N2- and C8-substituted oligodeoxynucleotides with enhanced thrombin inhibitory activity in vitro and in vivo.

Article abstract of DOI:10.1021/jm970434d

2'-Deoxyguanosine (G) analogues carrying various hydrophobic substituents in the N2 and C8 positions were synthesized and introduced through solid-phase synthesis into 15-mer oligodeoxynucleotide, GGTTGGTGTGGTTGG, which forms a chairlike structure consist

Full text of DOI:10.1021/jm970434d

2234
J . Med. Chem. 1998, 41, 2234-2242
N2- a n d C8-Su bstitu ted Oligod eoxyn u cleotid es w ith En h a n ced Th r om bin
In h ibitor y Activity in Vitr o a n d in Vivo
Gong-Xin He,* Steven H. Krawczyk, S. Swaminathan, Regan G. Shea, J oseph P. Dougherty, Terry Terhorst,
Veronica S. Law, Linda C. Griffin, Steven Coutre´, and Norbert Bischofberger
Gilead Sciences, 353 Lakeside Drive, Foster City, California 94404
Received J uly 1, 1997
2′-Deoxyguanosine (G) analogues carrying various hydrophobic substituents in the N2 and C8
positions were synthesized and introduced through solid-phase synthesis into 15-mer oligode-
oxynucleotide, GGTTGGTGTGGTTGG, which forms a chairlike structure consisting of two
G-tetrads and is a potent thrombin inhibitor. The effects of the substitutions at N2 and C8 of
the G-tetrad-forming G residues on the thrombin inhibitory activity are relatively small,
suggesting that these substitutions cause relatively small perturbations on the chairlike
structure formed by the oligodeoxynucleotide. Introduction of a benzyl group into N2 of G6
and G11 and naphthylmethyl groups into N2 of G6 increased the thrombin inhibitory activity,
whereas other substituents in these positions had almost no effect or decreased the activity.
Particularly, the oligodeoxynucleotide carrying a 1-naphthylmethyl group in the N2 position
of G6 showed an increase in activity by about 60% both in vitro and in vivo. Substitutions on
the N2 position of other G residues had little effect or decreased the activity. Introduction of
a relatively small group, such as methyl and propynyl, into the C8 positions of G1, G5, G10, and
G
14 increased the activity, presumably due to the stabilization of a chairlike structure, whereas
introduction of a large substituent group, phenylethynyl, decreased the activity, probably due
to the steric hindrance.
In tr od u ction
Thrombin is a serine protease with multiple functions
in hemostasis. Its roles in coronary heart disease and
other thrombotic disorders have promoted efforts toward
the identification of specific inhibitors1 and an under-
standing of their interactions with this enzyme.2
A report from our laboratory disclosed the selection
of DNA aptamers by screening from a library of 1013
96-mer single-stranded oligodeoxynucleotides (ODNs).
The consensus sequence GGTTGGTGTGGTTGG (1) was
found to be a potent inhibitor of thrombin with an EC50
of 20 nM in a purified fibrinogen clotting assay.3 Sub-
sequent studies in cynomolgus monkeys demonstrated
that 1 is a potent and rapid-acting anticoagulant with
an in vivo half-life of approximately 2 min.4 Moreover,
in an ex vivo model of arterial thrombosis in a rabbit
aorta, 1 was found to inhibit clot-bound thrombin,
whereas heparin was ineffective.5 The rapid onset of
action, the short in vivo half-life, and the inhibitory
activity of clot-bound thrombin suggest that 1 may be
medically useful, e.g., as an anticoagulant in cardio-
pulmonary bypass surgeries.
The three-dimensional solution structure of 1 has
been solved using NMR techniques.6-8 As shown in
Figure 1, the chairlike structure adopted by this oli-
godeoxynucleotide consists of two G-tetrads connected
by two TT loops and a single TGT loop. In the structure,
two of the diagonally opposed 2′-deoxyguanosine resi-
dues within each of the G-tetrads adopt a syn conforma-
tion about their glycosidic bonds (G1, G10 and G5, G14
in Figures 1 and 2).
F igu r e 1. Schematic drawing of the NMR solution structure
of the 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG (1).
deoxyguanosine residues (G1, G2, G5, G6, G10, G11, G14,
and G15) as well as T4 and T13 in 1 by an abasic nu-
cleoside analogue residue can significantly decrease the
thrombin inhibitory activity of the compound (>500-
fold), while replacement of the other residues has a
relatively small effect (10-fold decrease for T3, G8, T9,
or T12) or no effect (T7) on the activity. The variation
in activity reflects the magnitude of structural disrup-
tion afforded by removal of each base, confirming the
importance of the two G-tetrads in maintaining the
active form of the molecule.
In an effort to increase the potency of 1, we have
pursued attachments which could interact with throm-
bin and increase the potency. The structure of 1 shows
that the G-tetrad-forming 2′-deoxyguanosine (G) resi-
dues, which form the core part of the structure, have
two positions (N2 and C8) available for attaching one or
more groups pointing away from the chairlike structure
Previous structure-activity relationship studies9 show
that replacing any of the eight G-tetrad-forming 2′-
S0022-2623(97)00434-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/27/1998

N2- and C8-Substituted Oligodeoxynucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2235
activity of the 15-mer analogues. As compared to the
purified fibrinogen clotting assay conducted in the
previous study,9 prothrombin time (PT) is measured in
human plasma and more directly related to the natural
event occurring in vivo.16 To eliminate the variation
caused by the measurements conducted on different
days or by using different lots of reagents, relative
prothrombin time (relative PT) was determined, which
is the activity of the test ODN relative to 1 measured
concurrently.
Introduction of a benzyl group into the N2 position of
the G residues in 1 led to different effects on the
thrombin inhibitory activity (Table 1). Substitutions on
G6 (ODN 105) and G11 (ODN 107) cause a moderate
increase in the activity (30-40%), while substitution on
G
10 (ODN 106) slightly increased the activity (20%). On
the other hand, however, substitutions on G5 (ODN 103)
and G14 (ODN 108) significantly decreased the activity,
and the substitutions at other positions produced almost
no effect. Moreover, introduction of a phenylethyl group
or a phenylpropyl group on G6 (ODN 109 or 111,
respectively) or G11 (ODN 110 or 112, respectively) had
little effect on the activity.17
The 1-naphthylmethyl group on G6 of 113 increased
the activity by 60%, while 2-naphthylmethyl group on
G6 of 116 gave a 30% increase (Table 2). However, the
4′-biphenylmethyl and 3′-biphenylmethyl on G6 or G11
had almost no effect on the activity (ODNs 119, 120,
122, and 123). For the naphthylmethyl groups and
biphenylmethyl groups, double substitutions on both G6
and G11 always decreased the activity (ODNs 115, 118,
121, and 124). Single incorporation of the adamantyl-
methyl group on G6 or G11 (ODN 125 or 126, respec-
tively) also decreases the activity.
As shown in Table 3, single incorporation of the
methyl group at C8 of G1, G5, or G10 in 127, 128, or 129,
respectively, had almost no effect on the activity.
However, 130 carrying one methyl group on C8 of G14
showed increased activity. In 131, when four methyl
groups were introduced simultaneously into G1, G5, G10,
and G14, the activity was increased by 60%. Attaching
a middle-size propynyl group into G1, G5, G10, or G14
(ODN 132, 133, 134, or 135, respectively) resulted in a
more pronounced increase of the activity. However, the
propynyl substitution lost its advantage over the methyl
substitution when all four substitutions were made at
once (ODN 136). Moreover, adding the larger phenyl-
ethynyl group showed little effect in single substitution
(ODNs 137, 138, 139, or 140, respectively) and led to
decreased activity in the quadruple substitution (ODN
141).
F igu r e 2. Top G-tetrad (top) and bottom G-tetrad (bottom)
in the chairlike structure of 1 shown in Figure 1, labeled with
the conformation about each glycosidic bond.
(Figure 2). In this paper, we report the results of our
studies of ODN analogues, in which modified G residue-
(s), carrying substituents on the N2 or C8 position, have
been introduced into the G-tetrads. It was found that
substitutions at certain G residues lead to moderate
increases in the thrombin inhibition. The increased
activities for the substitutions on C8 positions may be
explained by the stabilization of syn conformation of the
G residues, while the increased activities for the sub-
stitutions on N2 positions may be due to the interaction
with thrombin.
Resu lts
Syn th esis. N2-Substituted G derivatives were pre-
pared through a 2-fluoro-2′-deoxyguanosine intermedi-
ate (Scheme 1).10 After O6-protection with a p-nitro-
phenylethyl group, the 2-amino group was converted to
fluoro through diazotization with tert-butyl nitrite in
hydrogen fluoride-pyridine.11 From 2-fluoro intermedi-
ate 5, N2-substituted G derivatives were prepared with
the corresponding primary amines.
Synthesis of 8-methyl-2′-deoxyguanosine as well as
the corresponding ODNs was reported previously.9
8-Propynyl-2′-deoxyguanosine and 8-(phenylethynyl)-2′-
deoxyguanosine were prepared through the palladium-
mediated coupling of 8-bromo-2′-deoxyguanosine12 with
the corresponding acetylene derivatives (Scheme 2).13,14
The modified G monomers were incorporated into
ODN analogues with standard solid-phase DNA chem-
istry utilizing the phosphoramidite method.15
The increased thrombin inhibitory activity of a sub-
stituted ODN was further confirmed in vivo in monkeys.
Figure 3 shows the time course of the ODN-mediated
anticoagulation (PT) during a constant-rate iv infusion
at different concentrations of 1 or 113 which carries
1-naphthylmethyl group on N2 of G6. Both compounds
showed a clear dose response, and 113 had an increased
activity compared to 1 without losing the rapid-acting
and short half-life feature of the anticoagulant.
Discu ssion
Th r om bin In h ibitor y Activity. A prothrombin
assay was used to evaluate the thrombin inhibitory
In a previous paper,9 we have shown that the throm-
bin inhibitory activity of 1 can be significantly decreased

2236 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
He et al.
Sch em e 1a
a
Reagents: (a) p-NO2C6H4CH2CH2OH, PPh3, DEAD, in 1,4-dioxane; (b) NH3 in MeOH; (c) t-Bu-ONO, in 50% Py-HF; (d) DMT-Cl, in
Py; (e) R-NH2, in DMF, (f) DBU, in Py; (g) (Me2CH)2NPCl(OCH2CH2CN), EtN(CHMe2)2, in CH2Cl2.
Ta ble 1. N2-Substituted G Oligomers and Their Activities:
Benzyl, Phenylethyl, and Phenylpropyl Substitutions
Sch em e 2a
on N2 and C8 cause different effects on the activity, and
these effects can be rationalized as follows.
Previous studies using a purified fibrinogen clotting
assay showed that introducing a methyl group into the
C8 positions of syn-G residues of 1 increases the activity,
while substitutions at the C8 positions of anti-G residues
decrease the activity.9 In the present study, the results
obtained from the PT assay conducted in human plasma
for ODNs carrying methyl and propynyl substitutions
confirm this conclusion. It is known that the presence
of a substituent group on the C8 position can stabilize
the syn conformation of the G residue.18 Recently, it
was reported that introducing a bromine atom into the
C8 position of the syn-G residues can stabilize the
G-tetrads as well as the chairlike structure.19 There-
fore, the activity increase obtained by introducing a
methyl or a propynyl group into the C8 positions of the
syn-G residues of 1 can be explained by the stabilization
of the G-tetrads and consequently the chairlike struc-
ture. Since the chairlike structure does not show any
change in its UV spectrum upon melting, we have
measured the melting point (Tm) of the chairlike struc-
a
Reagents: (a) MeCtCH or PhCtCH, Et3N, Pd catalyst, in
DMSO; (b) TMS-Cl, in Py; (c) isobutyric anhydride, in Py, quenched
by aq NH3; (d) DMT-Cl, in Py; (e) EtN(CHMe2)2, (Me2CH)2NPCl-
(OCH2CH2CN), in CH2Cl2.
by replacing one of the eight G-tetrad-forming G resi-
dues with a base-modified deoxynucleotide residue, such
as 2′-deoxyinosine or 7-deaza-2′-deoxyguanosine. These
deoxynucleotide residues are unable to form the hydro-
gen bond required for the formation of the G-tetrad and
consequently cause significant disruption to the chair-
like structure. In this study, the effects of the substitu-
tions at N2 and C8 of the G-tetrad-forming G residues
of 1 on the thrombin inhibitory activity are relatively
small, suggesting that these substitutions cause rela-
tively small perturbations on the chairlike structure
formed by the ODN. However, different substitutions
1
ture formed by 1 and its derivative 131 using H NMR

N2- and C8-Substituted Oligodeoxynucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2237
Ta ble 2. N2-Substituted G Oligomers and Their Activities:
Naphthylmethyl, Biphenylmethyl, and Adamantylmethyl
Substitutions
F igu r e 3. Time course of OND-mediated anticoagulation (PT)
during a constant-rate iv infusion of 15-mer 1 (b) or modified
15-mer 113 (O) in cynomolgus monkeys. Each OND was
infused into two animals over a period of 45 min. The infusion
rate was 0.15 mg/kg/min in the time period of 0-15 min, 0.3
mg/kg/min in 15-30 min, and 0.6 mg/kg/min in 30-45 min.
Blood samples were collected from each animal at different
time points and assayed.
tion. In the quadruple substitutions, the steric effect
of the substituent groups becomes more serious. There-
fore, the propynyl substitution showed the same effect
as the methyl substitution, while the phenylethynyl
substitution showed decreased activity.
Ta ble 3. C8-Substituted G Oligomers and Their Activities:
Methyl, Propynyl, and Phenylethynyl Substitutions
The other attachment site studied is N2 of the
G-tetrad-forming G residues.17 The substitutions on
this position provide different effects from the C8
substitutions. As shown in Table 1, the highest increase
in the thrombin inhibitory activity was obtained from
105 and 107 (30-40%), which carry a benzyl group on
G6 and G11, respectively. Since both substituents on G6
and G11 are located on the backside of the chairlike
structure (Figures 1 and 2), pointing toward the outside,
they may have certain interactions with thrombin. In
a separate study using neutral formacetal linkage to
replace negatively charged phosphodiester linkage, we
also found that the backside of the chairlike structure
is involved in the binding with thrombin.20
On the other hand, however, the substitutions on G5
and G14 of 103 and 108, respectively, decrease the
activity. In fact, both substituents on G5 and G14 are
located in the bottom TT loops where the structure is
very crowded due to the phosphate backbone (see bottom
G-tetrad in Figure 2). Therefore, substituents on these
two positions may destabilize the structure due to the
steric hindrance, resulting in the decreased activity.
Since G6 and G11 were identified as favorable posi-
tions, different attachments on these two positions were
studied by either increasing the number of methylene
groups or adding another phenyl ring to the attachment.
As shown in Tables 1 and 2, however, single substitution
of these attachments has a relatively small effect on the
activity except for naphthylmethyl groups on G6. For
the 1-naphthylmethyl group, the 60% increase of the
activity may be explained by the similar interaction with
thrombin as achieved by the benzyl group discussed
above. However, the double substitution of these groups
on both G6 and G11 decreases the activity, probably due
to the steric hindrance. The same explanation may also
under the same conditions.6 The melting point was
increased from 55 °C for 1 to about 70 °C for 131,
suggesting an improved stability of the chairlike struc-
ture formed by 131.
As shown in Table 3, for the single substitutions, the
middle-size propynyl group usually provides better
stabilization of the chairlike structure compared to the
small methyl group, except for the single methyl sub-
stitution at C8 of G14 in 130 which had a similar increase
in the activity as the single-propynyl substitution. How-
ever, further increase in the size of the substituent
group as in the large phenylethynyl group does not pro-
vide any increase of the stability. The result may be
explained by the steric hindrance. Thus, the steric hin-
drance of the large group diminishes the effect of sta-
bilization by having a substituent group on the C8 posi-

2238 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
He et al.
fibrometer using citrated plasma (Sigma C7916 or freshly spun
1500g, 10 min) and thromboplastin/calcium (Sigma T7405) at
final concentrations of 2 and 4 µM for ODNs in plasma. The
assay was performed in duplicate at each concentration. The
clotting time for the control sample was in the range of 10-
13 s. Prothrombin time was the percentage ratio of the clotting
time of test ODN to that of the control sample. Relative
prothrombin time was the activity of test ODN relative to
unmodified 1 measured at the same day and calculated with
the following equation:
apply to the results obtained from the single substitu-
tion of the bulky adamantylmethyl group.
Different from most other thrombin inhibitors, the
oligodeoxynucleotides described in this study inhibit the
thrombin activity not through binding in the active site.
Instead, an anionic binding exosite on the thrombin
molecule has been identified as a putative binding site
for 1.21 Therefore, the inhibition constant (Ki) cannot
be determined for 1 by measuring the inhibitory effect
on the cleavage rate of a small chromogenic substrate.
By using surface plasmon resonance technology,22 we
were able to measure the dissociation constant (Kd) for
1 and 113. In this technology, the interaction between
the oligodeoxynucleotide and thrombin was monitored
by immobilizing thrombin on a sensor chip and injecting
1 across the sensor surface. As a result, Kd ) 1.4 nM23
for 1 and Kd ) 0.8 nM for 113. The result is consistent
with the PT result, although the increase in Kd is also
relatively small.
As shown in Figure 3, 131 has increased in vivo
thrombin inhibitory activity compared to 1 but main-
tains a rapid onset of action and a short half-life. In
addition, the increase (∼60%) in the in vivo activity of
131 is consistent with the increase in the in vitro
activity as well as the increase in binding described
above. Therefore, like 1, the in vivo thrombin inhibitory
activity of 131 should result from the full-length oli-
godeoxynucleotide and not from its degradation prod-
ucts. These results are further supported by previous
pharmacokinetic studies,24,25 showing that the rapid
clearance of the oligodeoxynucleotide in vivo is mainly
due to irreversible tissue uptake and that nuclease
degradation in the blood (mainly 3′-exonuclease) only
accounts for approximately 22% of the total clearance
of 1 in vivo. Therefore, a modification on one of the G
residues described in this study will not significantly
affect the in vivo pharmacokinetic behavior of the
compound.
relative PT ) (PT1 - 100)/(PT2 - 100)
where PT1 represents the prothrombin time of test ODN and
PT2 represents the prothrombin time of unmodified 1. In the
experiment, PT2 was about 150 at 2 µm and 200 at 4 µm. The
results shown in Tables 1-3 are the average values for the
data measured at different concentrations of ODNs mentioned
above.
Con sta n t Ra te In fu sion of ODN a n d Dose Resp on se
in Vivo. The time course of the ODN-mediated anticoagula-
tion during a constant-rate iv infusion and the dose response
of 15-mer 1 or modified 15-mer 113 were studied using
cynomolgus monkeys by measuring PT.4 The animals used,
as well as the experiment setting, were the same as those
reported previously.4 Each ODN was infused into two animals
over a period of 45 min. The infusion rate was 0.15 mg/kg/
min in the time period of 0-15 min, 0.3 mg/kg/min in 15-30
min, and 0.6 mg/kg/min in 30-45 min. Blood samples were
collected from each animal at different time points and
assayed.
Syn th esis of Oligom er s. ODN analogues were synthe-
sized by using standard solid-phase DNA chemistry on con-
trolled pore glass (CPG) support with the phosphoramidite
method15 on a Biosearch DNA synthesizer. The standard
building blocks suitable for the solid-phase synthesis, such as
3′-O-[(diisopropylamino)(cyanoethoxy)phosphino]-5′-O-(4,4′-
dimethoxytrityl)-N2-isobutyryl-2′-deoxyguanosine and 3′-O-
[(diisopropylamino)(cyanoethoxy)phosphino]-5′-O-(4,4′-di-
methoxytrityl)thymidine, as well as CPG supports loaded with
the corresponding building blocks were purchased from Glen
Research (Sterling, VA). The CPG supports loaded with the
modified building blocks were prepared by standard methods.27
ODNs were purified by polyacrylamide gel electrophoresis
(PAGE) (ca. 1 µmol) or reversed-phase HPLC (SepTech PRP-1
column) (g10 µmol), followed by desalting with a Sephadex
NAP-25 column. ODNs were analyzed for purity (g90%) and
chemical integrity by PAGE, ion-exchange HPLC, and base
composition analysis. Qualitative PAGE analysis involved
inspection of acrylamide gels (25 × 16 × 0.05 cm, 20%
acrylamide, 1:19 cross-linked, 7 M urea, Tris/boric acid/EDTA
buffer) using UV shadowing or staining with Stains-ALL.
Mobilities were compared to that of oligothymidine standards
and unmodified 15-mer 1. Ion-exchange HPLC analysis was
performed on a Waters 600 E system with UV detection at
254 nm using a Dionex Nucleopac PA-100 column. The elution
gradient was linear from 75 mM LiCl/5 mM LiOH to 1.25 M
LiCl/5 mM LiOH in 25 min. To confirm the presence of the
expected modified nucleoside components, ODNs were com-
pletely digested to nucleoside monomers using snake venom
phosphodiesterase, P1 nuclease, and alkaline phosphatase.
The digested samples were evaluated via reversed-phase
HPLC (Amicon 10 µm, C18, 25 cm × 4.6 mm column, 37 °C,
2% acetonitrile to 35% acetonitrile in 35 min, constant 50 mM
monobasic potassium phosphate). After the correction for
extinction coefficient differences using the factors of 0.608 and
1.00 for G and T, respectively, the peak areas obtained were
used to calculate the relative base composition for each ODN.
ODN extinction coefficients were calculated by the method
reported by Tinoco,28 and unmodified 15-mer 1 has ꢀ ) 143 300
based on the method. The N2-substituted and C8-substituted
G residues were treated as G in the calculation. Since most
of the modified ODNs under consideration contain only one
modified base, the error introduced by the treatment can be
expected to be less than the systematic error of the assay.
In conclusion, the effects of the substitutions at N2
and C8 of the G-tetrad-forming G residues on the
thrombin inhibitory activity are relatively small, sug-
gesting that these substitutions cause relatively small
perturbations on the chairlike structure formed by the
ODN. Introducing a small methyl group or a middle-
size propynyl group into the C8 position(s) of the syn-G
residues increases the activity presumably due to the
stabilization of the chairlike structure. On the other
hand, introducing a 1-naphthylmethyl group into the
N2 position of G6 increases the activity by about 60%
both in vitro and in vivo probably due to the increased
interaction with thrombin.
Exp er im en ta l Section
Gen er a l. Column chromatography was performed using
silica gel 60 from EM science, and thin-layer chromatography
was performed on Kieselgel 60 F254 aluminum plates. Solvents
used were Burdick & J ackson B&J Chrompure HPLC grade
and were dried over 4A molecular sieves. Commercially avail-
able chemical reagents were purchased from Aldrich Chemical
Co. unless noted otherwise. 1H and 31P NMR spectra were
obtained on a General Electric QE-300 at 300.6 and 121.7
MHz, respectively, using tetramethylsilane internal standard
for proton and 5% phosphoric acid in D2O external standard
(in capillary) for phosphorus. J values are listed in hertz (Hz).
Rela tive P r oth r om bin Tim e (P T). The prothrombin time
assay (PT) was carried out by the standard method4,26 on a

N2- and C8-Substituted Oligodeoxynucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2239
Syn th esis of Mon om er s. N2-Su bstitu ted 2′-Deoxygu a -
n osin e Der iva t ives. 6-O-(4-Nit r op h en et h yl)-2′-d eoxy-
gu a n osin e (4). A suspension of 2′-deoxyguanosine (3 g, 11.3
mmol) and acetic anhydride (5.0 g, 49 mmol) in 40 mL of
pyridine and 30 mL of DMF was heated at 75 °C for 4 h to
give a clear solution. TLC showed the completion of the
reaction. After cooling, the solution was concentrated to
dryness, and the solid residue obtained was washed twice with
2-propanol to give 3.2 g of 3′,5′-di-O-acetyl-2′-deoxyguanosine
(3) (yield, 81%) as a white solid. The structure was confirmed
) 6.5, 2H, -CH2-), 2.8 and 2.5 (2m, 2H, 2′-Hab), 1.91 (d, J )
3, 1H, 3′-OH).
5′-O-(4,4′-Dim e t h oxyt r it yl)-N 2-b e n zyl-2′-d e oxygu a -
n osin e (6a ). A solution of 5′-O-(4,4′-dimethoxytrityl)-6-O-(4-
nitrophenethyl)-2-fluoro-2′-deoxyguanosine (5) (2 g, 2.8 mmol)
and benzylamine (3.2 g, 30 mmol) in 50 mL of dry DMF was
stirred at room temperature for 24 h. When the reaction was
completed, the solution was concentrated under vacuum and
the residue obtained was dissolved in 300 mL of CH2Cl2. After
being washed with 100 mL of 5% aqueous NaHCO3 solution,
the organic solution was dried over Na2SO4 and concentrated.
The crude product obtained was purified with silica gel
chromatography using CH2Cl2/MeOH/Et3N ) 95/5/1 (v/v) to
give 1.9 g of 5′-O-(4,4′-dimethoxytrityl)-6-O-(4-nitrophenethyl)-
N2-benzyl-2′-deoxyguanosine as a pale-yellow foam in 84%
yield.
1
by H NMR (DMSO-d6).
3′,5′-Di-O-acetyl-2′-deoxyguanosine (3) (3.2 g, 9.1 mmol) was
suspended in 100 mL of dioxane. After triphenylphosphine
(3.6 g, 13.7 mmol) and 4-nitrophenethyl alcohol (2.3 g, 13.7
mmoL) were added, the suspension was heated at 80 °C for
30 min. Then, the suspension was cooled to 60 °C, and diethyl
azodicarboxylate (2.4 g, 13.7 mmol) was added dropwise. The
resultant mixture was heated at 60 °C for another 2 h, and a
clear-yellow solution was obtained. After cooling, the solution
was concentrated. The residue was dissolved in 300 mL of
CH2Cl2, and the organic solution obtained was washed with
200 mL of water and dried over Na2SO4. The solution was
concentrated, and the residue obtained was purified with silica
5′-O-(4,4′-Dimethoxytrityl)-6-O-(4-nitrophenethyl)-N2-ben-
zyl-2′-deoxyguanosine (1.9 g, 2.3 mmol) was dissolved in 30
mL of dry pyridine. After 2.3 g of DBU was added, the solution
obtained was stirred at room temperature for 15 h. Then, the
solution was concentrated under vacuum, and the residue
obtained was dissolved in 300 mL of CH2Cl2. After being
washed with 100 mL of 0.5 M aqueous citric acid solution and
then 100 mL of 5% aqueous NaHCO3 solution, the organic
solution was dried over Na2SO4 and concentrated. The crude
product obtained was purified with silica gel chromatography
using CH2Cl2/MeOH/Et3N ) 90/10/1 (v/v) to give 1.1 g of 5′-
O-(4,4′-dimethoxytrityl)-N2-benzyl-2′-deoxyguanosine (6a ) as
a white-yellow foam in 73% yield. 1H NMR (DMSO-d6): δ
10.59 (s, 1H, NH), 7.78 (s, 1H, 8-H), 7.1-7.4 (m, 9H, Ar-H),
6.80 (m, 5H, NH + Ar-H), 6.17 (t, J ) 6, 1H, 1′-H), 5.29 (d, J
) 4, 1H, 3′-OH), 4.33 (m, 3H, 3′-H, N-CH2-Ph), 3.91 (m, 1H,
4′-H), 3.70 (s, 6H, -OCH3), 3.0-3.3 (m, 2H, 5′-Hab), 2.7 and
2.2 (2m, 2H, 2′-Hab).
gel chromatography using CH2Cl2/MeOH ) 95/5 (v/v).
A
yellow viscous oil was obtained, and 1H NMR showed that it
contained mainly the desired product, 3′,5′-di-O-acetyl-6-O-
(4-nitrophenethyl)-2′-deoxyguanosine, contaminated by a small
amount of triphenylphosphine oxide. The crude product
thus obtained was dissolved in 150 mL of 1 M NH3/metha-
nol solution. The solution was stirred at room temperature
for 15 h. After the reaction was completed, the solvent was
evaporated and the residue was purified with silica gel
chromatography using CH2Cl2/MeOH ) 90/10 (v/v) to give
3.0 g of 6-O-(4-nitrophenethyl)-2′-deoxyguanosine (4) as a
pale-yellow foam in 79% yield. 1H NMR (DMSO-d6): δ 8.17
(d, J ) 8, 2H, Ar-H), 8.06 (s, 1H, 8-H), 7.61 (d, J ) 8, 2H,
Ar-H), 6.44 (bs, 2H, NH), 6.19 (t, J ) 6.5, 1H, 1′-H), 5.26
(bs, 1H, 3′-OH), 4.98 (bs, 1H, 5′-OH), 4.65 (t, J ) 6.5, 2H,
-CH2-), 4.34 (m, 1H, 3′-H), 3.81 (m, 1H, 4′-H), 3.4-3.6 (m,
2H, 5′-Hab), 3.24 (t, J ) 6.5, 2H, -CH2-), 2.6 and 2.2 (2m,
2H, 2′-Hab).
Compounds 6b-h were prepared with the same procedure
as above using compound 5 and 10 equiv of the corresponding
1
amines. The amine used, the product yield, and the H NMR
data of the product are as follows.
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-(2-p h en ylet h yl)-2′-d e-
oxygu a n osin e (6b). Phenethylamine; yield: 85%. 1H NMR
(DMSO-d6): δ 7.79 (s, 1H, 8-H), 7.1-7.4 (m, 9H, Ar-H), 6.75
(t, J ) 9, 4H, Ar-H), 6.31 (t, J ) 5, 1H, NH), 6.21 (t, J ) 6,
1H, 1′-H), 5.32 (d, J ) 4, 1H, 3′-OH), 4.40 (m, 1H, 3′-H), 3.93
(m, 1H, 4′-H), 3.69 (s, 6H, -OCH3), 3.23 (m, 2H, N-CH2-),
3.0-3.2 (m, 2H, 5′-Hab), 2.71 (t, J ) 8, 2H, -CH2-Ph), 2.8
and 2.2 (2m, 2H, 2′-Hab).
5′-O-(4,4′-Dim et h oxyt r it yl)-6-O-(4-n it r op h en et h yl)-2-
flu or o-2′-d eoxygu a n osin e (5). Dry pyridine (4 g) was added
dropwise at -30 °C to 10 g of hydrogen fluoride-pyridine
(containing about 70% HF in pyridine; Aldrich) under stirring
to prepare 50% hydrogen fluoride-pyridine. To a suspension
of 6-O-(4-nitrophenethyl)-2′-deoxyguanosine (4) (2 g, 4.8 mmol)
in 14 g of 50% hydrogen fluoride-pyridine prepared above at
-30 °C was added 98% tert-butyl nitrite (0.7 g, 6 mmol)
dropwise, and the resultant mixture was stirred at -30 °C
for 2 h. After the bubbling stopped, a clear-yellow solution
was obtained. The solution was poured into 250 mL of ice-
water, and the mixture was extracted with CH2Cl2 (5 × 100
mL). The solvent used was removed to give 6-O-(4-nitrophen-
ethyl)-2-fluoro-2′-deoxyguanosine (2 g, 98%) as a pale-yellow
foam, which was used in the subsequent reaction without
further purification.
4,4′-Dimethoxytrityl chloride (2.0 g, 5.9 mmol) was added
to a solution of 6-O-(4-nitrophenethyl)-2-fluoro-2′-deoxygua-
nosine (2 g, 4.8 mmol) in 30 mL of dry pyridine. The solution
was stirred at room temperature for 15 h, and 5 mL of MeOH
was added. The resultant solution was concentrated, and the
residue obtained was dissolved in 300 mL of CH2Cl2. After
being washed with 100 mL of 5% aqueous NaHCO3 solution,
the organic solution was dried over Na2SO4 and concentrated.
The crude product obtained was purified with silica gel
chromatography using CH2Cl2/MeOH/Et3N ) 100/5/1 (v/v) to
give 2.5 g of 5′-O-(4,4′-dimethoxytrityl)-6-O-(4-nitrophenethyl)-
2-fluoro-2′-deoxyguanosine (5) as a pale-yellow foam with 72%
yield. 1H NMR (CDCl3): δ 8.17 (d, J ) 8.5, 2H, Ar-H), 8.03
(s, 1H, 8-H), 7.49 (d, J ) 8, 2H, Ar-H), 7.1-7.4 (m, 9H, Ar-
H), 6.80 (d, J ) 8.5, 4H, Ar-H), 6.37 (t, J ) 6.5, 1H, 1′-H),
4.83 (t, J ) 6.5, 2H, -CH2-), 4.67 (m, 1H, 3′-H), 4.12 (m, 1H,
4′-H), 3.78 (s, 6H, -OCH3), 3.2-3.5 (m, 2H, 5′-Hab), 3.29 (t, J
5′-O-(4,4′-Dim e t h oxyt r it yl)-N 2-(3-p h e n ylp r op yl)-2′-
d eoxygu a n osin e (6c). 3-Phenylpropylamine; yield: 62%. 1H
NMR (DMSO-d6): δ 10.42 (bs, 1H, NH), 7.78 (s, 1H, 8-H), 7.1-
7.4 (m, 9H, Ar-H), 6.78 (t, J ) 9, 4H, Ar-H), 6.30 (bs, 1H,
NH), 6.17 (t, J ) 6, 1H, 1′-H), 5.31 (d, J ) 4, 1H, 3′-OH), 4.40
(m, 1H, 3′-H), 3.93 (m, 1H, 4′-H), 3.70 (s, 6H, -OCH3), 3.0-
3.2 (m, 4H, N-CH2-, 5′-Hab), 2.56 (t, J ) 8, 2H, -CH2-Ph),
2.7 and 2.2 (2m, 2H, 2′-Hab), 1.7 (m, 2H, -C-CH2-C-).
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-(1-n a p h t h ylm et h yl)-2′-
d eoxygu a n osin e (6d ). 1-Naphthylmethylamine; yield: 89%.
1H NMR (DMSO-d6): δ 10.45 (bs, 1H, NH), 7.8-8.1 (m, 3H,
Ar-H), 7.82 (s, 1H, 8-H), 7.1-7.6 (m, 13H, Ar-H), 6.83 (bs,
1H, NH), 6.76 (t, J ) 9, 4H, Ar-H), 6.22 (t, J ) 6, 1H, 1′-H),
5.26 (d, J ) 4, 1H, 3′-OH), 4.7-4.9 (m, 2H, -N-CH2-Ar), 4.28
(m, 1H, 3′-H), 3.91 (m, 1H, 4′-H), 3.68 (s, 6H, -OCH3), 3.0-
3.2 (m, 2H, 5′-Hab), 2.7 and 2.2 (2m, 2H, 2′-Hab).
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-(2-n a p h t h ylm et h yl)-2′-
d eoxygu a n osin e (6e). 2-Naphthylmethylamine; yield: 78%.
1H NMR (DMSO-d6): δ 10.68 (bs, 1H, NH), 7.8-8.0 (m, 3H,
Ar-H), 7.80 (s, 1H, 8-H), 7.1-7.6 (m, 13H, Ar-H), 6.92 (t, J
) 5, 1H, NH), 6.77 (t, J ) 9, 4H, Ar-H), 6.19 (t, J ) 6, 1H,
1′-H), 5.30 (d, J ) 4, 1H, 3′-OH), 4.52(d, J ) 5, 2H, -N-CH2-
Ar), 4.34 (m, 1H, 3′-H), 3.92 (m, 1H, 4′-H), 3.69 (s, 6H, -OCH3),
3.0-3.2 (m, 2H, 5′-Hab), 2.7 and 2.2 (2m, 2H, 2′-Hab).
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-(4-b ip h en ylm et h yl)-2′-
d eoxygu a n osin e (6f). 4-Biphenylmethylamine; yield: 73%.
1H NMR (DMSO-d6): δ 10.64 (bs, 1H, NH), 7.80 (s, 1H, 8-H),
7.1-7.7 (m, 18H, Ar-H), 6.84 (t, J ) 5, 1H, NH), 6.78 (t, J )

2240 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
He et al.
9, 4H, Ar-H), 6.19 (t, J ) 6, 1H, 1′-H), 5.30 (d, J ) 4, 1H,
3′-OH), 4.40 (d, J ) 5, 2H, -N-CH2-Ar), 4.36 (m, 1H, 3′-H),
3.93 (m, 1H, 4′-H), 3.69 (s, 6H, -OCH3), 3.0-3.2 (m, 2H, 5′-
Hab), 2.7 and 2.2 (2m, 2H, 2′-Hab).
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-(3-b ip h en ylm et h yl)-2′-
d eoxygu a n osin e (6g). 3-Biphenylmethylamine; yield: 42%.
1H NMR (CDCl3): δ 12.2 (bs, 1H, NH), 7.9 (bs, 1H, NH), 7.49
(s, 1H, 8-H), 7.0-7.4 (m, 18H, Ar-H), 6.74 (t, J ) 9, 4H, Ar-
H), 6.09 (m, 1H, 1′-H), 4.48 (m, 2H, -N-CH2-Ar), 4.33 (m,
1H, 3′-H), 3.95 (m, 1H, 4′-H), 3.70 (s, 6H, -OCH3), 3.6 and 3.2
(2m, 2H, 5′-Hab), 2.5 and 2.1 (2m, 2H, 2′-Hab).
5′-O-(4,4′-Dim eth oxytr ityl)-N2-(1-a d a m a n tylm eth yl)-2′-
deoxygu an osin e (6h ). 1-Adamantylmethylamine; yield: 86%.
1H NMR (DMSO-d6): δ 10.32 (bs, 1H, NH), 7.77 (s, 1H, 8-H),
7.1-7.4 (m, 9H, Ar-H), 6.78 (t, J ) 9, 4H, Ar-H), 6.30 (bs,
1H, NH), 6.17 (t, J ) 6, 1H, 1′-H), 5.30 (d, J ) 4, 1H, 3′-OH),
4.37 (m, 3H, 3′-H), 3.91 (m, 1H, 4′-H), 3.70 (s, 6H, -OCH3),
3.0-3.2 (m, 2H, 5′-Hab), 2.8 (m, 2H, N-CH2-), 2.7 and 2.2
(2m, 2H, 2′-Hab), 1.4-1.9 (m, 15H, -CH- and -CH2- in ad).
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-dim eth oxytr ityl)-N2-ben zyl-2′-deoxygu an osin e (7a).
2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.8 g, 3.6
mmol) was added dropwise to a solution of 5′-O-(4,4′-dimeth-
oxytrityl)-N2-benzyl-2′-deoxyguanosine (6a ) (1.1 g, 1.7 mmol)
and N,N-diisopropylethylamine (0.7 g, 5 mmol) in 30 mL of
dry CH2Cl2 at 0 °C. The solution obtained was stirred at 0 °C
for 2 h and at room temperature for another 4 h. Then, 5 mL
of methanol was added to the solution. After the mixture was
stirred at room temperature for 10 min, 300 mL of CH2Cl2
was added. The solution obtained was washed with 100 mL
of 5% aqueous NaHCO3 solution and dried over Na2SO4. The
solvent was removed under reduced pressure at room tem-
perature. The crude product obtained was purified with silica
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im e t h oxyt r it yl)-N 2-(4-b ip h e n ylm e t h yl)-2′-d e -
oxygu a n osin e (7f). 3′-O-[(Diisopropylamino)(cyanoethoxy)-
phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(4-biphenylmeth-
yl)-2′-deoxyguanosine (7f) was prepared from 5′-O-(4,4′-
dimethoxytrityl)-N2-(4-biphenylmethyl)-2′-deoxyguanosine (6f)
by the method described above. Yield: 56%. 31P NMR
(CDCl3): δ 148.97, 149.28.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im e t h oxyt r it yl)-N 2-(3-b ip h e n ylm e t h yl)-2′-d e -
oxygu a n osin e (7g). 3′-O-[(Diisopropylamino)(cyanoethoxy)-
phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(3-biphenylmethyl)-
2′-deoxyguanosine (7g) was prepared from 5′-O-(4,4′-dimeth-
oxytrityl)-N2-(3-biphenylmethyl)-2′-deoxyguanosine (6g) by the
method described above. Yield: 50%. 31P NMR (CDCl3): δ
148.97, 149.38.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im et h oxyt r it yl)-N2-(1-a d a m a n t ylm et h yl)-2′-d e-
oxygu a n osin e (7h ). 3′-O-[(Diisopropylamino)(cyanoethoxy)-
phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(1-adamantylmethyl)-
2′-deoxyguanosine (7h ) was prepared from 5′-O-(4,4′-dimeth-
oxytrityl)-N2-(1-adamantylmethyl)-2′-deoxyguanosine (6h ) by
the method described above. Yield: 80%. 31P NMR (CDCl3):
δ 148.78, 149.50.
C8-Su bstitu ted 2′-Deoxygu a n osin e Der iva tives. 8-(1-
P r opyn yl)-2′-deoxygu an osin e (9a). A suspension of 8-bromo-
2′-deoxyguanosine (8) (3.24 g, 9.36 mmol) in 50 mL of DMF
containing triethylamine (5 mL, 35.9 mmol), cuprous iodide
(0.38 g, 2.0 mmol), and tetrakis(triphenylphosphine)palladium
(1.16 g, 1.0 mmol) was saturated with propyne at 5 °C. The
mixture was stirred in a sealed flask at room temperature for
72 h and then concentrated. The residue was triturated with
75 mL of methanol, and the solid formed was collected by
filtration, washed with methanol and ether, and dried under
vacuum to yield 2.28 g (80%) of a tan solid which was
contaminated with triphenylphosphine as shown by 1H NMR.
However, this material was found to be suitable for use in the
subsequent reaction. The analytical sample was obtained by
chromatography purification on silica gel using methylene
chloride/methanol (4:1, v/v) followed by crystallization from
water/methanol (4:1, v/v) to give 9a as an amorphous solid.
1H NMR (DMSO-d6): δ 10.7 (s, 1H, NH), 7.8 (m, 2.5H,
contaminant), 6.5 (bs, 2H, NH2), 6.2 (dd, 1H, 1′-H), 5.2 (d,
1H, 3′-OH), 4.8 (t, 1H, 5′-OH), 4.3 (m, 1H, 3′-H), 3.8 (m, 1H,
4′-H), 3.4-3.7 (m, 2H, 5′-Hab), 2.1 and 3.0 (2m, 2H, 2′-Hab),
2.1(s, 3H, propyne CH3). Anal. (C13H15N5O4‚0.9H2O) C, H, N.
8-(1-P h en yleth yn yl)-2′-d eoxygu a n osin e (9b). A suspen-
sion of 8-bromo-2′-deoxyguanosine (8) (1.66 g, 4.8 mmol) in 25
mL of DMSO containing triethylamine (2.5 mL, 18 mmol) and
phenylacetylene (0.7 mL, 6.38 mmoL) was treated with bis-
(triphenylphosphine)palladium dichloride (0.2 g, 0.28 mmol).
The mixture was heated at 85 °C for 14 h and then concen-
trated. The residue was triturated with 100 mL of acetonitrile,
and the solid formed was collected by filtration and crystallized
from 100 mL of water/acetonitrile (9:1, v/v). After filtration,
gel chromatography using CH2Cl2/MeOH/Et3N
) 95/3/1
(v/v). The white foam product obtained was redissolved in
20 mL of CH2Cl2, and the solution was added dropwise to
200 mL of hexane under vigorous stirring. The precipitate
formed was collected by filtration and dried under vacuum to
give 1.1 g of 3′-O-[(diisopropylamino)(cyanoethoxy)phosphino]-
5′-O-(4,4′-dimethoxytrityl)-N2-benzyl-2′-deoxyguanosine as a
white powder in 75% yield. 31P NMR (DMSO-d6): δ 148.40,
148.70.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im eth oxytr ityl)-N2-(2-p h en yleth yl)-2′-d eoxygu a -
n osin e (7b). 3′-O-[(Diisopropylamino)(cyanoethoxy)phosphino]-
5′-O-(4,4′-dim et h oxyt r it yl)-N2-(2-ph en ylet h yl)-2′-deoxy-
guanosine (7b) was prepared from 5′-O-(4,4′-dimethoxytrityl)-
N2-(2-phenylethyl)-2′-deoxyguanosine (6b) by the method de-
scribed above. Yield: 48%. 31P NMR (CDCl3): δ 148.44,
148.80.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-dim eth oxytr ityl)-N2-(3-ph en ylpr opyl)-2′-deoxygu a-
n osin e (7c). 3′-O-[(Diisopropylamino)(cyanoethoxy)phosphino]-
5′-O-(4,4′-dim et hoxyt rit yl)-N2-(3-ph en ylpropyl)-2′-deoxy-
guanosine (7c) was prepared from 5′-O-(4,4′-dimethoxytrityl)-
N2-(3-phenylpropyl)-2′-deoxyguanosine (6c) by the method
described above. Yield: 70%. 31P NMR (CDCl3): δ 148.72,
149.13.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im e t h oxyt r it yl)-N 2-(1-n a p h t h ylm e t h yl)-2′-d e -
oxygu a n osin e (7d ). 3′-O-[(Diisopropylamino)(cyanoethoxy)-
phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(1-naphthylmethyl)-
2′-deoxyguanosine (7d ) was prepared from 5′-O-(4,4′-dimeth-
oxytrityl)-N2-(1-naphthylmethyl)-2′-deoxyguanosine (6d ) by the
method described above. Yield: 49%. 31P NMR (CDCl3): δ
148.68, 149.05.
3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im e t h oxyt r it yl)-N 2-(2-n a p h t h ylm e t h yl)-2′-d e -
oxygu a n osin e (7e). 3′-O-[(Diisopropylamino)(cyanoethoxy)-
phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(2-naphthylmethyl)-
2′-deoxyguanosine (7e) was prepared from 5′-O-(4,4′-dimeth-
oxytrityl)-N2-(2-naphthylmethyl)-2′-deoxyguanosine (6e) by the
method described above. Yield: 60%. 31P NMR (CDCl3): δ
148.75, 149.05.
a
second crop precipitated from the filtrate, which was
combined with the crystallized precipitate, and the combined
materials were recrystallized from 75 mL of water/acetonitrile
(9:1, v/v) to afford 0.58 g of 8-(1-phenylethynyl)-2′-deoxygua-
nosine (9b ) as an amorphous solid in 33% yield. 1H NMR
(DMSO-d6): δ 10.9 (s, 1H, NH), 7.5-7.8 (m, 5H, phenyl), 6.6
(bs, 2H, NH2), 6.3 (dd, 1H, 1′-H), 5.4 (d, 1H, 3′-OH), 4.9 (t,
1H, 5′-OH), 4.5 (m, 1H, 3′-H), 3.8 (m, 1H, 4′-H), 3.5-3.7 (m,
2H, 5′-Hab), 2.2 and 3.3 (2m, 2H, 2′-Hab). Anal.
(C18H17N5O4‚1.75H2O) C, H, N.
5′-O-(4,4′-Dim eth oxytr ityl)-N2-isobu tyr yl-8-(1-pr opyn yl)-
2′-d eoxygu a n osin e (10a ). A suspension of 8-(1-propynyl)-
2′-deoxyguanosine (9a ) (2.5 g, 8.2 mmol) in 100 mL of pyridine
was treated with 15 mL (118 mmol) of chlorotrimethylsilane.
The mixture was stirred for 15 min, and then, 5 mL (30 mmol)
of isobutyric anhydride was added. After the mixture was
stirred for 3 h, 20 mL of water and 10 mL of concentrated
ammonium hydroxide were added. The mixture was stirred
for 1 h and then concentrated. The residue was triturated with

N2- and C8-Substituted Oligodeoxynucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2241
100 mL of water, and the solid formed was collected by
filtration and washed with ethanol (2 × 20 mL) and ether (2
× 20 mL) to afford 2.18 g (71%) of a tan solid. A solution of
this solid (2.0 g, 5.33 mmol) in 50 mL of pyridine was treated
with 2.16 g (6.38 mmol) of 4,4′-dimethoxytrityl chloride. After
the mixture was stirred for 20 min, 20 mL of methanol was
added and the mixture was concentrated. The residue was
partitioned between 100 mL of ethyl acetate and 100 mL of
water. The organic layer was separated, washed with water
and brine, dried over sodium sulfate, and then concentrated.
The residue was triturated with ether, and the solid formed
was collected by filtration to give 2.73 g of 5′-O-(4,4′-dimeth-
oxytrityl)-N2-isobutyryl-8-(1-propynyl)-2′-deoxyguanosine (10a )
as a yellow amorphous solid with 63% yield. 1H NMR (DMSO-
d6): δ 11.30 and 12.04 (2bs, 2H, NH), 6.6-7.4 (m, 13H, phenyl),
6.38 (dd, 1H, 1′-H), 5.23 (d, 1H, 3′-OH), 4.44 (m, 1H, 3′-H),
3.97 (m, 1H, 4′-H), 3.65 and 3.67 (2s, 6H, OCH3), 3.39 (m,
2H, 5′-Hab), 2.23 and 3.06 (2m, 2H, 2′-Hab), 2.69 (m, 1H,
isobutyryl CH), 2.03 (s, 3H, propyne CH3), 1.07 (m, 6H,
isobutyryl CH3).
5′-O-(4,4′-Dim eth oxytr ityl)-N2-isobu tyr yl-8-(1-p h en yl-
eth yn yl)-2′-d eoxygu a n osin e (10b). 5′-O-(4,4′-Dimethoxy-
trityl)-N2-isobutyryl-8-(1-phenylethynyl)-2′-deoxyguanosine (10b)
was prepared from 8-(1-phenylethynyl)-2′-deoxyguanosine (9b)
by the method described above as a yellow amorphous solid
with 83% yield. 1H NMR (DMSO-d6): δ 11.48 and 12.14 (2bs,
2H, NH), 6.6-7.6 (m, 18H, phenyl), 6.53 (dd, 1H, 1′-H), 5.31
(d, 1H, 3′-OH), 4.49 (m, 1H, 3′-H), 4.02 (m, 1H, 4′-H), 3.68
and 3.69 (2s, 6H, OCH3), 3.06 and 3.40 (2m, 2H, 5′-Hab), 2.37
and 3.12 (2m, 2H, 2′-Hab), 2.75 (m, 1H, isobutyryl CH), 1.12
(m, 6H, isobutyryl CH3).
and 3.1 (2m, 2H, 2′-Hab), 0.8-1.2 (m, 18H, isobutyryl CH3,
isopropyl CH3).
Ack n ow led gm en t. This work was supported in part
by SBIR Grant No. 1R43 HL484311-01. G.-X.H. ex-
presses gratitude to Brian C. Froehler, Gilead Sciences
Inc., for his valuable comments and suggestions during
the preparation of the manuscript.
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3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im eth oxytr ityl)-N2-isobu tyr yl-8-(1-p r op yn yl)-2′-
d eoxygu a n osin e (11a ). A solution of 5′-O-(4,4′-dimethoxy-
trityl)-N2-isobutyryl-8-(1-propynyl)-2′-deoxyguanosine (10a ) (1
g, 1.48 mmol) in 15 mL of methylene chloride and 0.75 mL
(5.38 mmol) of triethylamine was treated with 0.75 g (3.37
mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.
The mixture was stirred for 20 min, and 1 mL of methanol
was added. The resulting mixture was concentrated. The
residue obtained was triturated with 2 mL of ethyl acetate,
and the solid formed was removed by filtration. The filtrate
was concentrated, and the residue was chromatographed on
a 2.5 cm 20 cm silica column, first eluting with 500 mL of ethyl
acetate (1% triethylamine, v/v) and then with ethyl acetate/
methanol (9:1, 1% triethylamine, v/v). The fractions contain-
ing the product were pooled and concentrated. The residue
was coevaporated with ethyl acetate (3 × 5 mL) and then
dissolved in 5 mL of ethyl acetate. This solution was dripped
into 200 mL of hexane under vigorous stirring. The resulting
precipitate was collected by filtration, washed with hexane,
and dried in vacuo to give 0.63 g of 3′-O-[(diisopropylamino)-
(cyanoethoxy)phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-isobu-
tyryl-8-(1-propynyl)-2′-deoxyguanosine (11a ) as a white amor-
phous solid with 21% yield. 1H NMR (DMSO-d6): δ 11.2 and
12.1 (2bs, 2H, NH), 6.6-7.6 (m, 13H, phenyl), 6.4 (m, 1H, 1′-
H), 4.5 (m, 1H, 3′-H), 4.1 (m, 1H, 4′-H), 3.7 (s, 6H, OCH3),
3.2-3.6 (2m, 4H, 5′-Hab, -OCH2CH2CN), 2.6 and 2.7 (2m, 3H,
isobutyryl CH, -OCH2CH2CN), 2.4 and 3.2 (2m, 2H, 2′-Hab),
2.0 (s, 3H, propyne CH3), 0.9-1.2 (m, 18H, isobutyryl CH3,
isopropyl CH3).
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3′-O-[(Diisop r op yla m in o)(cya n oeth oxy)p h osp h in o]-5′-
O-(4,4′-d im et h oxyt r it yl)-N2-isob u t yr yl-8-(1-p h en ylet h -
yn yl)-2′-d eoxygu a n osin e (11b). 3′-O-[(Diisopropylamino)-
(cyanoethoxy)phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-isobu-
tyryl-8-(1-phenylethynyl)-2′-deoxyguanosine (11b) was pre-
pared from 5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryl-8-(1-
phenylethynyl)-2′-deoxyguanosine (10b) by the method described
above as a white amorphous solid in 25% yield. 1H NMR
(DMSO-d6): δ 11.4 and 12.2 (2bs, 2H, NH), 6.6-7.6 (m, 18H,
phenyl), 6.5 (m, 1H, 1′-H), 4.5 (m, 1H, 3′-H), 4.2 (m, 1H, 4′-
H), 3.7 (s, 6H, OCH3), 3.2-3.6 (2m, 4H, 5′-Hab, -OCH2CH2-
CN), 2.6 and 2.7 (2m, 3H, isobutyryl CH, -OCH2CH2CN), 2.5
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carrying a 1-naphthylmethyl group on N2 position of G15 was
synthesized and tested (data not shown). It has a slightly
decreased activity compared to 1.
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