiv), Na2CO3 (2 M aq solution,
15 equiv) and iodide 620 or iodide 19 (vide infra). The reaction mix-
ture was heated at 120 °C for 500 s under microwave irradiation
and then filtered through a pad of celite. The filtrate was concen-
trated under reduced pressure and the residue was purified by pre-
parative RP-HPLC eluting with MeCN/water containing 0.1% TFA to
give the title compound as a solid.
5.3. Synthesis of triazole 9
Trimethylsilylmethyl azide (3.0 equiv) was added to a 0.25 M
5.1.1. Pyrazole 7a
solution of 5-ethynyl-7-(20-C-methyl-b-
D-ribofuranosyl)-7H-pyr-
Solid (65%). 1H NMR (300 MHz, DMSO-d6) d 13.14 (s, 1H), 10.65
(br s, 1H), 8.60 (br s, 1H), 8.40 (s, 1H), 8.38 (s, 1H), 7.93 (br s, 1H),
6.63 (br s, 1H), 6.20 (s, 1H), 4.04 (d, J = 9.1 Hz, 1H), 3.99–3.87 (m,
2H), 3.75 (m, 1H), 0.78 (s, 3H); MS (ES+) C15H18N6O4 requires:
346. Found: 347 [M+H]+. HRMS (ESI) m/z Calcd for C15H19N6O4
347.1462, measured 347.1457. RP-HPLC method 1, tR = 1.41 min;
purity 99.3%; method 2, tR = 5.25 min; purity 98.2%.
rolo[2,3-d]pyrimidin-4-amine 824 in a 1:1 v:v mixture of water
t
and BuOH containing
L-ascorbic acid sodium salt (0.5 equiv) and
copper(II) sulfate pentahydrate (0.05 equiv). The heterogeneous
mixture was stirred at 50 °C overnight, cooled to RT and concen-
trated under reduced pressure. The residue was treated with 1 M
aq solution of NaOH (5.0 equiv) in a 1:1 v:v mixture of MeOH
and H2O. The resulting mixture was stirred at 50 °C for 2 h and
then concentrated under reduced pressure. Purification by pre-
parative RP-HPLC eluting with MeCN/water containing 0.1% TFA
gave the title compound as a solid (21%). 1H NMR (300 MHz,
DMSO-d6) d 8.48 (s, 1H), 8.40 (s, 1H), 8.20 (s, 1H), 6.23 (s, 1H),
4.17 (s, 3H), 3.98–3.85 (m, 3H), 3.75 (m, 1H), 0.79 (s, 3H); 19F
NMR (300 MHz, DMSO-d6) d ꢂ73.90; MS (ES+) C15H19N7O4 re-
quires: 361. Found: 362 [M+H]+. HRMS (ESI) m/z Calcd for
5.1.2. Pyrazole 7b
Solid, 55%; 1H NMR (300 MHz, DMSO-d6) d 8.44 (s, 1H), 7.91, (s,
1H), 7.73 (m, 1H), 7.80 (s, 1H), 6.22 (s, 1H), 4.02 (d, J = 9.3 Hz, 1H),
3.96–3.81 (m, 2H), 3.69 (m, 1H), 0.78 (s, 3H); MS (ES+) C15H18N6O4
requires: 346. Found: 347 [M+H]+. HRMS (ESI) m/z Calcd for
C15H19N6O4 347.1462, measured 347.1455. RP-HPLC method 1,
tR = 1.26 min; purity 99.0%; method 2, tR = 4.27 min; purity 99.2%.
C15H20N7O4 362.1571, measured 362.1563. RP-HPLC method 1,
tR = 1.44 min; purity 97.6%; method 2, tR = 5.33 min; purity 96.3%.
5.1.3. Pyrazole 22b
(68%) 1H NMR (400 MHz, CD3CN/D2O) d 8.19 (bd, J = 17.1 Hz,
2H), 7.72 (br s, 1H), 6.63 (br s, 1H), 6.47 (d, J = 17.1 Hz, 1H), 4.26
(bd, J = 25.1 Hz, 1H), 4.05 (br s, 2H), 3.87 (m, 1H), 1.08 (d,
J = 22.4 Hz, 3H); 19F NMR (400 MHz, CD3CN/D2O) d ꢂ162.64; MS
(ES+) C15H17FN6O3 requires: 348. Found: 349 (M+H+). HRMS (ESI)
m/z Calcd for C15H18FN6O3 349.1426, measured 349.1419. RP-HPLC
method 1, tR = 1.52 min; purity 99.1%.
5.4. Synthesis of carbonitrile intermediate 10
To a solution of iodide 620(50 mg, 0.123 mmol) in DMF (0.8 mL)
were added zinc cyanide (22 mg, 0.18 mmol) and Pd(PPh3)4
(14 mg, 0.012 mmol) and the resulting solution was heated for
15 min at 150 °C under microwave irradiation. The reaction mix-
ture was then allowed to cool to RT, filtered through a pad of Celite
and concentrated to dryness. The residue was purified by Silica gel
chromatography eluting with 3–10% MeOH in DCM to afford the ti-
tle compound (90%) as a solid; 1H NMR (400 MHz, D2O) d 8.45 (s,
1H), 8.43 (s, 1H), 6.40 (s, 1H), 4.22–4.16 (m, 1H), 4.15–4.08 (m,
2H), 3.99–3.92 (m, 1H), 0.94 (s, 3H); MS (ES+) C13H15N5O4 requires
305.1. Found: 306 [M+H]+.
5.2. General procedure for the Stille coupling and selected
examples
PdCl2(PPh3)2 (0.1 equiv) was added to a 0.1 M solution of tri-n-
butylstannyl)-heterocycle (3.0 equiv) and either iodide 620 or

M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
4809
5.5. Synthesis of oxadiazole 11
(300 MHz, DMSO-d6) d 11.00–10.63 (br s, 1H), 9.06–8.49 (br s,
1H), 8.38 (s, 1H), 8.22 (s, 1H), 6.24 (s, 1H), 4.01–3.81 (m, 3H),
3.74 (dd, J = 5.1, 12.6 Hz, 1H), 0.79 (s, 3H); 1H NMR (300 MHz,
DMSO-d6) d 9.02 (br s, 1H), 8.91 (s, 1H), 8.35 (s, 1H), 8.15 (br s,
1H), 6.23 (s, 1H), 4.12–3.85 (m, 3H), 3.70 (m, 1H), 2.45 (s, 3H),
0.78 (s, 3H); MS (ES+) C15H18N6O5 requires 362. Found: 363
[M+H]+. HRMS (ESI) m/z Calcd for C15H19N6O5 363.1411, measured
363.1405. RP-HPLC method 1, tR = 1.57 min; purity 98.2%.; method
2, tR = 6.15 min; purity 98.8%.
Hydroxylamine hydrochloride (1.5 equiv) and triethylamine
(2.0 equiv) were added to a 0.2 M solution of carbonitrile 10 in
ethanol. The resulting mixture was heated at 50 °C for 6 h, cooled
to RT and concentrated under reduced pressure to yield 4-amino-
0
N -hydroxy-7-(2-C-methyl-b-
D-ribofuranosyl)-7H-pyrrolo[2,3-d]
pyrimidine-5-carboximidamide as a solid [MS (ES+) C13H18N6O5 re-
quires: 338.1. Found: 339 [M+H]+]. The solid residue was sus-
pended in triethyl orthoformate (3.0 equiv), treated with BF3ꢀEt2O
(0.3 equiv) and heated at 100 °C for 15 min. After cooling to RT,
the mixture was diluted with water and concentrated under re-
duced pressure. The residue was diluted with DCM (0.1 M), cooled
to 0 °C and treated with a 1 M solution of BBr3 in DCM (6.0 equiv).
After stirring for 3 h at RT the reaction mixture was diluted at 0 °C
with MeOH, treated with a 2 M solution of ammonia in MeOH and
concentrated under reduced pressure. The residue was purified by
preparative RP-HPLC eluting with MeCN/water containing 0.1%
TFA to give the title compound as a solid (25%). 1H NMR
(300 MHz, DMSO-d6) d 9.78 (s, 1H), 8.76 (s, 1H), 8.62–8.04 (m,
2H), 8.37 (s, 1H), 6.23 (s, 1H), 4.04 (d, J = 9.2 Hz, 1H), 3.99–3.85
(m, 2H), 3.72 (m, 1H), 0.77 (s, 3H); MS (ES+) C14H16N6O5 requires:
348. Found: 349 [M+H]+. HRMS (ESI) m/z Calcd for C14H17N6O5
349.1255, measured 349.1259. RP-HPLC method 1, tR = 1.40 min;
purity 96.6%; method 2, tR = 5.17 min; purity 95.8%.
5.8. Synthesis of oxadiazole 15
Steps 1 and 2: 4-Methylmorpholine (1.0 equiv) was added to a
cooled (0 °C) 0.2 M solution of carboxylic acid 12 in THF containing
tert-butyl hydrazinecarboxylate (1.0 equiv) and 1H-benzotriazol-
1-ol hydrate (2.0 equiv). After 5 min stirring at 0 °C, N,N0-dicyclo-
hexylcarbodiimide (1.0 equiv) was added. The reaction mixture
was stirred for 1 h at 0 °C and for further 12 h at RT. The mixture
was then cooled to 0 °C and filtered through a short pad of Celite.
The filtrate was diluted with DCM, washed with s.s. NaHCO3, brine
and dried over Na2SO4. The residue was evaporated to dryness un-
der reduced pressure, dissolved in 4 M HCl in dioxane (25 equiv)
and stirred at RT for 6 h. The reaction mixture was evaporated un-
der reduced pressure and partitioned between water/Et2O. The
aqueous layer was brough to pH 7 by addition of s.s. NaHCO3, ex-
tracted with Et2O and concentrated under reduced pressure. The
residue was purified by RP-HPLC (Atlantis T3, 19 ꢁ 150 mm,
5.6. Synthesis of carboxylic acid internediate 12
5 lm) eluting with MeCN/water containing 0.1% TFA to give carbo-
Carbonitrile 10 (140 mg, 0.46 mmol) was suspended in 3 M aq
HCl (1.5 mL) and heated at reflux for a total of 18 h. The reaction
mixture was concentrated under reduced pressure and the residue
was co-evaporated several times with Et2O to yield the corre-
sponding unprotected carboxylic acid as a yellow solid. The latter
hydrazide 14 as a white solid (67%); 1H NMR (300 MHz, DMSO-d6)
d 11.00–10.63 (br s, 1H), 9.06–8.49 (br s, 1H), 8.38 (s, 1H), 8.22 (s,
1H), 6.24 (s, 1H), 4.01–3.81 (m, 3H), 3.74 (dd, J = 5.1, 12.6 Hz, 1H),
0.79 (s, 3H); MS (ES+) C13H18N6O5 requires 338.32. Found: 339
[M+H]+.
was then dissolved in acetic acid (925
l
l) and treated with acetyl
Step 3: BF3ꢀEt2O (0.2 equiv) was added dropwise to a 0.5 M
chloride (197 l, 2.78 mmol), and the resulting reaction mixture
l
solution of 4-amino-7-(2-C-methyl-b-D-ribofuranosyl)-7H-pyrrol-
was stirred at RT for 18 h. The mixture was concentrated under re-
duced pressure and the residue was partitioned between DCM and
H2O. The pH of the aqueous layer was adjusted to neutral by care-
ful addition of s.s. NaHCO3 and extracted with DCM. The organic
layer was washed with brine, dried over Na2SO4 and concentrated
under reduced pressure to give the title compound (201 mg,
0.446 mmol, 96% over two steps) as a yellow solid; 1H NMR
(300 MHz, DMSO-d6) d 8.19 (s, 1H), 8.00 (s, 1H), 6.61 (s, 1H), 5.53
(d, J = 6.4 Hz, 1H), 4.48–4.32 (m, 3H), 2.16–2.06 (m, 9H), 1.33 (s,
3H); MS (ES+) C19H22N4O9 requires: 450. Found: 451 [M+H]+.
o[2,3-d]pyrimidine-5-carbohydrazide (1.0 equiv) in DMF contain-
ing triethyl orthoformate (2.0 equiv). The reaction mixture was
heated at 70 °C for 180 min, cooled to RT and diluted with water.
The mixture was then treated with aqueous 1 M HCl stirring for
20 min at RT (pH ꢃ2) and then treated with aq 6 M NH4OH and
stirred for further 30 min at RT (pH ꢃ8). The reaction mixture
was evaporated under reduced pressure and purified by prepara-
tive RP-HPLC eluting with MeCN/water containing 0.1% TFA to give
the title compound 15 (25%) as a white fluffy solid. 1H NMR
(300 MHz, DMSO-d6) d 9.34 (s, 1H), 8.66 (s, 1H), 8.31 (s, 1H), 6.22
(s, 1H), 4.17–3.82 (m, 3H), 3.78–3.67 (m, 1H), 0.78 (s, 3H); (MS
(ES+) C14H16N6O5 requires 348. Found: 349 [M+H]+.
5.7. Synthesis of oxadiazole 13
To a mixture of carboxylic acid 12 (200 mg, 0.44 mmol), HOBT
5.9. Synthesis of 20-deoxy-20-fuoro-7-iodo intermediate 19
(1.36 mg, 8.88 lmol), DIPEA (388 ll, 2.220 mmol) and acetamide
oxime (36.2 mg, 0.488 mmol) in DMF (4.5 mL) was added TBTU
(150 mg, 0.466 mmol) and the resulting reaction mixture was stir-
red for 14 h at RT. The mixture was then quenched with water and
extracted with DCM. The collected organic layers were washed
with brine, dried over Na2SO4 and concentrated under reduced
pressure. The solid residue was dissolved in DMF (5.0 mL) and
heated at 140 °C for 3 h. The reaction mixture was then cooled to
RT, diluted with DCM and washed with brine. The organic layers
were dried (Na2SO4) and concentrated under reduced pressure.
The brown oil residue was dissolved in MeOH (10 mL) and treated
with 1 M aq NaOH (1.33 mL, 1.33 mmol). After 2 h, the reaction
mix was concentrated under reduced pressure, the pH was ad-
justed to neutrality by addition of 1 M aq HCl and purified by prep
HPLC (Atlantis T3, 19 ꢁ 150 mm; eluent from 5% to 80% then to
95% CH3CN in water + 0.1% TFA) to afford the title compound as
an off-white solid (74.7 mg, 35% over three steps); 1H NMR
DIAD (2.8 equiv) was added to a 0.05 M solution of Ph3P
(3.0 equiv) in acetonitrile and the resulting mixture was stirred
for 30 min at 0 °C. The solution was then added via cannula into
a 0.1 M solution of 20-deoxy-20-fluoro-2-methyl-
D-ribofuranose
intermediate 1725 (1.0 equiv) in acetonitrile at ꢂ40 °C. The result-
ing pale brown suspension was stirred overnight at RT. The reac-
tion was quenched with AcOEt and the organic phase was
washed with water, brine and dried over Na2SO4. The volatiles
were removed under reduced pressure and the residue was puri-
fied by flash chromatography (gradient elution from 0% to
5% AcOEt/Hexane then isocratic elution 5% AcOEt/Hexane) to
obtain 4-chloro-7-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-2-methyl-
b-D-ribofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 18-b as
the first eluting anomer. The latter was dissolved in a 7 M solution
of NH3 in MeOH (0.01 M) and the resulting mixture was stirred
overnight at 110 °C in a closed vessel. The volatiles were then

4810
M. E. Di Francesco et al. / Bioorg. Med. Chem. 20 (2012) 4801–4811
removed under reduced pressure and the residue was purified by
flash chromatography eluting with MeOH:DCM 1:9 to give the title
compound as white foam (24%). 1H NMR (400 MHz, CD3CN/D2O) d
8.14 (s, 1H), 7.61 (s, 1H), 6.35 (d, J = 18.5 Hz, 1H), 4.20 (dd, J = 24.0,
9.4 Hz, 1H), 3.98–3.95 (m, 2H), 3.78 (dd, J = 12.0, 2.8 Hz, 1H), 1.00
(d, J = 22.6 Hz, 3H); 19F NMR (400 MHz, CD3CN/D2O) d ꢂ160.89;
MS (ES+) C12H14FIN4O3 requires: 408. Found: 409 (M+H+).
(d, J = 9.4 Hz, 1H), 3.17 (m, 6H), 2.92 (m, 18H), 1.82–1.68 (m, 6H),
1.48–1.29 (m, 18H), 0.98–0.87 (m, 9H), 0.84 (s, 3H); 31P NMR
(121 MHz, D2O, 300 K) d ꢂ10.45, ꢂ11.15 (m, 2P), ꢂ22.92 (t,
J = 19.6 Hz, 1P); MS (ESꢂ) C14H19N6O14P3 requires: 588.0. Found:
587 [MꢂH]ꢂ.
5.11.4. Oxadiazole 39
1H NMR (300 MHz, D2O, 300 K) d 9.29 (s, 1H), 8.34 (br s, 1H),
8.03 (s, 1H), 6.42 (d, J = 18.6 Hz, 1H), 4.61 (m, 1H), 4.44 (m, 1H),
4.31 (m, 1H), 3.14 (m, 6H), 2.89 (m, 18H), 1.78–1.66 (m, 6H),
1.43–1.27 (m, 18H), 1.04 (d, J = 23.1 Hz, 3H), 0.95–0.84 (m, 9H);
31P NMR (121 MHz, D2O, 300 K) d ꢂ10.60 (d, J = 19.4 Hz, 1P),
ꢂ11.12 (d, J = 19.4 Hz, 1P), ꢂ22.99 (t, J = 19.4 Hz, 1H); 19F NMR
(282 MHz, D2O, 300 K) d ꢂ161.35 (s, 1F); MS (ESꢂ) C14H18FN6O13P3
requires: 590.0. Found: 589 [MꢂH]ꢂ, 611 [M+NaꢂH]ꢂ.
5.9.1. Oxadiazole 21
The title compound was obtained in 27% isolated yield follow-
ing the same procedure described for oxadiazole 11, using 20-
deoxy-20-fluoro-carbonitrile 19 instead of carbonitrile 10. 1H
NMR (400 MHz, CD3CN/D2O) d 9.07 (s, 1H), 8.53 (s, 1H), 8.18 (s,
1H), 6.38 (d, J = 16.9 Hz, 1H), 4.13 (dd, J = 24.0, 8.2 Hz, 1H), 3.94–
3.88 (m, 2H), 3.71 (m, 1H), 0.96 (d, J = 22.6 Hz, 1H); 19F NMR
(400 MHz, CD3CN/D2O) d ꢂ163.26; MS (ES+) C14H15FN6O4 requires:
350. Found: 351 (M+H+). HRMS (ESI) m/z Calcd for C14H16FN6O4
351.1218, measured 351.1213. RP-HPLC method 1, tR = 1.54 min;
purity 96.5%.
5.11.5. Oxazole 40
1H NMR (300 MHz, D2O, 300 K) d 8.28 (br s, 1H), 7.99 (s, 1H),
7.92 (s, 1H), 7.21 (s, 1H), 6.43 (d, J = 17.8 Hz, 1H), 4.63 (m, 1H),
4.52–4.29 (m, 3H), 3.22–3.07 (m, 6H), 2.90 (m, 18H), 1.80–1.64
(m, 6H), 1.44–1.26 (m, 18H), 1.06 (d, J = 22.8 Hz, 3H), 0.96–0.83
(m, 9H); 31P NMR (121 MHz, D2O, 300 K) d ꢂ10.36 (d, J = 19.6 Hz,
1P), ꢂ11.23 (d, J = 19.6 Hz, 1P), ꢂ23.07 (t, J = 19.6 Hz, 1P); 19F
5.10. Synthesis of NMP prodrug 23
To a 0.1 M solution of nucleoside 7a in dry THF at ꢂ78 °C was
added dropwise t-BuMgCl (1.0 M solution in THF, 2.2 equiv). The
reaction mixture was stirred at ꢂ78 °C for 5 min and then at 0 °C
NMR (282 MHz, D2O, 300 K)
C
d
ꢂ162.22 (s, 1F); MS (ESꢂ)
15H19FN5O13P3 requires: 589.0. Found: 588 [MꢂH]ꢂ.
for further 30 min.
L-alanine-N-chlorophenoxyphosphinyl-ethyl
ester was then added dropwise (1.0 M solution in dry THF,
1.5 equiv) at 0 °C, and the resulting mixture was stirred at RT for
60 min and quenched with 2 ml of s.s NH4Cl. The volatiles were re-
moved under reduced pressure and the residue was purified by RP-
5.11.6. Pyrazole 41
1H NMR (300 MHz, D2O, 300 K) d 8.23 (br s, 1H), 7.74 (s, 1H),
7.70 (s, 1H), 6.96 (s, 1H), 6.30 (d, J = 17.2 Hz, 1H), 4.70 (m, 1H),
4.50–4.25 (m, 3H), 3.14 (m, 6H), 2.89 (m, 18H), 1.80–1.63 (m,
6H), 1.45–1.26 (m, 18H), 0.98 (d, J = 22.6 Hz, 3H), 0.94–0.82 (m,
9H); 31P NMR (121 MHz, D2O, 300 K) d ꢂ9.88 (d, J = 19.4 Hz, 1P),
ꢂ11.24 (d, J = 19.4 Hz, 1P), ꢂ22.82 (t, J = 19.4 Hz, 1P); 19F NMR
(282 MHz, D2O, 300 K) d ꢂ162.93 (s, 1F); MS (ESꢂ) C15H20FN6O12P3
requires: 588.0. Found: 587 [MꢂH]ꢂ.
HPLC (Atlantis T3, 19 ꢁ 150 mm, 5
lm) eluting with MeCN/H2O
containing 0.1% TFA to give the title compound. 1H NMR
(600 MHz, CD3CN + D2O, 300 K) d 11.86 (br s, 1H), 10.87 (br s,
1H), 8.96 (s, 1H), 8.18 (s, 1H), 7.86–7.82 (m, 1H), 7.58–7.54 (m,
1H), 7.36–7.31 (m, 2H), 7.25–7.15 (m, 3H), 6.81–6.76 (m, 1H),
6.32–6.30 (m, 1H), 4.55–4.49 (m, 1H), 4.47–4.40 (m, 1H), 4.18–
4.13 (m, 1H), 4.18–3.97(m, 4H), 3.96–3.90 (m, 1H), 1.27–1.25 (m,
3H), 1.14–1.12 (m, 3H), 0.85 (s, 3H). 31P (243 MHz. CD3CN+D2O)
d 3.85, 3.49. MS (ES+) C26H32N7O8P requires 601.2. Found: 602
[M+H]+.
Acknowledgment
We greatfully acknowledge Nadia Gennari, Monica Bisbocci and
Sergio Altamura for the enzymatic and cellular assays.
5.11. Selected NTP examples (for a general procedure for the
NTP synthesis see Ref. 31)
References and notes
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5.11.1. Oxazole 32
1H NMR (300 MHz, D2O, 300 K) d 8.37 (m, 1H), 8.03–7.91 (m,
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5.11.2. Pyrazole 34
1H NMR (300 MHz, D2O, 300 K) d 8.25 (br s, 1H), 7.79 (s, 1H),
7.71 (s, 1H), 7.00 (s, 1H), 6.11 (s, 1H), 4.71 (m, 1H), 4.43 (m, 1H),
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5.11.3. Oxadiazole 33
1H NMR (300 MHz, D2O, 300 K) d 9.33 (s, 1H), 8.41 (m, 1H), 8.09
(s, 1H), 6.23 (s, 1H), 4.61 (m, 1H), 4.55 (m, 1H), 4.33 (m, 1H), 4.16

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