Synthesis of reactive cytidine derivatives as building blocks for cross-linking oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2005.04.083

6-Vinylcytidine derivative (1) possessing good Michael acceptor properties has been synthesized through C-6 formylation and subsequent Wittig reaction. In view of introducing the reactive nucleoside into the oligonucleotide sequence, protection of the vin

Full text of DOI:10.1016/j.tetlet.2005.04.083

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4361–4364
Synthesis of reactive cytidine derivatives as building blocks
for cross-linking oligonucleotides
Marco Radi, Claudia Mugnaini, Elena Petricci, Federico Corelli* and Maurizio Botta*
`
Dip. Farmaco Chimico Tecnologico, Universita degli Studi di Siena., Via A. Moro, 53100 Siena, Italy
Received 3 March 2005; revised 13 April 2005; accepted 20 April 2005
Available online 10 May 2005
Abstract—6-Vinylcytidine derivative (1) possessing good Michael acceptor properties has been synthesized through C-6 formylation
and subsequent Wittig reaction. In view of introducing the reactive nucleoside into the oligonucleotide sequence, protection of the
vinyl group as ethylthio derivative was proved to be effective for the masking and subsequent regeneration of the reactive vinyl
moiety.
Ó 2005 Elsevier Ltd. All rights reserved.
After decades and hundreds of million of dollars spent
on antisense research, currently a large number of anti-
NHBz
6-vinyl substituent
as Michael acceptor
N
to increase ODNs
hybridization properties
sense compounds are in preclinical and clinical trials,
particularly in the cancer, cardiovascular and infectious
disease fields.1 Main drawbacks for the use of natural
oligonucleotides as therapeutical agents are the unsatis-
factory binding affinity, the instability against cellular
nucleases, the insufficient membrane penetration and
the low bioavailability.2
O
N
TBDMSO
O
2'-O-MOE substituent
to increase ODNs
TBDMSO
O
OMe
nuclease resistance
1
Figure 1. 20-O-MOE-6-vinylcytidine derivative.
In order to overcome the problems associated with the
binding affinity and the endonuclease digestion, our
work was mainly focused on the synthesis of a modified
monomer able to confer nuclease resistance and better
binding properties once inserted into a specific oligo-
nucleotide sequence. Based on the good Michael acceptor
properties showed by some 6-vinylpyrimidine deriva-
tives recently synthesized in our group,3 we planned to
synthesize the cytidine derivative 1 (Fig. 1): the 6-vinyl
group could be able to confer good binding properties
through Michael addition reaction while the 20-O-
MOE moiety is known to confer nuclease resistance
properties.4
mity of the target site. While natural nucleosides exist
predominantly in the anti conformation referred to the
glycosyl bond, the introduction of a C-6 substituent on
the nucleobase constrains the molecule into a non-natu-
ral syn conformation.5 The introduction of the vinyl
group in C-6 position of a cytidine derivative could
therefore constrain the resulting molecule in an unnatu-
ral syn conformation, thus allowing a cross-linking reac-
tion with the amino group of a guanosine in the target
sequence (Fig. 2).
Even if 6-substituted pyrimidine oligonucleotides have
proved to bring about duplex destabilization,6 this class
of compounds have not been thoroughly studied; more-
over, the introduction of a reactive moiety in C-6 posi-
tion could provide interesting insights into duplex base
interactions.
In order to provide efficient interaction with the target
site, reactive groups within the oligonucleotide sequence
should have high reactivity and the ability to be in proxi-
Keywords: Nucleosides; Oligonucleotides; Michael addition; Cross-
linking.
In order to synthesize the target building block 1, we ini-
tially envisaged to use the Stille reaction according to a
procedure reported for the introduction of the 6-vinyl
*
Corresponding authors. Tel.: +39 (0)577234306; fax: +39
(0)577234333; e-mail: botta@unisi.it
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.083

4362
M. Radi et al. / Tetrahedron Letters 46 (2005) 4361–4364
Anti
1D NOE difference spectrometry (NOEDS) experiments
Syn
conformation
conformation
of 1 (Scheme 2) confirmed the expected syn conforma-
tion of the glycosyl bond since proton H01 showed net
NOE enhancement upon irradiation of proton H010. With
this result in hand, we evaluated the ability of 1 to give a
Michael addition reaction with a guanosine amino
group (Scheme 3).
NH2
O
N
N
O
N
HN
H2N
N
O
N
O
O
O
Target
sequence
Guanosine monohydrate 5 was first protected as t-butyl-
dimethylsilyl derivative 6 and then reacted in CH2Cl2
with compound 1 in a 1:1 stoichiometric ratio. Under
these experimental conditions, the slow formation of
the adduct 7 was detected after 12 h through MS analy-
sis. However, compound 7 could be obtained in 35%
yield after only 2 h carrying out the same reaction in
the presence of an acidic catalyst, such as 10-camphor-
sulfonic acid. According to the work developed by Sasa-
ki and co-workers,12 we tried to enhance the alkylating
properties of compound 1 towards the guanosine amino
group by introducing an ethylsulfonyl moiety on the vi-
nyl group (Scheme 4).
MeO
Antisense
oligonucleotide
Figure 2. Hypothetical cross-linking reaction between the oligo-
nucleotide bearing the Michael acceptor nucleoside and the guanosine
of the target sequence.
moiety into uridine derivatives.7 To this aim, compound
2 (Scheme 1), synthesized according to a literature pro-
cedure,8 was initially protected in a two-step reaction
obtaining 3 in quantitative yield.
However, the next lithiation and electrophilic substitu-
tion never gave in our hands the 6-tributylstannyl or
the 6-iodo derivatives for the subsequent Stille reaction.
We ascribed the failure of the above described function-
alization to the steric bulk of the electrophiles and there-
fore, the use of the Wittig reaction on the less hindered
6-formyl derivative was planned.9 Compound 3 was first
lithiated and then reacted with methyl formate thus
affording the corresponding 6-formyl derivative 4,
whose formation was followed by HPLC–MS analysis.10
Retrosynthetically, the desired compound 8 could be ob-
tained by reaction of 9 with a base and subsequent sulfur
oxidation, while 9 could arise from bromine displace-
ment in 10 obtained by bromination of the double bond
NHBz
N
O
N
O
H1''
4%
H1'
'
H2
After a simple filtration on silica, the 40:60 mixture of
compounds 3 and 4 was reacted with the suitable Wittig
reagent to give the 6-vinyl derivative 1 in a good 60%
yield.11 Unreacted 3 was recovered from the reaction
mixture and reused in the formylation reaction to in-
crease the overall yield of 1.
TBDMSO
TBDMSO
23%
O
1
MeO
Scheme 2. Important NOE correlations.
NHBz
N
NH2
N
O
O
N
N
N
N
NH
NH
NH2
N
O
N
O
N
N
NH2
TBDMSO
HO
HO
TBDMSO
i
i. ii.
O
O
O
O
OH OH
TBDMSO
OTBDMS
TBDMSO
O
OH
O
OMe
OMe
6
2
3
5
ii.
iii.
NHBz
N
NHBz
N
O
N
NHBz
N
N
N
N
N
HN
O
O
O
CHO
N
O
O
O
N
H
TBDMSO
TBDMSO
OTBDMS
TBDMSO
iv.
O
TBDMSO
OTBDMS
TBDMSO
O
TBDMSO
O
OMe
OMe
TBDMSO
O
OMe
1
4
Scheme 1. Reagents and conditions: (i) TBDMSCl, imidazole, DMF,
rt, 4 h, 99%; (ii) (C6H5CO)2O, DMF, MW 160 °C, 15 min, 99%; (iii)
LDA, THF, ꢀ78 °C, 3 h then HCOOMe, ꢀ78 °C, 3 h; (iv) N-BuLi,
THF, CH3P(C6H5)3Br, rt, 3 h, 60%.
7
Scheme 3. Reagents and conditions: (i) TBDMSCl, imidazole, DMF,
rt, 24 h, 58%; (ii) 1, CH2Cl2, CSA, rt, 2 h, 45%.

M. Radi et al. / Tetrahedron Letters 46 (2005) 4361–4364
4363
moted HBr elimination, was highlighted. In order to
verify, if 12 is the precursor of 11, compound 10 was re-
acted with DBU only, obtaining in this way the mono-
bromo derivative 12, which showed the same retention
time as the hypothesized monobrominated intermediate
and whose 1H NMR spectra clearly showed the presence
of two geminal vinyl protons.14 Finally, compound 11
was obtained by reacting 12 with ethanethiol in CH3CN
at ꢀ10 °C.15 According to these results and in the ab-
sence of more details regarding the reaction mechanism
at the moment, we can only exclude the progress of the
reaction through an initial elimination of bromine from
10 followed by Michael addition. Compound 11 could
also be obtained in a comparable yield by simple reac-
tion of 1 with ethanethiol at room temperature, thus
highlighting the good Michael acceptor properties of 1
also towards thiols.
NHBz
N
NHBz
N
N
O
O
O
O
N
SEt
TBDMSO
TBDMSO
S
Et
SEt
O
O
TBDMSO
TBDMSO
O
O
OMe
OMe
8
9
NHBz
N
NHBz
N
N
N
O
O
O
O
Br
TBDMSO
TBDMSO
Br
TBDMSO
TBDMSO
O
O
OMe
OMe
1
10
This result prompted us to consider 11 as a masked pre-
cursor of the reactive vinyl derivative 1. Therefore, com-
pound 11 was first deprotected to give 13, which was
then oxidized to the corresponding sulfone 14 by means
Scheme 4. Retrosynthetic approach for the synthesis of the ethyl-
sulfonyl derivative 8.
of OxoneÒ (Scheme 6). Subsequent treatment of the
16
crude sulfone with 0.25 M NaOH gave a one-pot depro-
tection of the amino group and b-elimination affording
15 in 87% yield.
NHBz
N
O
O
N
Br
TBDMSO
Br
iii.
In conclusion, a facile synthesis of the 6-vinylcytidine
derivative 1 as well as its Michael acceptor properties
have been described. Moreover, an ethylthio moiety
was proved to be effective for the protection of the vinyl
group and easily removable under mild alkaline condi-
tions after selective oxidation with OxoneÒ. This stra-
tegy could be favourably used during the subsequent
oligonucleotide synthesis thus avoiding unwanted side
reactions on the reactive vinyl moiety. Further studies
on the introduction of compound 13 into a specific oli-
gonucleotide sequence are underway.
i.
1
TBDMSO
O
NHBz
N
OMe
10
N
O
O
v.
TBDMSO
Br
ii.
NHBz
N
TBDMSO
O
OMe
N
12
O
O
SEt
TBDMSO
NHBz
NHBz
iv.
TBDMSO
O
OMe
N
N
11
Et
O
O
O
N
SEt
O
O
N
S
O
RO
HO
Scheme 5. Reagents and conditions: (i) Br2, CCl4, ꢀ10 °C, 1.5 h, 87%;
(ii) DBU, EtSH, CH3CN, ꢀ10 °C, 2 h, 80%; (iii) DBU, CH3CN,
ꢀ10 °C, 2 h, 48%; (iv) EtSH, CH3CN, ꢀ10 °C, 1 h, 75%; (v) EtSH,
CH2Cl2, rt, 2 h, 84%.
ii.
RO
O
HO
O
OMe
OMe
14
11 R = TBDMS
13 R = OH
i.
of 1. Compound 1 was therefore first reacted with bro-
mine in carbon tetrachloride at ꢀ10 °C to give the dibro-
mide 10 (Scheme 5). Quite surprisingly, the subsequent
bromine substitution to obtain the key intermediate 9
gave the monoethylthio derivative 11 as the only pro-
duct which was fully characterized through 1H, 13C
NMR, HPLC–MS and COSY experiments.13
iii.
NH2
N
N
O
O
HO
HO
O
To better understand the result thus obtained, the reac-
tion progress was monitored by HPLC–MS immediately
after the addition of ethanethiol. In this way, the
presence in the reaction mixture of both 11 and a mono-
bromo derivative 12, probably arising from DBU pro-
OMe
15
Scheme 6. Reagents and conditions: (i) TBAF, THF, rt, 1 h, 87%; (ii)
OxoneÒ, MeOH/H2O, rt, overnight; (iii) NaOH 0.25 M, rt, 2 h, 80%.

4364
M. Radi et al. / Tetrahedron Letters 46 (2005) 4361–4364
11. Procedure for the preparation of 1: Asolution of BuLi
Acknowledgements
(1.6 M in hexane, 1.28 mL, 2.04 mmol) was added to a
suspension of methyltriphenylphosphonium bromide
(729 mg, 2.04 mmol) in THF (10 mL) kept at ꢀ78 °C.
The mixture was stirred at 0 °C for 30 min, followed by the
addition of a solution of 4 (270 mg, 0.41 mmol) in THF
(10 mL). The reaction mixture was stirred for 1 h at room
temperature, then a saturated NH4Cl solution was added
followed by EtOAc. The organic phase was washed
successively with water and brine then dried over anhy-
drous Na2SO4. Evaporation of the solvent gave a crude
oil, which was chromatographed on a silica gel column
(eluent: CHCl3/MeOH, 98/2) to give pure 1 (169 mg,
60%).
This work was supported by the LSHB-CT-2003-503480
(European Union project ÔTargeting Replication and
Integration of HIVÕ, TRIoH) grant and by MIUR
(Rome) as a project within the PRIN 2003. One of us
(M.B.) wishes to thank the Merck Research Labora-
tories for the 2004 Academic Development Program
(ADP) Chemistry Award.
References and notes
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residue was dissolved in CHCl3. The organic phase was
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of 10 (25 mg, 0.038 mmol) in THF (5 mL), DBU (13 lL,
0.084 mmol) was added at ꢀ10 °C. Stirring was continued
at ꢀ10 °C for 30 min and at room temperature for 5 h.
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evaporated under reduced pressure. The residue was
dissolved in CH2Cl2 and then washed successively with
water, brine and dried over anhydrous Na2SO4. Evapo-
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3
(708 mg, 1.12 mmol) in anhydrous THF (20 mL) was
added dropwise to a freshly prepared solution of LDA
(14.54 mmol) in anhydrous THF (20 mL) kept at ꢀ78 °C.
After 4 h, HCOOMe (896 lL, 29.08 mmol) was added and
the reaction mixture was stirred for 3 h at ꢀ78 °C, then
allowed to warm to room temperature, diluted with
EtOAc and quenched by the addition of NH4Cl saturated
solution. The organic phase was washed successively with
water and brine then dried over anhydrous Na2SO4.
Evaporation of the solvent gave 4 as white foam which
was used without further purification.
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solution of 12 (556 mg, 0.84 mmol) in CH2Cl2 (20 mL),
ethanethiol (125 lL, 1.69 mmol) was added and the
reaction mixture was stirred for 1 h at room temperature.
After evaporation of the solvent under reduced pressure,
the resulting residue was chromatographed on a silica gel
column (eluent: CHCl3/MeOH, 98/2) to give 11 (456 mg,
75%) as a pure compound.
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