Synthesis and antiviral properties of novel tetracyclic nucleoside inhibitors of hepatitis C NS5B polymerase

Article abstract of DOI:10.1080/15257770.2012.707344

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC50 = 0.1M, a level of intrinsic potency similar to that of previous and current clinical candidates.

Full text of DOI:10.1080/15257770.2012.707344

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Synthesis and Antiviral Properties of
Novel Tetracyclic Nucleoside Inhibitors
of Hepatitis C NS5B Polymerase
M. Emilia Di Francesco a , Salvatore Avolio a , Gabriella Dessole a
,
Uwe Koch a , Marco Pompei a , Vincenzo Pucci a , Michael Rowley a
Vincenzo Summa a
&
a Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.p.A.,
Merck Research Laboratories Rome, Pomezia, Italy
Version of record first published: 21 Aug 2012.
To cite this article: M. Emilia Di Francesco , Salvatore Avolio , Gabriella Dessole , Uwe Koch ,
Marco Pompei , Vincenzo Pucci , Michael Rowley & Vincenzo Summa (2012): Synthesis and Antiviral
Properties of Novel Tetracyclic Nucleoside Inhibitors of Hepatitis C NS5B Polymerase, Nucleosides,
Nucleotides and Nucleic Acids, 31:8, 592-607
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Nucleosides, Nucleotides and Nucleic Acids, 31:592–607, 2012
Copyright ꢀC Merck Sharp & Dohme Corp
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.707344
SYNTHESIS AND ANTIVIRAL PROPERTIES OF NOVEL TETRACYCLIC
NUCLEOSIDE INHIBITORS OF HEPATITIS C NS5B POLYMERASE
M. Emilia Di Francesco, Salvatore Avolio, Gabriella Dessole, Uwe Koch,
Marco Pompei, Vincenzo Pucci, Michael Rowley, and Vincenzo Summa
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.p.A., Merck Research Laboratories
Rome, Pomezia, Italy
As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV
2
NS5B polymerase, we report the discovery of a novel series of 2ꢁ-C-Methyl-ribose nucleoside deriva-
tives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform
for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs)
was established, enabling a solid understanding of the SAR relationship within the series. By this
approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cel-
lular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC50
0.1 µM, a level of intrinsic potency similar to that of previous and current clinical candidates.
=
Keywords HCV polymerase; nucleoside inhhibitor; nucleoside triphosphate; 2ꢁ-C-
methyl-ribose; 7-deaza-adenine
INTRODUCTION
Hepatitis C virus (HCV) infection is one of the most common blood-born
chronic infections, affecting an estimated 170 million people.[1] More than
70% of patients infected with HCV develop chronic HCV infection, which
often evolves into cirrhosis and ultimately in hepatocellular carcinoma.[2]
Until very recently, the standard of care (SOC) treatment of HCV infection
relied solely on a combination of PEGylated Interferon-α (PEG-IFNα) and
ribavirin,[3] and has been associated with limited efficacy (approximately
50% of the treated patients achieved sustained virologic response, SVR),
long treatment periods; and several side effects such as fatigue, influenza-
like symptoms, neutropenia, anemia, and depression.[4]
Received 23 April 2012; accepted 25 June 2012.
The authors gratefully acknowledge Nadia Gennari, Monica Bisbocci, and Sergio Altamura for the
enzymatic and cellular assays.
Address correspondence to M. Emilia Di Francesco, Institute for Applied Cancer Science, MD Ander-
son Cancer Center, 1901 East Road, Houston, TX 77054, USA. E-mail: medifrancesco@mdanderson.org
592

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
593
Over the past two decades, the improved understanding of the HCV
life cycle has boosted interest in the development of direct-acting antiviral
(DAA) agents that interfere with HCV replication by targeting essential vi-
rally encoded nonstructural enzymes.[5] Among these, the NS3/4A protease
and the NS5B polymerase have been the focus of intense drug discovery
efforts, which resulted in the identification of several clinical candidates
and culminated with the recent approval of the first two HCV NS3 protease
inhibitors—telaprevir and boceprevir.[6,7]
The viral RNA-dependent RNA polymerase (RdRp) NS5B plays a piv-
otal role in HCV replication, being responsible first for the synthesis of the
negative strand copy of the RNA genome and subsequently for the positive
strand RNA copies of the progeny virus.[8] Both nucleoside inhibitors (NIs)
and nonnucleoside inhibitors (NNIs) of NS5B have been described and
are currently advancing in late-stage clinical development.[9] NIs offer the
advantage of being pan-genotype inhibitors, as they target the highly con-
served RdRp active site.[10] Moreover, NIs offer a higher genetic barrier to
resistance compared with NS3 inhibitors and NS5B nonnucleoside agents,
holding great promise for future combination therapies.[11] Clinical proof
of concept has been achieved first with valopicitabine (1, Figure 1), later
discontinued due to an unfavorable risk/benefit profile.[12] Several NIs and
prodrugs thereof have subsequently shown significant efficacy in patients
and in primate models of HCV, including mericitabine (RG-7128, 2),[13]
PSI-7977 (3),[14] and MK-0608 (4).[15a,b]
NIs can be considered as prodrugs, as they need to be converted into
the corresponding nucleoside triphosphate (NTP) via a step-wise process
catalyzed by cellular kinases.[10] The NTP anabolyte must be recognized by
the RdRp and incorporated into the growing nucleic acid chain.[10] The
particular structural modification within the sugar moiety then prevents the
addition of further nucleotides, thus terminating chain elongation. The in-
troduction of a methyl group in the 2ꢁ-position of the ribose ring is among
O
NH2
N
NH2
N
NH
O
P
OPh
O
O
N
N
O
N
O
N
H
HO
N
R1O
O
O
O
O
2'
2'
2'
HO
OH
HO
F
R2O
R3
valopicitabine (1) mericitabine, RG-7128 (2)
PSI-7977 (3)
MK-0608 (4)
R3 = F
R1, R2
R1, R3 = H, OH
O
O
H2N
=
R2
=
FIGURE 1 Nucleoside derivatives that entered the clinic.

594
M. E. Di Francesco et al.
the most successful structural modifications leading to chain termination,
and several of the most advanced known NIs rely on this particular struc-
tural feature for their antiviral activity.[16] Among these, the 7-deaza-2ꢁ-C-
methyladenosine analog MK-0608 (4, Figure 1) has shown high inhibitory
potency against HCV replication in vitro (NTP IC50 = 0.1 µM; inhibition of
RNA replication in a subgenomic replicon assay EC50 = 0.3 µM) combined
with efficient conversion to NTP in vivo and excellent pharmacokinetic prop-
erties in preclinical species.[15a] Oral or intravenous administration of MK-
608 to HCV-infected chimpanzees resulted in robust and sustained reduction
of viral load (≥3 log) with no viral breakthrough due to the emergence of
resistant viral strain.[15b]
Following this discovery, extensive investigations have been conducted
to identify further 2ꢁ-C-Me-ribose derivatives with similar antiviral properties
to MK-0608.[17a,b] This paper describes our efforts to investigate novel 7-
deaza-2ꢁ-C-methyladenosine analogs as NIs of HCV NS5B, leading to the
identification of a novel class of tetracyclic analogs with high levels of intrinsic
inhibitory potency. Conversion of every novel nucleoside analog prepared
into the corresponding NTP has been a key feature of our approach, and has
proven essential for a thorough understanding of the SAR relationship of the
NIs investigated. Synthetic challenges toward the preparation of nucleoside
analogs and their NTPs will be described, as well as their biological properties
and insights for potential future development of this novel class of HCV NS5B
NI.
SYNTHESIS
In the context of our quest for 2ꢁ-Me-ribose derivatives with similar
antiviral properties to MK-0608 (4), we set out to evaluate 7-deaza-2ꢁ-C-
methyladenosine analogs and initially focused our investigations on 7-aryl
and 7-heteroaryl substituted 2ꢁ-C-Me-ribose analogs. We also established a
platform for a reliable synthesis and simplified purification of the corre-
sponding NTP analogs, thus enabling a timely evaluation of their inhibitory
properties.
Nucleoside Analogs
With the goal of 7-aryl and 7-heteroaryl substituted 7-deaza-adenine
analogs in mind, we initially pursued a practical synthesis of the key iodi-
nated intermediate 10 (Scheme 1), which was initially attempted by direct
iodination of the fully functionalized MK-0608 (4). The combination of mod-
est yield of conversion (13–18%), challenging purification procedures of the
desired product, and the complex synthesis required to assemble the starting
material 4 prompted us to explore alternative routes.[18] We turned next to

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
595
Cl
N
Cl
I
(a)
N
N
Cl
N
I
N
H
N
N
O
H
N
6
(c)
N
TolO
O
TolO
TolO
O
(b)
OH
OH
TolO
OH
9
O
TolO
TolO
7
8
MeO
N
N
NH2
NH2
R =
I
R
a
b
c
d
N
N
(d)
S
(e)
RB(OH)2
e
N
HO
N
N
HO
N
O
O
MeSO2
f
HO
OH
N
HO
10
11a-f
OH
HO2C
SCHEME 1 Reaction conditions: (a) NIS, DMF, 70%; (b) MsCl, Et3N, DCM, 30◦C; (c) NaH, THF/DMA,
50◦C, 55%; (d) 7N NH3/MeOH, 110◦C, 91%; (e) Pd(PPh3)4, Na2CO3, H2O/dioxane, 100◦C, 50–80%.
a more convergent 3-step route that proved to be reproducible and scalable,
and allowed us to assemble the required intermediate 10 in practical yields
starting from readily available starting materials. Treatment of 6-chloro-7-
deazapurine with N-iodosuccinimide afforded the iodinated nucleobase 6
in good yield after crystallization.[19] The latter was then deprotonated with
NaH and progressed to 9 via a key β-selective glycosidation step envisaging
the stereospecific opening of epoxide 8, which in turn was formed in situ
upon treatment of the 2ꢁ-Me-ribose derivative 7 with mesylchloride and tri-
ethylamine.[18] Displacement of the 6-chloro with ammonia in MeOH with
concomitant removal of the p-methyl-benzoyl protecting groups afforded
then the required 10 in 91% yield after purification. With a practical syn-
thesis of the 7-iodo building block 10 in hand, we turned our attention to
the synthesis of the planned 7-aryl and 7-heteroaryl derivatives. We initially
targeted a limited set of structurally diverse boronic acids to explore the
antiviral properties of nucleosides and NTPs in this series. We were pleased
to find that the reaction of 10 in the standard Suzuki–Miyaura coupling
conditions with the initial set of chosen boronic acids afforded the desired
nucleoside analogs in 11a-f in good yields (50–80%) and without requiring
further protecting group manipulations (Scheme 1).[20,21]
As a potential extension of this initial work, we envisaged the possibility
of connecting the 7-aryl substituent and the 6-NH2 moiety within a cyclic
structure, as in the lactam analogs 13 (Scheme 2). On the basis of the many
successful precedents for the Suzuki–Miyaura coupling between interme-
diate 10 and a variety of aryl- and heteroaryl-boronic acids, we initially at-
tempted the synthesis of 13a via a two-step sequence involving cross-coupling
of 10 with the (2-carboxylphenyl)-boronic acid, followed by intramolecular

596
M. E. Di Francesco et al.
O
O
H
NH2
Ar/Het
Ar/Het
NH
NH2
N
I
7
6
N
N
(a)
(b)
13a
13b
N
HO
N
N
N
O
HO
HO
N
N
O
O
S
CHO
S
CHO
or
B(OH)
HO
OH
2
B(OH)
2
HO
OH
12a/b
HO
OH
10
Ar/Het
NH
6
N
7
14a
14b
N
(c)
HO
N
O
S
HO
OH
SCHEME 2 Reaction conditions: (a) Pd(PPh3)4, Na2CO3, H2O/dioxane, 100◦C; (b) NaClO2, isobutene,
tBuOH, K2HPO4, 20% over two steps; (c) NaCNBH3, AcOH, 24% over two steps.
amide coupling. The unexpected failure of the Suzuki–Miyaura coupling
prompted us to explore alternative routes. Fortunately, the reaction of
10 with (2-formylphenyl)-boronic acid in the usual conditions resulted in
straightforward formation of the intermediate aldehyde 12, which was then
progressed directly to the desired lactam 13a by Lindgren oxidation[22] to
the corresponding carboxylic acid and concomitant intramolecular amide
formation. In a similar manner, the tetracyclic thiophene lactam analog 13b
was obtained starting from coupling between 10 and 2-formylthienyl boronic
acid, followed by the one-step oxidation/intramolecular cyclization.
Intrigued by this novel class of tetracyclic NI, we set out to investigate the
role of the amide moiety on the antiviral potency. The corresponding cyclic
amines 14a and 14b were targeted first, and obtained in straightforward
manner by intramolecular reductive amination of the aldehyde intermedi-
ates 12a/b (Scheme 2).
Nucleoside Triphospate Analogs (NTPs)
Central to our approach toward the identification of potent inhibitors
of HCV NS5B polymerase has been the ability of preparing the correspond-
ing NTP of every targeted novel nucleoside analog. Toward this end, we
established a reliable protocol that, although relying mainly on known tech-
nology for the synthesis of the NTP species, introduced greatly simplified
purification procedures and thus proved amenable to the rapid exploration
required for our SAR studies. As described in Scheme 3, the unprotected
nucleoside was converted to the corresponding NTP following general syn-
thetic methods such as the Ludwig[23] or the Broom[24] procedures. These
are one-pot, three-step processes involving activation of the nucleoside by
treatment with trimethylphosphate and POCl3, followed by addition of ei-
ther tri-n-butyl ammonium pyrophosphate or tri-n-butyl ammonium phos-
phate and final hydrolysis of the resulting trimethaphosphate intermediate

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
597
H
N
H
N
R
N
R
N
O
Cl P O
Cl
HO
(a)
(b)
N
N
O
N
N
O
HO
HO
HO
HO
11a-f
13a-b
14a-b
H
N
N
R
H
N
N
N
O
O
P
O
O
P
R
P
O
O
O
O
HO P O P O P
O
N
O
N
O
(c)
O
OH OH OH
O
N
O
O
15a-f
16a-b
17a-b
HO
HO
HO
HO
−
SCHEME 3 Reaction conditions: (a) POCl3, (MeO)3PO, 0◦C or RT; (b) [Bu3NH+]H2PO4, NBu3 or
[Bu3NH+]4.P2O74−, NBu3, DMF, 0◦C or RT; (c) 1M Et3NHHCO3, RT; then solid phase extraction (SPE)
and RP-HPLC; 10–50%.
with triethyl ammonium bicarbonate. The described procedures, as well
as other NTP synthesis protocols,[25] have typically been associated with
time-consuming purification procedures of very complex reaction mixtures,
involving lengthy ion exchange column chromatography (up to 6 hours),
resulting in large solvent volumes (1–2L) and requiring a final step of reverse
phase (RP) HPLC. In contrast, our revised protocol relied on the quick pu-
rification (ca. 15 minutes) of the reaction mixture by solid phase extraction
(SPE) with a polymeric mixed mode sorbent (e.g. Strata X-AW SPE, a RP
and weak anion exchange resin), resulting in elimination of the majority of
inorganic phosphate salts as well as of the nonreacted nucleoside and nucle-
oside monophosphate species. A much simplified mixture of the di- and the
desired tri-phosphate was then eluted in a very small volume, and progressed
to C18 RP HPLC for final purification (full experimental details are covered
in Section 5). This improved protocol proved to be a robust platform to
access the vast majority of the required NTP in practical amounts, typically
ensuring >80% success rate, with yields of the purified NTPs in the 10–46%
range.

598
M. E. Di Francesco et al.
RESULTS AND DISCUSSION
The described nucleoside analogs 11a-f, 13a–b, and 14a–b were tested
for their ability to affect the replication of HCV RNA in cultured hepatoma
cells HBI10A containing a subgenomic HCV Replicon.[26] In all cases, the
compounds were found to be significantly less potent than the unsubstituted
7-deaza-adenosine analog MK-0608, and showed low micromolar levels of an-
tiviral potency (EC50 = 15 − >100 µM, Table 1). A lack of antiviral potency
in the cellular assay, or marginal levels thereof, can often be the result of an
inefficient intracellular phosphorylation process, and does not necessarily re-
flect a poor inhibitory potency of the NTP against HCV polymerase. Indeed,
in some cases, the antiviral potency can be restored with a kinase bypass such
as the use of a monophosphate prodrug.[27] In view of these considerations
and undeterred by the initial replicon data, we pursued the synthetic con-
version of our nucleoside analogs into the corresponding NTPs, and their
profiling against HCV NS5B polymerase as the most direct and reliable way
to assess the inhibitors’ intrinsic potency in order to fully appreciate the SAR
relationships of the targeted Nuc analogs. With the possible exception of the
pyridyl analog 15d (Entry 4, Table 1), which inhibits NS5B polymerase with
IC50 = 6.9 µM, none of the other NTP analogs bearing a 7-aryl or -heteroaryl
group showed significant levels of inhibitory potency (compounds 15a–c
and 15e–f, IC50 = 24 − >100 µM, Table 1). This result suggested that the
initial lack of antiviral potency observed with the corresponding nucleosides
in the replicon assay was mostly due to the poor intrinsic potency against the
NS5B polymerase rather than an inefficient phosphorylation process, and in-
deed questioned the opportunity of further probing 7-aryl and 7-heteroaryl
substitutions toward the identification of novel HCV Nuc inhibitors.
Interestingly, an unexpected finding prompted us to further pursue our
investigation in this area. Despite the modest cellular potency associated
with the lactam analog 13a (EC50 = 23.8 µM, Entry 7, Table 1) and in line
with our strategy based on NTPs as key to a reliable SAR, we progressed
13a to the corresponding NTP 16a. To our surprise, 16a proved to be an
extremely potent inhibitor of HCV NS5B with IC50 = 0.1 µM, a more than
1000× boost compared to the NTP of the corresponding 7-phenyl analog
15a and equipotent to the NTP of the unsubstituted 7-deaza-adenosine ana-
log MK-0608. Encouraged by this finding, we focused on the synthesis of
a few selected analogs to confirm this initial result and understand the key
structural features associated with the observed boost in potency. The closely
related thiophene analog
13b and the corresponding NTP 16b (Entry 8, Table 1) substantiated the
first finding, and were found to be indeed very similar to the phenyl lactam
analogs 13a and 16a, both in terms of cellular and enzymatic activity (EC50
33.3 µM and IC50= 0.12 µM, respectively). With the aim of investigating the
=

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
599
TABLE 1 (a) Cellular activity determined in the HCV biscistronic replicon assay using HBI10A cells
stably transfected with genotype 1b HCV replicon RNA;[26] EC50 are measured in the presence of 10%
Fetal Bovine Serum (FBS); EC50 values are the mean of ≥3 experiments, standard deviation was within
20% of the reported value; (b) enzyme potency measured in a NS5B polymerization assay, using an
heteromeric RNA template;[28] IC50 values are the mean of ≥3 experiments, standard deviation was
within 20% of the reported value
NH2
R
NH2
N
R
N
O
P
O
P
O
P
HO
O
O
O
N
N
N
HO
O
N
O
OH OH OH
HO
OH
HO
OH
Compound
nucleoside/NTP
HCV replicon8
EC50 (|iM)
HCV NS5B polymeraseb
IC50 (|iM)
Entry
R
1
11a/15a
56.5
100
S
2
3
11b/15b
11c/15c
15.0
50
21.4
100
HO C
2
MeO
N
4
5
11d /15d
11e/15e
100
6.9
40.8
100
MeSO
N
2
N
6
7
11f /15f
13a/16a
100
100
O
23.8
0.10
NH
N
N
N
O
S
8
13b/ 16b
33.3
0.12
NH
N
N
N
9
14a/17a
14b/17b
46.2
21
28.0
7.2
NH
N
N
N
S
10
NH
N
N
N

600
M. E. Di Francesco et al.
FIGURE 2 Molecular model of lactam 16a (a) and amine 17a (b) in the context of NS5B polymerase
active site. The template RNA-chain is represented on the right-hand side, the growing RNA chain and
the incoming NTP on the left coordinated to the Mg2+ ions; NS5B RNA polymerase not shown.
role of the lactam moiety in the observed boost in potency within these tetra-
cyclic 7-arylsubstituted analogs, we next turned our attention to the cyclic
amine analogs 14a and 14b. These compounds were lacking the carbonyl
group but still maintained the key H-bond donor typical of the natural purine
nucleobases, and thus could potentially retain the H-bond network with the
template RNA chain within the active site of the polymerase enzyme. When
converted to the corresponding NTPs 17a and 17b, the tetracyclic amines
proved to be significantly poorer inhibitors of NS5B polymerase, compared
to the corresponding lactams 16a and 16b, suggesting that the presence of
the lactam moiety is a required feature to maintain the intrinsic inhibitor
potency (17a IC50 = 28 µM; 17b IC50 = 7.2 µM; Entries 9-10, Table 1).
Intrigued by this result, we modeled the NTPs of the lactam 16a and amine
17a within the active site of HCV NS5B polymerase (Figure 2). The mod-
eling suggested that while the presence of the lactam moiety might impart
to the nucleobase of 16a, a completely planar structure, in the case of the
amine 17a the planarity appears to be lost, potentially resulting in a steric
clash with the preceding nucleotide in the growing RNA chain and thus in
a suboptimal interaction within the active site of the enzyme.
The hypothesis that planarity might play a key role in the intrinsic po-
tency of these novel lactam analogs can explain the striking loss of potency
of the amine analogs. In fact, compounds are almost devoid of inhibitory
potency despite the presence of an NH-group which, according to the mod-
eling shown, seems able to retain the capability of engaging in key H-bond
interactions with the template RNA-chain, similarly to the lactam analogs
and to the natural purines.
The identification of this novel series of tetracyclic lactam inhibitors of
NS5B polymerase with excellent levels of intrinsic enzymatic potency war-
rants further investigation. The lack of antiviral potency shown by the nucle-
oside analogs 13a and 13b in the cellular assay might be due to the inefficient
conversion to the required NTP by cellular kinases, and can hopefully be

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
601
overcome by the use of monophosphate prodrugs as a kinase bypass, an
amply precedented approach that has recently proved successful also in the
clinical settings with compounds such as PSI-7977 (3, Figure 1).[14]
CONCLUSIONS
With the goal of identifying novel 2ꢁ-C-Me-ribose nucleoside derivatives as
novel inhibitors of HCV NS5B polymerase, we focused our investigations on
the 7-deaza-adenine analogs substituted with 7-aryl or 7-heteroaryl groups. In
order to gain a solid understanding of the SAR relationship within the series,
we set up a reliable and practical protocol that allowed us to convert every
nucleoside analog prepared into the corresponding NTP, thus enabling a
thorough evaluation of the intrinsic antiviral potency of the novel nucleoside
analogs. By this approach, we identified the novel tetracyclic lactam analogs
13a and 13b, the NTPs of which, 16a and 16b, are excellent inhibitors of
NS5B with IC50 = 0.1 µM. This level of intrinsic potency is comparable
or better to that of previous and current clinical candidates, and warrants
further investigation. Future work will include extended SAR studies in the
series and efforts to restore cellular potency by a kinase bypass approach
with the use of monophosphate prodrugs.
EXPERIMENTAL SECTION
1H-NMR spectra were obtained on Bruker spectrometers 300 MHz,
400 MHz, or 600 MHz as specified within the text. MS analyses were per-
formed on a Waters micromass ZQ instrument equipped with a Waters Ac-
quity UPLC system. The solvent system used was MeCN/H2O with 0.1%
formic acid. All reagents and solvents were obtained from commercial
sources.
Synthesis of 5-iodo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (10)
Step 1: To a suspension of 6-chloro-7-deazapurine (2g, 13 mmol) in DCM
(100 mL) was added N-iodosuccinimmide (3.2 g, 14.3 mmol) and the result-
ing mixture was stirred at room temperature for 6 hours. The suspension
was filtered, the mother liquors were concentrated under reduced pressure,
and the residue was recrystallized from MeOH to give 6-chloro-7-iodo-7-
1
deazapurine 618 as an off-white solid (2.48 g, 8.87 mmol, 70%); H NMR
(400 MHz, DMSO-d6) δ 12.91 (bs, 1H), 8.56 (s, 1H), 7.89 (s, 1H); MS (ES+)
C6H3ClIN3 requires: 279, found: 280 (M+H)+.
Step 2: To a solution of diol 719 (0.26 g, 0.64 mmol) in DCM (3 mL)
was added triethylamine (0.1 mL, 0.72 mmol), followed by methanesulfonyl
chloride (0.056 mL, 0.72 mmol) and the resulting mixture was stirred at 30◦C
for 1 hour. The mixture was then diluted with DCM (5 mL), washed with

602
M. E. Di Francesco et al.
brine, dried (Na2SO4), and partially concentrated under reduced pressure
(cold bath) to ca. half of the initial volume. Anhydrous toluene (2 mL) was
then added and the remaining DCM was removed under reduced pressure.
The resulting solution of epoxide 8 in toluene (ca. 0.3 M) was used directly
in the subsequent glycosidation step.
Step 3: NaH (14 mg of 60% dispersion in oil, unwashed, 0.36 mmol) was
added to a solution of 6-chloro-7-iodo-7-deazapurine 6 (200 mg, 0.72 mmol)
in THF (1.5 mL) and N,N-dimethylacetamide (1.0 mL) at 0◦C. After gas evo-
lution had ceased, the mixture was stirred at room temperature for further
15 minutes. N,N-dimethylacetamide (1.0 mL) was added, followed by the
solution of epoxide 8 in toluene (2 mL, ca. 0.3 M). The resulting mixture
was stirred at 50◦C for 8 hours, cooled to room temperature, and parti-
tioned between EtOAc and brine. The organic layer was separated, dried
(Na2SO4), and concentrated under reduced pressure. The residue was puri-
fied by SiO2 gel chromatography (gradient elution, 10–40% EtOAc) to give
the glycosidated intermediate 9 as a pale yellow foam (232 mg, 0.35 mmol,
55%).
Step 4: Intermediate 9 (204 mg, 0.31 mmol) was taken up in 5 mL of
a 7N solution of NH3 in methanol and heated at 110◦C in an autoclave
for 16 hours. The reaction mixture was then cooled to room temperature,
the volatiles were removed under reduced pressure, and the residue was
purified by SiO2 gel chromatography (5% MeOH in DCM) to give the title
1
compound 10 as an off-white solid (114 mg, 0.28 mmol, 91%); H NMR
(400 MHz, CD3CN/D2O) δ 8.11 (s, 1H), 7.62 (s, 1H), 6.14 (s, 1H), 4.04 (bd,
J = 10.0 Hz, 1H), 3.92 (m, 2H), 3.74 (bd, J = 12.0 Hz, 1H), 0.77 (s, 3H);
MS (ES+) C12H15IN4O4 requires: 406, found: 407 (M+H)+.
General Procedure for Synthesis of Analogs 11a-f and Selected Examples
Pd(PPh3)4 (0.1 eq) was added to a 0.1 M solution of 5-iodo-7-(2-C-methyl-
β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, boronic acid (1.5
eq) and Na2CO3 (2M aq. solution, 15 eq) in dioxane. The reaction mix-
ture was heated at 100◦C for 3 hours and then filtered through a pad
of celite. The filtrate was concentrated under reduced pressure and the
residue was purified by preparative RP-HPLC eluting with MeCN/water con-
taining 0.1% TFA to give the title compounds as solids (Typical yields ranged
50–80%).
1
Nucleoside 11a: H NMR (400 MHz, CD3CN) δ 8.14 (s, 1H), 7.52–7.31
(m, 6H), 6.22 (s, 1H), 4.04 (d, J = 9.1 Hz, 1H), 3.98–3.88 (m, 2H), 3.77 (m,
1H), 0.84 (s, 3H); MS (ES+) C18H20N4O4 requires: 356, found: 357 (M+H)+.
1
Nucleoside 11b: H NMR (400 MHz, D2O) δ 8.22 (s, 1H), 7.62–7.54
(m, 2H), 7.31–7.22 (m, 2H), 6.38 (s, 1H), 4.28–4.08 (m, 3H), 3.95 (m,
1H), 0.96 (s, 3H); MS (ES+) C16H18N4O4S requires: 362, found: 363
(M+H)+.

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
Tetracyclic Lactam Analogs 13a and 13b
603
To a solution of 5-iodo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 10 in dioxane (ca. 0.2 M) was added Pd(Ph3P)4 (0.1
eq), followed by (2-formyl-aryl)boronic acid (1.5 eq) and Na2CO3 (2M aque-
ous solution, 3 eq) and the resulting mixture was heated at 100◦C for 1 hour.
The solution was allowed to cool to room temperature and then filtered
through a pad of celite. The filter cake was washed with MeOH and the
filtrate was concentrated under reduced pressure. The residue was dissolved
in a 1/1 v/v mixture of tert-butanol and 2-methyl-2-butene, a solution of
NaClO2 (10 eq) and KH2PO4 (7.5 eq) in water was added, and the reaction
mixture was stirred at room temperature for 8 hours. The volatiles were re-
moved under reduced pressure and the residue was purified by preparative
RP-HPLC eluting with MeCN/water containing 0.1% TFA to give the title
compounds.
13a: 1H NMR (400 MHz, D2O/CD3CN) δ 11.00 (s, 1H), 8.52 (s, 1H), 8.42
(s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.60 (m, 1H),
7.34 (m, 1H), 6.22 (s, 1H), 4.11 (m, 1H), 4.00–3.89 (m, 2H), 3.78 (m, 1H),
0.79 (s, 3H); MS (ES+) C19H18N4O5 requires: 382, found: 383 (M+H)+.
1
13b: H NMR (600 MHz,DMSO-d6) δ 11.07 (s, 1H), 8.39 (s, 1H), 8.20
(s, 1H), 8.02 (d, J = 4.63 Hz, 1H), 7.74 (d, J = 4.63 Hz, 1H), 6.19 (s, 1H),
5.31–5.16 (m, 3H), 4.05–4.00 (m, 1H), 3.97–3.92 (m, 1H), 3.92–3.87 (m,
1H), 3.79–3.74 (m, 1H) 0.79 (s, 3H); MS (ES+) C17H16N4O5S requires: 388,
found: 389 (M+H)+.
Tetracyclic Amine Analogs 14a and 14b
Pd(Ph3P)4 (0.1 eq) was added to a solution of 5-iodo-7-(2-C-methyl-β-
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine 10 (1.0 eq) (2-formy-
aryl)boronic acid (1.5 eq) and sodium carbonate (2M aqueous solution, 3
eq) in dioxane and the resulting mixture was heated at 100◦C for 1 hour. The
solution was allowed to cool to room temperature and then filtered through
a pad of celite. The filter cake was washed with MeOH and the filtrate
was concentrated under reduced pressure. The dark residue obtained was
dissolved in MeCN and acetic acid (0.5 eq) was added, followed by molecular
sieves. The resulting suspension was stirred for 5 minutes, NaBH3CN (1.2 eq)
was then added, and the reaction mixture was stirred at room temperature
for 12 hours. The reaction was quenched with 1M aq. NaOH, the volatiles
were removed under reduced pressure, and the residue was purified by
preparative RP-HPLC eluting with MeCN/water containing 0.1% TFA to
give the title compound.
1
14a: H NMR (400 MHz, D2O) δ 8.28 (s, 1H), 8.12 (s, 1H), 7.67 (m,
1H), 7.50 (m, 1H), 7.42–7.36 (m, 2H), 6.39 (s, 1H), 4.57 (d, J = 5.2 Hz,
2H), 4.28–4.16 (m, 3H), 4.05 (m, 1H), 0.98 (s, 3H); MS (ES+) C19H20N4O4
requires: 368, found: 369 (M+H+).

604
M. E. Di Francesco et al.
14b: 1H NMR (600 MHz, D2O/CD3CN) δ 8.24 (s, 1H), 7.96 (s, 1H), 7.37
(d, J = 5.33 Hz, 1H), 7.30 (d, J = 5.33 Hz, 1H), 6.26 (s, 1H), 4.69 (s, 2H),
4.10 (d, J = 8.84 Hz, 1H), 4.03–3.96 (m, 2H), 3.85–3.82 (m, 1H), 0.86 (s,
3H). MS (ES+) C17H18N4O4S requires: 374, found: 375 (M+H+).
General Procedure for the Synthesis of NTPs
Neat POCl3 (2.5 eq) was added dropwise via a syringe to a 0.15 M solu-
tion of the appropriate nucleoside (previously dried by coevaporation with
pyridine and toluene) in trimethyl phosphate (stored over sieves) at 0◦C or
room temperature. After stirring the resulting mixture for 2 hours at 0◦C or
at room temperature, a 0.5 M solution of bis tributylammonium pyrophos-
phate (6.0 eq) and tributylamine (5.0 eq) in DMF was added in one portion
to the reaction mixture under vigorous stirring. After vigorous stirring for 1
minute at 0◦C or room temperature, triethylammonium hydrogencarbonate
buffer (1 M aq., 50 eq, pH = 7.5) was added to the reaction mixture, which
was then stirred for further 3 hours at room temperature and concentrated
under reduced pressure (cold bath). The residue was dissolved in water and
the triphosphate was recovered by anion exchange SPE with a buffer sys-
tem of 0.5 M TEAB (pH = 7.5), followed by RP-HPLC purification (mobile
phase: 10 mM dimehtylhexylammonium bicarbonate, pH = 8.0 / MeCN or
MeOH) to afford the title compounds as tris dimethylhexylammonium salts
(colorless oils). Typical yields ranged from 10 to 46%.
1
NTP 15a: obtained in 30% yield; H NMR (300 MHz, D2O) δ 8.28 (s,
1H), 7.60–7.42 (m, 6H), 6.35 (s, 1H), 4.52 (m, 1H), 4.38 (m, 1H), 4.34–4.25
(m, 2H), 3.40 (bs, 1H), 3.18–3.06 (m, 6H), 3.14–3.02 (m, 18H), 1.82–1.65
(m, 6H), 1.42–1.26 (m, 18H), 0.95–0.82 (m, 12H). 31P NMR (121 MHz, D2O,
300 K) δ –23.20 (t, J = 20.1 Hz, 19.6, 1P), –11.25 (d, J = 19.0 Hz, 1P), –10.32
(d, J = 19.9 Hz, 1P). MS (ES−) m/z 595 (M-H)−.
1
NTP 15b: obtained in 16% yield; H NMR (300 MHz, D2O) δ 8.33 (s,
1H), 7.56–7.22 (m, 2H), 7.20–7.16 (m, 2H), 6.29 (s, 1H), 4.55 (m, 1H),
4.40–4.21 (m, 3H), 3.18–3.10 (m, 6H), 3.04–2.80 (m, 18H), 1.82–1.62 (m,
6H), 1.41–1.20 (m, 18H), 0.92–0.81 (m, 12H). 31P NMR (121 MHz, D2O,
300 K) δ –23.04 (m, 1P), –11.25 (d, J = 19.6 Hz, 1P), –10.55 (d, J = 19.4 Hz,
1P). MS (ES−) m/z 601 (M-H)−.
NTP 15c: obtained in 10% yield; 1H NMR (300 MHz, D2O) δ 8.37 (s, 1H),
7.93–7.86 (m, 2H), 7.68–7.48 (m, 2H), 6.22 (s, 1H), 4.50 (m, 1H), 4.39–4.26
(m, 2H), 4.16 (m, 1H), 3.20–3.04 (m, 6H), 2.96–2.80 (m, 18H), 1.80–1.62
(m, 6H), 1.42–1.24 (m, 18H), 0.98–0.80 (m, 12H). 31P NMR (121 MHz, D2O,
300 K) δ –23.03 (m, 1P), –11.01 (d, J = 14.8 Hz, 1P), –10.60 (d, J = 15.7 Hz,
1P), MS (ES−) m/z 639 (M-H)−.
1
NTP 15d: obtained in 26% yield; H NMR (300 MHz, D2O) δ 8.30 (s,
1H), 8.13 (s, 1H), 7.86 (m, 1H), 7.51 (s, 1H), 6.98 (m, 1H), 6.25 (s, 1H),
4.48 (m, 1H), 4.36 (m, 1H), 4.28–4.20 (m, 2H), 3.96 (s, 3H), 3.16–3.07 (m,
6H), 2.94–2.80 (m, 18H), 1.78–1.61 (m, 6H), 1.39–1.22 (m, 18H), 0.94–0.82

Novel Tetracyclic Nucleoside HCV NS5B Inhibitors
605
(m, 12H). 31P NMR (121 MHz, D2O, 300 K) δ –22.86 (m, 1P), –11.09 (d, J =
14.9 Hz, 1P), –10.24 (d, J = 14.7 Hz, 1P). MS (ES−) m/z 626 (M-H)−.
1
NTP 15e: obtained in 16% yield; H NMR (300 MHz, D2O) δ 8.42 (s,
1H), 7.98–7.70 (m, 4H), 7.60 (m, 1H), 6.26 (s, 1H), 4.56 (m, 1H), 4.45–4.26
(m, 2H), 4.20 (m, 1H), 3.35 (s, 3H), 3.22–3.08 (m, 6H), 3.02–2.80 (m,
18H), 1.85–1.63 (m, 6H), 1.48–1.24 (m, 18H), 1.02–0.82 (m, 12H). 31P NMR
(121 MHz, D2O, 300 K) δ –22.98 (m, 1P), –11.19 (d, J = 19.8 Hz, 1P), –10.58
(d, J = 19.5 Hz, 1P). MS (ES−) m/z 673 (M-H)−.
1
NTP 15f: obtained in 16% yield; H NMR (300 MHz, D2O) δ 9.08 (s,
1H), 8.92 (s, 2H), 8.25 (s, 1H), 7.72 (s, 1H), 6.32 (s, 1H), 4.38 (m, 1H),
4.35–4.18 (m, 3H), 3.22–3.10 (m, 6H), 2.98–2.82 (m, 18H), 1.82–1.68 (m,
6H), 1.46–1.26 (m, 18H), 0.96–0.85 (m, 12H). 31P NMR (121 MHz, D2O,
300 K) δ –22.23 (m, 1P), –11.29 (d, J = 20.1 Hz, 1P), –10.79 (d, J = 19.8 Hz,
1P). MS (ES−) m/z 597 (M-H)−.
1
NTP 16a: obtained in 23% yield; H NMR (300 MHz, D2O) δ 8.34 (s,
1H), 8.16 (d, J = 8.4 Hz, 1H), 8.06–7.99 (m, 2H), 7.64 (m, 1H), 7.33 (m,
1H), 6.31 (s, 1H), 4.59 (m, 1H), 4.48 (m, 1H), 4.41–4.29 (m, 2H), 3.21–3.06
(m, 6H), 2.90 (bs, 18H), 1.79–1.64 (m, 6H), 1.42–1.25 (m, 18H), 0.94–0.83
(m, 12H). 31P NMR (121 MHz, D2O, 300 K) δ –23.09 (bt, J = 20.4 Hz, 19.6,
1P), –11.21 (d, J = 20.4 Hz, 1P), 10.76 (d, J = 19.6 Hz, 1P). MS (ES−) m/z
621 (M-H)−.
1
NTP 16b: obtained in 46% yield; H NMR (300 MHz, D2O) δ 8.25
(bs, 1H), 7.94–7.78 (m, 2H), 7.65–7.48 (m, 2H), 6.22 (bs, 1H), 4.53 (m,
1H), 4.44 (m, 1H), 4.35–4.24 (m, 2H), 3.15–3.05 (m, 6H), 2.90–2.74 (m,
18H), 1.82–1.64 (m, 6H), 1.38–1.22 (m, 18H), 0.94–0.82 (m, 12H). 31P NMR
(300 MHz, D2O, 300 K) δ –22.80 (t, J = 20.0 Hz, 1P), –11.40 (d, J = 19.9 Hz,
1P), 10.22 (d, J = 19.9 Hz, 1P). MS (ES−) m/z 627 (M-H)−.
1
NTP 17a: obtained in 10% yield; H NMR (300 MHz, D2O) δ 8.18 (s,
1H), 8.09 (s, 1H), 7.67 (m, 1H), 7.44–7.12 (m, 3H), 6.25 (s, 1H), 4.57 (m,
2H), 4.40–4.18 (m, 3H), 3.16–3.08 (m, 6H), 2.96–2.81 (m, 18H), 1.78–1.64
(m, 6H), 1.38–1.25 (m, 18H), 0.95–0.84 (m, 12H). 31P NMR (121 MHz, D2O,
300 K) δ –24.34 (m, 1P), –12.88 (d, J = 20.2 Hz, 1P), –11.02 (d, J = 19.4 Hz,
1P). MS (ES−) m/z 607 (M-H)−.
1
NTP 17b: obtained in 11% yield; H NMR (300 MHz, D2O) δ 8.45 (bs,
1H), 7.88 (s, 1H), 7.74 (m, 1H), 7.52 (m, 1H), 6.28 (s, 1H), 4.75–4.05 (m,
6H), 3.21–3.10 (m, 6H), 2.98–2.79 (m, 18H), 1.80–1.62 (m, 6H), 1.41–1.24
(m, 18H), 0.98–0.80 (m, 12H). 31P NMR (121 MHz, D2O, 300 K) δ –22.95
(m, 1P), –11.16 (d, J = 19.9 Hz, 1P), –10.61 (d, J = 19.3 Hz, 1P). MS (ES−)
m/z 613 (M-H)−.
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