Synthesis of 2′-N-formamido nucleosides and biological evaluation

Article abstract of DOI:10.1080/15257770903362164

The 2′-N-formamide derivatives of adenosine, cytidine, and 9-β-D-arabinofuranosyladenine were synthesized and tested (as triphosphate) for their substrate capacities for the HCV NS5B polymerase.

Full text of DOI:10.1080/15257770903362164

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Synthesis of 2′-N-Formamido Nucleosides
and Biological Evaluation
a
a
a
Mikhail Abramov , Marleen Renders & Piet Herdewijn
a
Laboratory of Medicinal Chemistry, Rega Institute for Medical
Research, K.U. Leuven, Leuven, Belgium
Published online: 24 Nov 2009.
To cite this article: Mikhail Abramov , Marleen Renders & Piet Herdewijn (2009) Synthesis of 2′-
N-Formamido Nucleosides and Biological Evaluation, Nucleosides, Nucleotides and Nucleic Acids,
28:11-12, 1042-1050, DOI: 10.1080/15257770903362164
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Nucleosides, Nucleotides and Nucleic Acids, 28:1042–1050, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903362164
ꢀ
SYNTHESIS OF 2 -N-FORMAMIDO NUCLEOSIDES AND BIOLOGICAL
EVALUATION
Mikhail Abramov, Marleen Renders, and Piet Herdewijn
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, K.U. Leuven,
Leuven, Belgium
ꢁ
2
The 2 -N-formamide derivatives of adenosine, cytidine, and 9-β-d-arabinofuranosyladenine
were synthesized and tested (as triphosphate) for their substrate capacities for the HCV NS5B
polymerase.
ꢁ
Keywords 2 -N -formamide derivatives; HCV drugs; RNA viruses; anti HCV nucleosides;
antiviral drug design
INTRODUCTION
The discovery of the hepatitis C virus[1] has activated research groups
in industry and academia to develop antiviral compounds targeting RNA
viruses. The current therapy uses a combination of pegylated interferon
alpha and ribavirin, but its effectiveness is limited and side effects may
[2]
be severe.
The research on potential hepatitis C virus (HCV) drugs
has been boosted by the development of in vitro and in vivo test systems
to screen compound.[ As far as nucleoside chemistry is concerned, the
most attractive target to develop anti-HCV nucleosides is the NS5B RNA
dependent RNA polymerase, which plays an essential role in viral replication
3]
[4]
and which has no equivalent in mammalian cells. The approach itself, that
is, selecting a modified nucleoside that is phosphorylated in the cell and, as
a triphosphate, inhibits the polymerase, is common in the field of antiviral
drug design. So far, only a limited number of nucleoside analogues has been
[5]
found to inhibit the HCV RNA dependent RNA polymerase. Most of these
Received 27 April 2009; accepted 22 September 2009.
The authors thank Gilead Sciences for providing us the NS5B ꢀ55 polymerase.
Address correspondence to Piet Herdewijn, Laboratory of Medicinal Chemistry, Rega Institute
for Medical Research, K.U. Leuven, Minderbroedersstraat 10, BE-3000, Leuven, Belgium. E-mail:
piet.herdewijn@rega.kuleuven.be
1
042

ꢁ
2
-N-formamido Nucleosides
1043
ꢁ
nucleoside analogues carry a 3 -hydroxyl group, which means that they are
most probably not absolute chain terminators. The presence of a substituent
ꢁ
ꢁ
at either the 2 - or the 4 -position is apparently sufficient to inhibit chain
elongation resulting in an antiviral effect. The specific recognition of
ꢁ
the 2 -position of nucleosides by the RNA dependent RNA polymerase is
required to distinguish between incoming nucleoside triphosphates (2 -
OH) and incoming deoxynucleoside triphosphates (2 -OH). 2 -OMe and
-deoxy-2 -fluoro2 -C-methylnucleosides as triphosphates have been identi-
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2
[
5a,h]
ꢁ
fied as inhibitors of the NS5B polymerase.
We have synthesized 2 -N-
formamido nucleosides and their triphosphates and we have evaluated these
ꢁ
triphosphates as potential inhibitors of NS5B. By formylating the 2 -NH
2
ꢁ
ꢁ
group of 2 -amino-2 -deoxynucleosides, the basic amino group is converted
into a slightly acidic 2 -substituent (pK of formamide: 23.5 ), however,
ꢁ
[6]
a
with considerable steric bulk.
RESULTS AND DISCUSSION
ꢁ
The 2 -formamido nucleosides and their triphosphates are very poor
ꢁ
ꢁ
described in literature. Only 2 -formamido-2 -deoxyuridine was prepared
by formylation of protected 2 -amino-2 -deoxyuridine with acetic formic
anhydride in moderate yield.
ꢁ
ꢁ
[7]
ꢁ
ꢁ
The synthesis of 2 -N -formamido-2 -deoxyadenosine (IV) is based on
the procedure of Robins.[ We used Markiewicz’s disiloxane group to
8]
[9]
ꢁ
ꢁ
regiospecifically mask the 5 - and 3 -hydroxyl groups of ara-A, leaving
the 2 -hydroxyl group free for sulfonylation (triflation) (see Figure 1).
Transient 2 -triflation with trifluoromethanesulfonyl chloride followed by
S 2 displacement at C2 using LiN gave the 2 -azidonucleoside in good
yield which was reduced to the 2 -amino derivative. To avoid a possible
formylation of exocyclic aminogroup of adenine nucleobase with acetic
ꢁ
ꢁ
ꢁ
ꢁ
N
3
ꢁ
ꢁ
formic anhydride the 2 -amino group was formylated using ammonium
[
10]
formate
as a more selective formylating reagent. Deprotection with
Et N.HF/NMP gave the desired N -formamido compound (IV) in 60% yield.
3
ꢁ
ꢁ
2
-N -Formamido-2 -deoxy-ara-adenosine (V) was synthesized in a similar way
starting from adenosine.
ꢁ
ꢁ
The synthesis of 2 -N -formamido-2 -deoxycytosine (VI) is based on the
[
11,12]
ꢁ
procedure described earlier.
Uridine was converted into its 2,2 -
The interaction of this compound with sodium
azide in the presence of dibenzo-18-crown-6 leads to the formation of
O-anhydroderivative.[
13]
ꢁ
ꢁ
ꢁ
ꢁ
2
-azido-2 -deoxyuridine which is protected at the 5 - and 3 -hydroxyl groups
[9]
with a disiloxane group. The uridine base was converted into cytosine
according to the published procedure
into the amino-derivative using a Staudinger reduction.
[11]
and azidonucleoside was reduced
[14]
The selective

1
044
M. Abramov et al.
NH2
N
NH2
NH2
N
N
N
N
N
N
N
vii,viii
i-vi
HO
HO
N
TPO
N
N
N
O
O
OH HN
IV
O
OH OH
OH HN
CHO
CHO
I
NH2
N
NH2
NH2
N
N
N
N
i-vi
N
vii,viii
HO
N
HO
N
TPO
N
N
N
N
O
O
OH HN
V
O
OH HN
II
OH OH
CHO
CHO
ꢁ
ꢁ
FIGURE 1 Synthesis of 2 -deoxy-2 -formamidonucleosides (IV, V) and its triphosphates (I, II). Reagents
◦
and conditions: (i) TIPS-Cl/Pyr; (ii) CF3SO2Cl, DMAP/DCM, 0 C; (iii) LiN3, DMF; (iv) H2, 5%
Pd(C); (v)HCOONH4, MeCN; (vi) Et3N.HF/THF; (vii) POCl3, PO(OMe)3; (viii) (Bu3NH)4P2O7,
Bu3N/TEAB.
aminogroup N -formylation using ammonium formate[10] followed by de-
protection gave the desired N -formamido compound (VI) in 75% yield.
[
15]
To obtain triphosphates (I–III) the one pot synthesis of Ludwig
as
ꢁ
the most straightforward method for the 5 -selective phosphorylation of
nucleosides (IV–VI) was applied.
The synthetic nucleoside triphosphate analogues were tested on their
substrate capacities for the HCV NS5B polymerase. In order to function as
an inhibitor of a polymerase, a compound has to be accepted as a substrate
by the polymerase and has to be incorporated in an elongating primer
O
N
NH2
N
NH2
N
NH
i-vii
viii,ix
HO
O
HO
N
O
TPO
N
O
O
O
O
OH OH
OH HN
OH HN
CHO
CHO
VI
III
ꢁ
ꢁ
FIGURE 2 Synthesis of 2 -deoxy-2 -formamido-β-D-ribofuranosylcytidine (VI) and its triphosphate (III).
(
◦
i) (PhO)2CO, 120 C; (ii) NaN3, dibenzo-18-crown-6; (iii) TIPS-Cl, Py; (iv) POCl3, 1,2,4-triazole,
NH3; (v) PPh3, H20-THF; (vi) HCOONH4, MeCN; (vii) Et3N.HF/NMP; (viii) POCl3, PO(OMe)3; (ix)
Bu3NH)4P2O7, Bu3N/TEAB.
(

ꢁ
2
-N-formamido Nucleosides
1045
FIGURE 3 Incorporation of the nucleotide analogues into an RNA duplex by the HCV NS5B
◦
polymerase after 30 minutes, 1 hour, and 2 hours at 30 C. The blanc reaction was carried out without
∗
the addition of triphosphate analogues. [NTP ] = 1 mM; [HCV NS5B pol] = 3.8 µM.
strand hybridized to a template sequence. The single nucleotide incorpora-
tion (SNI) assay was used to check the substrate capacities of the nucleoside
triphosphate analogues for the NS5B polymerase. In the SNI assay used,
a dinucleotide acts as the priming agent for the synthesis. Consequently,
the capacity of the compounds to interact during the transition from the
initiation to the elongation process of the RNA synthesis was checked.The
molecules were tested for their interference at a very early stage of the
ꢁ
ꢁ
viral replication. 4 -Azido-cytidine 5 -O-triphosphate was used as a reference
compound as it is known to be an efficient substrate of the NS5B polymerase
enzyme IC50 being 0.04 µM).[
5d]
The assay was carried out with the ꢀ55
(
variant of the NS5B polymerase. None of the three compounds was accepted
by the enzyme as a substrate.
CONCLUSIONS
Ammonium formate is a convenient selective reagent for the N-
ꢁ
ꢁ
formylation of 2 -amino-2 -deoxy nucleosides. As triphosphate, the synthe-
sized nucleosides were, however, not substrates for HCV NS5B polymerase.
In the past, many compounds were tested for their potential to inhibit
the HCV polymerase, but only a few exhibited activity. The number of
structural classes that can interfere with the enzyme function, seems to be
limited.
It remains, however, important to continue exploring this polymerase
as a target for modified nucleoside triphosphates. The work, as presented
here, may contribute to a better understanding of the SAR for nucleotides
in the polymerase field.

1
046
M. Abramov et al.
TABLE 1 Overview of the RNA primer and
template sequences used in the NS5B single
nucleotide incorporation assay
Nucleotide analogue
tested
Primer and template
sequences used
ꢁ
ꢁ
ꢁ
ꢁ
-GC-3
3 -CGUAAAAAAAC-5
5
ꢁ
ꢁ
A
C
3 -CGGAAAAAAAC-5
EXPERIMENTAL
General
For all reactions, analytical grade solvents were used. All moisture-
sensitive reactions were carried out in oven-dried glassware under N2.
Reagents and solvents were provided by Acros (Geel, Belgium), Sigma-
Aldrich (Bornem, Belgium), or Pharma Waldhof (Dusseldorf, Germany).
TLC: Precoated aluminium sheets (Fluka silica gel/TLC cards, 254 nm);
the spots were visualized with UV light. Column chromatography (CC):
1
13
31
ICN silica gel 63–200 60 P. H-, C- and P-NMR spectra: a Bruker Avance
00-MHz, or a Bruker Avance 500-MHz spectrometer (Bruker, Brussels,
3
Belgium). For sake of clarity, NMR signals of nucleoside-sugar-moiety H-
and C-atoms are indicated with primes. Exact mass measurements were
performed on a quadrupole time-of-flight mass spectrometer (Q-Tof-2, Mi-
cromass, Manchester, UK) equipped with a standard electrospray-ionization
(
ESI) interface; samples were infused in i-PrOH/H O 1 : 1 at 3 ml/min.
2
The GC dinucleotide primer was obtained from Sigma-Aldrich. All
template sequences were ordered from Eurogentec. An overview of the
primer and template sequences is given in Table 1.
Typical Procedure for the Synthesis of
ꢁ
ꢁ
2
-Deoxy-2 -formamidonucleosides
ꢁ
ꢁ
ꢁ
ꢁ
To
a
solution of 2 -amino-2 -deoxy-3 ,5 -O-(1,1,3,3-tetraisopropyl
disiloxane-1,3-diyl) nucleoside (1 mmol) in dry acetonitrile (5 mL) was
added anhydrous ammonium formate (1.5 mmol) and the resulting
◦
mixture was heated at 95 C (bath temperature) for 11 hours. Acetonitrile
was removed under reduced pressure. The residue was diluted with ethyl
acetate (20 mL) and washed with water (2 × 10 mL). The organic layer
was dried over anhydrous Na SO and then concentrated to dryness.
2
4
N-Methyl-2-pyrrolidinone (1800 µl), triethylamine (900 µl, 6.47 mmol)
and triethylamine tris-hydrofluoride (1200 µl, 7.37 mmol) was added and
the reaction mixture was stirred at RT for 30 min. The reaction mixture
was diluted with water (20 mL) and washed with dichloromethane (3 × 25

ꢁ
2
-N-formamido Nucleosides
1047
mL) The aqueous layer was concentrated using a rotary evaporator. The
crude product was purified by column chromatography on silica gel with
EtOH/CHCl (1:3 to 1:1).
3
ꢁ
ꢁ
1
2
-Deoxy-2 -formamido-β-D-ribofuranosyladenine. H NMR (D O): 8.30
2
(
s, 1H, H8), 8.19 (s, 1H, H2), 7.95 (s, 1H, H-CO), 6.01 (d, J = 8.2 Hz, 1H,
ꢁ
ꢁ
ꢁ
H1 ), 5.01 (dd, J = 8.0,5.7 Hz, 1H, H2 )4.41 (dd, J = 1.9,5.7 Hz, 1H, H3 ),
ꢁ
ꢁ
13
4
1
.26 (m, 1H, H4 ), 3.82 (m, 2H, H5 ). C NMR (D O) δ: 164.4(CHO),
2
ꢁ
49.2, 149.1(C2, C6), 148.3(C4), 141.6(C8), 119.0(C5), 87.4, 87.3 (C1 ,
ꢁ
ꢁ
ꢁ
ꢁ
+
C4 ), 70.4(C3 ), 61.6(C5 ), 54.4(C2 ). HRMS calcd for C H N O , [MH ]
11
14
6
4
2
95.1155, found, 295.1149.
ꢁ
ꢁ
1
2
-Deoxy-2 -formamido-arabinofuranosyladenine. H NMR (D O): 8.22
2
(
s, 1H, H8), 8.10 (s, 1H, H2), 7.79 (s, 1H, H-CO), 7.24 (s, 2H, NH ), 6.32 (d,
2
ꢁ
ꢁ
ꢁ
5
J = 6.8 Hz, 1H, H1 ), 5.61 (d, J = 6.0, 1H,OH ), 5.24(t, J = 5.2, 1H, OH ),
3
ꢁ
ꢁ
ꢁ
4
.65 (m, H2 )4.41 (m, 1H, H3 ), 4.42 (m, 1H, H4 ), 3.62–3.84 (m, 3H,
ꢁ
ꢁꢁ 13
H4, H5 H5 ). C NMR (DMSO-d6) δ: 161.3(CHO), 155.9(C6), 152.3(C2),
1
5
ꢁ
ꢁ
ꢁ
ꢁ
49.1(C4), 139.6(C8), 118.5(C5), 83.2, 81.8 (C1 , C4 ), 70.7(C3 ), 59.7(C5 ),
ꢁ
+
6.5(C2 ). HRMS calcd for C H N O , [MH ] 295.1155, found, 295.1150.
-Deoxy-2 -formamido-β-D-ribofuranosylcytidine. H NMR (D O): 8.02
s, 1H, CHO), 7.75 (d, 1H, J = 7.6 Hz, H6), 6.00 (m, 2H, H5 and H1 ), 4.49
11
14
6
4
ꢁ
ꢁ
1
2
2
ꢁ
(
(
ꢁ
ꢁ
dd, J = 7.6, 6.0 Hz, 1H, H2 ), 4.27 (dd, J = 6.0, 2.6 Hz, 1H, H4 ), 4,09 (dd,
ꢁ
ꢁ
ꢁꢁ 13
J = 7.6,2.6 Hz, 1H, H3 ), 3.75 (m, 2H, H5 , H5 ). C NMR (D O) δ: 165.6
2
ꢁ
(C4), 164.4 (CHO), 157.2(C2), 141.4(C6), 96.85 (C5), 87.4, 86.07 (C4 and
ꢁ
ꢁ
ꢁ
ꢁ
+
C1 ), 70.4 (C3 ), 61.3 (C5 ), 54.(C2 ). HRMS calcd for C H N O , [MH ]
10
14
4
5
271.1042, found 271.1038.
Typical Procedure for the Synthesis of
ꢁ
ꢁ
ꢁ
2
-deoxy-2 -Formamidonucleoside 5 -O-Triphosphate
Tetra(triethylammonium) Salts
After lyophilisation overnight the unprotected nucleoside was dissolved
◦
in trimethylphosphate (10 µl/mg nucleoside) and cooled to 0 C. Under
stirring phosphorous oxychloride (2 equivalents) was added. The mixture
◦
is kept at 0 C for several hours and followed up with a 2-propanol,
NH OH, water (6:3:1) TLC system. Subsequently 10 equivalents of tri-(tetra-
4
n-butylammonium)hydrogenpyrophosphate (1 M solution in DMF in the
presence of 0.3 M Bu N) was slowly added under argon. After two minutes
3
of vigorously stirring, the solution is pipetted into a 1 M TEAB solution at
pH 7.5. The mixture was stirred for 40 minutes, the solvent was removed and
the residue was lyophilized. Purification of the triphosphates was performed
on a Source 15Q ion exchange column (Amersham Biosciences, Diegem,
Belgium) using a TEAB gradient from 0 to 0.5 M in 30 minutes.
ꢁ
ꢁ
ꢁ
2
-Deoxy-2 -formamido-β-D-ribofuranosyladenine5 -O-triphosphate tetra
31
(
triethyl-ammonium) salt. P NMR (D O): -9.00 (d, γ -P); −11.30 (d, α-P);
2

1
048
M. Abramov et al.
−
−
22.83 (t, β-P). HRMS calcd for C H N O P [MH ] 532.9994, found
11
17
6
13 3
5
32.9996.
2
ꢁ
ꢁ
ꢁ
-Deoxy-2 -formamido-β-D-ribofuranosylcytidine5 -O-triphosphate tetra
31
(
-
triethylammonium) salt. P NMR (D O): −10.25 (d, γ -P); −11.54 (d, α-P);
2
−
23.16 (t, β-P). HRMS, calcd for C H N O P , [MH ] 508.9881, found
10
17
4
14 3
5
08.9887.
ꢁ
ꢁ
ꢁ
2
-Deoxy-2 -formamido-arabinofuranosyladenine 5 -O-triphosphate tetra
31
(
triethyl-ammonium) salt. P NMR (D O): −10.90 (d, γ -P); −11.52 (d, α-P);
2
−
−
23.36 (t, β-P). HRMS calcd for C H N O P , [MH ] 532.9994, found
11
17
6
13 3
5
32.9996.
33
P-Labelling of the primer. An unlabelled primer (100 pmol) was mixed
with 33P-γ -ATP (2.5 µl of a 370 MBq/ml (10 mCi/ml) solution, Perkin
Elmer), 45 units of T4 polynucleotide kinase (Amersham Biosciences),
◦
kinase buffer, and water. After incubation of the mixture at 37 C for 1 hour,
◦
the kinase was inactivated by heating the solution at 75 C for 10 minutes. A
ꢁ
4
µM 5 -labelled solution of primer in water was obtained.
ꢁ
HCV NS5B Polymerase reaction. After labelling the 5 -end of the primer
strand the oligonucleotide was added to a cold solution of the primer
sequence, hybridized to a template strand and mixed with reaction buffer
(
1× concentrate: 50 mM HEPES (pH 7.3), 5 mM MgCl and 10 mM DTT).
2
The polymerase was added and the solution (2× concentrate) was put on
◦
an electric heating block at 30 C. A solution of nucleoside triphosphate (2×
◦
concentrate) was added and the reaction was again incubated at 30 C. Final
concentrations were 20 µM for the primer and the template strand, 1 mM
for the triphosphate analogue and 3.8 µM for the polymerase. After 30
minutes, 1 hour, and 2 hours an aliquot of the reaction mixture was removed
and mixed with a double volume of gel loading solution containing 90%
formamide and 0.05% of both the colouring agents bromofenolblue and
xylenecyanol. Analysis of the reaction was carried out through loading of
a 2 µl sample on a 25% polyacrylamide gel in a TBE buffer containing 7
M urea, 100 mM Tris–borate, 2.5 mM EDTA running buffer, pH 8.3. By
applying a power of 70W until the lowest blue dye was 70% down on the
gel the non-extended primer was separated from the extended primer. The
gel was exposed to a phosphor screen overnight after which the screen is
scanned with a phosphorimager. The percentage of incorporation was deter-
mined by integration using the Optiquant image analysis software (Perkin
Elmer, Zaventem, Belgium).
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