Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues

Article abstract of DOI:10.1021/acs.jmedchem.5b01933

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-in

Full text of DOI:10.1021/acs.jmedchem.5b01933

Article
pubs.acs.org/jmc
Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and
Pyrimidine C‑Nucleosides as Favipiravir (T-705) Analogues
,
†
‡
‡
‡
†
†
Guangyi Wang,* Jinqiao Wan, Yujian Hu, Xiangyang Wu, Marija Prhavc, Natalia Dyatkina,
†
†
†
†
†
†
Vivek K. Rajwanshi, David B. Smith, Andreas Jekle, April Kinkade, Julian A. Symons, Zhinan Jin,
Jerome Deval, Qingling Zhang, Yuen Tam, Sushmita Chanda, Lawrence Blatt,
and Leonid Beigelman*
†
†
†
†
†
,†
†Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California 94080, United States
‡Department of Medicinal Chemistry, WuXi AppTec, Shanghai 200131, P.R. China
*
S Supporting Information
ABSTRACT: Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant
morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely
approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-
resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action.
Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its
riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed,
prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral
mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells,
and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are
also presented.
5
nfluenza viruses are responsible for seasonal epidemics and
occasional pandemics, which cause significant morbidity and
peramivir is used by IV route only. Thus, the worldwide spread
of drug-resistant influenza strains poses an urgent need for
novel antiviral drugs, particularly with a different mechanism of
action. Current drug discovery continues NA and M2 inhibitor
optimization via structure based drug design to solve the drug
I
mortality. Influenza occurs globally with an annual attack rate
1
estimated at 5%−10% in adults and 20%−30% in children.
Worldwide, these annual epidemics are estimated to result in
about 3 to 5 million cases of severe illness, and about 250,000
to 500,000 deaths. Although safe and effective influenza
vaccines are available, they may be less effective among the
10
resistance issue. Recently, VX-787, a potent inhibitor of the
influenza virus replication complex PB2 subunit, was disclosed,
which is currently in phase II clinical development. Inhibitors
targeting the influenza endonuclease have also been reported.
11
1
12
elderly in preventing illness. Moreover, vaccines need to be
reformulated each year due to the genetic variability of the
virus, and they are not always protective; in addition, a rapidly
emerging influenza pandemic cannot be contained by
vaccination. There are currently two classes of widely
approved anti-influenza drugs: M2 channel blockers (amanta-
From the drug discovery standpoint, it would be ideal to
develop broad-spectrum anti-influenza drugs that are active
against all strains of influenza, thus circumventing the need for
combination therapy. Among this class are ribavirin and T-705
2
13
(
favipiravir). The latter, in the brand name of Avigan, has
been approved in Japan as an oral anti-influenza drug.
Favipiravir was found to have potent anti-influenza activity in
cell-based assays, and its riboside (2) triphosphate inhibited the
dine and rimantadine) and neuraminidase inhibitors (oselta-
2
−5
mivir, zanamivir, laninamivir, and peramivir).
Due to
widespread resistance to the class, the M2 ion channel
13,14
5
−7
influenza polymerase.
favipiravir acts via lethal viral mutagenenesis.
Additional studies suggested that
inhibitors are not recommended currently for therapy.
Resistance to the only orally bioavailable drug oseltamivir was
1
5,17,18
As part of
8
,9
dominant in the 2007−2008 influenza season.
Low
bioavailability and their nasal route of administration limit the
use of zanamivir and laninamivir in severely ill patients, while
Received: December 14, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b01933
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Figure 1. Favipiravir and C-nucleoside analogues.
a
Scheme 1
a
Regents and conditions: (a) LDA, THF, −78 °C, 54%; (b) BF -Et O, Et SiH, 0 °C to rt, 24%; (c) NH OH, H O , MeOH, 50%; (d) NH F,
3
2
3
4
2
2
4
MeOH, 67%; (e) i. I , PPh , py, overnight, 51%; ii. DBU, THF, 60 °C, overnight, 89%; (f) NIS, TEA-3HF, MeCN, 67%; (g) i. BzCl, py, 75%; ii.
2
3
mCPBA, Bu NOH-TFA, 67%; (h) NH /MeOH, 72%; (i) i. 2,2-dimethoxypropane, TsOH, acetone, 84%; ii. NaOMe, dioxane, 28%; (j) H O , NH ,
4
3
2
2
3
MeOH/H O, 73%; (k) EtSNa, DMF, 40 °C, 55%; (l) HCl, MeOH, 24% for both 3d and 3e.
2
broad influenza programs at Alios, we investigated a variety of
nucleoside analogues as potential anti-influenza agents. One
series of the analogues investigated in our laboratories are
pyridine, pyridazine, and pyrimidine C-nucleoside, which retain
the carboxamide moiety of favipiravir. Like ribavirin and
favipiravir, the C-nucleosides are expected to mimic both
adenosine and guanosine via its two orientations of the
carboxamide when they base-paired with an RNA template.
Therefore, lethal viral mutagenesis may result from this series of
compounds. The goal of this drug discovery program is to
identify new chemical entities which can possess broad-
spectrum antiviral activity via a similar mechanism of action
to those of favipiravir and ribavirin. In this article, we report the
synthesis and in vitro anti-influenza activity of pyridine,
pyridazine, and pyrimidine C-nucleosides 3-8 (Figure 1). The
inhibitory effects of the 5′-triphosphate of 3c on viral
polymerases as well as the metabolic properties of 3c are also
presented.
CHEMISTRY
■
Synthesis of 3c, 3d, 3e, and 8 is shown in Scheme 1.
19
Compound 9 was coupled with the pyridine derivative 10 via
a nucleophilic addition in the presence of LDA to give 11.
Removal of the 1′-hydroxyl was achieved by reduction with
triethylsilane in the presence of a strong Lewis acid, which also
removed isopropylidene concomitantly. The isolated yield of
12 was relatively low, partly due to the generation of both α-
and β-1′-epimers. Conversion of the β-isomer 12 to the amide
13 was effected by an oxidative hydrolysis, and the latter was
subjected to desilylation to give 3c. Compound 3c was
B
DOI: 10.1021/acs.jmedchem.5b01933
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converted to the 4′,5′-olefin 14 by iodination at the 5′-position
and subsequent HI elimination in the presence of a base.
Introducing 4′-fluorine on a nucleoside was first reported by
similar procedure as described in Scheme 1. The 1′-OH of 22a
was converted to the 1′-O-acetate, which was reduced by
Et SiH more effectively than the 1′-OH. The resulting 23a was
3
20
23
Moffatt and co-workers using iodine and silver fluoride. Thus
far, most of the known 4′-fluoronucleosides were synthesized
using the same reagents. In addition to iodine and silver
fluoride, we also used NIS and TEA-3HF for introducing 4′-
fluoro to nucleosides, usually with better yields. Thus,
treatment of 14 with NIS and TEA-3HF yielded the 4′-
fluoro-5′-iodo intermediate 15 in good yield. Benzoylation of
oxidized to an N-oxide, which was treated with TMSCN to
give the nitrile 24a in good yield. Oxidative hydrolysis of 24a
yielded the amide, which was debenzylated with boron
trichloride to give 3f. Compound 3g was prepared from 22b
via the same sequence of reactions as described for 3f.
Compound 25a was reduced by catalytic hydrogenation to 25c,
which was debenzylated to give 3a. Compound 24b was
subjected to palladium-mediated coupling reactions to afford,
after oxidative hydrolysis and debenzylation, the methyl
derivative 3b, the vinyl derivative 3h, and the ethynyl derivative
3i, respectively, in good yields.
21
1
5 and subsequent oxidative hydrolysis yielded 16, which was
treated with ammonia to give 8 in good yield. Replacement of
the 3-fluorine of 12 with methoxy was complicated by 5′-O,C-
3-cyclization under strong basic condition, under which the 5′-
24
O-TBDPS group could be partially removed, therefore resulting
in a 5′-O,C-3-cyclized byproduct (not shown) and yielding 17
in only moderate yield. Oxidative hydrolysis of 17 yielded 18,
which was converted to 19 via demethylation and concomitant
desilylation by NaSEt. Treatment of 18 and 19 with HCl in
MeOH yielded 3d and 3e, respectively.
Synthesis of 4a is shown in Scheme 3. The lactone 27 was
reduced to the 1-OH product, which was treated with a
a
Scheme 3
Synthesis of 3a, 3b, 3f, 3g, 3h, and 3i is shown in Scheme 2.
Compounds 22a−b were prepared, respectively, by condensa-
22
tion of the lactone 20 with 3-halopyridines 21a−b by a
a
Scheme 2
a
Reagents and conditions: (a) i. DIBAL-H, toluene, −78 °C, 30 min,
9
4%; ii. Ph PCHCOOEt, CH CN, reflux, 16 h, 93%; (b) BuLi, −78
3
3
°
C, CH CN, 30 min, 69% ; (c) i. DMF-DMA, t-BuOCH(NMe ) , 100
3
2
2
°
C; ii. NH Ac, EtOH, reflux, 1 d, 56%; (d) i. 80% TFA/H O, 0 °C, 3
4
2
h, 50%; ii. 4 M HCl/MeOH, 60 °C, 2 d, 20%; (e) NH , THF, 100 °C,
3
1
5 h, 6%.
phosphonium ylide and yielded the ethyl ester 28 according to
25
a procedure for a similar compound. Reaction of 28 with
acetonitrile anion yielded the cyano derivative 29. Compound
30 was prepared by reaction of 29 with DMF-DMA/tert-
butoxybis(dimethylamino)methane and subsequent heating
with ammonium acetate by a procedure for a similar pyridone
26
compound. Compound 30 was converted to 31 by successive
removal of isopropylidene and benzyl groups. Compound 4a
was obtained by treatment with ammonia via amidine
formation and hydrolysis.
Synthesis of 4b and 4c is shown in Scheme 4. Compound
a
Reagents and conditions: (a) LDA, THF, −78 °C, 1 h; (b) LiHMDS,
27
3
2
was converted to the nitrile 33 in the same manner as 28.
Ac O, 70% (22a, 2 steps); (c) Et SiH, BF -Et O, DCM, 1 h, 51%
28
2
3
3
2
Treatment of 33 with TfN in the presence of triethylamine
3
(23a); (d) mCPBA, DCM, 2 h; (e) TMSCN, Et N, MeCN, reflux, 5 h,
3
yielded the diazo intermediate 34, which was subjected to a
5
1
8% (24a, 2 steps); (f) H O , K CO , DMSO, 1 h, 72% (25a); (g) H ,
2 2 2 3 2
reduction by PMe . After protection of 35 with Boc, the
3
0% Pd/C, 5 h, 36%; (h) BCl , DCM, −78 °C, 1 h, 62% (3f); (i)
3
resulting hydrazone 36 reacted with DMF-DMA to give a
mixture of the expected pyridazine derivative 37 and
unintended 38, which were separated by flash chromatography.
Compounds 37 and 38 were converted to the amides and then
AlMe , Pd(PPh ) , reflux, 10 h, 88%; (j) Bu SnCHCH , Pd(dppf)-
3
3
4
3
2
Cl , KF, dioxane, MW 120 °C, 0.5 h, 79%; (k) i. ethynyl-TMS,
2
Pd(PPh ) Cl , CuI, Et N, MW 135 °C, 2 h, 51%; ii. TBAF, THF, 5 h,
8
3
2
2
3
6%.
C
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Article
a
29
Scheme 4
known compound and then heated with concentrated sulfuric
acid to yield the dihydrooxazole 41. Treatment of 41 with BuLi
and subsequent reaction with the lactone 9 yielded the 1′-OH
product 42, which was reduced, via its 1′-OAc, by Et SiH.
3
Deprotection by acidic hydrolysis of the resulting 43 also
opened the dihydrooxazole ring and yielded the ester 44, which
was treated with ammonia to give 5a. Compound 43 was
readily converted to the 3-OMe product 45a, which was then
desilylated to 45b. The acidic deprotection of 45b yielded an
undesired byproduct 46, presumably via an intramolecular
amination of an intermediate similar to 47. Transamination of
4
6 with NH did not readily proceed as expected. Alternatively,
3
a controlled acidic hydrolysis of 45b and careful purification on
silica gel could provide 47, which was subjected to acetylation
under nonbasic conditions to yield a mixture of 48a and 48b.
Acetylation of NH could prevent the intramolecular amination.
2
Then, treatment of 48a and 48b with ammonia afforded, after
sugar deprotection, the desired 5b in good yield, which was
converted to 5c by a SN2 reaction. 3
0
Synthesis of 6 started with acid 49 (Scheme 6), which was
converted to the Weinreb amide 50 and then reacted with
TMS-ethylnyllithium to give 51. Cyclization of 51 with methyl
carbamimidothioate yielded the pyrimidine 52. The methylthio
was replaced, after oxidation, by a cyano group. The resulting
nitrile 53 was readily converted to 6 in the same manner as
described for 3f.
a
Reagents and conditions: (a) nBuLi, CH CN, 61%; (b) TfN , TEA,
3
3
MeCN, 36%; (c) PMe , THF, H O, 86%; (d) Boc O, py, 66%; (e)
DMF-DMA, THF, 12 h; (f) TEA, H O , MeOH, 83%; (g) 80% aq
TFA, 55% for 4b and 51% for 4c
3
2
2
2
2
Synthesis of 7 is shown in Scheme 7. The lactone 56,
31
prepared from 55, was coupled with 10 to afford the 1′-OH
product 57. Further treatment as described for 3f yielded 7.
To investigate the feasibility to bypass the first cellular
desilylated to give 4b and 4c, respectively, in the same manner
as described for 3c.
32
Synthesis of compounds 5a, 5b, and 5c is shown in Scheme
. Commercially available 39 was oxidized to the acid 40, which
phosphorylation of 3c, well-known bis-POM prodrug and
33
5
phosphoramidate prodrug were employed. Thus, the prodrug
59 and the prodrug 60 were prepared. For biochemical and
DMPK studies, nucleoside triphosphates 61 and 62 as well as
was converted to an amide by reaction of its anhydride form
with 2-amino-2-methylpropanol according to a procedure for a
a
Scheme 5
a
Reagents and conditions: (a) KMnO , H O, 12 h, 60%; (b) i. i-BuOC(O)Cl, NH CMe CH OH, 2 h, 38%; ii. H SO , 120 °C, 30 min, 55%; (c)
4
2
2
2
2
2
4
BuLi, THF, −78 °C, 2 h, 59%; (d) LiHMDS, Ac O, 88%; (e) BF -Et O, Et SiH, hexanes, 30 min, 36%; (f) 80% aq TFA, 30 min; (g) NH OH, 50
2
3
2
3
4
°
C, 12 h, 23% (2 steps); (h) NaOMe, dioxane, 6 h, 68%; (i) TBAF, THF, 1h; (j) 80% aq HCOOH, 4 h; 40%; (k) 2% TFA/DCM, 84%; (l) Ac O, 40
2
°C, 73%; (m) NH , MeOH, 80%; (n) TFA/H O (1:5), 80%; (p) EtSNa, DMF, 60 °C, 41%.
3
2
D
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a
Scheme 6
a
Reagents and conditions: (a) HATU, DIPEA, N,O-dimethylhydroxylamine, DCM, 8 h, 82%; (b) BuLi, TMS-acetylene, THF, −78 °C, then rt 3 h;
c) methyl carbamimidothioate-HCl, MeCN, 80 °C, 3 h, 32% (2 steps); (d) i. mCPBA, DCM, 3 h, 92%; ii. KCN, DMSO, overnight, 63%; (e) H O ,
(
2
2
NH OH, MeOH, 1 h, 57%; (f) BCl , DCM, 0 °C, 4 h, 41%.
4
3
a
Scheme 7
a
Reagents and conditions: (a) i. 80% TFA, 12 h, 90%; ii. PDC, DCM,
9
6
8
5/%; (b) LDA, THF −78 °C, 1 h, 37%; (c) i. LiHMDS, Ac O, rt,
2
5%; ii. Et SiH, BF −EtO, 1 h, 71%; (d) i. H O , K CO , DMSO, 1 h,
3
3
2
2
2
3
7%; ii. BCl , DCM, −78 °C, 1 h, 45%.
3
nucleoside monophosphate 63 were prepared from 3c and 2,
respectively, by general procedures.
IN VITRO ANTI-INFLUENZA ACTIVITY
Anti-influenza activity was obtained by using a neuraminidase
■
3
4
activity-based assay as described by Eichelberger et al. Madin-
Darby canine kidney (MDCK) epithelial cells were infected
with influenza strain A/WSN/33 (H1N1). All the compounds
in this report were tested for their inhibitory effects. Table 1
shows the compounds that have at least weak anti-influenza
activity while compounds with EC > 100 μM are not listed in
5
0
the table. As shown in Table 1, 3c has comparable activity to
2
7
favipiravir (1). The EC value of 1.9 μM for 3c is the average
5
0
of multiple repeats in the same assay. Compound 3c did not
exhibit cytotoxicity up to 400 μM. Although compound 3e
exhibited potent anti-influenza activity, it was almost equally
cytotoxic, which excludes the possibility for further develop-
ment as an anti-influenza agent. Compounds 3h and 3i, having
the same pyridine scaffold as 3c, showed only moderate
inhibition. Compounds 4a and 5a−c, in which the nitrogen of
the pyridine shifted position, showed little inhibition, with 5b
being weakly active. Compounds 4b and 4c, having a pyridazine
scaffold, showed little inhibitory effect. However, the
pyrimidine compound 6 showed significant activity. Com-
pounds 7 and 8, in which the sugars were modified at the 2′-
and 4′-positions by fluorination, had only weak activity.
Although 59, the bis-POM-prodrug of 3c, exhibited a good
activity, its potency did not exceed its parent compound 3c,
which suggested that 3c might be efficiently converted to its 5′-
monophosphate in cells. In contrast, 60, the phosphoramidate
prodrug of 3c, showed little inhibition (EC = 129 μM), which
50
might be ascribed to its lack of efficient conversion to the
monophosphate of 3c in MDCK cells.
INHIBITION OF INFLUENZA A POLYMERASE BY
THE 5′-TRIPHOSPHATE OF 3c (61)
■
The inhibitory effect of 61 on RNA synthesis catalyzed by the
recombinant influenza A polymerase (PA/PB1/PB2) complex
18
was performed as described previously. In the RNA synthesis
assay, the polymerase complex makes RNA products of ∼50
nucleotides (nt) in length when a 50-nt RNA template is used
E
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Table 1. Anti-Influenza Activity of 1, 2, and C-
a,b
Nucleosides
Compound
EC , μM
CC , μM
5
0
50
1
2
2.7
2.2
1.9
1.3
58
>400
>400
>400
2.0
3
3
3
3
4
5
6
7
8
5
c
e
h
i
152
36
98
c
b
91
291
88
207
8.7
87
>200
306
98
185
9
5.8
>400
a
Cell-based assay was performed in MDCK cells infected with A/
b
WSN/33 (H1N1). Each EC50 value is an average of n ≥ 2
determinations.
in the assay. As shown in Figure 2, 61 effectively inhibited the
RNA synthesis by the polymerase with an IC50 of 4.8 μM, a
Figure 3. Template directed nucleotide incorporation catalyzed by
influenza A polymerase. (A) Top, the reaction scheme using the U-
template to test the incorporation of 61 as an ATP analogue. Bottom,
the gel image of the reactions. (B) Top, the reaction scheme using the
C-template to test the incorporation of 61 as a GTP analogue. Bottom,
the gel image of the reactions. (C) 3c appears capable of base-pairing
with both cytidine and uridine.
INCORPORATION OF 61 INTO RNA AS THE A/G
ANALOGUE BY INFLUENZA POLYMERASE
■
Although 61 exhibited the inhibition of RNA synthesis in the
influenza polymerase assay, the inhibition was not necessarily
the only mechanism of action that contributed to the cell-based
anti-influenza activity. Similar to 2, compound 3c appears
capable of base-pairing with both cytidine and uridine (Figure
Figure 2. Polyacrylamide gel electrophoresis (PAGE) gel image and
the plot that demonstrates the inhibition of RNA synthesis by 61.
3
C); therefore, we tested whether 61 could be incorporated
into RNA by the influenza polymerase as an A or a G analogue
18
using a template-directed nucleotide incorporation assay. As
shown in Figure 3, A and B, 61 as substrate was indeed
incorporated into RNA with either U or C on the template.
When U was the nucleotide on the RNA template (Figure 3A),
similar level of inhibition as the 5′-triphosphate of 2 (62) (IC50
=
2.8 μM in our assay). As shown in Figure 3, 61 can be
61 was not only incorporated into the RNA, but also allowed
incorporated into RNA by the polymerase complex. As the
sugar moiety of 3c is an unmodified ribose with both 2′- and 3′-
hydroxyls, a complete chain termination was unlikely. However,
factors such as the unconventional base-pairing of 3c with the
RNA template and the orientation of the sugar moiety of 3c as
well as other unknown factors might significantly reduce the
incorporation efficiency of 3c and the next incoming
nucleotides, leading to the inhibition of full length RNA
synthesis in this in vitro assay. Previously, 62 was shown to
effectively terminate chain elongation when 62 was incorpo-
the incorporation of further nucleotides to form full length
products. As controls, the reactions with water and ATP yielded
a 9-mer and a 14-mer product, respectively. For the reaction
with water, the faint bands above the 9-mer were most likely
misincorporation products. For the reaction with 61, the 9-mer
was converted to longer RNA products, the majority of which
was 14-mer product, indicating that 61 was incorporated into
the RNA and the oligomer was further extended after its
incorporation. Its incorporation and extension profile was
similar to that of 62. The reaction with GTP served as a
control to indicate the level of GTP misincorporation opposite
U on the template. By comparison, 61 and 62 were completely
incorporated while GTP was only partially incorporated.
When C was the nucleotide on the template (Figure 3B), the
control reactions with water and GTP generated the expected
products. For 61, the 9-mer was completely converted to longer
18
18
rated into two consecutive positions in a growing RNA.
Similarly, two consecutive incorporations of 3c might play a
role in the inhibition of the chain elongation in a similar
manner.
F
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RNA products, the majority of which was 10-mer product,
indicating the incorporation of 61 into the RNA. The 14-mer
product was also formed, indicating further extension after the
incorporation of 3c. Again, the 61 incorporation and extension
profile was similar to that of 62. By comparison, misincorpora-
tion of ATP was incomplete. The reaction with 3′dGTP
showed the incorporation profile of an obligated chain
terminator: no further extension after the incorporation of
its prodrugs 59 and 60 in three cell types: A549, normal human
bronchiolar epithelial (NHBE), and MDCK cells according to a
36
standard procedure. In vitro NTP levels were obtained by
incubating each compound with each cell type for 24 h and
analyzing the cell extracts for the corresponding NTP
concentration. As shown in Table 3, a much higher level of
61 was observed in MDCK cells than in the other two cell types
for 3c while there were moderate to high levels of 5′-
monophosphate of 3c (63) in all three types of cells. The
results suggested that conversion of 63 to 61 was efficient only
in MDCK cells. The levels of 61 are correlated well with the
observed in vitro anti-influenza activity, indicating that 61 was
the active metabolite. Compounds 59 and 60, the prodrugs of
3c, were also tested for their nucleotide formation in the cells.
The bis-POM prodrug 59 yielded similar level of 61 in MDCK
cells as 3c and moderate to high level of 63 in all three types of
cells while the phosphoramidate prodrug 60 yielded low to
moderate level of 63 and below-detection level of 61.
Compared to metabolism of 3c to 63, conversion of 63 to
61 appears to be rate-limiting in NHBE and A549 cells. Other
five C-nucleosides (3a, 3f, 4c, 6 and 8) in Figure 1 were also
subjected to the same testing in A549 cells and they generated
either very low or below-detection levels of NTPs.
3′dGTP.
Incorporation of 61 and 62 into RNA sequence opposite
both U and C on the templates indicates that they can serve as
analogues of both ATP and GTP. Further extension after their
incorporation indicates that they can be incorporated and exist
in the RNA molecule. Such incorporation of an ambiguous
base-pairing nucleoside into the influenza viral RNA sequence
may introduce mutations to the genomic RNA during the next
rounds of viral RNA transcription/replication. It has been
shown that favipiravir exerted strong mutagenic effects on
influenza virus and norovirus that generated nonviable
1
5−17
viruses,
and the molecular basis of this effect was linked
14,18
to its ambiguous base-paring property.
Similarly, lethal viral
mutagenesis combined with polymerase inhibition could be the
primary mechanism for the observed antiviral activity of 3c in
the cell-based assay.
To assess in vivo nucleotide formation, female Balb/c mice
were dosed with compound 59 by IV route. The studies were
conformed to regulations and guidelines regarding animal care
ANTIVIRAL ACTIVITY SPECTRUM OF 61
The inhibitory effect of 61 on RNA synthesis catalyzed by other
■
37
and welfare. In vivo, high level of 3c was observed in blood
and high level of 63 was observed in the lung (Table 4).
viral RNA polymerases was also measured, using standard
14,35
internal procedures.
As shown in Table 2, the RdRp activity
Table 4. Formation of NTPs from 59 in Mouse
Table 2. Inhibition of Various RNA Polymerases
Conc after IV dose of 59
a
Viral polymerase
IAV pol
4.8
HCV NS5B
5.3
HRV16 pol
4.0
NV pol
3.5
Metabolites of 59
0.25 h
2 h
IC50 (μM)
3c (nM) in blood
63 (nM) in lung
61 (nM) in lung
1436
1764
BQL
422
a
IAV pol stands for influenza RNA polymerase, HCV NS5B is for
792
hepatitis C virus RNA polymerase; HRV16 pol is human rhinovirus
type 16 RNA polymerase; NV pol is for human norovirus RNA
polymerase.
BQL
However, the level of 61 was below the limit of quantitation in
the lung (60 nM), consistent with the low levels of 61 and the
weak anti-influenza activity in NHBE and A549 cells.
Conversion of 63 to 61, therefore, appeared to be a rate-
limiting step in the lung.
of hepatitis C virus, rhinovirus, and norovirus was almost
equally inhibited by 61 with an IC50 of 5.3, 4.0, and 3.5 μM,
respectively. These results indicate that 3c may have the
potential to be a broad-spectrum antiviral agent if it can be
phosphorylated to 61 in infected cells.
CONCLUSION
■
A series of novel pyridine, pyridazine, and pyrimidine C-
nucleosides as favipiravir analogues were synthesized, and 3c
demonstrated potent anti-influenza activity and an excellent
selectivity window in MDCK cells. The 5′-triphosphate of 3c
exhibited potent inhibition of RNA synthesis by influenza A
polymerase in an enzyme assay. Both influenza polymerase
inhibition and lethal viral mutagenesis are assumed to be
responsible for the potent anti-influenza activity. Compound 3c
METABOLIC PROPERTIES OF 3c AND ITS
PRODRUGS
In vivo nucleoside 5′-triphosphate (NTP) levels in the target
tissue are critical to in vivo efficacy of an antiviral nucleoside
polymerase inhibitor. In vitro NTP formation in appropriate
cell lines may provide the first assessment for predicting in vivo
NTP level. Thus, we measured the in vitro NTP level of 3c and
■
Table 3. Formation of NTPs from 3c and Its Prodrugs in Three Types of Cells and in Vitro Activity in Different Cells
NTP (pmol/million cells)
NMP (pmol/million cells)
Anti-influenza EC50 (μM)
a
b
Compd
NHBE
A549
MDCK
NHBE
A549
MDCK
NHBE
A549
MDCK
3
5
6
c
21.6
7.22
BLQ
11.6
5.00
BLQ
192
262
113
42.6
41.0
74.0
59.5
48.5
248
44.7
29.5
57.5
52.0
34.7
74.1
1.9
5.8
129
9
0
179
c
BLQ
NT
a
b
Cell-based assay was performed in NHBE cells infected with A/WSN/33 (H1N1). Cell-based assay was performed in A549 cells infected with A/
c
WSN/33 (H1N1). NT: not tested.
G
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
could be phosphorylated to its monophosphate 63 without the
need of monophosphate prodrugs in all three types of cells
used. The levels of its triphosphate 61 were good only in
MDCK cells while they were below levels of quantitation in
A549 and NHBE cells. Consistent with the in vitro results in
NHBE cells, conversion of 63 to 61 was identified as a rate
limiting step in the mouse lung. As 61 also showed inhibition of
other RNA viral polymerases, investigation into the potential of
MeOH (100 mL) was refluxed for 16 h and then concentrated.
Chromatography with 0.5% NH OH and 5% MeOH in DCM gave 3c
4
1
(
4
500 mg, 67%) as a white solid. H NMR (DMSO-d ) δ 8.38 (d, J =
.4 Hz, 1H, H-6), 7.98 (br s, 1H, NH), 7.80 (t, J = 4.4 Hz, 1H, H-5),
6
7
.63 (br s, 1H, NH), 5.25 (d, J = 6.0 Hz, 1H, H-1′), 5.01 (d, J = 4.8
Hz, 1H, 2′- or 3′−OH), 4.97 (d, J = 5.2 Hz, 1H, 2′- or 3′−OH), 4.89
(
3
t, J = 5.2 Hz, 1H, 5′−OH), 3.82−3.88 (m, 3H, H-2′, H-3′, H-4′),
19
.53−3.66 (m, 2H, H-5′). F NMR (DMSO-d ) δ −128.35 (d, J = 4.9
+
6
Hz). MS, m/z 272.9 (M + H) .
3
c as antiviral against various RNA viruses is underway.
Compound 14. A solution of 3c (0.83 g, 3.05 mmol), PPh (1.29
3
g, 4.88 mmol) and iodine (1.162 g, 4.57 mmol) in pyridine (10 mL)
was stirred overnight. The reaction was quenched with saturated
Na S O (30 mL). After workup, the residue was purified on silica gel
EXPERIMENTAL SECTION
■
2
2
3
Favipiravir (1) and its riboside (2) were prepared according to patent
applications from Toyama Chemical Co.
with 1% MeOH in DCM to give the 5′-iodo intermediate (590 mg,
51%), which was dissolved in THF (10 mL), followed by addition of
DBU (1.17 g, 7.7 mmol). The resulting mixture was stirred at 60 °C
overnight, then quenched with 1 N HCl. After workup, chromatog-
3
8,39
All commercially
obtained solvents and reagents were used as received. All solvents
used for chemical reactions were anhydrous grade, unless specifically
indicated. Structures of the target compounds in this work were
assigned by use of NMR spectroscopy and MS spectrometry. The
purities of all compounds were >95% as determined on an Agilent
1
raphy with 80% EA in PE gave 14 (350 mg, 89%) as a white solid. H
NMR (CD OD) δ 8.44 (m, 1H), 7.56 (m, 1H), 5.42 (d, J = 3.6 Hz,
3
1H), 4.51 (m, 1H), 4.42 (m, 1H), 4.28 (m, 1H), 4.10 (m, 1H).
Compound 15. To an ice-cold solution of 14 (360 mg, 1.42
mmol) in MeCN (5 mL) was added TEA-3HF (343 mg, 2.12 mmol),
followed by NIS (400 mg, 1.77 mmol). The resulting mixture was
stirred at rt overnight, then the precipitate was filtered and washed
with DCM to give the crude 15 (380 mg, 67%) as a white solid.
3-Fluoro-4-(4-fluoro-β-D-ribofuranosyl)-2-pyridinecarboxamide
(8). To a solution of 15 (380 mg, 0.95 mmol) in pyridine (4 mL) was
added BzCl (320 mg, 2.28 mmol) at 0 °C. The resulting mixture was
stirred at rt for 30 min, then quenched with water. After workup,
chromatography with 2% MeOH in DCM gave the 2′,3′-dibenzoyl
1
0
200 HPLC, XTerra 3.5 μm 4.6 × 150 mm MS C18 column, using
.04 (v/v) TFA in water and 0.02 (v/v) TFA in acetonitrile as mobile
1
phase. H NMR spectra were recorded on a Bruker Advance III (400
MHz) or a Varian 400MR (400 MHz) NMR spectrometer. Chemical
shifts are reported in parts per million (ppm, δ) using residual solvent
line as an internal reference. Splitting patterns are reported as s
(
(
singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s
broad singlet). Coupling constants (J) are reported in herz (Hz).
Mass spectrometric analyses for nucleosides were performed on an
Agilent 1200 HPLC with Agilent 6110/6140/1956C MSD mass
spectrometer using ESI as ionization, Phenomenex Luna C18 5 μm 5.0
intermediate (433 mg, 75%). An aqueous solution of Bu NOH (55%,
4
×
20 mm column; mobile phase: 0.04% (v/v) TFA in water and
.02%(v/v) TFA in acetonitrile, 40 °C, flow rate 0.4 mL/min. Mass
spectrometric analyses for nucleotides were performed on an Agilent
100 HPLC with API 2000 LC-MS/MS System using ESI as
3.95 mL, 7.9 mmol) was acidified with TFA to pH ∼ 4. To this
solution at 0 °C was added the dibenzoyl intermediate (200 mg, 0.33
mmol), followed by mCPBA (480 mg, 2.8 mmol). The resulting
mixture was stirred at rt overnight, then quenched with saturated
Na S O (30 mL). After workup, chromatography with 90% EA in PE
0
1
ionization, Synergi 75 × 2.0 mm, 4 μm Hydro-RP80 Å column, 50
mM TEAA in water and 50 mM in acetonitrile, flow rate 0.4 mL/min.
Workup procedures for most of chemical reactions are the same or
similar, therefore, unless specifically indicated, the workup refers to the
following procedure: the reaction mixture at 0 °C is quenched with
2
2
3
gave 16 (110 mg, 67%) as a white solid. A solution of 16 (120 mg,
0.24 mmol) in NH /MeOH (7 N, 10 mL) was stirred for 5 h and then
3
concentrated. The crude product was washed thoroughly with DCM
1
to give 8 (50 mg, 73%) as a white solid. H NMR (CD OD) δ 8.45 (br
3
water or 5% NaHCO , diluted with ethyl acetate or dichloromethane,
s, 1H, H-6), 7.80 (br s, 1H, H-5), 5.50 (br s, 1H, H-1′), 4.11−4.29 (m,
3
1
9
washed with 5% sodium bicarbonate and then with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated to dryness.
Unless specifically indicated, chromatography refers to a flash
chromatography on a silica gel column.
2H, H-2′, H-3′), 3.83 (m, 2H, H-5′a, H-5′b). F-NMR (CD OD) δ
3
+
−124.7, −127.4. MS, m/z 312.9 (M + Na) .
Compound 17. A suspension of 12 (3.0 g, 6.1 mmol), 2,2-
dimethoxypropane (4.2 g, 40.3 mmol) and TsOH-H O (0.3 g, 1.6
2
Compound 11. To a solution of 10 (5.72 g, 46.9 mmol) in THF
mmol) in acetone (50 mL) was stirred overnight, then quenched with
(
50 mL) at −78 °C was added LDA in THF (2.0 M, 23.5 mL). The
solid NaHCO to pH ∼ 7, and filtered to remove the salt. After
3
mixture was stirred at −78 °C for 30 min, and then 9 (20.0 g, 46.9
mmol) in THF (150 mL) was added. The resulting mixture was stirred
at −78 °C for 6 h, quenched with Saturated NH Cl, and extracted with
ethyl acetate. After workup, chromatography with 20% EA in PE gave
workup, chromatography with 2% MeOH in DCM gave the 2′,3′-
isopropylidene intermediate (3.2 g). A solution of the intermediate
(450 mg, 0.84 mmol) and NaOMe (177 mg, 3.2 mmol) in 1,4-dioxane
(5 mL) was stirred for 3 h, then quenched with aqueous HCl. The
4
1
1
4.0 g (54%) of 11 as a white solid. H NMR (CDCl ) δ 8.25−8.36
crude product was purified on a preparative TLC (silica gel, 40% EA in
3
1
(
5
m, 1H), 7.45−7.87 (m, 11H), 5.15−5.20 (m, 1H), 3.90−4.25 (m,
PE) to give 17 (130 mg, 28%) as a white solid. H NMR (CDCl ) δ
3
H), 2.75−2.84 (m, 1H), 2.29−2.41 (m, 1H), 1.07 (s, 9H).
8.24 (m, 1H), 7.63−7.68 (m, 5H), 7.38−7.41 (m, 6H), 5.18 (d, J = 4.8
Hz, 1H), 4.74 (dd, J = 3.6 Hz, 6.4 Hz, 1H), 4.47 (m, 1H), 4.22 (m,
1H), 4.14 (s, 3H), 4.00 (dd, J = 3.2 Hz, 11.2 Hz, 1H), 3.86 (dd, J = 3.6
Hz, 11.6 Hz, 1H) 1.62 (s, 3H), 1.34 (s, 3H), 1.05 (s, 9H).
Compound 12. To a mixture of 11 (14 g, 25.5 mmol), Et SiH
3
(
(
118 g, 101.7 mmol) in DCM (140 mL) at 0 °C was added BF -Et O
4.2 mL, 33.6 mmol). The mixture was stirred at 0 °C for 1 h, and
3 2
then more BF -Et O (11.2 mL, 90.7 mmol) was added. The mixture
Compound 19. To a solution of 17 (820 mg, 1.5 mmol) in MeOH
and water (10:1, 4 mL) was added aqueous ammonia (10%, 13 mL),
followed by H O (30%, 13 mL) at rt, and the resulting mixture was
3
2
was stirred at rt for 16 h. After workup, chromatography with 20−50%
EA in PE gave 12 (3.0 g, 24%) as a white solid.
2
2
Compound 13. To a mixture of 12 (2.7 g, 5.5 mmol) in MeOH/
stirred for 8 h, then quenched with Na SO . After workup,
2 3
H O (10:1, 8 mL) was added aqueous ammonia (10%, 20 mL),
chromatography with 80% EA in PE gave 18 (618 mg, 73%) as a
white solid. To a solution of EtSH (713 mg, 11.5 mmol) in DMF (18
mL) was added NaH (60% in mineral oil, 460 mg, 11.5 mmol) at 0 °C.
The mixture was stirred at 0 °C for 1 h and a solution of 18 (920 mg,
1.63 mmol) in DMF (10 mL) was added. The reaction mixture was
stirred at 40 °C for 4 h, then quenched with water. The crude product
2
followed by H O (30%, 8 mL) at rt. The mixture was stirred for 30
2
2
min, then quenched with saturated Na SO . After workup,
2
3
chromatography with 3% MeOH in DCM gave 13 (1.4 g, 50%) as a
1
white solid. H NMR (CD OD) δ 8.15 (d, J = 5.2 Hz, 1H), 7.82 (m,
3
1
(
H), 7.73 (m, 4H), 7.38−746 (m, 6H), 5.18 (d, J = 4.4 Hz, 1H), 4.20
m, 1H), 4.04−4.08 (m, 3H), 3.90 (m, 1H), 1.08 (s, 9H).
-Fluoro-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3c). A
solution of 13 (1.4 g, 2.75 mmol) and NH F (4.2 g, 11.3 mmol) in
was purified by RP-HPLC (0.1% HCOOH in water and MeCN) to
1
3
give 19 (282 mg, 55%) as a white solid. H NMR (DMSO-d ) δ 13.29
6
(s, 1H), 8.57 (s, 1H), 8.22 (s, 1H), 8.12 (d, J = 4.0 Hz, 1H), 7.65 (d, J
4
H
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
=
(
4.0 Hz, 1H), 5.12 (d, J = 2.8 Hz, 1H), 4.95 (t, J = 5.4 Hz, 1H), 4.63
m, 2H), 4.02 (m, 1H), 3.57 (m, 2H), 1.51 (s, 3H), 1.27 (s, 3H).
-Methoxy-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3d). A
4-(β-D-Ribofuranosyl)-2-pyridinecarboxamide (3a). A mixture of
25a (300 mg, 0.54 mol) and Pd−C (10%, 150 mg) in MeOH (10 mL)
3
was stirred under H balloon for 5 h. The catalyst was filtered through
2
solution of 18 (230 mg, 0.41 mmol) and concentrated hydrochloric
acid (0.02 mL) in MeOH (2 mL) was stirred at 40 °C for 5 h, then
silica gel pad and washed with MeOH. The filtrate was concentrated,
the residue was purified on silica gel (50% EA in PE) to give 25c (100
mg, 36%) as a white solid. MS, m/z 525.1 (M + H) . Compound 25c
+
neutralized to pH ∼ 7 with NH OH and concentrated. The residue
4
was purified by RP-HPLC (NH HCO in water and MeCN) to give
(100 mg, 0.19 mmol) was converted to 3a (30 mg, 62%) as a white
4
3
1
1
3
4
7
d (28 mg, 24%) as a white solid. H NMR (DMSO-d ): δ 8.28 (d, J =
solid by the same procedure as described for 3f. H NMR (CD
3
OD) δ
6
.8 Hz, 1H, H-6), 7.87 (s, 1H, NH), 7.69 (d, J = 4.4 Hz, 1H, H-5),
.51 (s, 1H, NH), 5.05 (br, 3H, 2′−OH, 3′−OH, 5′−OH), 4.98 (d, J
4.4 Hz, 1H, H-1′), 3.81−3.92 (m, 6H, H-2′, H-3′, H-4′, OMe), 3.79
8.57 (d, J = 5.2 Hz, 1H, H-6), 8.20 (t, J = 0.8 Hz, 1H, H-3), 7.65 (dd, J
= 5.2, 1.2 Hz, 1H, H-5), 4.78 (d, J = 2.8 Hz, 1H, H-1′), 4.01−4.05 (m,
=
2H, H-2′, H-3′), 3.70−3.83 (m, 3H, H-4′, H-5′a, H-5′b). MS, m/z
+
2
55.0 (M + H) .
(
1
dd, J = 3.0 Hz, 11.8 Hz, 1H, H-5′a), 3.57 (dd, J = 3.6 Hz, 11.6 Hz,
+
Compound 22b. Compound 22b (1.2 g, 20%) as a colorless syrup
was prepared from 21 (3.0 g, 19.1 mmol) and 20 (4.0 g, 9.6 mmol) by
the same procedure as described for 22a.
H, H-5′b). MS, m/z 306.9 (M + Na) .
-Hydroxy-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3e). By
3
the same procedure as described for 3d, 19 (49 mg, 0.16 mmol) was
1
Compound 23b. Compound 23b (650 mg, 34%) as a white solid
converted to 3e (10 mg, 24%) as a white solid. H NMR (CD OD) δ
3
was prepared from 22b (2.1 g, 3.4 mmol) by the same procedure as
8
=
.01 (d, J = 3.6 Hz, 1H, H-6), 7.65 (d, J = 3.6 Hz, 1H, H-5), 5.08 (d, J
3.6 Hz, 1H, H-1′), 3.96 (m, 1H, H2′), 3.80−3.93 (m, 3H, H-3′, H-
1
described for 23a. H NMR (CDCl ) δ 8.56 (s, 1H), 8.14 (d, J = 5.2
3
Hz, 1H), 7.69 (d, J = 5.2 Hz, 1H), 7.19−7.31 (m, 15H), 5.29 (d, J =
4
′, H-5′a), 3.67 (dd, J = 4.4 Hz, 12.0 Hz, 1H, H-5′b). MS, m/z 270.9
+
2.4 Hz, 1H), 4.70 (s, 2H), 4.45−4.56 (m, 3H), 4.31−4.39 (m, 2H),
(
M + H) .
4
.03 (dd, J = 4.4 Hz, 7.6 Hz, 1H), 3.85−3.90 (m, 2H), 3.65 (dd, J = 2.8
Compound 23a. To a stirred solution of 21a (2.0 g, 17.7 mmol)
in THF (50 mL) at −78 °C was added dropwise LDA in THF (2 M,
Hz, 10.8 Hz, 1H).
Compound 24b. Compound 23b (0.9 g, 1.6 mmol) was
8
.85 mL). The resulting mixture was stirred at this temperature for 30
14
converted to 24b (500 mg, 54%) as a white solid by the same
min, then a solution of 20 (3.7 g, 8.85 mmol) in THF (10 mL)
added dropwise. After stirring for one more hour at −78 °C, the
reaction mixture was quenched with aqueous NH Cl. After workup,
1
procedure as described for 24a. H NMR (CDCl ) δ 8.12 (d, J = 4.4
3
Hz, 1H), 7.95 (d, J = 4.8 Hz, 1H), 7.26 (m, 15H), 5.25 (s, 1H), 4.34−
4
4
5
.77 (m, 7H), 4.04 (m, 1H), 3.89 (m, 2H), 3.62 (m, 1H). MS, m/z
85.0 (M + H) .
Compound 25b. Compound 24b (250 mg, 0.43 mmol) was
chromatography with 20% EA in PE gave a 1′-hydroxy nucleoside (4.1
g, 87%) as colorless syrup. To a stirred solution of the 1′-hydroxy
nucleoside (2.1 g, 3.95 mmol) in THF (10 mL) was added dropwise
LiHMDS (1.0 M in THF, 5.9 mL) at rt, then stirring continued for 10
+
converted to 25b (150 mg, 58%) as a white solid by the same
+
procedure as described for 25a. MS, m/z 604.7 (M + H) .
min. To the above mixture was added dropwise Ac O (610 mg, 6.0
2
3
-Bromo-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3g).
mmol), then stirring continued for 20 min. More LiHMDS and Ac O
2
Compound 25b (150 mg, 0.25 mmol) was converted to 3g (50 mg,
were added until the reaction was completed. After workup,
1
6
1%) as a white solid by the same procedure as described for 3f. H
chromatography with 25% EA in PE gave the 1′-O-acetyl intermediate
NMR (CD OD) δ 8.46 (d, J = 5.2 Hz, 1H, H-6), 7.89 (d, J = 4.8 Hz,
3
2
2a (1.8 g, 80%). To a stirred solution of 22a (2.0 g, 3.5 mmol) in
1
3
H, H-5), 5.23 (d, J = 2.8 Hz, 1H, H-1′), 3.93−4.04 (m, 4H, H-2′, H-
DCM (20 mL) was added dropwise Et SiH (12.1 g, 104.7 mmol) and
3
′, H-4′, H-5′a), 3.79 (dd, J = 12.4, 4.0 Hz, 1H, H-5′b). MS, m/z 333.0
BF -Et O (743 mg, 5.23 mmol) at rt, and the mixture was stirred for 1
+
3
2
(M + H) .
h. The reaction was quenched with aqueous NaHCO . After workup,
3
Compound 26a. To a vigorously stirred solution of 24b (320 mg,
chromatography with 25% EA in PE gave 23a (910 mg, 56%) as a
0
.55 mmol) and Pd(PPh ) (31 mg, 0.027 mmol) in THF (5 mL)
1
3 4
white solid. H NMR (CDCl ) δ 8.48 (s, 1H), 8.19 (d, J = 4.8 Hz,
3
under nitrogen at rt was added Me Al (1 M in toluene, 1.1 mL, 1.1
3
1
H), 7.75 (d, J = 5.2 Hz, 1H), 7.25−7.33 (m, 15H), 5.41 (d, J = 2.4
mmol). The resulting mixture was refluxed for 10 h, then cooled,
Hz, 1H), 4.75 (d, J = 12.0 Hz, 1H), 4.70 (d, J = 12.0 Hz, 1H), 4.40−
poured into saturated NaH PO and extracted with EA. Chromatog-
2
4
4.60 (m, 3H), 4.39 (m, 2H), 4.05 (dd, J = 4.6 Hz, 7.8 Hz, 1H), 3.89−
3.98 (m, 2H), 3.69 (dd, J = 2.8 Hz, 10.8 Hz, 1H). MS, m/z 516.1 (M +
raphy with 30% EA in PE gave 26a (250 mg, 88%) as a white solid.
+
MS, m/z 521.1 (M + H) .
+
H) .
3
-Methyl-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3b). To a
Compound 24a. To a stirred solution of 23a (1.0 g, 1.94 mmol)
stirred solution of 26a (210 mg, 0.40 mmol) in DMSO (10 mL) were
in anhydrous DCM (10 mL) was added mCPBA (666 mg, 3.9 mmol)
at rt under nitrogen. The reaction mixture was stirred for 2 h, then
quenched with aqueous Na SO . After workup, chromatography gave a
added K CO (221 mg, 1.61 mmol) and H O (30%, 3.6 mL) at rt,
2
3
2
2
and the mixture was stirred for 1 h. Then the reaction was quenched
with Na SO . After workup, chromatography with 50% EA in PE gave
2
3
2
3
crude product, which was dissolved in acetonitrile (10 mL). TMSCN
1.62 g, 16.6 mmol) was added, followed by addition of TEA (1.65 g,
6.3 mmol). The resulting mixture was refluxed for 5 h, then cooled
and quenched with NaHCO . After workup, chromatography with
the amide product (150 mg, 69%) as white solid. The amide (135 mg,
(
1
0
.25 mmol) was converted to 3b (50 mg, 74%) as a white solid by the
1
same procedure as described for 3f. H NMR (CD OD) δ 8.35 (d, J =
3
3
5.2 Hz, 1H, H-6), 7.78 (d, J = 5.2 Hz, 1H, H-5), 5.13 (d, J = 4.8 Hz,
3
0% EA in PE gave 24a (0.6 g, 58%) as a white solid.
1
H, H-1′), 3.96−3.99 (m, 2H, H-2′, H-3′), 3.87−3.91 (m, 2H, H-4′,
Compound 25a. To a stirred solution of 24a (735 mg, 1.36
H-5′a), 3.77 (dd, J = 4.4 Hz, 12.0 Hz, 1H, H-5′b), 2.54 (s, 3H, Me).
MS, m/z 269.0 (M + H) .
+
mmol) in DMSO (20 mL) was added K CO (750 mg, 5.4 mmol) and
2
3
H O (30%, 12.3 mL) at rt. The resulting mixture was stirred for 1 h,
2
2
Compound 26b. To a stirred solution of 24b (700 mg, 1.19
mmol) in dioxane (2 mL) under nitrogen were added tributyl(vinyl)-
then quenched with Na SO . After workup, chromatography with 50%
2
3
EA in PE gave 25a (550 mg, 72%) as a white solid.
-Chloro-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3f). To a
stirred solution of 25a (220 mg, 0.39 mmol) in DCM (6 mL) at −78
C under nitrogen was added BCl (1.0 M in DCM, 4.7 mL). The
stannane (3.79 g, 12.0 mmol), Pd(dppf)Cl (40 mg) and KF (30 mg,
2
3
0.52 mmol). The reaction vessel was sealed and heated under
microwave at 120 °C for 30 min. After workup, chromatography with
30% EA in PE gave 26b (501 mg, 79%) as a white solid. MS, m/z
°
3
+
mixture was stirred at this temperature for 1 h, then quenched with
pyridine in MeOH. The solvent was removed and the residue purified
by RP-HPLC (NH HCO in water and MeCN) to give 3f (70 mg,
533.0 (M + H) .
4-(β-D-Ribofuranosyl)-3-vinyl-2-pyridinecarboxamide (3h). Com-
pound 26b (600 mg, 1.12 mmol) was converted to the amide product
(550 mg, 89%) as a white solid by the same procedure as described in
preparation of 3b.The amide (250 mg, 0.45 mmol) was converted to
4
3
1
6
2%) as a white solid. H NMR (CD OD) δ 8.45 (d, J = 5.2 Hz, 1H,
3
H-6), 7.92 (d, J = 4.8 Hz, 1H, H-5), 5.26 (d, J = 2.8 Hz, 1H, H-1′),
3
1
.92−4.01 (m, 4H, H-2′, H-3′, H-4′, H-5′a), 3.79 (dd, J = 12.0, 4.0 Hz,
3h (81 mg, 64%) as a white solid by the same procedure as described
+
1
H, H-5′b). MS, m/z 289.0 (M + H) .
for 3b. H NMR (CD OD) δ 8.42 (d, J = 5.2 Hz, 1H, H-6), 7.82 (d, J
3
I
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
=
5.2 Hz, 1H, H-5), 7.06 (dd, J = 17.6, 11.6 Hz, 1H, CHCH ), 5.55
mL) at rt was added P(CH ) (1.0 M in THF, 32.0 mL). The mixture
was stirred for 15 min, then quenched with water. After concentration,
2
3 3
(
1
=
dd, J = 1.6 Hz, 11.6 Hz, 1H, CHCHaHb), 5.49 (dd, J = 1.4 Hz,
7.8 Hz, 1H, CHCHaHb), 5.14 (d, J = 5.2 Hz, 1H, H-1′), 4.03 (t, J
chromatography (15% EA in PE) gave 35 (10.4 g, 86.2%) as a white
1
5.2 Hz, 1H, H-2′), 3.94−3.97 (m, 2H, H-3′, H-4′), 3.87 (dd, J =
solid. H NMR (CDCl ) δ 7.72−7.74 (m, 2H), 4.64−4.66 (m, 1H),
3
1
2.0, 2.8 Hz, 1H, H-5′a), 3.76 (dd, J = 12.0, 4.4 Hz, 1H, H-5′b). MS,
4.37−4.46 (m, 2H), 4.04−4.06 (m, 1H), 3.68−3.69 (m, 2H), 2.98−
3.20 (m, 2H), 1.53 (s,3H), 1.33 (s,3H), 0.89 (s, 3H), 0.05 (s, 6H).
Compound 37 and 38. A solution of 35 (10.0 g, 25.2 mmol) and
+
m/z 280.9 (M + H) .
Compound 26c. To a stirred solution of 24b (200 mg, 0.342
mmol) in TEA (1.0 mL) under nitrogen were added ethynyltrime-
thylsilane (334 mg, 3.42 mmol), CuI (10 mg) and Pd(PPh ) Cl (20
mg). The mixture was heated under microwave at 135 °C for 1 h. After
workup, chromatography with 30% EA in PE gave the 3-(TMS-
ethynyl)pyridine (105 mg, 51%) as a white solid. A solution of this
product (110 mg, 0.18 mmol) and TBAF (72 mg, 0.27 mmol) in THF
(Boc) O (5.6 g, 25.6 mmol) in pyridine (20 mL) was stirred at room
2
temperature for 3 h. After workup, chromatography (10% EA in PE)
gave 36 (8.3 g, 66%) as a white solid. A solution 36 (3.8 g, 7.6 mmol)
and DMF-DMA (4.1 g, 34.2 mmol) in THF (10 mL) was stirred at rt
for 12 h, treated with water, and stirred for 30 min. The solvent was
evaporated and the residue purified by chromatography (50% EA in
3
2
2
(
2
3 mL) was stirred at rt for 5 h. After workup, chromatography with
PE) to give 37 (385 mg) and 38 (238 mg), both as a white solid.
1
0% EA in PE gave 26c (80 mg, 86%) as a white solid. MS, m/z 531.1
Compound 37: H NMR (CDCl ) δ 8.65 (s, 1H), 5.09 (d, J = 3.2 Hz,
3
+
(
M + H) .
-Ethynyl-4-(β-D-ribofuranosyl)-2-pyridinecarboxamide (3i).
Compound 26c (160 mg, 0.30 mmol) was converted to 3i (31 mg,
1H) 4.67 (m, 2H), 4.38 (d, J = 2.0 Hz, 1H), 3.94 (dd, J = 2.2 Hz, 11.0
Hz, 1H), 3.76 (dd, J = 3.0 Hz, 11.4 Hz, 1H), 1.58 (s, 3H), 1.39 (1s,
3H), 0.79 (s, 9H), 0.02 (s, 3H), −0.02 (s, 3H). MS, m/z 408.1 (M +
3
1
+
1
3
7%) as a white solid by the same procedure as described for 3b. H
H) . Compound 38: H NMR (CDCl ): δ 8.05 (s, 1H), 5.07 (s, 1H),
3
NMR (CD OD) δ 8.51 (d, J = 4.8 Hz, 1H, H-6), 7.93 (d, J = 4.8 Hz,
4.59 (m, 2H), 4.18 (m, 1H), 3.97 (s, 3H), 3.88 (dd, J = 2.6 Hz, 11.4
Hz, 1H), 3.72 (dd, J = 3.6 Hz, 11.6 Hz, 1H) 1.58 (s, 3H), 1.32 (s, 3H),
0.87 (s, 9H), 0.05 (s, 6H). MS, m/z 421.9 (M + H) .
3
1
4
1
H, H-5), 5.36 (d, J = 3.2 Hz, 1H, H-1′), 4.23 (s, 1H, CCH), 3.92−
+
.03 (m, 4H, H-2′, H-3′, H-4′, H-5′a), 3.79 (dd, J = 12.0 Hz, 4.0 Hz,
+
H, H-5′b). MS, m/z 279.0 (M + H) .
3-Carbamoyl-5-(β-D-ribofuranosyl)-4−1H-pyridazinone (4b). To
a stirred solution of 37 (370 mg, 0.9 mmol) in anhydrous MeOH (3
mL) at rt was added H O (30%, 10 mL) and TEA (400 mg, 4.0
Compound 28. To a solution of 27 (13.5 g, 48.5 mmol) in toluene
(
90 mL) at −78 °C was added DIBAL-H (1 M in toluene, 73 mL).
2
2
The mixture was stirred at −70 °C for 30 min, then quenched with
acetone. After workup, chromatography (10% EA in PE) gave the
desired 1-OH product (12.8 g, 94%) as yellow oil. A mixture of the 1-
OH i nter medi a te (15. 4 g, 55 mmol) and e thyl
mmol). The resulting mixture was stirred for 12 h and then quenched
with aq. Na SO . After workup, chromatography (50% EA in PE) gave
2
3
the desired amide (320 mg, 83%), which was dissolved in 80% aq.
TFA (5 mL) and stirred at rt for 1 h. After removal of volatiles, the
residue was purified by RP-HPLC (NH HCO in water and MeCN)
(
(
triphenylphosphoranylidene)acetate (38.3 g, 110 mmol) in MeCN
250 mL) was refluxed for 16 h. The solvent was removed in vacuo and
4
3
to give 4b (112 mg, 55%, containing ∼10% of another isomer) as a
1
the residue was purified by chromatography (10% EA in PE) to give
white solid. H NMR (D O) δ 8.37 (s, 1H, H-6), 4.87 (d, J = 3.6 Hz,
2
2
8 (17.9 g, 93%) as a white solid.
Compound 30. To a solution of MeCN (5.0 g, 122 mmol) in
1H, H-1′), 4.02 (m, 1H, H-2′), 3.88 (m, 2H, H-3′, H-4′), 3.75 (m, 1H,
+
H-5′a), 3.60 (m, 1H, H-5′b). MS, m/z 272.1 (M + H) .
THF (65 mL) at −78 °C was added n-BuLi (2.5M, 44.8 mL), then the
solution was stirred at −78 °C for 30 min. To the above solution was
added 28 (17.9 g, 51 mmol) in THF (15 mL). The mixture was stirred
at −78 °C for 30 min. The reaction was quenched with AcOH (7.2
mL, 125 mmol). After workup, chromatography (20% EA in PE) gave
3-Carbamoyl-1-methyl-5-(β-D-ribofuranosyl)-4-pyridazinone
(4c). Compound 38 (220 mg, 0.52 mmol) was converted to 4c (76
mg, 51%) as a white solid by the same procedure as described for 4b.
1
H NMR (D O): δ 8.54 (s, 1H, H-6), 5.01 (d, J = 3.2 Hz, 1H, H-1′),
2
4.16, 4.06 (2 m, 1H, 2H, H-2′, H-3′, H-4′), 4.09 (s, 3H, Me), 3.93 (dd,
J = 1.6 Hz, 13.2 Hz, 1H, H-5′a), 3.77 (m, 1H, H-5′b). MS, m/z 286.0
2
9 (12.2 g, 69%) as a white solid. A solution of 29 (10.1 g, 29.24
+
mmol) and tert-butoxybis(dimethylamino)methane (5.1 g, 29.3 mmol)
in DMF-DMA (25 mL) was stirred at 100 °C for 16 h, then
concentared. The residue was dissolved in EtOH (60 mL) and
ammonium acetate (6.76 g, 87.7 mmol) was added. The mixture was
refluxed for 24 h and then concentrated. After workup, chromatog-
raphy (40% EA in PE) gave 30 (6.25 g, 56%) as a white solid.
(M + H) .
Compound 41. A solution of 39 (40.0 g, 0.2 mol) and KMnO4
(100 g, 0.6 mol) was refluxed for 12 h, then cooled to rt, and the
resulting precipitate filtered. The filtrate was concentrated to about
200 mL, then acidified with hydrochloric acid. The resulting white
precipitate was filtered to give 40 (28.0 g, 60%). To a solution of 40
(28.0 g, 128 mmol) in DCM (500 mL) at 0 °C were added TEA (19.3
g, 192 mmol) and isobutyl chloroformate (20 g, 147 mmol), followed
by 2-amino-2-methylpropanol (13.1 g, 147.0 mmol). The reaction
mixture was stirred at rt for 2 h, then quenched with water. After
workup, chromatography (20% EA in PE) gave an intermediate (12.0
g, 38%) as colorless oil, which was dissolved in concentrated H SO
3
-Carbamoyl-5-(β-D-ribofuranosyl)-4−1H-pyridinone (4a). A sol-
ution of 30 (3.0 g, 7.08 mmol) in 80% TFA (30 mL) was stirring at 0
C for 3 h, then concentrated to dryness. The residue was purified by
°
chromatography (2% MeOH in DCM) to give the 2′,3′−OH
intermediate (1.3 g, one isomer), which was dissolved in 4 M HCl/
MeOH (20 mL). The solution was stirred at 60 °C for 2 days and
concentrated to dryness. The residue was purified by RP-HPLC
2
4
(60 mL) and stirred at 120 °C for 30 min. The reaction mixture was
(
NH HCO in water and MeCN) to give 31 (200 mg, 10%) as a white
cooled to rt, and poured into NH OH (50 mL) at 0 °C. After workup,
4
3
4
solid. Compound 31 (150 mg, 0.6 mmol) in THF (10 mL) saturated
with NH in a sealed tube was stirred at 100 °C for 15 h. The solution
was concentrated and the residue was purified by RP- HPLC
chromatography (10% EA in EA) gave 41 (7.3 g, 55%) as a white
1
solid. H NMR (CDCl ) δ 8.23 (d, J = 5.2 Hz, 1H), 7.70 (t, J = 5.2 Hz,
3
3
1H), 4.13 (s, 2H), 1.39 (s, 6H).
(
NH HCO in water and MeCN) to give 4a (9.2 mg, 6%) as a
3-Fluoro-2-(β-D-ribofuranosyl)-4-pyridinecarboxamide (5a). To a
stirred solution of 41 (7.0 g, 25.7 mmol) in anhydrous THF (50 mL)
at −78 °C was added n-BuLi (1.3 M in hexane, 19.7 mL). The
resulting mixture was stirred at −78 °C for 30 min, then 9 (21.9 g,
51.4 mmol) in THF (20 mL) was added. The reaction mixture was
4
3
1
white solid. H NMR (CD OD) δ 8.54 (d, J = 1.6 Hz, 1H, H-2), 8.08
3
(
(
(
t, J = 3.4 Hz, 1H, H-6), 4.93 (dd, J = 0.8 Hz, 4.8 Hz, 1H, H-1′), 4.09
m, 1H, H-2′), 4.06 (t, J = 5.2 Hz, 1H, H-3′), 4.02 (m, 1H, H-4′), 3.84
dd, J = 3.0 Hz, 12.2 Hz, 1H, H-5′a), 3.70 (dd, J = 4.2 Hz, 12.2 Hz,
+
1
H, H-5′b). MS, m/z 271.1 (M + H) .
stirred at −78 °C for 2 h, then quenched with aqueous NH Cl. After
4
19
Compound 35. Compound 32 (54.0 g, 144.4 mmol) was
workup, chromatography (30% EA in PE) gave 42 (9.4 g, 59%) as a
white solid. Compound 42 (6.0 g, 9.7 mmol) was converted to 43 (1.8
g, 36%) as a white solid by the same procedure as described for 23a. A
solution of 43 (800 mg, 1.3 mmol) in aqueous TFA (80%, 5 mL) was
stirred at rt for 30 min, then concentrated. The residue was dissolved
in water, washed with DCM, and then concentrated to get 44 as a
syrup, which was dissolved in concentrated aqueous ammonia (50
converted to 33 (32.6 g, 61%) as a white solid by the same procedure
as described for 29. A solution of 33 (32.0 g, 80.7 mmol), TfN (22.6
g, 130.0 mmol) and py (13.7 g, 173 mmol) in MeCN (40 mL) was
stirred at rt for 3 h. The solvent was removed and the residue was
purified by chromatography (10% EA in PE) to give 34 (12.4 g, 36%)
as colorless oil. To a solution of 34 (12.0 g, 30.4 mmol) in THF (20
3
J
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mL) in a sealed tube. The solution was stirred at 50 °C for 12 h and
then concentrated. The residue was purified by RP-HPLC (NH HCO
in water and MeCN) to give 5a (83 mg, 23%) as a white solid. H
mmol), followed by N,O-dimethylhydroxylamine (0.5 g, 5 mmol). The
reaction mixture was stirred at rt for 8 h. After workup,
chromatography (50% EA in PE) gave 50 (1.8 g, 82%) as a white
solid. To a stirred solution of trimethylsilylacetylene (234 mg, 2.4
mmol) in THF (8 mL) at −78 °C was added dropwise n-BuLi (2.5 M
in hexane, 1 mL). The solution was stirred at −78 °C for 30 min, then
50 (900 mg, 1.8 mmol) in THF (5 mL) added. The reaction mixture
4
3
1
NMR (D O) δ 8.52 (d, J = 4.8 Hz, 1H, H-6), 7.73 (t, J = 5.2 Hz, 1H,
2
H-5), 5.34 (dd, J = 1.4 Hz, 5.8 Hz, 1H, H-1′), 4.42 (t, J = 5.6 Hz, 1H,
H-2′), 4.31 (t, J = 5.6 Hz, 1H, H-3′), 4.18 (dd, J = 3.2 Hz, 4.6 Hz, 1H,
H-4′), 3.94 (dd, J = 3.2 Hz, 12.4 Hz, 1H, H-5′a), 3.80 (dd, J = 4.6 Hz,
+
1
2.6 Hz, 1H, H-5′b). MS, m/z 273.1 (M + H) .
was stirred at rt for 3 h, then quenched with aqueous NH Cl. Usual
4
Compound 45a. A solution of compound 43 (1.0 g, 1.7 mmol)
workup yielded the crude 51, which was dissolved in MeCN (10 mL)
and NaOMe (918.0 mg, 17.0 mmol) in anhydrous 1,4-dioxane (50
and H O (5 mL), followed by addition of methyl carbamimidothioate
2
mL) was stirred at rt for 6 h. After workup, chromatography (20% EA
hydrochloride (464 mg, 3.7 mmol). The reaction mixture was stirred
1
in PE) gave 45a (710 mg, 68%) as colorless oil. H NMR (CDCl ) δ
at 80 °C for 3 h. After workup, chromatography (20% EA in PE) gave
3
1
8
1
1
6
.25 (s, 1H), 7.76−7.63, 7.31−7.39 (2m, 11H), 5.45 (d, J = 3.6 Hz,
H), 5.26 (dd, J = 3.6 Hz, 6.4 Hz, 1H), 4.84 (dd, J = 3.4 Hz, 6.2 Hz,
H), 4.13 (s, 2H), 3.86 (s, 3H), 3.69 (m, 2H), 1.60 (s, 3H), 1.37 (s,
H), 1.36 (s, 3H), 1.01 (s, 9H). MS, m/z 634.1 (M + 18) .
Compound 46. To a solution of 45a (0.61 g, 1.0 mmol) in THF
52 (400 mg, 32%) as a white solid. H NMR (CDCl ) δ 8.28 (d, J =
3
4.8 Hz, 1H), 7.23−7.41 (m, 16H), 5.08 (m, 1H), 4.82 (d, J = 12.0 Hz,
1H), 4.70 (d, J = 12.4 Hz, 1H), 4.49−4.59 (m, 3H), 4.32−4.14 (m,
2H), 4.11 (m, 1H), 3.91 (m, 1H), 3.85 (m, 1H), 3.64 (m, 1H), 2.52 (s,
3H).
+
(
10 mL) at 0 °C was added TBAF (1 M in THF; 2 mL, 2 mmol) and
4-(β-D-Ribofuranosyl)-2-pyrimidinecarboxamide (6). A solution of
52 (400 mg, 0.76 mmol) and mCPBA (400 mg, 2.3 mmol) in DCM
(10 mL) was stirred at rt for 3 h. After workup, chromatography (50%
EA in PE) gave an oxidized intermediate (390 mg, 92%) as a white
solid. A solution of the intermediate (350 mg, 0.625 mmol) and KCN
(60 mg, 0.92 mmol) in anhydrous DMSO (3 mL) was stirred at rt
overnight, then diluted with EA (15 mL). After workup, chromatog-
raphy (50% EA in PE) gave 53 (200 mg, 63%) as a white solid.
Compound 53 (200 mg, 0.4 mmol) was converted to 54 (120 mg,
57%) as a white solid by the same procedure as described for 13. A
the resulting mixture stirred at rt for 1 h. The reaction was quenched
with silica and evaporated to dryness. Chromatography on silica gel
with 4−10% MeOH in DMC gave 0.33 g (90%) of 45b. A solution of
4
5b (38 mg, 0.1 mmol) in 80% aq. HCOOH was stirred at rt for 4 h,
then concentrated and purified by RP-HPLC (A: 50 mM TEAA in
water, pH 7; B: 50 mM TEAA in MeCN, pH 7) to yield 46 (21 mg,
1
4
1
1
0%). H NMR (DMSO-d ) δ 8.35 (d, J = 4.8 Hz, 1H, H-6), 7.99 (br,
6
H, NH), 7.38 (d, J = 4.8 Hz, 1H, H-5), 5.06 (d, J = 5.2 Hz, 1H, H-
′), 4.23 (dd, J = 4.8 Hz, 5.2 Hz, 1H, H-2′), 4.01 (dd, J = 4.8 Hz, 5.2
Hz, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.80 (s, 3H, OMe), 3.58 (dd, J =
4
1
solution of 54 (100 mg, 0.19 mmol) and BCl (1 M in DCM, 1.9 mL)
3
.0 Hz, 11.6 Hz, 1H, H-5′a), 3.44 (s, 2H, CH OH), 3.40−3.44 (m,
in anhydrous DCM (5 mL) was stirred at 0 °C for 4 h, then quenched
with pyridine and MeOH. The solvent was removed and the residue
2
+
H, H-5′b), 1.28 (s, 6H, CMe ). MS, m/z 357.3 (M+1) .
2
Compound 47. A solution of 45b (0.23 g, 0.6 mmol) in 2% TFA
was purified by RP-HPLC (AcONH in water and MeCN) to give 6
4
1
in DCM (6 mL) stood at rt overnight, then concentrated and
coevaporated with toluene several times. Chromatography on silica gel
with 4−15% MeOH in DCM yielded 0.24 g (84%) of 47. H NMR
(20 mg, 41%) as a white solid. H NMR (CD OD): δ 8.86 (d, J = 5.1
3
Hz, 1H, H-6), 8.11 (br s, 1H, NH), 7.81 (d, J = 5.1 Hz, 1H, H-5), 7.77
(br s, 1H, NH), 5.28 (d, J = 5.5 Hz, 1H, H-1′), 4.89 (d, J = 5.5 Hz, 1H,
3′−OH), 5.85 (t, J = 5.5 Hz, 1H, 5′−OH), 4.72 (d, J = 4.3 Hz, 1H,
2′−OH), 3.99 (m, 1H, H-2′), 3.85 (m, 2H, H-3′, H-4′), 3.65 (m, 1H,
1
(
DMSO-d ) δ 8.48 (d, J = 4.8 Hz, 1H, H-6), 7.76 (d, J = 4.8 Hz, 1H,
6
H-5), 5.22 (d, J = 8.0 Hz, 1H, H-1′), 5.05 (dd, J = 4.0 Hz, 6.4 Hz, 1H,
+
H-2′), 4.98 (t, J = 5.6 Hz, 1H, 5′−OH), 4.73 (dd, J = 3.2 Hz, 6.4 Hz,
H-5′a), 3.51 (m, 1H, H-5′b). MS, m/z 256.1 (M + H) .
2
3
1
H, H-3′), 4.19 (s, 2H, C(O)OCH ), 4.02 (m, 1H, H-4′), 3.81 (s, 3H,
Compound 58. A solution of 55 (10.6 g, 30 mmol) in aqueous
TFA (80%, 100 mL) was stirred at rt for 12 h. Volatiles were
evaporated and the residue was purified by chromatography (20% EA
in PE) to give the 1-OH intermediate (9.0 g, 90%) as colorless oil. To
a solution of the intermediate (9.0 g, 27.0 mmol) in DMSO (60 mL)
2
OMe), 3.32 (m, 2H, H-5′a, H-5′b), 1.48, 1.27 (2s, 6H, CMe ), 1.21 (s,
6
2
+
H, CMe ). MS, m/z 397.3 (M+1) .
2
3
-Methoxy-2-(β-D-ribofuranosyl)-4-pyridinecarboxamide 5b. A
mixture of 47 (0.14 g, 0.35 mmol) in MeCN (1.5 mL) and acetic
anhydride (1.5 mL) was stirred at rt for 1 day, then concentrated and
purified on silica gel with 4−15% i-PrOH in DCM to give the N-
acetate 48a (68 mg, 40%) and the 5′-O, N-diacetate 48b (50 mg,
was added Ac O (40 mL) at rt, and the mixture was stirred at rt. for 12
2
h. After workup, the residue was purified by chromatography (10% EA
in PE) to give 56 (8.5 g, 95%) as a colorless syrup. To a stirred
solution of 3-fluoropicolinonitrile (2.9 g, 24.2 mmol) in THF (50 mL)
at −78 °C was added dropwise LDA (12.0 mL, 24.2 mmol). The
mixture was stirred at this temperature for 30 min. To the above
mixture was added a solution of 56 (8.0 g, 24.0 mmol) in THF (10
mL) at −78 °C, and stirring continued for 1h. The reaction was
3
3%), which were separately treated with methanolic ammonia (7 N, 3
mL) for 20 h. After evaporation of volatiles and coevaporation with
methanol, the residues were separately dissolved in 17% aqueous TFA
and stood for 2 h. After evaporation the residue was purified by RP-
HPLC (A: 50 mM TEAA in water; B: 50 mM TEAA in MeCN) to
yield 5b as a white solid (85 mg, 80%). H NMR (DMSO-d ): δ 8.35
1
6
quenched with aqueous NH Cl. After workup, chromatography (20%
4
(
d, J = 4.8 Hz, 1H, H-6), 7.94, 7.76 (2 br, 2H, NH ), 7.35 (d, J = 4.8
EA in PE) gave 57 (4.0 g, 37%) as a white solid. Compound 57 (4.0 g,
2
Hz, 1H, H-5), 5.07 (d, J = 5.2 Hz, 1H, H-1′), 4.25 (m, 1H, H-2′), 4.02
(
(
8.8 mmol) was converted to 58 (1.7 g, 44%) as a white solid by the
1
t, J = 5.2 Hz, 1H, H-3′), 3.85 (m, 1H, H-4′), 3.80 (s, 3H, OMe), 3.58
same procedure as described for 23a. H NMR (CD OD) δ 8.20 (d, J
3
dd, J = 4.2 Hz, 11.8 Hz, 1H, H-5′a), 3.43 (dd, J = 4.6 Hz, 11.8 Hz,
= 4.8 Hz, 1H), 8.01 (dd, J = 5.2 Hz, 5.6 Hz, 1H), 7.27−7.34 (m, 10H),
5.47 (d, J = 23.2 Hz, 1H), 5.16 (dm, J = 54.4 Hz, 1H), 4.69 (d, J = 12.0
Hz, 1H), 4.57 (d, J = 11.2 Hz, 1H), 4.49 (m, 2H), 4.24 (m, 1H), 4.12
(ddd, J = 3.8 Hz, 8.4 Hz, 10.8 Hz, 1H), 3.94 (dd, J = 2.0 Hz, 10.8 Hz,
1H), 3.69 (dd, J = 2.8 Hz, 11.2 Hz, 1H).
−
1
H, H-5′b). MS, m/z 283.4 (M-1) .
3
-Hydroxy-2-(β-D-ribofuranosyl)-4-pyridinecarboxamide 5c. A
mixture of 5b (45 mg, 0.11 mmol) and NaSEt (64 mg, 0.77 mmol)
in DMF (0. Six mL) were stirred at 60 °C for 2 h, then cooled down
and purified by RP-HPLC, gradient 0−60% B (A: 50 mM aqueous
3-Fluoro-4-(2-deoxy-2-fluoro-β-D-ribofuranosyl)-2-pyridinecar-
boxamide (7). Compound 58 (1.7 g, 3.8 mmol) was converted to the
amide (1.5 g, 87%) as described for compound 25a.The amide
intermediate (300 mg, 0.66 mmol) was converted to 7 (82 mg, 45%)
TEAA, B: 50 mM TEAA in MeCN) to yield triethylammonium salt of
1
5
c as a white solid (16 mg, 41%). H NMR (DMSO-d ) δ 8.90, 8.28
6
(
2 br, 2H, NH ), 8.04 (d, J = 4.8 Hz, 1H, H-6), 7.71 (d, J = 5.1 Hz,
2
1
1
H, H-5), 5.12 (d, J = 4.0 Hz, 1H, H-1′), 5.07 (br, 1H, 3-OH), 4.84 (d,
as a white solid by the same procedure as described for 3f. H NMR
J = 5.6 Hz, 3′−OH), 4.18 (dd, J = 4.4 Hz, 4.8 Hz, H-2′), 4.00 (dd, J =
(D O) δ 8.46 (br s, 1H, H-6), 7.85 (br s, 1H, H-5), 5.60 (d, J = 25.6
2
5
1
.6 Hz, 11.2 Hz, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.63 (dd, J = 3.6 Hz,
Hz, 1H, H-1′), 5.12 (dd, J = 3.6 Hz, 54.4 Hz, 1H, H-2′), 4.16−4.24
1.6 Hz, 1H, H-5′a), 3.45 (dd, J = 4.0 Hz, 11.6 Hz, 1H, H-5′b). MS,
(m, 2H, H-3′, H-4′), 4.05 (dd, J = 1.6 Hz, 12.8 Hz, 1H, H-5′a), 3.86
−
+
m/z 269.0 (M-1) .
(dd, J = 4.4 Hz, 12.8 Hz, 1H, H-5′b). MS, m/z 275.1 (M + H) .
22
Compound 52. To a suspension of 49 (2 g, 4.5 mmol) in DCM
Compound 59. A mixture of 3c (100 mg, 0.37 mmol), TsOH·
(
20 mL) were added DIPEA (1.7 g, 13.5 mmol) and HATU (2.1 g, 5.5
H O (7 mg, 0.037 mmol), and trimethoxymethane (390 mg, 3.7
2
K
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol) in MeCN (2 mL) was heated at 70 °C overnight. The mixture
μL 25 μM 2′-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid
(Sigma-Aldrich) dissolved in 33 mM MES, pH 6.5 (Emerald
Biosystems, Bainbridge Island, WA) was added to the cells. After
incubation for 45 min at 30 °C, reactions were stopped by addition of
150 μL stop solution (100 mM glycine, pH 10.5, 25% ethanol, all
Sigma-Aldrich). Fluorescence was measured with excitation and
emission filters of 355 and 460 nm, respectively, on a Victor X3
multilabel plate reader (PerkinElmer, Waltham, MA). Cytotoxicity of
uninfected parallel cultures was determined by addition of 100 μL
CellTiter-Gloreagent (Promega, Madison, WI), and incubation for 10
min at room temperature. Luminescence was measured on a Victor X3
multilabel plate reader.
was diluted with MeOH (5 mL) and aqueous NH (0.2 mL) and
3
stood at rt for 2 h. After evaporation, the residue was purified on silica
gel (5% MeOH in DCM) to give the 2′,3′-methoxymethylidene
derivative of 3c (68 mg, yield: 59%) as white foam. A mixture of
triethylammonium bis(pivaloxymethyl)phosphate (0.35 mmol, pre-
pared from 112 mg of bis(POM)phosphate and Et N) and the 2′,3′-
3
methoxymethylidene derivative of 3c (72 mg; 0.23 mmol) was
coevaporated with anhydrous toluene and then dissolved in THF (3
mL). Diisopropylethylamine (0.2 mL, 1.2 mmol), BOP-Cl (146 mg,
0
.57 mmol) and 3-nitro-1,2,4-triazole (66 mg, 0.57 mmol) were added,
and the resulting mixture stirred at rt for 1 h. After workup, the residue
was purified on silica gel with 3−10% i-PrOH in DCM. Thus, obtained
nucleotide prodrug was treated with 80% aqueous HCOOH (1 mL) at
rt for 15 min. The reaction mixture was concentrated and
coevaporated with a mixture of toluene and methanol containing
Inhibition of recombinant influenza A polymerase complex. The
determination of the half maximal inhibitory concentration, IC50, of
the compounds against the recombinant influenza polymerase
18
complex, PA/PB1/PB2, was performed as described previously. In
summary, each 10-μL reaction was performed at 37 °C for 40 min in a
reaction mixture containing reaction buffer (25 mM Tris-Cl, pH 7.5,
one drop of Et N. The residue was purified on silica gel with 4−10%
3
MeOH in DCM to yield 59 as white foam (62 mg, 47% for 2 steps).
1
H NMR (DMSO-d ) δ 8.37 (d, J = 5.7 Hz, 1H, H-6), 8.0 (brs, 1H,
100 mM NaCl, 5 mM MgCl , 0.5 mM EDTA, 2 mM DTT, 5%
2
6
NH), 7.65 (br s, 1H, NH), 7.63 (d, J = 5.7 Hz, 1H, H-5), 5.58 (m, 4H,
glycerol), 0.15 μM polymerase complex, 0.4 mM 5′-ApG primer, 1.5
μM 50-nt 3′vRNA template, 1.6 μM 15-nt 5′vRNA, 0.20 U/μL
RNaseIn, 500 μM UTP, ATP, and CTP, 1 μM GTP, 2.5 μCi
2
x OCH O), 5.43 (d, J = 5.5 Hz, 1H, H-1′), 5.22 (d, J = 5.5 Hz, 1H,
2
3
′−OH), 4.98 (d, J = 4.7 Hz, 1H, 2′−OH), 4.27 (m, 1H, H-5′a), 4.18
33
[
α- P]GTP and compounds at various concentrations. The reactions
(
1
5
m, 1H, H-5′b), 4.03 (m, 1H, H-4′), 3.82 (m, 2H, H-2′, H-3′), 1.13,
19
were stopped by a quench solution containing formamide with 50 mM
EDTA. The samples were incubated at 95 °C for 5 min. The samples
were run on a 15% denaturing PAGE gel (Invitrogen) at 190 V for 50
min. The gel was exposed to a storage phosphor screen and visualized
by a Typhoon scanner (GE healthcare). The amount of RNA products
was proportional to the intensity of the bands on the gel that were
quantified using the ImageQuant software (GE Healthcare).
.12 (2s, 18H, 2 x C(CH ) ). F-NMR (DMSO-d ) δ −127.73 (d, J =
3
3
6
.5 Hz). 31P NMR (CDCl ) δ −4.15 (s). MS, m/z 581.4 (M+1) .
+
3
Compound 60. To a stirred solution of the 2′,3′-O-methox-
ymethylidene derivative of 3c (32 mg, 0.1 mmol) in NMI (0.2 mL)
and MeCN (0.3 mL) at 0 °C was added (2S)-cyclohexyl 2-
(
0
(chloro(phenoxy)phosphoryl)amino)propanoate (1.0 M in THF,
.5 mL). The resulting solution was stirred at 30 °C for 5 h. After
The compound concentration at which the RNA products was
workup, the crude product was dissolved in 80% formic acid and
stirred for 3 h. Evaporation and coevaporation with toluene gave a
residue, which was dissolved in MeOH containing a few drops of
triethyamine and stood at 35 °C for 2 h. After evaporation, the residue
reduced by 50% (IC ) was calculated by fitting the data to the
50
equation,
max − min
Y = min +
was purified on silica gel with 5−10% MeOH in DCM to give 33 mg
(logIC50−X)·h
1
1 + 10
(
57%) of 60 as a white solid. H NMR (CD OD, two P-isomers) δ
3
8
5
.38, 8.32 (2d, J = 4.8 Hz, 1H, H-6), 7.80, 7.73 (2t, J = 4.8 Hz, 1H, H-
), 7.14−7.36 (m, 5H, Ph), 5.15 (m, 1H, H-1′), 4.71 (m, 1H, CH of
where Y corresponds to the percentage of inhibition (percentage of
RNA products reduced), Min is the minimal percentage of inhibition,
Max is the maximal inhibition at high concentration of compound, X
corresponds to the log of compound concentration, and h is the
Hillslope.
Ala), 4.29−4.49 (m, 2 H, H-2′, H-3′), 4.18 (m, 1H, H-1 of
cyclohexyl), 3.91−4.06 (m, 3H, H-4′, H-5′a, H-5′b), 3.75−3.88 (m,
1
H, NH of Ala), 1.22−1.84 (m, 10 H, 5xCH of cyclohexyl), 1.35, 1.32
2
+
(
2d, J = 7.2 Hz, Me of Ala). MS, m/z 582.6 (M + H) , 604.9 (M +
Nucleotide incorporation assay using recombinant influenza A
polymerase complex. The recombinant polymerase complex
catalyzed the primer extension reaction using 5′-pApG as primer, a
14-nt synthetic RNA oligonucleotide as template and the 15-nt
5′vRNA as promoter. The 14-nt RNA template and the 15-nt 5′vRNA
promoter form a panhandle structure that was required for RNA
+
19
Na) . F-NMR (CD OD, two P-isomers) δ −127.81 (d J = 4.5 Hz),
3
31
−
127.86 (d, J = 4.1 Hz). P NMR (CD OD, two P-isomers) δ 3.88
3
(
s), 3.75 (s).
Preparation of NTPs 61 and 62. Compound 61 and 62 were
prepared from 3c and 2, respectively, by a general method described in
36
18
our previous publiocation. The purities of 61 and 62 were >95%,
replication activity of the influenza polymerase complex. To study if
determined on an Agilent 1100 HPLC, 50 mM TEAA in water and 50
61 can be incorporated opposite U or C on the template, nucleotide
incorporation assays using specific templates were performed
(sequences shown in Figure 3). A typical reaction was performed at
37 °C in a reaction mixture containing reaction buffer (40 mM Tris-
mM TEAA in acetonitrile as mobile phase.
1
6
1: H NMR (D O) δ 8.35 (d, J = 4.8 Hz, 1H, H-6), 7.83(dd, J =
2
4
.8, 5.2 Hz, 1H, H-5), 5.15 (d, J = 5.2 Hz, H-1′), 4.08−4.23 (m, 5H,
31
H-2′, H-3′, H-4′, H-5′). P NMR (D O) δ −4.15 (d, J = 19.1 Hz,),
HCl, pH7, 20 mM NaCl, 5 mM MgCl , and 2 mM DTT), 0.15 μM
2
2
−
10.77 (d, J = 18.8 Hz,), −20.60 (dd, J = 19.1, 18.8 Hz,). MS, m/z
polymerase complex, 1 μM 15-nt 5′vRNA promoter, 2.5 μM 14-nt
−
5
11.0 (M-1) .
RNA template, 0.4 mM 5′-pApG primer, 0.2 U/μL RNaseIn, 25 μM
1
33
6
2: H NMR (D O) δ 8.30 (d, J = 5.2 Hz, 1H, H-5), 5.95 (d, J = 1.2
UTP, 25 μM CTP, 0.033 μM [α- P]CTP, and the testing NTPs or
2
31
Hz, 1H, H-1′), 4.21−4.30 (m, 5H, H-2′, H-3′, H-4′, H-5′a, H-5′b).
P
NTP analogues at 100 μM. The replication reactions were stopped
after 35 min by mixing with 2x volume of the formamide quench
solution containing 50 mM EDTA. The quenched reactions were
denatured at 95 °C for 3 min and then were loaded onto a 22.5%
denaturing polyacrylamide gel with 7 M urea (National Diagnostics,
Atlanta, GA). The electrophoresis was performed at 80 W using a
Sequi-Gen GT system from Bio-Rad (Hercules, CA).
NMR (D O) δ −5.47 (d, J = 18.9 Hz,), −11.27 (d, J = 19.4 Hz,),
2
19
−
4
21.00 (dd, J = 19.4, 18.9 Hz,). F-NMR (D O) δ −102.94 (d, J =
.9 Hz,). MS, m/z 528.1 (M-1) .
2
−
Antiviral and cytotoxicity assay. The antiviral assay is based on
2
6
a neuraminidase activity assay as described by Eichelberger et al. In
brief, Madin-Darby canine kidney epithelial cells (MDCK, ATCC)
5
4
were plated at a density of 1 × 10 cells/ml (1 × 10 cells/well) in
assay media (DMEM supplemented with 0.3% FBS, 1% penicillin/
streptomycin and 1% DMSO) in 96-weel plates. After 24 h, serially
diluted compounds were added to cells and incubated for an additional
In vivo nucleotide formation of 3c. The studies were conducted
37
at WuXi AppTec and conformed to the regulation and guidelines
regarding animal care and welfare: The study protocols were reviewed
by and approved by WuXi AppTec’s Institutional Animal Care and
Use Committee (IAUCUC) prior to the study initiation. Female Balb/
c mice (N = 2) were fasted overnight before administrated
intravenously with the prodrug 59. The prodrug was formulated as a
2
4 h. Cells were infected with 250 IU/well of Influenza strains A/
WSN/33 (H1N1) (Virapur, San Diego CA) and incubated for 20 h at
7 °C, 5% CO . The cell culture supernatant was aspirated off and 50
3
2
L
DOI: 10.1021/acs.jmedchem.5b01933
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
solution of in 10% DMSO/60% PEG400/30%water and administrated
at 2 mg/kg. While the animals were under anesthesia with isoflurane,
blood samples were collected at 0.25 and 2 h post dose into tubes
(8) Cheng, P.; Leung, T.; Ho, E.; Leung, P.; Ng, A.; Lai, M.; Lim, W.
Oseltamivir and amantadine-resistant influenza viruses A (H1N1).
Emerging Infect. Dis. 2009, 15, 966−968.
containing K EDTA as the anticoagulant. Following an immediate
(9) Samson, M.; Pizzorno, A.; Abed, Y.; Boivin, G. Influenza virus
resistance to neuraminidase inhibitors. Antiviral Res. 2013, 98, 174−
185.
2
plasma protein precipitation treatment, the supernatant of the blood
samples was analyzed for 3c concentrations using a LC/MS/MS
method. After the terminal blood collection, lung tissue was removed
immediately and flash-frozen into liquid nitrogen to prevent any ex
vivo degradation of the phosphorylated metabolites. The frozen tissue
was homogenized in an extraction solution in a sample tube set in a
dry ice/ethanol bath to maintain cold temperature. The supernatant of
the lung extracts was subjected to LC/MS/MS analysis for the
concentrations of 63 and 61.
(10) Du, J.; Cross, T. A.; Zhou, H.-X. Recent progress in structure-
based anti-influenza drug design. Drug Discovery Today 2012, 17,
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111−1120.
11) Byrn, R. A.; Jones, S. M.; Bennett, H. B.; Bral, C.; Clark, M. P.;
Jacobs, M. D.; Kwong, A. D.; Ledeboer, M. W.; Leeman, J. R.; McNeil,
C. F.; Murcko, M. A.; Nezami, A.; Perola, E.; Rijnbrand, R.; Saxena, K.;
Tsai, A. W.; Zhou, Y.; Charifson, P. S. Preclinical activity of VX-787, a
first-in-Class, orally bioavailable inhibitor of the influenza virus
polymerase PB2 Subunit. Antimicrob. Agents Chemother. 2015, 59,
ASSOCIATED CONTENT
■
1
(
569−1582.
*
S
Supporting Information
12) DuBois, R. M.; Slavish, P. J.; Baughman, B. M.; Yun, M.-K.; Bao,
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01933.
J.; Richard, J.; Webby, R. J.; Webb, T. R.; White, S. W. Structural and
biochemical basis for development of influenza virus inhibitors
targeting the PA endonuclease. PLoS Pathog. 2012, 8, e1002830.
(
13) Furuta, Y.; Gowen, B. B.; Takahashi, K.; Shiraki, K.; Smee, D. F.;
A table with molecular formula strings and the associated
biochemical and biological data (CSV)
Barnard, D. L. Favipiravir (T-705), a novel viral RNA polymerase
inhibitor. Antiviral Res. 2013, 100, 446−454.
(14) Jin, Z.; Tucker, K.; Lin, X.; Kao, C. C.; Shaw, K.; Tan, H.;
AUTHOR INFORMATION
Symons, J.; Behera, I.; Rajwanshi, V. K.; Dyatkina, N.; Wang, G.;
Beigelman, L.; Deval, J. Biochemical evaluation of the inhibition
properties of Favipiravir and 2′-C-methylytidine triphosphates against
human and mouse norovirus RNA polymerases. Antimicrob. Agents
Chemother. 2015, 59, 7504−7516.
■
Corresponding Authors
For G.W.: phone, 650-635-5506; E-mail, gwang47@ITS.JNJ.
com.
For L.B.: phone, 650-635-5508; E-mail, lbeigelm@ITS.JNJ.
com.
*
*
(
15) Arias, A.; Thorne, L.; Goodfellow, I. Favipiravir elicits antiviral
mutagenesis during virus replication in vivo. eLife 2014, 3, e03679.
16) Oestereich, L.; Ludtke, A.; Wurr, S.; Rieger, T.; Munoz-Fontela,
C. Stephan Gunther. Successful treatment of advanced Ebola virus
(
̈
̃
Notes
̈
The authors declare no competing financial interest.
infection with T-705 (favipiravir) in a small animal model. Antiviral
Res. 2014, 105, 17−21.
(17) Baranovich, T.; Wong, S. S.; Armstrong, J.; Marjuki, H.; Webby,
R. J.; Webster, R. G.; Govorkova, E. A. T-705 (favipiravir) induces
lethal mutagenesis in influenza A H1N1 viruses in vitro. J. Virol. 2013,
ACKNOWLEDGMENTS
■
The authors would like to thank Vladimir Serebryany for a
general procedure for preparing 4′-fluoronucleosides using NIS
and TEA-3HF and Ishani Behera for testing 61 in HCV NS5B
and HRV16 polymerase assays.
8
(
7, 3741−3751.
18) Jin, Z.; Smith, L. K.; Rajwanshi1, V. K.; Kim, B.; Deval, J. The
Ambiguous base-pairing and high substrate efficiency of T-705
(
Favipiravir) ribofuranosyl 5′-triphosphate towards influenza A virus
ABBREVIATIONS USED
■
polymerase. PLoS One 2013, 8, e68347.
IBX, 2-iodoxybenzoic acid; DMTrCl, 4,4′-dimethoxytrityl,
chloride; MMTrCl, 4-methoxytrityl chloride; TBDPSCl, tert-
butyldiphenylchlorosilane; DMP, Dess−Martin periodinane;
TEA, triethylamine; TPSCl, 2,4,6-triisopropylbenzenesulfonyl
chloride; EDCI, 1-ethyl-3-(3-(dimethylamino)propyl)-carbodii-
mide hydrochloride; PE, petroleum ether; EA, ethyl acetate
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J. Med. Chem. XXXX, XXX, XXX−XXX