Synthesis and antiviral evaluation of the 2'-c-methyl branched derivative of a nucleoside analog inhibitor of RNA viral infections, T-1106

Article abstract of DOI:10.1135/cccc2011089

An example of a 2'-C-methyl branched nucleoside analogue bearing 3,4-dihydro-3-oxopyrazine-2-carboxamide as the base, namely 4-(2-C-methyl- β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide, is reported. This compound was synthesized following a Vorbrueggen's glycosylation procedure in a few steps. When evaluated in cell culture experiments against a broad range of viruses, this compound did not exhibit any significant antiviral effect or cytotoxicity.

Full text of DOI:10.1135/cccc2011089

T-1106, 2′-C-Methyl Branched Derivative
1327
SYNTHESIS AND ANTIVIRAL EVALUATION OF THE 2′-C-METHYL
BRANCHED DERIVATIVE OF A NUCLEOSIDE ANALOG INHIBITOR
OF RNA VIRAL INFECTIONS, T-1106
a1
a2
a3,b
Claire PIERRA , Clément COUNOR , Richard STORER
and
a4,c,
Gilles GOSSELIN
*
a
Idenix Pharmaceuticals, Medicinal Chemistry Laboratories IDENIX Sarl,
Cap Gamma, 1682 Rue de la Valsière, BP 50001, 34189 Montpellier Cedex 4, France;
1
2
e-mail: pierra.claire@idenix.com, counor.clement@idenix.com,
3
4
richard.storer@summitplc.com, gosselin.gilles@idenix.com
b
c
Current Address: Summit Corporation plc, 91 Milton Park, Abingdon, Oxfordshire OX14 4RY,
England
UMR 5247 CNRS-UM1-UM2 (IBMM), Université Montpellier 2, CC 1704, 34095 Montpellier
Cedex 5, France
Received April 27, 2011
Accepted May 31, 2011
Published online October 30, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his
outstanding contributions to the area of nucleic acid chemistry.
An example of a 2′-C-methyl branched nucleoside analogue bearing 3,4-dihydro-3-oxo-
pyrazine-2-carboxamide as the base, namely 4-(2-C-methyl-β-D-ribofuranosyl)-3-oxo-
3
,4-dihydropyrazine-2-carboxamide, is reported. This compound was synthesized following
a Vorbrüggen’s glycosylation procedure in a few steps. When evaluated in cell culture exper-
iments against a broad range of viruses, this compound did not exhibit any significant anti-
viral effect or cytotoxicity.
Keywords: T-1106; 2′-C-Methyl branched derivative; Antiviral evaluation; Hepatitis C virus;
Nucleosides; Antiviral agents; Biological activity.
Although efficacious drugs have been developed to treat infections caused
1
2
by DNA viruses and to control retroviruses infections , no specific antiviral
drugs are available for the prevention or the treatment of infections caused
by the majority of RNA viruses. Yet, RNA viruses, whose more than 350 spe-
cies have been described and which are classified, according to their ge-
nome structure and expression modalities, as single-stranded positive RNA
+
–
(
(
ssRNA ), single-stranded negative RNA (ssRNA ), and double-stranded RNA
dsRNA) viruses, are major human pathogens causing millions of deaths
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1327–1333
2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011089
©

1
328
Pierra, Counor, Storer, Gosselin:
3
annually . Recently, a nucleoside analogue [2-oxo-1-(β-D-ribofuranosyl)-
pyrazine-3-carboxamide (1), T-1106; Chart 1], discovered and synthesized
4
by Toyama Chemical Co., Ltd. , was shown to have antiviral activity
+
against three viruses of the ssRNA Flaviviridae family, that is, Bovine Virus
5
6
Diarrhoea Virus (BVDV, a Pestivirus) , Yellow Fever (YF, a Flavivirus) , and
5
Hepatitis C Virus (HCV, a Hepacivirus) , as well as against a virus of the
–
7
ssRNA Bunyaviridae family, that is, Punta Toro Virus (PTV, a Phlebovirus) .
On the other hand, and following the discovery that a series of branched-
sugar ribonucleosides could act as potential chemotherapeutic agents
against HCV 8 and against flaviviruses and pestiviruses , we have previously
shown that 2′-C-methylribonucleoside derivatives in the pyrimidine series
9
1
0
are potent and broad-spectrum anti-RNA virus agents . In continuation of
1
1
our interest in 2′-C-methyl-branched nucleoside derivatives , here we re-
port the synthesis of 4-(2-C-methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydro-
pyrazine-2-carboxamide (2), the 2′-C-methyl-branched derivative of T-1106
(
Chart 1), as well as the results of its antiviral evaluations. It is noteworthy
1
2
that during the course of our current work , the synthesis of 2 has been re-
1
3
ported by another author . However, in that communication, the ap-
proach implemented for the synthesis of 2 was slightly different from our
approach, and no results of antiviral evaluations have been given.
CHART 1
RESULTS AND DISCUSSION
Synthesis
For the synthesis of 4-(2-C-methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydro-
pyrazine-2-carboxamide (2), first the pyrazine base 5 was prepared in good
1
4
yield following a procedure described in the literature , via 2-amino-
propanediamide 4 15 and starting from the commercially available diethyl-
amino malonate 3 (Scheme 1). Then, glycosylation of 5 with 1,2,3,5-
O-tetrabenzoyl-2-C-methyl-β-D-ribofuranose (6)16 under Vorbrüggen-type
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1327–1333

T-1106, 2′-C-Methyl Branched Derivative
1329
SCHEME 1
Synthesis of 3-oxo-3,4-dihydropyrazine-2-carboxamide (5)
per-silylation conditions17 led to the benzoylated nucleoside derivative 7 as
an inseparable α/β anomeric mixture (Scheme 2). Treatment with methan-
olic ammonia of this anomeric mixture, followed by purification on reverse
phase column chromatography, allowed the isolation of the pure desired
4
-(2-C-methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide
(
2) and of its α-anomer (compound 8) in 10 and 58% yields, respectively.
SCHEME 2
Synthesis of 4-(2-C-methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide (2)
and its α-anomer 8
Antiviral Evaluations
4
-(2-C-Methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide
(
2) and its α-anomer 8 were evaluated in cell-based assays (following meth-
1
0a
ods described in ref. ) against viruses representative of three genera of the
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1
330
Pierra, Counor, Storer, Gosselin:
+
ssRNA Flaviviridae family, that is, Pestivirus (Bovine Virus Diarrhoea Virus),
Flavivirus (Yellow Fever, Dengue and West Nile Viruses) and Hepacivirus
+
(
HCV), as well as against one genus of the ssRNA Picornaviridae family,
Enterovirus (Coxsackie Virus B2 and Poliovirus Sabin-1). Both anomers 2
–
and 8 were also tested against genera representative of three ssRNA virus
families, Paramyxoviridae, that is, Pneumovirus (Respiratory Syncytial Virus),
Rhabdoviridae, that is, Vesiculovirus (Vesicular Stomatitis Virus), Orthomyxo-
viridae, that is, Influenzavirus (Influenza A), against one genus of the dsRNA
Reoviridae family, that is, Orthoreovirus (Reo-1) and against viruses represen-
tative of two dsDNA virus families, Poxviridae (Vaccinia Virus) and Herpes-
viridae (Herpes Simplex Virus). Unfortunately, for all these viruses, the new
nucleoside analogues 2 and 8 showed neither antiviral activity nor cyto-
toxicity at the highest concentration tested (generally 100 µM).
SUMMARY AND CONCLUSION
4
-(2-C-Methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide
(
2) was synthesized as an example of a 2′-C-methyl branched pyrazine
nucleoside analogue. Unfortunately, when evaluated in cell culture experi-
ments against a broad range of viruses, this compound exhibited no signifi-
cant antiviral effect or cytotoxicity. Several factors could be responsible for
the inactivity of 2-oxo-1-(2-C-methyl-β-D-ribofuranosyl)pyrazine-3-carbox-
amide (2), such as its inability to enter cells or to serve as a substrate for
intracellular enzymes catalyzing phosphorylation, or perhaps a lack of inhi-
1
8
bition of the viral polymerases by its triphosphate . Further research, be-
yond the scope of this work, would be needed to support these hypotheses.
EXPERIMENTAL
General Procedure
1
H NMR spectra were recorded at ambient temperature on a Bruker AC 300 or 400 MHz
1
spectrometer. H NMR chemical shifts (δ-scale) are quoted in ppm referenced to the residual
solvent peak [dimethyl sulfoxide (DMSO-d ) set at 2.49 ppm, D O set at 4.79 ppm]; coupling
6
2
constants J are given in Hz. The accepted abbreviations are as follows: s, singlet; d, doublet;
t, triplet, m, multiplet. FAB mass spectra were recorded in the positive- or negative-ion mode
on a JEOL DX 300 mass spectrometer operating with a JMA-DA 5000 mass data system and
using a mixture of glycerol (G) and thioglycerol (T) (1:1, v/v) as the matrix. Melting points
were determined in open capillary tubes with a Büchi B-545 apparatus and are uncorrected.
UV spectra were recorded on a Uvikon XS spectrophotometer. Thin-layer chromatography
(
TLC) was performed on precoated aluminium sheets of silica gel 60 F254 (Merck, Art. 5554),
visualization of products being accomplished by UV absorbance and by charring with 10%
ethanolic sulfuric acid on heating. Column chromatography was carried out on silica gel 60
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1327–1333

T-1106, 2′-C-Methyl Branched Derivative
1331
(
Merck, Art. 9385). Evaporation of solvents was carried out in a rotary evaporator under re-
duced pressure. All moisture sensitive reactions were carried out under rigorously anhydrous
conditions under argon atmosphere using oven-dried glassware. Solvents were dried and dis-
tilled prior to use and solids were dried over P O under reduced pressure. Analytical high-
2
5
performance liquid chromatography (HPLC) studies were carried out on a Waters Associates
unit (multisolvent delivery system, 717 autosampler injector, 996 photodiode array detector
and a Millenium data workstation) using a reverse-phase analytical column (Nova-Pak
Silica 60 Å 4 µm, C18, 150 × 3.9 mm).
2
-Aminopropanediamide 4 15
To a solution of diethylaminomalonate hydrochloride 3 (9 g, 42.5 mmol) in water (10 ml)
was added solid sodium hydrogen carbonate until pH > 7. After extraction with ethyl ace-
tate, the organic phase was separated and evaporated under reduced pressure. Then, the resi-
due was taken into a methanolic ammonia solution (7 N) and the reaction mixture heated in
a high pressure reactor at 80 °C overnight. After evaporation of the solvent under reduced
pressure, the crude residue was precipitated in methanol. The solid was filtered and washed
successively with methanol and diethyl ether to afford compound 4 (3.5 g, 70%). FAB > 0
+
– 1
m/z (GT) 118 (M + H) ; FAB < 0 m/z (GT) 116 (M – H) . H NMR (400 MHz, DMSO-d ): 7.40
6
and 7.24 2 s, 6 H, D O exchangeable (3 NH ); 3.75 s, 1 H (CH).
2
2
3
-Oxo-3,4-dihydropyrazine-2-carboxamide (5)14
To a suspension of compound 4 (3.5 g, 30 mmol) in water (24 ml) glyoxal (10 g, 36 mmol)
was added. The reaction mixture was stirred at 90 °C for 3 h, then cooled down to room
temperature. After neutralization with 15% NH OH solution until pH > 7, H O was added
4
2
2
dropwise at 0 °C and the reaction mixture was stirred at room temperature for 1 h. The pre-
cipitate was filtered, washed with acetone and crystallized in water to give the desired pure
+
pyrazine compound 5 (2.65 g, 63%), m.p. 269–271 °C. FAB > 0 m/z (GT) 140 (M + H) ;
–
1
FAB < 0 m/z (GT) 138 (M – H) . H NMR (400 MHz, DMSO-d ): 13.30 s, 1 H, D O exchange-
6
2
able (NH); 8.7 and 7.9 2 s, 2 H, D O exchangeable (NH ); 8.1 m, 2 H (H-5 and H-6). HPLC:
2
2
R = 0.98 min (100% H O over a 10 min period, flow rate of 1 ml/min), λ
= 265 nm. UV
t
2
max
(
H O): λ
= 344 nm (ε 6800), λmin = 279 nm (ε 670), λmax = 229 nm (ε 8000), λmin = 207
2
max
nm (ε 3000).
1
,2,3,5-Tetra-O-benzoyl-2-C-methyl-α-(and β)-D-ribofuranose (6)
This compound was prepared according to a published procedure16. The corresponding
β-anomer was obtained by crystallization from hexanes and ethyl acetate. Yields and
1
6
physico-chemical characteristics were in accordance with those reported in the literature
.
4
3
-(2,3,5-Tri-O-benzoyl-2-C-methyl-α-(and β)-D-ribofuranosyl)-
-oxo-3,4-dihydropyrazine-2-carboxamide (7)
A suspension of 3-oxo-3,4-dihydropyrazine-2-carboxamide (5; 300 mg, 2.1 mmol) in anhy-
drous acetonitrile (7.0 ml) was treated with N,O-bis(trimethylsilyl)acetamide (1.04 ml,
4
.24 mmol) and brought to 90 °C for 1 h. The resulting solution was allowed to cool to am-
bient temperature, peracylated sugar 6 (600 mg, 1.0 mmol) was added, followed cautiously
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1327–1333

1
332
Pierra, Counor, Storer, Gosselin:
with stannic chloride (0.41 ml, 3.5 mmol), and heated to reflux for 2 h. After addition of a
cold saturated solution of hydrogen carbonate, the solid was filtered over Celite. The filtrate
was diluted with ethyl acetate, washed with a solution of saturated sodium hydrogen car-
bonate and brine, dried over sodium sulfate, and evaporated under reduced pressure. Silica
gel column chromatography of the residue using 2% of methanol in dichloromethane af-
forded the title compound 7 (396 mg, 66%) as a α/β (80:20) anomeric mixture. FAB > 0 m/z
+
+
+
–
–
(
GT) 598 (M + H) , 459 (Sugar) , 105 (Bz) ; FAB < 0 m/z (GT) 596 (M – H) , 121 (BzO) .
1
H NMR (400 MHz, DMSO-d ): 8.5–7.3 m, 19 H (H-5, H-6, Bz, NH ); 6.9 s, 0.2 H (H-1′β);
6
2
6
4
.83 s, 0.8 H (H-1′α); 6.01 d, 1 H J = 3.0 (H-3′); 5.6 m, 0.8 H (H-4′α); 5.0 m, 0.2 H (H-4′β);
.9–4.7 m, 2 H (H-5′, H-5”); 2.2 s, 3 H (CH ).
3
4
4
-(2-C-Methyl-β-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide (2) and
-(2-C-methyl-α-D-ribofuranosyl)-3-oxo-3,4-dihydropyrazine-2-carboxamide (8)
A solution of the α/β anomeric mixture 7 (396 mg, 0.66 mmol) in methanol (13 ml) pre-
saturated with ammonia at 0 °C was stirred at room temperature overnight, and evaporated
to dryness. The residue was purified by reverse phase column chromatography (0–5% aceto-
nitrile in water) to provide pure 2 (19 mg, 10%) and pure 8 (109 mg, 58%) which were
lyophilized in water.
+
+
β-Anomer 2. FAB > 0 m/z (GT) 286 (M + H) , 140 (Base + 2 H) ; FAB < 0 m/z (GT) 284 (M –
–
–
1
H) , 138 (Base) . H NMR (300 MHz, D O): 8.14 d, 1 H J = 4.3 (H-6); 7.67 d, 1 H, J = 4.3
2
(
H-5); 6.22 s, 1 H (H-1′); 4.1–3.7 m, 4 H (H-3′, H-4′, H-5′, H-5”); 1.01 s, 3 H (CH ). HPLC:
3
R = 3.3 min (0–50% CH CN in H O over a 15 min period, flow rate of 1 ml/min), λ =
t
3
2
max
2
30 nm. UV (H O): λmax = 350 nm (ε 9900), λmin = 279 nm (ε 920), λmax = 228 nm
2
(
ε 10400), λmin = 207 nm (ε 5600).
α-Anomer 8. FAB > 0 m/z (GT) 286 (M + H) , 140 (Base + 2 H) . FAB < 0 m/z (GT) 284 (M –
+
+
–
–
1
H) , 138 (Base) . H NMR (400 MHz, DMSO-d ): 8.39 and 7.72 2 s, 2 H D O exchangeable
6
2
(
NH ); 7.66 d, 1 H, J = 4.3 (H-6); 7.54 d, 1 H, J = 4.3 (H-5); 6.22 s, 1 H (H-1′); 5.3–4.7 m, 3 H
2
D O exchangeable (OH-2′, OH-3′, OH-5′); 4.1 m, 1 H (H-4′); 3.8 m, 1 H (H-3′); 3.7 m, 1 H
2
(
H-5′); 3.5 m, 1 H (H-5′′); 1.25 s, 3 H (CH ). HPLC: R = 3.09 min (0–50% CH CN in H O
3
t
3
2
over a 15 min period, flow rate of 1 ml/min), λmax = 230 nm. UV (H O): λ
= 355 nm
2
max
(
ε 8300), λmin = 281 nm (ε 500), λmax = 228 nm (ε 8200), λmin = 205 nm (ε 3300).
We gratefully acknowledge Dr. M. Liuzzi (Idenix Senior Director Virology, Laboratory Cooperativo
Idenix-Universita di Cagliari, Italy) and his collaborators for the biological results. We are indebted to
K. Barry (Corporate Communications Specialist, Idenix Pharmaceuticals Inc.) for critical reading of
the manuscript.
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