Comparing in vitro and in vivo activity of 2′-O-[2-(methylamino)-2- oxoethyl]- and 2′-O-methoxyethyl-modified antisense oligonucleotides

Article abstract of DOI:10.1021/jm701537z

A number of 2′-O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2′-O-MOE (2′-O-methoxyethyl)- and 2′-O-Me (2′-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2′-O-modification, namely 2′-O-[2-(methylamino)- 2-oxoethyl] (2′-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2′-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2′-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2′-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2′-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2′-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

Full text of DOI:10.1021/jm701537z

2766
J. Med. Chem. 2008, 51, 2766–2776
Comparing In Vitro and In Vivo Activity of 2′-O-[2-(Methylamino)-2-oxoethyl]- and
2′-O-Methoxyethyl-Modified Antisense Oligonucleotides
Thazha P. Prakash,* Andrew M. Kawasaki, Edward V. Wancewicz, Lijiang Shen, Brett P. Monia, Bruce S. Ross,
Balkrishen Bhat, and Muthiah Manoharan
Department of Medicinal Chemistry and Antisense Core Research, Isis Pharmaceuticals Inc., 1896 Rutherford Road, Carlsbad,
California 92008
ReceiVed December 9, 2007
A number of 2′-O-modified antisense oligonucleotides have been reported for their potential use in
oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2′-O-MOE
(2′-O-methoxyethyl)- and 2′-O-Me (2′-O-methyl)-modified ASOs (antisense oligonucleotides). We now report
the synthesis and biological activity of another 2′-O-modification, namely 2′-O-[2-(methylamino)-2-oxoethyl]
(2′-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to
complementary RNA similar to 2′-O-MOE-modified ASOs as compared to first-generation antisense
oligodeoxynucleotides. The ASO modified with 2′-O-NMA reduced expression of PTEN mRNA in vitro
and in vivo in a dose-dependent manner similar to 2′-O-MOE modified ASO. Importantly, toxicity parameters
such as AST, ALT, organ weights, and body weights were found to be normal similar to 2′-O-MOE ASO-
treated animal models. The data generated in these experiments suggest that 2′-O-NMA is a useful modification
for potential application in both antisense and other oligonucleotide-based drug discovery efforts.
Introduction
The first marketed antisense drug fomivirsen was a 21-mer
phosphorothioate oligodeoxynucleotide (PS-DNA).1,2 This first-
generation chemistry offered a number of advantages, such as
ease of synthesis, sufficient nuclease resistance for parenteral
administration, activation of RNase H for cleaving the target
RNA, and desired binding to cellular and serum proteins for
uptake, absorption and distribution.2 However, upon rigorous
analysis, PS-DNAs were found to have some limitations with
regard to their broad use as antisense therapeutics because of
their relatively short in vivo half-life, low binding affinity to
Figure 1. (a) Antisense oligonucleotides with gapmer design having
2′-modified “wings” at the 3′ and 5′-end flanking a central 2′-deoxy
gap region for RNase H activity. (b) 2′-O-NMA- and 2′-O-MOE-
modified RNA structures.
RNA (ca. 1 °C < DNA per modified nucleotide), and nonspe-
cific binding to some cellular proteins.
In order to improve the in vivo profile of antisense oligo-
nucleotides (ASOs), hundreds of chemical modifications have
been synthesized and evaluated for biophysical and biochemical
properties.3–5 Antisense molecules modified at the 2′-O-position
of the sugar either at one or both termini (gapmers, Figure 1)
emerged as leading second-generation chemistries for clinical
applications.1–8 In particular, 2′-O-methoxyethyl (2′-O-MOE,
Figure 1)-modified gapmer ASOs successfully demonstrated
effective inhibition of many viral and cellular gene products,
both in vitro and in vivo.2 Therefore, the 2′-O-MOE chemistry
became one of the most successful modifications to advance to
clinical use because of significant improvement in metabolic
stability, cellular absorption, and protein binding properties. A
2′-O-MOE ASO targeting ApoB (mipomersen) is in the final
stage of clinical trials for treatment of cardiovascular disease.9
oligonucleotide, when hybridized to its complementary RNA,
exhibited improved Tm compared to unmodified DNA. Interest-
ingly, the Tm enhancement was found to be similar to the
corresponding 2′-O-MOE-modified oligonucleotide.10 Further-
more, the oligonucleotides having 2′-O-NMA modification at
the 3′-end exhibited enhanced exonuclease stability compared
to 2′-O-MOE-modified oligonuclotide.10 The high-resolution
crystal structure of an oligonucleotide with 2′-O-NMA substit-
uents revealed an extended C3′-endo conformation, either the
carbonyl oxygen or the amino nitrogen trap water molecule
between the phosphate group and the sugar. This conformation
allows high affinity binding to mRNA and protects against
nuclease degradation.11 These attributes warranted further
investigation of this chemistry in pharmacologically active
sequence models.
The aim of this study was to investigate the in vitro and in
vivo potency of 2′-O-NMA gapmer ASO and compare its
potency with the 2′-O-MOE gapmer chemistry. For this purpose,
we first developed an efficient method to synthesize 2′-O-NMA-
modified adenosine, guanosine, 5-methylcytidine, and 5-me-
thyluridine nucleosides and the corresponding 3′-phosphora-
midites (see below). We then successfully incorporated these
Among a number of other 2′-O-modifications tested, 2′-O-
NMA (Figure 1) was selected for further evaluation because of
its interesting biophysical properties. In our previous report, we
showed favorable hybridization properties and nuclease stability
of 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA)-modified
oligonucleotides and their analogues.10 The 2′-O-NMA-modified
* To whom correspondence should be addressed. Tel: 760-603-2590.
Fax: 760-603-2446. E-mail: tprakash@isisph.com.
10.1021/jm701537z CCC: $40.75
2008 American Chemical Society
Published on Web 04/10/2008

Comparison of ActiVity of Modified Antisense Oligonucleotides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2767
Scheme 2a
Scheme 1a
a Key: (i) (a) triethylamine, TMSCl, 1,2,4-triazole, CH3CN, POCl3, (b)
NH4OH-dioxane; (ii) benzoic anhydride, DMF; (iii) 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite, 4,5-dicyanoimidazole, CH3CN, rt; (iv)
succinic anhydride, pyridine, DMAP, rt; (v) TBTU, 4-methylmorpholine,
DMF, CPG, rt.
a Key: (i) 6, BH3-THF, NaHCO3, 150 °C; (ii) DMTCl, pyridine, DMAP,
rt; (iii) 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, 4,5-
dicyanoimidazole, CH3CN, rt; (iv) succinic anhydride, pyridine, DMAP,
rt; (v) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU), 4-methylmorpholine, DMF, CPG, rt.
The synthesis of the 5-methylcytidine phosphoramidite 2 is
illustrated in Scheme 2. Compound 9 was transiently silylated
at the 3′-O- position with TMSCl and triethylamine in anhydrous
acetonitrile at -10 to 0 °C. The trimethylsilyl derivative was
converted to the triazole derivative with 1,2,4-triazole, POCl3,
and Et3N.15 Subsequent treatment with aqueous NH3 in dioxane
at ambient temperature for 1 h gave the 5-methylcytidine
analogue 11 in 69% yield. The exocyclic amino group was
protected with a benzoyl group16 with benzoic anhydride in
anhydrous DMF to afford 12 (89% yield). Finally, phosphity-
lation at the 3′-O- position gave the phosphoramidite 2 (85%)
in good overall yield. A portion of compound 12 was converted
in to the functionalized solid support 13b (53 µmol/g) as
described above.
monomers into gapmer ASOs targeting PTEN mRNA. Activity
of the 2′-O-MOE modification in this sequence was reported.12
For comparison, we also synthesized 2′-O-MOE-modified ASO.
These were then evaluated for their ability to reduce PTEN
mRNA in vitro and in vivo, and the data from this study are
discussed in this report.
Key to the synthesis of the 2′-O-NMA purine nucleosides
involved the regioselective alkylation of the 2′-hydroxyl group
of unprotected nucleosides. There are many published reports
on this subject involving activated17–20 and inactivated21,22
electrophiles. The degree of selectivity encompasses a wide
range and appears to depend on the nature of the substrate and/
or the electrophile. For our purposes, methyl 2-bromoacetate
provided the two carbon synthon which could be transformed
into the desired 2′-O-functionality. The esters of 2-bromoacetate
have been used as electrophiles where the sugar moiety of
nucleosides has been selectively protected.23,24 When we reacted
unprotected adenosine or 2-,6-diaminopurin-9-yl-riboside (eq
2; key: (i) DMF, methyl 2-bromoacetate, NaH) with methyl
2-bromoacetate under basic conditions at low temperatures (-40
°C), we observed a highly regioselective alkylation of the 2′-
vs 3′-hydroxyl group in 9:1 or better ratios. This regioselective
alkylation afforded 2′-O-(methoxycarbonylmethylene)adenosine
(16a, eq 2) or 2′-O-(methoxycarbonylmethylene)-2-aminoad-
enosine (17a, eq 2) in about 75% yield. The regioselectivity in
both cases was confirmed by 2D NMR (TOCSY). In contrast,
when alkylation of 2,6-diaminopurin-9-ylriboside with methyl
iodide was carried out under similar experimental conditions, a
2:1 mixture of the 2′-O- and 3′-O-methyl regioisomers was
obtained. It appears that the highly regioselective alkylation of
the unprotected purine ribosides with methyl 2-bromoacetate
is somehow influenced by the effects of the carbonyl group
adjacent to the reactive site in this SN2 reaction.25
Results and Discussion
Chemistry. The synthesis of 2′-O-NMA-5-methyluridine and
its phosphoramidite was achieved as described in Scheme 1.
The synthesis started with preparation of 2-hydroxy-N-methyl-
acetamide (6) from ethyl glycol and methylamine in 97% yield
(eq 1; key: (i) aqueous CH3NH2/THF). The ring opening13 of
the 2,2′-anhydro-5-methyluridine 7 with a borate ester of
2-hydroxy-N-methylacetamide 6 at 150 °C furnished 2′-O-[2-
(methylamino)-2-oxoethyl] 5-methyluridine (2′-O-NMA-5MeU)
8 in 32% isolated yield. The compound 8 was then selectively
protected at the 5′-position by reaction with 4,4′-dimethoxytrityl
chloride (DMTCl) in pyridine to yield compound 9 (94% yield).
Phosphitylation of compound 9 at the 3′-position with 2-cya-
noethyl N,N,N′,N′-tetraisopropylphosphorodiamidite in the pres-
ence of 4,5-dicyanoimidazole in CH2Cl2 afforded the corre-
sponding phosphoramidite building block 1 in 84% yield. The
compound 9 was also converted to the 3′-O-succinyl derivative
10a and loaded on to the aminoalkyl controlled pore glass (CPG,
Scheme 1) according to the standard synthetic procedure14 to
yield the functionalized solid support 10b (55 µmol/g).
(1)

2768 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Prakash et al.
Scheme 3a
(2)
The alkylation reaction was reproducible on a multigram scale
(25 g) and the small amounts of 3′-O-isomer formed as a side
product were easily separated by chromatography at this stage
or the subsequent step. The development of the robust large-
scale synthetic method for purine precursors 16a and 17a in a
cost-effective manner was helpful to the successful in vivo
evaluation of this chemistry.
a Key: (i) aqueous CH3NH2, THF; (ii) TMSCl, pyridine, benzoyl chloride,
NH3OH, H2O; (iii) DMTCl, pyridine; (iv) 2-cyanoethyl N,N,N′,N′-tetra-
isopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide,
CH3CN, rt; (v) succinic anhydride, CH2Cl2, DMAP, (C2H5)3N, rt; (vi) O-(7-
azabenzotriazol-1-yl)-N,N,,N′,N′-tetramethyluronium hexafluorophosphate
(HATU), 4-methylmorpholine, DMF, CPG, rt.
The 2′-O-(methoxycarbonylmethylene)adenosine (16a) was
treated with aqueous N-methylamine in THF to yield 18
(Scheme 3, 95% yield). The exocyclic amino group of com-
pound 18 was benzoylated under transient protection condi-
tions26 to obtain 19 (Scheme 3, 80%). The nucleoside 19 was
then selectively protected at the 5′-position by reaction with
DMTCl in pyridine to furnish 20 in 65% yield. Phosphitylation
of compounds 20 at the 3′-position with 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite in the presence of N,N-
diisopropylamine tetrazolide in CH3CN afforded the corre-
sponding phosphoramidite 3 in 70% yield. A portion of
compound 20 was converted to functionalized solid support 22
(53 µmol/g) in a manner similar to that described earlier for
other derivatives.
Scheme 4a
The compound 17a was treated with 40% aqueous methyl-
amine to yield 2′-O-[2-(methylamino)-2-oxoethyl]-2-aminoad-
enosine, which was subsequently converted to 2′-O-[2-
(methylamino)-2-oxoethyl]guanosine 23 (Scheme 4) on treatment
with adenosine deaminase27–30 in aqueous buffer at pH 7.5 in
good yield (61%). The exocyclic amino group of compound
23 was protected with isobutyryl group under transient protec-
tion conditions26 to yield 24 (Scheme 3, 81%). It was then
converted to 5′-O-DMT derivative 25 (64% yield). Phosphity-
lation of compound 25 at the 3′-position with 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphorodiamidite in the presence of
N,N-diisopropylamine tetrazolide in CH3CN afforded the phos-
phoramidite 4 in 80% yield. A portion of compound 25 was
loaded on solid support 26b (51 µmol/g).
2′-O-NMA-Modified PTEN Gapmer ASO Synthesis. The
2′-O-MOE ASOs 27 and 29 (Table 1) were synthesized ac-
cording to literature procedure.31 The oligonucleotides 28 and
30 (Table 1) were synthesized on a solid-phase DNA synthesizer
using the phosphoramidites 1-4. A 0.1 M solution of the
phosphoramidites in anhydrous acetonitrile was used for the
synthesis. Phenylacetyl disulfide (PADS)32 was used as
the sulfurization agent for the syntheses.
a (i) (a) aqueous CH3NH2, THF, (b) adenosine deaminase, 100 mM
sodium phosphate, pH 7.5, rt; (ii) TMSCl, Py, isobutyryl chloride, H2O;
(iii) DMTCl, pyridine; (iv) 2-cyanoethyl N,N,N′,N′-tetraisopropylphospho-
rodiamidite, N,N-diisopropylammonium tetrazolide, CH3CN, rt; (v) succinic
anhydride, pyridine, DMAP, rt; (vi) HATU, 4-methylmorpholine, DMF,
CPG, rt.
Solid supports were treated with 4% methylamine in aqueous
ammonia (28–30 wt %) and kept at room temperature for 24 h
to release the ASO from the solid support as well as remove all
protecting groups from exocyclic amino groups of the bases

Comparison of ActiVity of Modified Antisense Oligonucleotides
Table 1. 2′-O-NMA- and 2′-O-MOE-Modified ASOsa
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2769
no.
sequences
chemistry
target
27
28
29
30
5′ C&T&G& C&T&A GCC TCT GGA T&T&T& G&A& 3′
5′ C/T/G/ C/T/A GCC TCT GGA T/T/T/ G/A/ 3′
5′ T&C&C& A&G&C ACT TTC TTT T&C&C& G&G& 3′
5′ T/C/C/ A/G/C ACT TTC TTT T/C/C/ G/G/ 3′
2′-O-MOE
2′-O-NMA
2′-O-MOE
2′-O-NMA
PTEN
PTEN
control
control
a Backbone chemistry ) phosphorothioates, C ) 2′-deoxy-5-methylcytidine, A/ ) 2′-O-NMA-adenosine, C/ ) 2′-O-NMA-5-methylcytidine, G/ ) 2′-
O-NMA-guanosine, U/ ) 2′-O-NMA-5-methyluridine, A& ) 2′-O-MOE-adenosine, C& ) 2′-O-MOE-5-methylcytidine, G& ) 2′-O-MOE-guanosine, U&
2′-O-MOE-5-methyluridine. Control: target mFAS.
)
Table 2. Tm, IC50, and Mass Spectral Data of 2′-O-NMA- and
2′-O-MOE-Modified ASOs
a
no. Tm (°C) IC50 (nM) calcd mass found mass
chemistry
27
28
29
30
67.9
68.2
66.8
68.5
43
38
7215.2
7345.2
7154.2
7284.2
7213.9
7342.8
7153.1
7282.4
2′-O-MOE
2′-O-NMA
2′-O-MOE
2′-O-NMA
a Tm conditions: see the Experimental Section.
and phosphorothioate linkages. This modified deprotection
procedure was used instead of the traditional method (aqueous
ammonia at 55 °C) to prevent any transamidation reaction at
2′-position of 2′-O-NMA ASOs. The ASOs were then purified
by high-pressure liquid chromatography (HPLC) on a strong
anion-exchange column and desalted using HPLC on a reversed-
phase column. The ASOs were characterized by HPLC coupled
mass spectrometry (Table 2).
Figure 2. Reduction of PTEN mRNA in AML12 cells after trasfection
with 2′-O-MOE- (27) and 2′-O-NMA-modified (28) ASOs and 2′-O-
MOE- (29) and 2′-O-NMA-modified (30) control (mFAS) ASOs.
Biology. 2′-O-NMA- and 2′-O-MOE-Modified ASOs Ex-
hibited Similar Binding Affinity to Target RNA. In order to
evaluate the activity of inhibition of RNase H mediated gene
expression, a 2′-O-NMA gapmer ASO 28 (Table 1) targeting
mouse PTEN mRNA expression12 was synthesized. For com-
parison, previously characterized12 2′-O-MOE gapmer ASO 27
was also synthesized (Table 1). Additionally, we also synthe-
sized 2′-O-NMA and 2′-O-MOE modified control ASOs 29 and
30 (Table 1) which targeted mouse FAS mRNA.
First, we wanted to know the effect of 2′-O-NMA modifica-
tion on the binding affinity of gapmer ASOs to target RNA.
We therefore determined the Tm of ASOs 28 and 30 (Table 2)
using a 20-mer complementary RNA. For comparative analysis,
Tm of 2′-O-MOE-modified ASOs 27 and 29 (Table 2) were also
determined. In an earlier report, we had shown that 2′-O-NMA
and 2′-O-MOE modification improves the affinity of oligo-
nucleotide to complementary RNA compared to unmodified
oligonucleotides.10 The 2′-O-NMA- (28, 30) and 2′-O-MOE-
modified (27, 29) ASOs (Table 2) showed similar Tm when
duplexes with sequence matched RNA. These data suggest that
ASOs with 2′-O-NMA and 2′-O-MOE modifications were
expected to have similar binding affinity to target mRNA.
Effect of 2′-O-NMA-Modified ASO (28) on PTEN mRNA
Expression in r Mouse Liver 12 (AML-12) Cells. In order to
evaluate the in vitro activity, the 2′-O-NMA-modified gapmer
ASO 28 (Table 1) targeting PTEN mRNA expression12 was
synthesized. The oligonucleotide 28, when transfected to the
AML-12 cell, efficiently reduced the expression of PTEN
mRNA in a concentration-dependent manner (Figure 2) with
an IC50 of 38 nM (Table 2), and potency was similar to 2′-O-
MOE gapmer 27 (IC50 43 nM, Table 2). The control ASOs 29
and 30, with 2′-O-MOE and 2′-O-NMA modifications, respec-
tively, did not reduce PTEN expression.
Figure 3. Inhibition of PTEN mRNA expression with 2′-O-MOE- (27)
and 2′-O-NMA-modified (28) ASOs and control ASOs with 2′-O-MOE
(29) and 2′-O-NMA (30) modifications in mouse liver.
O-NMA-modified ASO reduced the PTEN mRNA expression
in a dose-dependent manner, whereas the control ASOs 29 and
30 did not reduce the mRNA expression even at high dose
(Figure 3). Consistent with cell culture data, no significant
difference in antisense potency was observed between 2′-O-
NMA- (28, ED50 30 mg kg-1) and 2′-O-MOE-modified (27,
ED50 24 mg kg-1) ASOs (Figure 3). However, efficacy of 2′-
O-MOE ASO was slightly better than 2′-O-NMA ASO (Figure
3). More work will be needed to see if the observed difference
in efficacy is real or an experimental error. When analyzed by
Western blot, a similar dose dependent decrease in protein level
was found in liver samples treated with 2′-O-NMA- and 2′-O-
MOE-modified ASOs (Figure 4).
In order to asses the liver function in animals treated with
2′-O-NMA and 2′-O-MOE ASOs, plasma transaminase levels
were examined after the last dose. The AST and ALT levels
were within the normal range for all animals (Figure 5). No
heaptotoxic events were observed in the H & E staining of the
liver section from the animals treated with 2′-O-NMA or 2′-
O-MOE ASOs at 50 mg/kg dose (Figure 6). There was no
apoptosis or mitotsis or infiltrates seen with animals treated with
these ASOs. The organ weights of the animals treated with 2′-
Inhibition of PTEN mRNA and Protein Expression in
Mouse Liver after Systemic Administration of 2′-O-NMA-
Modified ASO (28). In order to compare the activity of 2′-O-
MOE and 2′-O-NMA ASOs in animal models, mice were treated
either with ASO 27 or 28 at 10, 25, and 50 mg kg-1 once a
week for 3 weeks. Treatment of mice with 2′-O-MOE- and 2′-

2770 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Prakash et al.
O-NMA and 2′-O-MOE ASOs were normal at the doses used
in this study.
Conclusions
In conclusion, we have developed an efficient method for
the synthesis of 2′-O-NMA-modified adenosine, guanosine,
5-methylcytidine, and thymidine phosphoramidites.
We also demonstrate that these synthetic routes could be used
for multigram scale synthesis of 2′-O-NMA nucleosides and
the corresponding phosphoramidites. The ASO synthesis bearing
2′-O-NMA-modified nucleotides proceeded smoothly using
normal solid-phase conditions. This chemistry was then evalu-
ated for in vitro and in vivo potency. The potency of the 2′-
O-NMA-modified gapmer ASO was similar to the second-
generation 2′-O-MOE ASO. Our study demonstrates that 2′-
O-NMA is a suitable modification for RNase H based antisense
therapeutics.33 This modification may also be useful for newly
emerging areas such as siRNA and micro RNA based thera-
peutics, diagnostics, and gene target validation.
Figure 4. Inhibition of PTEN protein expression with 2′-O-MOE- (27)
and 2′-O-NMA-modified (28) ASO and control ASOs with 2′-O-MOE
(29) and 2′-O-NMA (30) modifications in mouse liver.
Experimental Section
General Procedures. Solvents used were of anhydrous grade
and were stored under nitrogen at all times. The 2′-deoxynucleoside
phosphoramidites and reagents for oligonucleotide synthesis were
procured from Glen Research, Inc., Sterling, VA. All other starting
materials and reagents were purchased from Aldrich Chemical
Co. and were used without further purification. Thin-layer
chromatography was performed on precoated plates (silica gel
60 F254, EM Science, Gibbstown, NJ) and visualized with UV
light and spraying with a solution of p-anisaldehyde (6 mL),
H2SO4 (8.3 mL), CH3COOH (2.5 mL) in C2H5OH (227 mL)
followed by charring. 1H NMR spectra were referenced using
internal standard (CH3)4Si and 31P NMR spectra using external
standard 85% H3PO4. Mass spectra were recorded by the College
of Chemistry, University of California, Berkeley, CA.
[5′-O-(4,4′-Dimethoxytrityl-2′-O-[2-(methylamino)-2-oxoethyl]-
5-methyluridine]-3′-[(2-cyanoethyl)-N,N-diisopropyl]phosphor-
amidite (1). 5′-O-(4,4′-Dimethoxytrityl)- 2′-O-[2-(methylamino)-
2-oxoethyl]-5-methyluridine9(33.60g,53.20mmol)wascoevaporated
with anhydrous acetonitrile (150 mL). To the residue were added
anhydrous CH2Cl2 (200 mL), 2-cyanoethyl-N,N,N′,N′-tetraisopro-
pylphosphorodiamidite (16.83 g, 55.90 mmol) and dicyanoimidazole
(1.9 g, 16.00 mmol), and the solution was stirred at ambient
temperature for 24 h under argon atmosphere. The reaction mixture
was diluted with CH2Cl2 (400 mL) and washed with saturated
aqueous NaHCO3 (500 mL). The aqueous layer was back-extracted
with CH2Cl2 (100 mL). The combined organic layer was dried
(Na2SO4) and concentrated. The residue was dissolved in CH2Cl2
(100 mL) and purified by silica gel column chromatography. The
product was eluted with a gradient starting with ethyl acetate–
Figure 5. Plasma transaminase level for 2′-O-MOE (27) and 2′-O-
NMA (28) ASOs treated mice.
Figure 6. Histopathology of liver sections from mice treated 2′-O-MOE (B, 27) and 2′-O-NMA (C, 28) at 50 mg kg-1. Panel A shows histopathology
of liver treated with saline. H & E stain shows that livers from the animals treated with 2′-O-MOE (B, 27) or 2′-O-NMA (C, 28) ASOs did not
exhibit any significant hepatotoxicity.

Comparison of ActiVity of Modified Antisense Oligonucleotides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2771
hexanes-triethylamine (80:20:1), then straight ethyl acetate, and
then ethyl acetate-MeOH (9:1) to yield 1 (37.0 g, 84%): 31P NMR
(80 MHz, CDCl3) δ 149.7, 150.7; HRMS (FAB) m/z calcd for
C43H55N5O10P+ 832.3687, found 832.3696.
[4-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl-2′-O-[2-(methylamino)-
2-oxoethyl]-5-methylcytidine]-3′-[(2-cyanoethyl)-N,N-diisopro-
pyl]phosphoramidite (2). Compound 2 (41.6 g, 85%) was syn-
thesized from compound 12 (38.5 g, 52.4 mmol), CH2Cl2 (150 mL),
2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (16.58 g,
55.00 mmol) and dicyanoimidazole (1.87 g, 15.70 mmol) using
the procedure used for the synthesis of compound 1: 31P NMR (80
MHz, CDCl3) δ 149.8, 151.2; HRMS (FAB) m/z calcd for
C50H60N6O10P+ 935.46520, found 935.4638.
2′-O-[2-(Methylamino)-2-oxoethyl]-5-methyluridine (8). To an
8 L Parr stainless steel pressure reactor were added borane in
tetrahydrofuran (720 mL, 1.5 M, 1070.00 mmol), NaHCO3 (6.45
g, 76.00 mmol), and 2-hydroxy-N-methylacetamide 6 (400 g,
4490.00 mmol). The mixture was stirred uncovered until the
evolution of hydrogen gas subsided (ca. 5 min). 2,2′-Anhydro-5-
methyluridine 7 (215.90 g, 899.00 mmol) was added, and the vessel
was sealed and heated with stirring to 150 °C (internal temperature
with pressure ca. 110 psi). The reaction was heated for 48 h more
and then allowed to cool to ambient temperature. The dark solution
was decanted and concentrated under reduced pressure (50 °C, 20
mm) to dark oil. Water (1 L) was added, and the mixture was heated
to 100 °C for 30 min to hydrolyze the borate ester. The mixture
was concentrated, and the residue was dissolved in a mixture of
CH2Cl2-acetone-CH3OH (1 L, 40:10:1) and applied onto a silica
gel column (2.5 kg). The product was eluted with a gradient of
1-6% CH3OH in CH2Cl2-acetone (40:10). After concentration,
the residue was crystallized from CH3OH to yield 8 (95.5 g, 32%)
as a white solid: mp 193–194 °C; 1H NMR (200 MHz, DMSO-d6)
δ 1.77 (s, 3 H), 2.65 (d, J ) 4.7 Hz, 3 H), 3.45–3.82 (m, 2 H),
3.83–4.15 (m, 5H), 5.22 (t, J ) 5.0 Hz, 1 H), 5.40 (d, J ) 6.3 Hz,
1 H), 5.85 (d, J ) 3.8 Hz, 1 H), 7.82 (s, 1 H), 7.87 (d, J ) 4.6 Hz,
1 H), 11.37 (br s, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 12.2,
25.6, 60.0, 68.2, 69.3, 82.2, 84.1, 86.4, 109.1, 136.1, 150.5, 163.8,
169.7; MS (ES) m/z 328.1 [M - H]-; HRMS (TOF MS ES) m/z
[6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl-2′-O-[2-(methylamino)-
2-oxoethyl]adenosine]-3′-[(2-cyanoethyl)-N,N-diisopropyl]phos-
phoramidite (3). 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphospho-
rodiamidite (2.41 mL, 7.59 mmol) was added dropwise into a
solution of the nucleoside 20 (3.76 g, 5.06 mmol) and diisopro-
pylamine tetrazolide (0.87 g, 5.06 mmol) in anhydrous CH3CN (25
mL). The reaction mixture was stirred at room temperature under
argon atmosphere for 10 h. The reaction mixture was diluted with
EtOAc (200 mL), extracted with saturated aqueous NaHCO3 (2 ×
100 mL), and then washed with brine (100 mL). The organic phase
dried over MgSO4, filtered, and concentrated. The residue was
dissolved in CH2Cl2 (5 mL) and added to a vigorously stirred hexane
(250 mL), and precipitate formed was collected by decanting the
supernatant. The precipitation processes was repeated once more
to yield 3 (3.34 g, 70%): 31P NMR (80 MHz, CDCl3) δ 151.08
and 151.41; HRMS (FAB) m/z calcd for C50H58N8O9P+ 945.4064,
found 945.4054.
[5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoet-
hyl]-2-N-isobutyrylguanosine]-3′-[(2-cyanoethyl)-N,N-diisopro-
pyl]phosphoramidite (4). Compound 25 (25.0 g, 34.5 mmol) and
diisopropylamine tetrazolide (1.77 g, 10.00 mmol) were coevapo-
rated with anhydrous acetonitrile (150 mL). To the resulting oil
were added anhydrous acetonitrile (170 mL) and 2-cyanoethyl-
N,N,N′,N′- tetraisopropylphosphorodiamidite (11.46 g, 38.00 mmol),
and the mixture was stirred at ambient temperature for 24 h under
argon atmosphere. The reaction mixture was concentrated to an
oil and then partitioned between CH2Cl2 (300 mL) and saturated
aqueous NaHCO3 (300 mL). The aqueous layer was back-extracted
with CH2Cl2 (100 mL). The combined organic layer was dried
(Na2SO4) and concentrated to an oil under reduced pressure. The
residue was purified by silica gel column chromatography and eluted
with a gradient (0–10%) of methanol in EtOAc-triethylamine (99:
1). Appropriate product-containing fractions were combined, con-
centrated under reduced pressure, and coevaporated with CH3CN
(200 mL). The foam was dissolved in a mixture of EtOAc-CH2Cl2
(1:1, 40 mL), and then hexanes were slowly added until cloudiness
was maintained. After 20 min, a precipitate formed which was
collected, washed with hexanes-EtOAc-CH2Cl2 (8:1:1), and dried
to yield 4 (14.46 g, 45.35%). The filtrate was concentrated to yield
10 g (31.36%) of 4 as a foam: 31P NMR (80 MHz, CDCl3) δ 146.9,
149.5; HRMS (FAB) m/z calcd for C47H60N8O10P+ 927.4170, found
927.4160.
–
calcd for C13H18N3O7 328.1145, found 328.1132.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoethyl]-
5-methyluridine (9). 2′-O-[2-(Methylamino)-2-oxoethyl]-5-me-
thyluridine 8 (93.0 g, 282.00 mmol) was coevaporated with
anhydrous pyridine (500 mL) and then dissolved in the same (1
L). 4,4′-Dimethoxytrityl chloride (97 g, 286.00 mmol) was added
in four portions over 2 h. The reaction mixture was stirred for an
additional 1 h at room temperature under argon atmosphere. TLC
indicated a complete reaction (Rf of starting nucleoside 0.25, product
0.60, in CH2Cl2-acetone-methanol, 20:5:3). The reaction was
concentrated under reduced pressure to thick oil and then redis-
solved in a mixture of EtOAc and saturated aqueous NaHCO3 (1
L each). The organic layers were separated. The aqueous layer was
extracted with more EtOAc (200 mL), and the combined organic
layer was washed with more NaHCO3 solution (200 mL). The
organic layer was dried (Na2SO4), concentrated, and then redis-
solved in CH2Cl2 (200 mL). The solution purified by silica gel
column chromatography (2.5 kg) and eluted with a gradient of
CH3OH (0-10%) in EtOAc to yield 9 (167.5 g, 94%) as a white
1
foam: H NMR (300 MHz, DMSO-d6) δ 1.41 (s, 3 H), 2.64 (d, J
) 4.7 Hz, 3 H), 3.20–3.40 (m, 2 H), 3.74 (s, 6 H), 4.0–4.4 (m,
5H), 5.45 (d, J ) 7.1 Hz, 1 H), 5.84 (d, J ) 3.2 Hz, 1 H), 6.90 (d,
J ) 8.8, 4 H), 7.3–7.5 (m, 9 H), 7.50 (s, 1 H), 7.86 (q, J ) 4.3, 4.7
Hz, 1 H), 11.43 (br s, 1 H); 13C NMR (75 MHz, CDCl3) δ 12.0,
26.0, 55.4, 61.9, 68.6, 70.0, 83.6, 84.2, 86.0, 88.6, 111.4, 113.5,
127.3, 128.2, 128.3, 130.3, 135.3, 135.5, 135.6, 144.5, 151.4, 158.9,
164.4, 170.8; MS (ES) m/z 630.2 [M - H]-; HRMS (TOF MS
-
ES) m/z calcd for C34H36N3O9 630.2452, found 630.2448.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoethyl]-
5-methyluridine 3′-O-Succinate (10a). Compound 9 (5 g, 7.92
mmol) was mixed with succinic anhydride (1.58 g, 15.8 mmol)
and DMAP (0.483 g, 3.95 mmol). The mixture was dried over P2O5
2-Hydroxy-N-methylacetamide (6). Aqueous methylamine (40
wt %, 825 mL, 10630.00 mmol) was added to THF (1 L), and the
solution was cooled to 10 °C. Ethyl glycolate 5 (500 g, 4800.00
mmol) was added slowly with stirring at a rate so as to maintain
the internal temperature between 15 and 20 °C (ca. 1 h). The
reaction mixture was concentrated under reduced pressure to an
oil. The oil was coevaporated with anhydrous acetonitrile. Diethyl
ether (1 L) was added to the oil, which caused a white solid to
form exothermically (Caution: add slowly to prevent ether from
boiling over). After stirring, the slurry was filtered and the resulting
solid was washed with diethyl ether (500 mL) and then hexanes (2
× 500 mL) and then dried to yield 6 (417 g, 97%) as white irregular
crystals: mp 64–66 °C; 1H NMR (200 MHz, DMSO-d6) δ 2.80 (d,
ꢀ
in vacuo overnight at 40 C. The dried material was dissolved in
anhydrous pyridine (30 mL), and the reaction mixture was stirred
at room temperature under argon atmosphere overnight. The
reaction mixture was diluted with CH2Cl2 (150 mL) and washed
with 20% citric acid (ice cold, 100 mL). The citric acid layer was
then washed with 20 mL of CH2Cl2. The combined organic phase
was dried over Na2SO4 and evaporated to foam. It was then
coevaporated with 50 mL of CH3CN and 3 mL of triethylamine to
1
give 10a (5.79 g, 97%) as a yellow foam: H NMR (300 MHz,
CDCl3) δ 1.37 (s, 3 H), 2.61 (m, 4 H), 2.82, (d, J ) 4.8 Hz, 3H),
3.22–3.36 (m, 1 H), 3.62–3.55 (m, 1 H), 3.80 (s, 6 H), 4.16 (d, J
) 2.1 Hz, 2H), 4.31 (m, 2H), 5.45 (t, J ) 4.3 Hz, 1 H), 6.07 (d, J
) 5.5 Hz, 1 H), 6.85 (d, J ) 10.7, 4 H), 7.03 (q, J ) 4.8 Hz, 1 H),
7.23–7.38 (m, 9 H), 7.60 (s, 1 H), 10.00 (br s, 1 H); 13C NMR (75
J ) 37 Hz, 2 H), 4.00 (s, 3 H), 4.4 (br s, 1 H), 7.0 (br s, 1 H); 13
C
NMR (75 MHz, DMSO-d6) δ 25.6, 61.9, 173.4; HRMS (EI) m/z
+
calcd for C3H7NO2 89.0477, found 89.0477.

2772 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Prakash et al.
MHz, CDCl3) δ 11.9, 26.0, 29.0, 29.4, 55.4, 62.7, 70.7, 71.4, 81.1,
81.4, 86.5, 87.6, 112.2, 113.6, 127.5, 128.3, 130.3, 135.1, 135.2,
144.0, 151.2, 159.0, 164.6, 170.0, 171.6, 175.5; MS (ES) m/z 730.2
[M - H]-.
NaHCO3 (2 L). The organic layer was separated, dried (Na2SO4),
and concentrated under reduced pressure to white foam. The residue
was purified by silica gel column chromatography and eluted with
1
EtOAc-hexanes 4:1 to yield 12 (80.20 g, 89.4%): H NMR (200
MHz, DMSO-d6) δ 1.63 (s, 3H), 2.67 (d, J ) 4.7 Hz, 3H),
3.20–3.40 (m, 2H), 3.77 (s, 6H), 4.0–4.5 (m, 5H), 5.57 (d, J ) 7.4
Hz, 2H), 5.90 (d, J ) 2.2 Hz, 1H), 6.94 (d, J ) 8.0 Hz, 4H),
7.23–7.62 (m, 12 H), 7.80 (br s, 1H), 7.95 (s, 1H), 8.18 (br s, 2 H),
13.07 (br s, 1H); 13 C NMR (75 MHz, CDCl3) δ 12.5, 25.4, 54.9,
61.4, 68.5, 69.6, 82.8, 83.4, 86.5, 88.1, 111.7, 112.9, 126.8, 127.8,
128.8, 129.4, 129.8, 132.1, 134.9, 135.0, 136.4, 136.3, 144.0, 148.0,
158.4, 158.3, 159.4, 170.2, 170.9; HRMS (TOF MS ES) m/z calcd
5′-O-Dimethoxytrityl-2′-O-[2-(methylamino)-2-oxoethyl]-5-
methyluridine 3′-O-Succinyl CPG (10b). Compound 10a (0.2 g,
0.27 mmol) was dissolved in DMF (0.60 mL). 2-(1H-Benzotriazole-
1-yl)-1,2,3-tetramethyluronium tetrafluoroborate (TBTU, 0.81 g,
0.25 mmol) and 4-methylmorpholine (0.50 g, 0.49 mmol) were
added. The mixture was vortexed until it became a clear solution.
Anhydrous DMF (2.2 mL) and activated CPG (1.11 g, 115.2 µmol
g-1) were added and allowed to shake on a shaker for 18 h. The
mixture was filtered and the functionalized CPG washed thoroughly
with DMF, CH2Cl2, and diethyl ether. The functionalized CPG was
dried over P2O5 in vacuo. To cap the unfunctionalized sites, it was
mixed with the capping reagents (2 mL of 10% pyridine, 10% acetic
anhydride in THF and 2 mL of N-methylimidazole in THF) and
allowed to shake for 2 h. It was then filtered and washed with
acetonitrile and diethyl ether to give the functionalized CPG. After
-
for C41H41N4O9 733.2717, found 733.2718.
4-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl-2′-O-[2-(methylamino)-
2-oxoethyl]-5-methylcytidine 3′-O-Succinate (13a). Compound
13a (0.2 g, 71%) was synthesized from compound 12 (0.25 g, 0.38
mmol), succinic anhydride (0.057 g, 0.57 mmol), DMAP (0.023 g,
0.19 mmol), CH2Cl2 (1 mL), and triethylamine (0.106 mL, 0.76
mmol) using the procedure used for the synthesis of compound
drying in vacuo, the loading capacity is determined (53 µmol g-1
)
10a: H NMR (200 MHz, CDCl3) δ 1.56 (s, 3H), 2.63 (m, 4H),
1
using standard dimethoxytrityl assay.34
2.83 (d, J ) 4.8 Hz, 3H), 3.34–3.64 (m, 2H), 3.73 (s, 3H), 3.79 (s,
4H), 4.00–4.34 (m, 3H), 5.41 (t, J ) 4.4 Hz, 1H), 6.16 (d, J ) 5.6
5′-O-(4,4′-Dimethoxytrityl-2′-O-[2-(methylamino)-2-oxoethyl]-
5-methylcytidine (11). A 10 L jacketed reaction vessel was
equipped with a mechanical stirrer, thermometer, argon line, and
addition funnel and connected to a recirculating heater/chiller.
Compound 9 (112.40 g, 178.00 mmol) was coevaporated with
anhydrous acetonitrile (1 L) and then the residue dissolved in the
same (4 L). Triethylamine (396 mL, 2850.00 mmol) was added,
and the solution was cooled to –10 °C. Trimethylsilyl chloride (113
mL, 890.00 mmol) was dropped in over 15 min. The solution was
warmed to 0 °C and maintained for 1 h. TLC indicated a complete
reaction (Rf of starting material 0.20, TMS intermediate 0.65,
EtOAc-CH3OH, 95:5). 1,2,4-Triazole (123 g, 1780.00 mmol) was
added as a solid. The reaction mixture was cooled to –12 °C, and
phosphorus oxychloride (49.7 mL, 534.00 mmol) was dropped in
slowly so as to maintain the temperature below –10 °C (ca. 30
min). The reaction was allowed to warm to 35 °C over 2 h to give
a reddish suspension. TLC indicated a ca. 95+% complete reaction
(Rf of TMS U intermediate 0.65, TMS triazole intermediate 0.35
with a glow in long UV light, EtOAc-CH3OH, 95:5). Water (2 L)
and EtOAc (3 L) were added. After the mixture was stirred and
allowed to separate, the aqueous layer was removed and the organic
layer was washed with more water (3 L) as before. The organic
layer was concentrated under reduced pressure to foam. The foam
was dissolved in dioxane (1 L). Aqueous ammonium hydroxide
(50 mL, 28–30 wt %) was added in two portions over 1 h. (Note:
To reduce the possibility of transamidation, a minimum amount of
ammonia was used and the reaction progress was followed by TLC).
TLC indicated a ca. 95+% complete reaction (Rf of TMS triazole
intermediate 0.65, unblocked product 0.25, CH2Cl2-acetone-
CH3OH, 20:5:3). The reaction mixture concentrated to foam and
dissolved in a minimum amount of CH2Cl2 and purified by silica
gel column chromatography. The product was eluted with a 0–15%
CH3OH in CH2Cl2-acetone 4:1 to yield 11 (77.3 g, 69%) as a
Hz, 1H), 6.94 (d, J ) 8.4 Hz, 4H), 7.24–7.56 (m, 12 H), 7.74 (s,
13
1H), 8.25–8.28 (m, 2 H);
C NMR (50 MHz, CDCl3) δ 12.8,
25.8, 28.7, 29.0, 55.2, 62.0, 69.3, 70.4, 71.1, 81.4, 81.5, 86.9, 87.4,
113.0, 113.3, 127.3, 128.1, 129.9, 130.0, 132.5, 134.8, 135.8, 136.8,
143.9, 148.4, 158.8, 159.3, 169.7, 171.5, 174.7, 179.4; MS (API-
EI) m/z 857.2 [M + Na]+.
4-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-[2-(methylamino)-
2-oxoethyl]-5-methylcytidine 3′-O-Succinyl CPG (13b). Com-
pound 13b (55 µmol g-1) was synthesized from compound 13a
(0.2 g, 0.24 mmol), DMF (0.64 mL), 2-(1H-benzotriazole-1-yl)-
1,2,3-tetramethyluronium tetrafluoroborate (TBTU, 0.83 g, 0.26
mmol), 4-methylmorpholine (0.53 g, 0.52 mmol), anhydrous DMF
(2.2 mL), and activated CPG (1.13 g, 115.2 µmol/g) using the
procedure used for the synthesis of compound 10b.
2′-O-(Methoxycarbonylmethylene)adenosine (16a). Adenosine
14 (25.00 g, 93.50 mmol) was dissolved in anhydrous DMF (900
mL). The solution was cooled to –50 °C, and NaH (60% dispersion
in mineral oil, 4.86 g, 122.00 mmol) was added in two portions.
The reaction mixture was allowed to warm to –30 °C and stirred
for 30 min under argon atmosphere. After the reaction mixture was
cooled to –50 °C again, methyl 2-bromoacetate (11.5 mL, 122.00
mmol) was added dropwise, and the reaction mixture was allowed
to warm to ambient temperature. After the mixture was stirred at
ambient temperature for 1 h, MeOH (100 mL) was added, the
reaction mixture was stirred for 10 min, and the solvent was
evaporated in vacuo to give a foam. The product was coevaporated
with EtOAc to afford 16a (14.23 g, 45%) as a crude solid which
was of sufficient purity for use in the following reaction. A small
portion of the product was purified by column chromatography and
characterized: 1H NMR (200 MHz, DMSO-d6) δ 3.48 (s, 3H),
3.56–3.64 (m, 2H), 4.0 (m, 1H), 4.11–4.24 (dd, J ) 16.6, 11.2 Hz,
2H), 4.38 (m, 1H), 5.63 (m, 1H), 5.29 (d, J ) 4.8 Hz, 1H), 5.40
(m, 2H), 6.02 (d, J ) 6.4 Hz, 1H), 7.37 (m, 2H), 8.12 (s, 1H), 8.28
(s, 1H); 13C NMR (75 MHz, CDCl3) δ 52.7, 63.5, 69.4, 71.2, 84.9,
88.0, 89.3, 121.4, 141.3, 148.7, 152.6, 156.4, 172.4; HRMS (FAB)
m/z calcd for C13H17N5O6 + H+ 340.1257, found 340.1257. 2D-
1H NMR (TOCSY) confirmed 2′-O-alkylation.
2′-O-(Methoxycarbonylmethylene)-2,6-diaminopurin-9-yl Ri-
boside (17a). 2,6-Diaminopurin-9-yl riboside 15 (25.00 g, 88.60
mmol) was dissolved in anhydrous DMSO (90 mL) with gentle
heating to give a brown solution which was diluted with anhydrous
DMF (355 mL). The reaction mixture was cooled to 5 °C, NaH
(60% oil dispersion, 6.20 g, 155.00 mmol) was added, and the
reaction mixture was allowed to warm to ambient temperature and
stirred for 1 h. The suspension was cooled to –40 °C in an
CH3CN-CO2 bath, and methyl 2-bromoacetate (14.29 mL, 155.00
mmol) was added slowly. The reaction mixture was allowed to
slowly warm to ambient temperature over 1 h, and after 17 h, glacial
AcOH (6 mL) was added dropwise to give a solution with pH 4.
1
white foam: H NMR (300 MHz, DMSO-d6) δ 1.43 (s, 3H), 2.65
(d, J ) 4.7 Hz, 3H), 3.20–3.40 (m, 2H), 3.74 (s, 6H), 3.84–3.86
(m, 1H,), 4.04–4.13 (m, 2H), 4.19–4.32 (m, 2H), 5.41 (d, J ) 7.6
Hz, 1H), 5.80 (d, J ) 1.8 Hz, 1H), 6.81 (br s, 2H), 6.92 (d, J ) 8.9
Hz, 4H), 7.21–7.52 (m, 9H), 7.49 (s, 1H), 7.93 (m, 1H); 13 C NMR
(75 MHz, CDCl3) δ 12.8, 26.1, 55.4, 61.9, 68.9, 70.3, 83.0, 84.5,
86.9, 89.6, 102.2, 113.5, 127.3, 128.2, 128.4, 130.3, 130.4, 135.7,
135.8, 138.3, 144.7, 156.2, 158.8, 158.9, 165.7, 171.1; MS (ES)
m/z 629.2 [M - H]-.
4-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-[2-(methylamino)-
2-oxoethyl]-5-methylcytidine (12). Compound 11 (77.3 g, 123.00
mmol) and benzoic anhydride (33.25 g, 147.00 mmol) were
dissolved in anhydrous DMF (350 mL). The resulting solution was
stirred at ambient temperature for 20 h under argon atmosphere.
TLC indicated a complete reaction (Rf of starting material 0.15,
product 0.80, EtOAc-CH3OH, 9:1). The reaction mixture was
diluted with EtOAc (1.2 L) and washed with saturated aqueous

Comparison of ActiVity of Modified Antisense Oligonucleotides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2773
The solvent was evaporated in vacuo (1 Torr) at 45 °C to give an
amorphous mass to which CH2Cl2 (400 mL) was added. The
mixture was mechanically stirred for 15 min to give a suspension
to which hexanes (400 mL) was slowly added. The suspension stood
for 15 min, after which time the solids settled. The supernatant
was decanted, and the solid was washed with hexanes (3 × 200
mL) and then dissolved in H2O (250 mL). The aqueous layer was
extracted with diethyl ether (3 × 100 mL), which resulted in an
emulsion within the organic phase. The organic phase was then
washed with H2O (2 × 100 mL), the aqueous portions were
combined, and the H2O was evaporated in vacuo at 45 °C to give
a residue which was utilized for the following reaction. A small
portion of product was dissolved in MeOH, adsorbed onto silica
and was purified by column chromatography using 15% MeOH in
CH2Cl2 to yield 17a as a white solid: 1H NMR (200 MHz, DMSO-
The reaction mixture was stirred at room temperature for 10 h under
argon atmosphere. The solvent was removed under reduced pressure
to get an oil. The oil was partitioned between ethyl acetate (200
mL) and water (200 mL). The aqueous phase was extracted with
ethyl acetate (2 × 50 mL). Combined ethyl acetate layer dried over
MgSO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography and eluted with
MeOH/CHCl3 (5/95, v/v) to afford 20 (6.24 g, 65%): 1H NMR
(300 MHz, DMSO-d6) δ 2.62 (d, J ) 4.7 Hz, 3H), 3.27–3.21 (m,
2H), 3.71 (s, 6H), 4.18–4.04 (m, 3H), 4.57 (m, 1H), 4.70 (t, J )
4.8, 3.8 Hz, 1H), 5.53 (d, J ) 6.8 Hz, 1H), 6.29 (d, J ) 3.5 Hz,
1H), 6.84–6.80 (m, 4H), 7.36–7.16 (m, 9H), 7.67–7.52 (m, 3H),
7.89 (q, J ) 3.0, 4.7 Hz, 1H), 8.04 (d, J ) 7.2 Hz, 2H), 8.56 (s,
1H), 8.70 (s, 1H), 11.21 (s, 1H); 13C NMR (75 MHz, CDCl3) δ
26.0, 55.4, 63.3, 70.2, 70.6, 81.7, 84.1, 87.0, 87.7, 113.5, 127.2,
128.0, 128.1, 128.3, 129.2, 129.3, 130.2, 133.1, 133.3, 133.7, 135.7,
141.7, 144.6, 147.6, 151.5, 152.0, 152.3, 158.8, 164.9, 170.0, 170.5;
MS (ES) m/z 743.2 [M - H]-; HRMS (TOF MS ES) m/z calcd
d6) δ 3.53 (s, 3H), 3.55 (m, 2H), 3.92 (m, 1H), 4.20 (q J ) 16.8
,
Hz, 2H), 4.29 (m, 1H), 4.52 (m, 1H), 5.21 (d, J ) 2.5 Hz, 1H),
5.39 (m, 1H), 5.73 (br s, 2H), 5.87 (d, J ) 6.8 Hz, 1H), 6.76 (br
s, 2H), 7.91 (s, 1H); 13C NMR (50 MHz, CD3OD) δ 52.4, 63.4,
68.6, 71.1, 83.4, 88.0, 88.7, 115.0, 139.1, 152.0, 157.7, 161.3, 172.6;
–
for C41H39N6O8 743.2829, found 743.2838.
6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl-2′-O-[2-(methylamino)-
2-oxoethyl]adenosine-3′-O-succinate (21). Compound 20 (1.5 g,
2.03 mmol) was mixed with succinic anhydride (0.4g, 4.03 mmol)
and DMAP (0.13 g, 1.04 mmol). The mixture was dried over P2O5
in vacuo overnight at 40 °C. The dried mixture was dissolved in
anhydrous CH2Cl2 (6 mL) and triethyamine (1.12 mL, 8.66 mmol)
was added with stirring at ambient temperature under argon
atmosphere for 4 h. The reaction mixture was diluted with CH2Cl2
(40 mL) and washed with 10% citric acid (ice cold, 50 mL)
followed by water (50 mL). The organic phase was dried (anhydrous
Na2SO4) and evaporated in vacuo. The resulting foam was purified
by silica gel column chromatography and eluted with MeOH/
CH2Cl2/pyridine (1/8.9/0.1, v/v/v) to yield 21 (1.71 g, 98%) as a
+
HRMS (FAB) m/z calcd for C13H18N6O6 355.1362, found
355.1366.
2′-O-[2-(Methylamino)-2-oxoethyl]adenosine (18). Compound
16a (89.63 g, 155.15 mmol) was dissolved in THF (1300 mL),
N-methylamine (310 mmol, 350 mL, of 40 wt % solution in water)
was added, and the reaction mixture was stirred at ambient
temperature for 18 h. The mixture was concentrated to oil which
was dissolved in EtOAc (420 mL). The organic phase was washed
with water (3 × 350 mL) and brine (1 × 350 mL), dried (Na2SO4),
and evaporated under reduced pressure to yield 18 (84.80 g, 95%)
1
as a white solid: H NMR (200 MHz, DMSO-d6) δ 2.60 (d, J )
4.7 Hz, 3H), 3.60–3.53 (m, 1H), 3.72–3.66 (m, 1H), 3.97 (s, 2H),
3.99–4.02 (m, 1H), 4.33 (q, J ) 4.7, 4.3, 5.1 Hz, 1H), 4.50 (t, J )
5.1 Hz, 1H), 5.35 (t, J ) 4.9 Hz, 1H), 5.48 (d, J ) 5.3 Hz, 1H),
6.07 (d, J ) 5.1 Hz, 1H), 7.34 (s, 2H), 7.83 (q, J ) 4.1, 4.5 Hz,
1H), 8.14 (s, 1H), 8.36 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ
25.0, 61.1, 69.2, 69.3, 82.2, 85.4, 86.0, 119.3, 139.5, 148.9, 152.5,
156.1, 169.2; MS (ES) m/z 337.1 [M - H]-; HRMS (TOF MS
1
white foam: H NMR (200 MHz, CDCl3) δ 2.63–2.79 (m, 7H),
3.38–3.45 (m, 1H), 3.53–3.60 (m, 1H), 3.79 (s, 6H), 3.90–4.15 (m,
2H), 4.38 (m, 1H), 4.95 (m, 1H), 5.49 (m, 1H), 6.21 (d, J ) 6.7
Hz, 1H), 6.83 (d, J ) 8.9 Hz, 4H), 6.99 (q, J ) 5.3 Hz, 1H),
7.22–7.42 (m, 9H), 7.50–7.63 (m, 3H), 8.04 (d, J ) 6.7 Hz, 2H),
8.25 (s, 1H), 8.70 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 25.6,
29.0, 29.2, 55.1, 62.9, 70.3, 71.7, 80.5, 82.4, 86.6, 87.0, 113.2,
127.0, 128.1, 128.6,129.9, 132.7, 133.3, 135.2, 142.0, 144.1, 149.8,
151.7, 152.0, 152.3, 158.6, 165.0, 169.3, 171.4, 174.8; MS (ES)
m/z 842.9 [M - H]-.
-
ES) m/z calcd for C13H17N6O5 337.1260, found 337.1260.
6-N-Benzoyl-2′-O-[2-(methylamino)-2-oxoethyl]adenosine (19).
Compound 18 (84 g, 146.00 mmol) was dissolved in anhydrous
pyridine (500 mL) and cooled in an ice bath. Chlorotrimethylsilane
(70 mL, 582.60 mmol) was added dropwise, and the reaction
mixture was stirred for 30 min at 0 °C under argon atmosphere.
To the reaction mixture benzoyl chloride (68 mL, 582.60 mmol)
was added dropwise. The ice bath was removed after 30 min, and
the reaction mixture was stirred for 3 h at room temperature under
argon atmosphere. The reaction mixture was cooled in an ice bath.
Water (100 mL) was added followed with aqueous NH3 (28–30 wt
%, 100 mL). After 1 h, the reaction mixture was partitioned between
water and EtOAc (600/600 mL). The aqueous layer was extracted
with EtOAc (2 × 500 mL). The EtOAc layer was washed with
brine (500 mL), dried (anhydrous MgSO4), filtered, and evaporated
in vacuo to oil. The resulting crude material was purified by silica
gel column chromatography using Et2N/MeOH/EtOAc/CHCl3 (2/
6-N-Benzoyl-5′-O-(4.4′-dimethoxytrityl)-2′-O-[2-(methylamino)-
2-oxoethyl]adenosine 3′-O-Succinyl CPG (22). Compound 22
(70.3 µmol g-1) was synthesized from compound 21 (1.0g, 1.18
mmol), DMF (16 mL), HATU (0.45 g, 1.18 mmol), diisopropyl-
ethylamine (0.63 g, 0.81 mL,4.84 mmol), anhydrous DMF (33 mL),
and activated CPG (7.00 g, 115.2 µmol/g) using the procedure
described for the synthesis of compound 10b.
2′-O-[2-(Methylamino)-2-oxoethyl]guanosine (23). Compound
17a (100 g, 280.0 mmol) was dissolved in water (300 mL) and
N-methylamine (40 wt % solution in water, 87.6 mL, 1010.00
mmol) was added in one portion with mechanical stirring at ambient
temperature. After 4 h, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in a phosphate buffer
(0.1 M, 1 L, pH 7.0). Adenosine deaminase (1.5 g) was added,
and the mixture was mechanically stirred very slowly at 37 °C for
18 h. TLC indicated about a 60% complete reaction and the pH
had risen to 8.5. The pH was adjusted to 7.0 with phosphoric acid
and another portion of enzyme (0.5 g) was added. After 24 h, the
reaction was complete, and a precipitate formed was collected
1
5/60/33, v/v/v/v) as the eluent to yield 19 (79.32 g, 72.22%): H
NMR (300 MHz, DMSO-d6) δ 2.62 (d, J ) 4.7 Hz, 3H), 3.77–3.56
(m, 2H), 4.08–3.99 (m, 3H), 4.39 (q, J ) 4.9, 5.4 Hz, 1H), 4.59 (t,
J ) 4.8 Hz, 1H), 5.16 (t, J ) 5.6 Hz, 1H), 5.56 (d, J ) 5.8 Hz,
1H), 6.24 (d, J ) 4.7 Hz, 1H), 7.68–7.53 (m, 3H), 7.90 (q, J )
4.2, 4.7 Hz, 1H), 8.05 (d, J ) 7.2 Hz, 2H), 8.72 (s, 1H), 8.77 (s,
1H), 11.21 (br s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 25.1, 60.8,
69.0, 69.4, 82.2, 85.3, 85.9, 125.5, 128.8, 132.4, 133.3, 142.9, 150.4,
151.7, 151.9, 165.6, 169.2; MS (ES) m/z 441.1 [M-H]-; HRMS
1
washed with water and dried to yield 23 (60.9 g, 61% yield): H
NMR (300 MHz, DMSO-d6) δ 2.62 (d, J ) 4.7 Hz, 3 H), 3.66–3.51
(m, 2H), 3.95–3.91 (m, 1H), 3.97 (s, 2H), 4.25 (m, 1H), 4.34 (t, J
) 5.1, 1H), 5.07 (t, J ) 5.4 Hz, 1H), 5.44 (d, J ) 4.9, 1H), 5.85
(d, J ) 5.4, 1H), 6.48 (s, 2H) 7.83 (q, J ) 4.6, 1H), 7.95 (s, 1H),
10.59 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 25.1, 61.0, 69.1,
69.3, 82.4, 84.6, 85.0, 116.7, 135.3, 151.0, 153.8, 156.7, 169.2;
MS (ES) m/z 353.1 [M - H]-; HRMS (TOF MS ES) m/z calcd
-
(TOF MS ES) m/z calcd for C20H21N6O6 441.1523, found
441.1518.
6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-[2-(methylamino)-
2-oxoethyl]adenosine (20). Compound 19 (5.7g, 12.90 mmol) was
coevaporated with pyridine (3 × 50 mL) and the residue dissolved
in anhydrous pyridine (129 mL). 4,4′-Dimethoxytrityl chloride (5.24
g, 15.46 mmol) was added in two portions to the reaction mixture.
-
for C13H17N6O6 353.1210, found 353.1208.

2774 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Prakash et al.
2′-O-[2-(Methylamino)-2-oxoethyl]-2-N-isobutyrylgua-
nosine (24). Compound 23 (55.85 g, 158.00 mmol) was coevapo-
rated with anhydrous pyridine (2 × 200 mL) and then the residue
dissolved in anhydrous pyridine (700 mL). Trimethylsilyl chloride
(100 mL, 790.00 mmol) was dropped in at a rate as to maintain
the internal temperature between 20 and 25 °C. The reaction was
allowed to stir at room temperature for 90 min under argon
atmosphere. The reaction was cooled to –15 °C, and isobutyryl
chloride (83.25 mL, 790.00 mmol) was added at a rate as to
maintain the internal temperature between –10 to 0 °C. The reaction
was allowed to stir at ambient temperature for 18 h. The reaction
mixture was cooled to 0 °C, and water (50 mL) was added to quench
the reactions. After 10 min, the solvent was removed under reduce
127.2, 128.1, 128.3, 130.2, 135.9, 137.5, 144.8, 148.3, 156.0, 158.8,
171.3, 179.9; MS (ES) m/z 725.2 [M - H]-.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoethyl]-
2-N-isobutyrylguanosine 3′O-Succinyl CPG (26b). Compound
26b (68.3 µmol/g) was synthesized from compound 26a (1.0g, 1.21
mmol), DMF (16 mL), HATU (0.462 g, 1.21 mmol), diisopropy-
lethylamine (0.63 g, 0.81 mL, 4.84 mmol), anhydrous DMF (35
mL), and activated CPG (7.13 g, 115.2 µmol/g) according to the
procedure used for the synthesis of compound 10b.
2′-O-[2-(Methylamino)-2-oxoethyl] (2′-O-NMA)-Modified ASO
Synthesis. The synthesis of 2′-O-NMA ASO was performed on
ABI 394 DNA synthesizer or MilliGene/Biosearch 8800 reagent
delivery module for 2 µmol scale or 150 µmol scale, respectively.
The standard phosphoramidites and solid supports were used for
incorporation of A, T, G, and 5-methyl C residues. A 0.1 M solution
of the phosphoramidites 1-4 in anhydrous CH3CN was used for
the synthesis. The oligonucleotides were synthesized on prederiva-
tized CPG support 22 or 26b. For the coupling step, the 2′-O-NMA
phosphoramidites 1-4 were delivered 9-fold excess for 2-µmol
scale synthesis or 4 fold excess for 150-µmol scale over the solid
support and phosphoramidite condensation was carried out for 10
min. All other steps in the protocol supplied by the manufacturer
were used. PADS (0.2 M) in 1:1 3-picoline/CH3CN was used as a
sulfurization reagent with 2 min contact time. Solid-support bound
ASOs were cleaved in a two-step procedure. In the first step, solid
support was suspended in aqueous ammonia (28–30 wt %, 2.7 mL
for 2 µmol or 81 mL for 150 µmol scale synthesis) at room
temperature for 2 h. In the second step, aqueous methylamine (40
wt % in water, 0.3 mL for 2 µmol or 9 mL for 150 µmol scale
synthesis) was added, and the resulting mixture was allowed to
stay at room temperature for an additional 24 h. The solid supports
were filtered and washed with water. The combined filtrate and
the washing were concentrated and the residues obtained were then
purified by high-pressure liquid chromatography on a strong anion-
exchange column (Amersham Bioscience, Mono Q, 10/100, A )
100 mM ammonium acetate in 30% aqueous CH3CN, B ) 1.5 M
NaBr in A, 0–60% of B in 60 min, flow 1.5 mL min-1). The ASOs
were desalted by HPLC on a reversed-phase column to yield 28
and 30 in an isolated yield of 30–35% based on the loading of the
solid support. ASOs were characterized by ion-pair HPLC-MS
analysis with an Agilent 1100 MSD system.
Cell Culture Assay. AML12 (R mouse liver 12) cell line was
obtained from the American Type Culture Collection (ATCC,
Manassas, VA). AML12 cells were grown in T-75 flasks in
complete growth media, a 1:1 mixture of Dulbecco’s modified
Eagle’s medium and F12 medium (DMEM/F12, Invitrogen, Carls-
bad, CA) supplemented with 5 µg mL-1 of insulin, 5 µg mL-1 of
transferrin, 5 ng mL-1 of selenium, 40 ng mL-1 of dexamethasone,
and 10% fetal bovine serum (FBS, HyClone) until 70–80%
confluent. Cells were washed twice in 10 mL of DMEM. Oligo-
nucleotides 27–30 were transfected with the indicated concentration
of oligonucleotides using 3 µg mL-1 Cytofectin in OptiMEM and
then incubated at 37 °C for 4 h followed by replacement of
transfection media with complete growth media. After 24 h, cells
were harvested, and total mRNA was isolated from tissue culture
cells using Qiagen 96-well RNeasy plates. RNA was analyzed for
PTEN and cyclophilin A RNA levels. PTEN RNA levels normal-
ized to those of cyclophilin A were express as percent untreated
control (% UTC). Each treatment was performed in triplicate. IC50
values were determined using GraphPad Prism software by fitting
the data to a sigmoidal dose–response curve (variable slopes) using
a defined top of 100% and bottom of 0%.
pressure and the residue was dissolved in
a mixture of
CH2Cl2-acetone-MeOH (500 mL, 40:10:3). A precipitate formed
was collected and identified as inorganic salts. The filtrate was
loaded on to a silica gel column (800 g) and eluted with
CH2Cl2-acetone-MeOH (40:10:3 to 4:1:1 ration) to yield 24 (53.5
1
g, 81%): H NMR (300 MHz, DMSO-d6) δ 1.12 (d, J ) 6.8 Hz,
6H), 2.61 (d, J ) 4.7 Hz, 3H), 2.82–2.73 (m, 1H), 3.59 (q, J )
12.0 Hz, 2H), 3.97–3.98 (m, 2H), 4.00 (s, 2H), 4.30 (d, J ) 3.7
Hz, 1H), 4.43 (t, J ) 5.1 Hz, 1H), 5.08 (s, 1H), 5.53 (d, J ) 4.2
Hz, 1H), 5.96 (d, J ) 5.6 Hz, 1H), 7.87 (q, J ) 4.6 Hz, 1H), 8.27
(s, 1H), 11.65 (s, 1H), 12.09 (s, 1H); 13C NMR (75 MHz, DMSO-
d6) δ 18.8, 19.5, 25.1, 34.7, 60.9, 69.1, 69.3, 82.4, 84.7, 85.3, 120.2,
137.5, 148.2, 154.7, 156.7, 169.2, 180.1; MS (ES) m/z 422.12 [M
- H]-.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoethyl]-
2-N-isobutyrylguanosine (25). Compound 24 (42.5 g, 101.00
mmol) was coevaporated with anhydrous pyridine (2 × 200 mL)
and then redissolved in the same (500 mL). 4,4′-Dimethoxytrityl
chloride (37.63, 1110.00 mmol) was added in one portion, and the
reaction mixture was stirred at room temperature under argon
atmosphere for 18 h. The reaction was quenched by the addition
of methanol (20 mL) and then concentrated under reduced pressure
to thick oil. The oil was partitioned between EtOAc (500 mL) and
1 M aqueous NaHCO3 solution (500 mL). The organic layer was
dried (anhydrous Na2SO4) and concentrated and the residue obtained
was purified by silica gel column chromatography and eluted with
a gradient of MeOH in EtOAc (0 to 10%) to yield 25 (47 g, 64%)
as a white foam: 1H NMR (300 MHz, DMSO-d6) δ 1.12 (dd, J )
2.8, 4.0 Hz, 6H), 2.83–2.71 (m, 1H), 2.62 (d, J ) 4.7 Hz, 3H),
3.29–3.15 (m, 2H), 3.72 (s, 6H), 4.18–4.06 (m, 2H), 4.41 (q, J )
5.6 Hz, 1H), 4.50 (t, J ) 4.7 Hz, 1H), 5.46 (d, J ) 6.3, 1H), 6.06
(d, J ) 4.1 Hz, 1H), 6.84–6.79 (m, 4H), 7.34–7.08 (m, 9H), 7.84
(q, J ) 4.7 Hz, 1H), 8.13 (s, 1H), 11.58 (s, 1H), 12.10 (s, 1H); 13
C
NMR (75 MHz, DNSO-d6) δ 19.0, 26.1, 36.3, 37.5, 55.4, 63.5,
69.7, 70.4, 82.4, 83.4, 84.0, 86.7, 87.6, 113.4, 121.7, 127.2, 128.1,
128.3, 130.2, 135.9, 148.3, 156.0, 158.8, 171.3, 179.9; MS (ES)
m/z 725.2 [M - H]-; HRMS (TOF MS ES) m/z calcd for
–
C38H41N6O9 725.2935, found 725.2936.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-[2-(methylamino)-2-oxoethyl]-
2-N-isobutyrylguanosine 3′-O-Succinate (26a). Compound 25 (5
g, 6.9 mmol) was mixed with succinic anhydride (2.76 g, 27.6
mmol) and DMAP (0.42 g, 3.4 mmol). The mixture was dried over
P2O5 in vacuo overnight at 40 °C. The mixture was dissolved in
anhydrous pyridine (60 mL) and stirred at ambient temperature
under argon atmosphere for 4 h. The reaction was diluted with
CH2Cl2 (300 mL) and washed with 10% citric acid (ice cold, 300
mL) and water (300 mL). The organic phase was dried over
anhydrous sodium sulfate and evaporated in vacuo. The resulting
foam was purified by silica gel column chromatography using
MeOH/CH2Cl2/pyridine (1/8.9/0.1, v/v/v) as the eluent to yield 26a
Animal Treatment. Male BALB/c mice (6–8 weeks old, Charles
River, Wilmington, MA) were housed four to a cage under
conditions meeting National Institutes of Health regulations and
AAALAC accreditation.35 Oligonucleotides 27-30 were admin-
istered in 0.9% NaCl by intraperitoneal (i.p.) injection according
to the indicated dose levels once a week for three weeks. The
animals were maintained at a constant temperature of 23 °C and
were fed standard laboratory diet. Animal weight was recorded prior
to each dosing throughout the live phase of the study. Mice were
1
(5.19 g, 71%,): H NMR (300 MHz, CDCl3) δ 0.91 (d, J ) 6.8
Hz, 3H), 1.05 (d, J ) 6.8 Hz, 3H), 2.67 (m, 4H), 2.76 (d, J ) 4.7
Hz, 3H), 3.20 (dd, J ) 3.1, 7.9 Hz, 1H), 3.56–3.49 (m, 2H), 3.77
(s, 6H), 4.23–4.11 (m, 2H), 4.36–4.25 (m, 2H), 5.03 (t, J ) 4.9,
5.2, 1H), 5.86 (d, J ) 4.9 Hz, 1H), 6.91–6.77 (m, 4H), 7.54–7.08
(m, 10H), 7.84 (q, J ) 4.7 Hz, 1H), 7.86 (s, 1H), 8.99 (s, 1H),
12.05 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 19.0, 26.1, 36.3,
55.4, 63.5, 69.7, 70.4, 82.4, 83.4, 84.0, 86.7, 87.6, 113.4, 121.7,

Comparison of ActiVity of Modified Antisense Oligonucleotides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2775
(3) Freier, S. M.; Altmann, K. H. The Ups and Downs of Nucleic Acid
Duplex Stability: Structure-Stability Studies on Chemically-modified
DNA:RNA Duplexes. Nucleic Acids Res. 1997, 25, 4429–4443.
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anesthetized and sacrificed 2 days after administration of the final
dose. Serum was isolated from whole blood obtained via cardiac
puncture. Alanine aminotransferase (ALT) and aspartate ami-
notransferase (AST) levels were determined using an Olympus
AU400e bioanalyzer. Liver and spleen weight were determined.
The effect of the compound on organ weights, normalized to body
weight, was expressed relative to those of the saline treated group.
RNA Analysis. Total RNA was extracted from mouse liver by
immediate homogenization of the tissue in 4 M guanidinium
isothiocyanate/8% ꢁ-mercaptoethanol followed by ultracentrifuga-
tion through a cesium chloride gradient. RNAs (20–40 µg) were
resolved in 1.2% agarose gels containing 1.1% formaldehyde and
transferred to nylon membranes. The blots were hybridized with a
radiolabeled mouse PTEN cDNA probe as described.36 Probes
hybridized to mRNA transcripts were visualized and quantified
using a PhosphorImager (Molecular Dynamics). After the blots of
the radiolabeled probe were stripped, they were reprobed with
radiolabeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH)
cDNA to confirm equal loading. Data was expressed as a percentage
of PTEN mRNA level in saline-treated control after normalization
to G3PDH.
Western Blot Analysis. Frozen tissue samples were homog-
enized in RIPA buffer (PBS containing 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS) containing Complete protesase inhibitors
(Roche, Palo Alto, CA). Protein concentrations were determined
by Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA). Equal
quantities of total protein from each sample were separated on a
10% PAGE gel (Invitrogen) and subsequently transferred to a PVDF
membrane (Invitrogen). Following transfer, membranes were
initially blocked at room temperature in 5% nonfat dry milk in TBS
(Tris-buffered saline) and then incubated with PTEN antibody (Cell
Signaling, Beverly, MA) at a 1:1000 dilution overnight at 4 °C.
After washing, the blots were incubated with horseradish peroxidase
(HRP)-conjugated antirabbit secondary antibody (BD Biosciences/
PharMingen, San Diego, CA) at 1:10000 dilution at room temper-
ature. The blot was developed using ECL Plus Western Blotting
Detection System (GE Healthcare Amersham, Buckinghamshire,
UK). Protein bands were visualized by exposure to film; band
intensities were quantified by densitometry.
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therapeutics. Curr. Top. Med. Chem. 2007, 7, 641–649.
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Inamati, G.; Lesnik, E.; Owens, S.; Ross, B.; Prakash, T. P.;
Manoharan, M. Probing the Influence of Stereoelectronic Effects on
the Biophysical Properties of Oligonucleotides: Comprehensive Analy-
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of apolipoprotein B and low-density lipoprotein cholesterol by short-
term administration of an antisense inhibitor of apolipoprotein B.
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Lett. 2003, 5, 403–406.
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Hematoxylin and Eosin (H & E) Staining. Tissue samples were
fixed in formalin for 24 h and then changed to 70% ethanol until
blocked. Further dehydration and processing of tissue samples were
conducted using a Leica ASP300 tissue processor. Tissues were
embedded in paraffin, and 4 µm sections were mounted on positive
charged glass slides. The samples were then stained for hematoxylin
and eosin (H & E) using a Leica Autostainer XL.
Tm Analysis. The thermal stability of the duplexes formed by
oligonucleotides with the 2′-O-NMA- and 2′-O-MOE-modified
gapmer ASOs 27-30, and complementary RNA was studied by
measuring the UV absorbance versus temperature curves as
described previously.37 Each sample contained 100 mM Na+, 10
mm phosphate, 0.1 mM EDTA, 4 µM oligonucleotides, and 4 µM
complementary length matched RNA. Each Tm reported was an
average of two experiments.
Acknowledgment. We thank Dr. Eric E. Swayze for his
valuable suggestions and Dr. Dan P. Cook for his support for
this project.
Supporting Information Available: 31P NMR and HRMS
(FAB) mass spectra of compounds 1–4. Copies of 1H and 13C NMR
spectra and HRMS of compounds 8, 9, 12, 18-20, 23, and 25. LC
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