Nucleosides with self-complementary hydrogen-bonding motifs: Synthesis and base-pairing studies of two nucleosides containing the imidazo[4,5-d]pyridazine ring system

Article abstract of DOI:10.1016/j.bmc.2006.05.043

Synthesis and base-pairing studies of two 2′-deoxyribonucleosides, containing a common heterocyclic base, 7(4)-amino-5(6)H-imidazo[4,5-d]pyridazin-4(7)one (1 and 2), have been reported. The synthesis was accomplished by base-promoted deoxyribosylation of

Full text of DOI:10.1016/j.bmc.2006.05.043

Bioorganic & Medicinal Chemistry 14 (2006) 6359–6367
Nucleosides with self-complementary hydrogen-bonding
motifs: Synthesis and base-pairing studies of two nucleosides
containing the imidazo[4,5-d]pyridazine ring system
Ravi K. Ujjinamatada,a Robin L. Paulman,b Roger G. Ptakb and
Ramachandra S. Hosmanea,*
aLaboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
bInfectious Disease Research Department, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA
Received 31 March 2006; revised 20 May 2006; accepted 23 May 2006
Available online 9 June 2006
Abstract—Synthesis and base-pairing studies of two 20-deoxyribonucleosides, containing a common heterocyclic base, 7(4)-amino-
5(6)H-imidazo[4,5-d]pyridazin-4(7)one (1 and 2), have been reported. The synthesis was accomplished by base-promoted deoxyri-
bosylation of ethyl 5(4)-cyanoimidazole-4(5)-carboxylate (6), followed by ring-closure with hydrazine hydrate. The 1H NMR-based
base-pair studies were conducted using DMF-d7 as a solvent by measuring changes in chemical shifts of the amino, hydrazide, imid-
azole H-2, and the sugar H-10 protons of the nucleosides with variations in concentrations and temperatures. Large downfield chem-
ical shifts were observed for the NH, NH2, and to a lesser extent for the H-10 protons when the temperature was lowered from 25 to
0 ꢀC, and then further down to ꢀ50 ꢀC in 10 degree intervals. The observed experimental data are consistent with the results of
molecular modeling studies. Nucleoside 2 exhibited low level antiviral activity against HIV-1 in CEM-SS cells with an IC50 of
89.2 lM. No cellular toxicity was observed at the highest concentration of the compound tested.
ꢁ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
report by Matsuda and coworkers on the formation of
self-complementary base-pairs by the designed imidazo-
pyridopyrimidine nucleosides.29 However, the latter are
5:6:6-fused tricyclic ring systems that have little, if any,
resemblance to the natural 5:6-fused purines. In this
context, a self-pairing monocyclic or bicyclic heterocy-
clic base that closely mimics a natural pyrimidine or a
purine would be of considerable interest. We report
herein the design, synthesis, and preliminary base-pair-
ing studies of two isomeric nucleoside analogues, 1
and 2, possessing a common, self-pairing nucleobase
with a 5:6-fused heterocyclic ring system, and has the
structural resemblance of a purine. Also reported are
the results of in vitro anti-HIV screening studies that
showed that while 2 is a weak inhibitor of the viral rep-
lication, 1 is completely inactive.
There has been a surge of interest recently in unnatural
nucleic acid bases with novel base-pairing and hydro-
phobic characteristics as probes for fidelity and function
of DNA polymerases1–5 as well as for potential expan-
sion of the genetic code.6–25 Much attention is focused
on the design of unnatural Watson–Crick base-pair
partners that resemble a purine in one strand and a
pyrimidine in the other strand of a double-helix, a classic
example being the often-quoted isoG:isoC pair reported
several years ago by Benner and coworkers.26–28 By con-
trast, little effort is devoted to the design of molecules
which can effectively form self-complementary base-
pairs, substituting for both a purine and a pyrimidine,
and thus the same nucleobase occupying both strands
of the double-helix. A notable exception is a recent
2. Results and discussion
Keywords: Nucleoside analogues; Imidazo[4,5-d]pyridazine; Synthesis;
Base-pair studies using 1H NMR, anti-HIV activity.
The target nucleosides are 1 and 2, which contain the
common heterocyclic base 7(4)-amino-5(6)H-imi-
dazo[4,5-d]pyridazin-4(7)one. The nucleobase has four
*
Corresponding author. Tel.: +1 410 455 2520; fax: +1 410 455
1148; e-mail: hosmane@umbc.edu
0968-0896/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.043

6360
R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
the sodium salt glycosylation procedure,35 was quantita-
O
4
NH2
3
N
4
3
N
tive, and gave the desired isomeric nucleosides 8 (39%)
and 9 (61%), which were separated by silica gel flash
chromatography. The identities of the two isomers were
established by carrying through the synthesis to the tar-
get nucleosides 1 and 2, which could be easily distin-
guished by 1H NMR NOE studies (see below). The
toluoyl protecting groups of the sugar moieties of 8
and 9 were removed by treatment with tert-butylamine
in methanol. This reaction step also resulted in a com-
plete exchange of the ethyl ester group for methyl, form-
ing 10 (93%) or 11 (95%), respectively. The latter, upon
reaction with hydrazine hydrate in methanol at ice-cold
temperature, provided the intermediate 12 or 13, which,
without isolation and purification, was treated with
sodium ethoxide in ethanol at reflux to obtain the target
nucleoside 1 (76%) or 2 (81%), respectively. While the
1H and 13C NMR, and mass spectral data, as well as
elemental microanalyses, were consistent with their as-
signed structures, yet they did not provide a clear dis-
tinction between the two isomeric nucleosides 1 and 2.
The two structures were unequivocally assigned using
Nuclear Overhauser Effect Spectroscopy (NOESY).
The proximity of the exocyclic NH2 protons with the
anomeric H-10 proton was detected in 1 but not in 2,
thus providing a conclusive evidence for their structural
identities.
5
5
NH
N
2
2
6NH
1
N
1
N
6
N
7
5'
5'
7
O
O
HO
HO
1'
1'
NH2
O
4'
4'
2'
2'
3'
3'
HO
HO
2
1
possible sites for base-pairing, including the two H-bond
donor sites (NH2 and NH), and two H-bond acceptor
sites (@N and @O).
The two nucleosides are each capable of base-pairing
with the self or with each other. Our molecular modeling
studies (see Fig. 1) reveal that only three of the four pos-
sible sites in each can effectively participate in the Wat-
son–Crick base-pairing at any one moment, be it with
the self or with each other. Furthermore, base-pairing
with the self, for example, 1:1 (Fig. 1A) or 2:2
(Fig. 1B), would transpose the two respective backbone
sugars into a rare motif befitting a parallel stranded dou-
ble-helix.30 The intermolecular base-pairing between 1
and 2 (Fig. 1C), on the other hand, results in an anti-
parallel strand array, analogous to that of a natural B-
DNA duplex.30 The side-views of the complexes between
1:1 (Fig. 1A) and 1:2 (Fig. 1C) show near-perfect, flat,
coplanar structures containing the two base-paired
nucleobases, while the homo complex 2:2 (Fig. 1B) re-
veals a slight bend in the center. The two sugar residues
on each end of the complexes protrude themselves in
either a parallel (Fig. 1A and B) or anti-parallel
(Fig. 1C) orientation, depending upon whether the
base-pairing is between 1:1, 2:2 or 1:2, respectively.
Base-pair studies of 1, 2, and 1 + 2 were conducted
400 MHz 1H NMR spectrometer with
using
a
1
DMF-d7 as a solvent. H NMR spectroscopy is ideally
suited for monitoring base-pair hydrogen-bonding
between nucleosides36–38 as well as for differentiation
between base-pairing and stacking interactions.37,39
In most cases, upfield chemical shifts of ring protons
are observed upon vertical stacking of bases, whereas
base-pairing leads to downfield shifts of the participat-
ing protons.39 The change in chemical shifts of the
exocyclic amino and the endocyclic imino protons
would provide the evidence for base-pair interactions,
but it is necessary to ascertain that the observed shifts
are not due to interactions of nucleosides with the sol-
vent or due to self-association stacking interactions
between bases. Therefore, the chemical shifts of both
1 and 2 were individually monitored in dilute solu-
tions at various concentrations ranging from 0.01 to
0.08 M and at two different temperatures, 25 and
0 ꢀC. The results (see Tables I–IV in Supplementary
material) show that there is little, if any, concentra-
tion-dependent stacking self-association in these dilute
solutions as monitored by the chemical shifts of the
NH, NH2, imidazole H-2, or anomeric H-10 signals,
all of which were shifted slightly downfield rather than
upfield. While there was only a small gradual change
(0.3–7.7 Hz) in concentration-dependent base-pair
interactions at both 25 and 0 ꢀC in going from 0.01
to 0.08 M, a vast temperature-dependent downfield
shift was observed for each of the NH and NH2 sig-
nals (and to a lesser extent, for the H-2 signal) in
going from 25 to 0 ꢀC. For example, while the ob-
served maximum differences in chemical shifts for 1
at 25 and 0 ꢀC in going from 0.01 to 0.08 M are
3.5 Hz (NH) and 7.7 Hz (NH), respectively, a large
Several years ago, Townsend and coworkers reported an
elegant synthesis of nucleoside 3, the ribose analogue of
2, in several steps commencing from the imidazole deriv-
ative 4.31 A drawback of the synthesis, however, is the
poor yield (17%) in the final base-catalyzed transforma-
tion of the oxadiazole ring of 4 into the target imi-
dazo[4,5-d]pyridazine ring system of 3. We have
devised an alternative, more efficient synthesis to access
not only its deoxy analogue 2, but also the latter’s regio-
isomer 1, both of which are required for our studies. We
used classical chemical methods of glycosylation of a
suitably substituted imidazole derivative to synthesize
both 1 and 2.
The synthesis of 1 and 2 starts from the commercially
available 4,5-dicyanoimidazole (5) (Scheme 1), which
was converted to the corresponding cyano-ester (6) by
ethanolysis with concentrated sulfuric acid.32 Deoxyri-
bosylation of 6 with the a-chlorosugar 7,33,34 employing
NH2
CH3
N
N
N
N
O
N
NH
N
O
HO
O
N
H
C
N
OH
HO
3
4

R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
6361
(front View)
A
B
(front View)
(Side View)
(Side View)
(front View)
C
(Side View)
Figure 1. Self-complementary base-pair arrays of nucleosides 1 and 2: (A) between 1 and 1, (B) between 2 and 2, and (C) between 1 and 2.
difference in chemical shifts was observed for each of
the NH, NH2, and H-2 signals of 1 in going from 25
to 0 ꢀC. These values range from 48 to 52 Hz (NH),
36 to 39 Hz (NH2), and 15 to 17 Hz (H-2). Interest-
small, although somewhat higher than those for 1, with
the largest values of 9.6 Hz (NH) and 12.7 Hz (NH) at
25 and 0 ꢀC, respectively. As the observed shifts are
all downfield, they can once again be attributed to
intermolecular base-pairing rather than vertical stack-
ing interactions. As with 1, a dramatic difference in
chemical shifts was observed for each of the NH,
NH2, and H-2 signals in going from 25 to 0 ꢀC. These
values range from 46 to 49 Hz (NH), 62 to 65 Hz
(NH2), and 12 to 13 Hz (H-2). The concentration-
ingly, there was
a
slight upfield shift (ꢀ1.2 to
ꢀ1.4 Hz) of H-10 signals when the temperature was
lowered from 25 to 0 ꢀC. This is not too surprising
in view of the fact that the lower temperature would
result in a restricted rotation of the NH2 protons at
position-7 of 1, causing steric constraints in the vicin-
ity of H-10. In corroboration of this notion is the ab-
sence of such upfield shifts in the H-10 signal of 2
where the NH2 group is directed away from H-10.
1
dependent H NMR studies were then conducted using
equimolar mixtures of 1 and 2 (see Tables V and VI in
the Supplementary material). A graph of chemical
shifts in Hz versus concentration in M for the NH
and NH2 signals of 1 and 2 in a mixture of 1 + 2 at
both 25 and 0 ꢀC are shown in Figures 2A and B. As
The concentration-dependent differences in chemical
shifts for nucleoside 2 in 0.01–0.08 M range are also

6362
R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
O
p-TolO
O
Cl
NC
N
EtO
N
(7)
p-TolO
EtOH/H2SO4
Reflux/48 h
N
H
N
H
NC
NaH/CH3CN
RT/2h
NC
6
5
O
CN
N
N
N
OEt
OEt
N
CN
O
O
p-TolO
p-TolO
O
p-TolO
p-TolO
9 (61%)
8 (39%)
1). Chromatographic Separation
2). t-Butylamine/MeOH/RT/48 h
93%
O
95%
CN
O
N
N
N
OMe
OMe
N
O
HO
CN
O
HO
HO
HO
11
10
NH2NH2.H2O
MeOH/5OC/30 min
NH2NH2.H2O
MeOH/5 OC/30 min
O
NH2
CN
N
N
N
NH
NH
N
NH2
CN
O
O
HO
HO
O
HO
HO
12
13
NaOEt/EtOH
Reflux/8 h
NaOEt/EtOH
Reflux/8 h
O
NH2
N
N
NH
N
N
NH
N
N
O
O
HO
HO
NH2
O
HO
HO
(76%)
(81%)
2
1
Scheme 1.
is evident from the graphs, there is a considerable
change in concentration-dependent chemical shifts of
the NH protons (see Fig. 2A) in the region 0.001–
0.08 M (DHz for 1 = 28.8 and 26.5 Hz; DHz for
2 = 28.8 and 36.2 Hz at 25 and 0 ꢀC, respectively).
While the two NH signals of 1 and 2 are coalesced
at 25 ꢀC at all concentrations, they appear as two
distinct lines at 0 ꢀC. However, as was the case with
the individual components of the mixture, there is a
sharp downfield jump in chemical shifts of NH
at each concentration in going from 25 to 0 ꢀC
(DHz for 1 = 44–48 Hz; DHz for 2 = 52–59.6 Hz). The

R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
6363
1
Variable temperature H NMR studies were then con-
ducted using 0.08 M concentration for each of 1, 2,
and 1 + 2 at 10 degree intervals in the range from 0 to
ꢀ50 ꢀC (see Figs. 3–5) by monitoring the change in
chemical shifts of the same NH, NH2, imidazole H-2,
and anomeric H-10 signals as before. The results (see Ta-
bles VII–IX in Supplementary material) reveal dramatic
downfield shifts for the NH, NH2, and H-2 protons,
ranging by as much as 114 and 90 Hz, respectively, for
the NH and NH2 of 1, 103 and 157 Hz, respectively,
for the NH and NH2 of 2, 103 and 89 Hz, respectively,
for the NH and NH2 of 1 in 1 + 2, and 115 and
156 Hz, respectively, for the NH and NH2 of 2 in
1 + 2. Obviously, there is a sudden and sharp downfield
jump in chemical shifts of both NH and NH2 protons in
going from 25 to 0 ꢀC, which then steadily falls further
downfield as the temperature is decreased. Also, while
the NH signals of 1 and 2 are coalesced at 25 ꢀC, they
begin to separate when the temperature is gradually low-
ered. Apparently, there is a fast exchange of the two NH
protons among the four ring N atoms present in the six-
membered ring of 1 and 2 at 25 ꢀC. The two NH2 sig-
nals, by contrast, are separated even at 25 ꢀC, and the
temperature-dependent change in chemical shifts of
NH2 is more pronounced in 2 than in 1, possibly
because of the steric constraints imposed by the sugar
H-10 of 1 in base-pairing, as described earlier.
A
NH of compd. 2 at 0 ºC
4715
4695
4675
4655
4635
NH of compd. 1 at 0 ºC
Hz
NH of compd. 1 or 2 at 25 ºC
(signals coalesce)
0.001 0.002 0.02
0.04
0.06
0.08
Conc. (M)
B
2410
2360
2310
2260
2210
NH2 of compd. at 0 ˚C
2
NH2 of compd. at 25 ˚C
2
Hz
compd.
NH2 of
at 0 ˚C
1
NH2 of compd. at 25 ˚C
1
0.001 0.002
0.02
0.04
0.06
0.08
Conc. (M)
A
Figure 2. Concentration-dependent 1H NMR studies of a mixture of
compounds 1 and 2 at 25 and 0 ꢀC: (A) NH signals of 1 and 2, and (B)
NH2 signals of 1 and 2 in DMF-d7.
4790
NH of compd. 1
4740
Hz
concentration-dependent chemical shifts of the NH2
protons (see Fig. 2B), by contrast, are considerably less
pronounced than those of NH protons in the region
0.002–0.06 M (DHz for 1 = 11.8 and 12.4 Hz; DHz for
2 = 11.3 and 11.8 Hz at 25 and 0 ꢀC, respectively). Fur-
thermore, the NH2 signals of 1 and 2 have two distinct-
ly separate absorptions even at 25 ꢀC, the signal of 2
appearing farther downfield than that of 1. The most
conspicuous feature of the NH2 absorptions is the
sharp downfield jump in chemical shifts at each con-
centration in going from 25 to 0 ꢀC (DHz for 1 = 39–
41 Hz; DHz for 2 = 64–66 Hz). The concentration-de-
pendent chemical shifts of the H-2 protons of 1 and
2 in a mixture of 1 + 2 are rather small in the range
0.001–0.08 M (DHz = 9–10 Hz at both 25 and 0 ꢀC).
The changes in chemical shifts of the same signals at
each concentration in going from 25 to 0 ꢀC, while
noticeable (DHz for 1 = 16–17 Hz; DHz for 2 = 12–
14 Hz), are still considerably smaller than those for
NH and NH2 protons. Likewise, the concentration-de-
pendent chemical shifts of the H-10 signals of 1 and 2
in the range 0.001–0.08 M at both 25 and 0 ꢀC are also
small (DHz ꢁ 4 Hz). Once again, as was the case with
studies of individual nucleosides described earlier, there
was a slight upfield shift of the H-10 signals of 1
(DHz = ꢀ0.9 to ꢀ2.5 Hz) and a slight downfield shift
of the same signal in 2 (DHz = 1.9–3.2 Hz) in going
from 25 to 0 ꢀC.
4690
4640
25
0
-10
-20
˚C
-30
-40
-50
B
2350
2300
2250
2200
NH of compd. 1
2
Hz
25
0
-10
-20
˚C
-30
-40
-50
Figure 3. Temperature-dependent 1H NMR chemical shifts of (A) NH
and (B) NH2 protons of nucleoside 1 in DMF-d7.

6364
R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
A
A
4850
4795
4745
4695
4645
NH of compd. 2
4800
NH of compd. 2
Hz
Hz
4750
4700
4650
NH of compd. 1
25
0
-10
-20
˚C
-30
-40
-50
25
0
-10
-20
˚C
-30
-40
-50
2570
2520
2470
2420
2370
2320
B
2575
2525
2475
2425
2375
2325
2275
2225
B
NH of compd. 2
2
NH of compd. 2
2
Hz
Hz
NH of compd. 1
2
25
0
-10
-20
˚C
-30
-40
-50
25
0
-10
-20
˚C
-30
-40
-50
Figure 4. Temperature-dependent 1H NMR chemical shifts of (A) NH
and (B) NH2 protons of nucleoside 2 in DMF-d7.
Figure 5. Temperature-dependent 1H NMR chemical shifts of (A) NH
and (B) NH2 protons of nucleosides 1 and 2 in an equimolar mixture of
1 + 2 in DMF-d7.
The temperature-dependent downfield chemical shifts of
the H-2 signal of 1 and 2 in the range from 0 to ꢀ50 ꢀC
in the above experiments are also significant as revealed
by the values of 40 Hz for 1, 29 Hz for 2, 39 Hz for 1 in
1 + 2, and 30 Hz for 2 in 1 + 2. Once again, a conspicu-
ous observation concerned the H-10 signal of 1 both in
an isolated run and in a 1 + 2 mixture, which showed
an upfield rather than a downfield shift.
1
respectively. The H NMR-based base-pair studies are
in agreement with the results of molecular modeling.
Compound 2 is a weak inhibitor of HIV-1 in vitro, while
1 is inactive.
4. Experimental
Finally, as mentioned under Section 1, nucleoside 2
exhibited a weak antiviral activity against HIV-1 in
CEM-SS cells with a 50% inhibitory concentration
(IC50) of 89.2 lM and a 50% cytotoxic concentra-
tion (TC50) of >200 lM, the highest concentration
tested.
4.1. Base-pair studies using H NMR spectroscopy
All samples were directly prepared in 5 mL NMR
tubes using 0.5 mL DMF-d7 in concentrations rang-
ing 0.001–0.08 M. Variable temperature (25 to
ꢀ50 ꢀC) 1H NMR experiments were conducted with
a 400 MHz NMR spectrometer (JEOL JNM-ECX)
following the manufacturer’s procedures and instruc-
tions. Briefly, the procedure involves generation of a
constant flow of nitrogen gas by heating liquid
nitrogen in the metal Dewar. The gas is supplied to
the surroundings of the sample via the probe heater.
The desired temperature can be adjusted by heating
the nitrogen gas with the probe heater and
controlling its power. Tetramethylsilane (TMS) was
used as an internal reference standard for NMR
measurements.
3. Conclusion
We have designed and successfully synthesized two
isomeric nucleosides 1 and 2, containing a common
heterocyclic base, 7(4)-amino-5(6)H-imidazo[4,5-d]pyr-
idazine-4(7)one, in three steps commencing from ethyl
5(4)imidazole-4(5)carboxylate (6). Our molecular mod-
eling studies suggest that 1 and 2 can form stable
base-pair complexes with the self (homo) or with each
other (hetero), in a parallel or anti-parallel orientation,

R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
6365
4.2. Ethyl 5-cyano-1-[(20-deoxy-30,50-di-O-p-toluoyl)-b-D-
erythropento-furanosyl]imidazole-4-carboxylate (8) and
ethyl 4-cyano-1-[(20-deoxy-30,50-di-O-p-toluoyl)-b-D-ery-
thropento-furanosyl]imidazole-5-carboxylate (9)
roform/methanol, 10:1) showed the completion of reac-
tion. Deprotected methyl ester 10 was isolated by flash
silica gel chromatography, using 15:1 chloroform/meth-
anol solvent system. Yield 1.56 g, 93%; mp 145 ꢀC; Rf
0.29 (chloroform/methanol, 10:1); IR 3354, 3110, 2238,
To a solution of 632 (3.30 g, 0.02 mol) in anhydrous ace-
tonitrile (175 mL), sodium hydride (60% in oil, 2 g,
50 mmol) was added and the mixture was stirred at rt
under nitrogen atmosphere for 30 min. 2-Deoxy-3,
5-di-O-p-toluoyl-a-D-erythropento-furanosyl chloride
(7)33,34 (7.76 g, 0.02 mol) was added portionwise over a
period of 1 h. After the addition was complete, the reac-
tion mixture was allowed to stir at rt for 2 h. It was then
filtered and evaporated under reduced pressure. The res-
idue was purified by flash chromatography on a silica gel
column using a mixture of 4:1 hexanes/ethyl acetate as
an eluant for isolation of 8 and 9. The spectral and
analytical data for 8 and 9 are as follows:
1725, 1551, 1439, 1240 cmꢀ1 1H NMR (DMSO-d6,
;
400 MHz), d 8.41 (s, 1H, H-2), 6.16 (t, J = 5.96 Hz,
1H, 10H), 5.41–5.40 (d, J = 4.6 Hz, 1H, OH), 4.94 (t,
J = 5.64 Hz, 1H, OH), 4.33–4.31 (m, 1H, CH), 3.86–
3.84 (d, 1H, CH), 3.84 (s, 3H, CH3), 3.56–3.45 (m, 1H,
20-H), 2.61–2.36 (m, 2H, CH2); 13C NMR (DMSO-d6,
100 MHz) d 160.9, 141.0, 140.3, 110.5, 107.6, 89.1,
87.1, 70.5, 60.4, 52.6, 40.5. Mass (FAB) m/z 268.20
(MH+). Anal. Calcd for C11H13N3O5: C, 49.44; H,
4.90; N, 15.72. Found: C, 49.42; H, 4.96; N, 15.46.
4.6. Methyl 4-cyano-1-[(20-deoxy)-b-D-erythropento-
furanosyl]imidazole-5-carboxylate (11)
4.3. Compound 8
Compound 9 (5.17 g, 0.01 mol) was placed in anhydrous
methanol (50 mL) and tert-butylamine (3.65 g, 0.05 mol)
was added, and the reaction mixture was stirred at room
temperature for 48 h, when a TLC (chloroform/metha-
nol, 10:1) showed the completion of reaction. Deprotec-
ted methyl ester 11 was isolated by silica gel flash
column chromatography, using 20:1 CHCl3–MeOH sol-
vent system. Yield 2.46 g, 93%; mp 131 ꢀC; Rf 0.38
(chloroform/methanol, 10:1); IR 3361, 3109, 2241,
Yield 3.71 g, 39%; mp 69 ꢀC; Rf 0.11 (2:1 hexane/ethyl
acetate); IR 2235, 1716, 1612, 1268, 1178, 1096, 1019,
1
752 cmꢀ1; H NMR (CDCl3, 300 MHz), d 7.94 (s, 1H,
H-2), 7.92 (d, J = 3.6 Hz, 2H, ArH), 7.82 (d,
J = 7.82 Hz, 2H, ArH), 7.24 (dd, J = 8.1 Hz, 4H,
ArH), 6.29 (dd, J = 6 + 3 Hz, 1H, 10H), 5.69–5.67 (m,
1H, 30H), 4.76–4.63 (m, 3H, 40, 50 and 500-H), 4.43 (q,
J = 6.9 Hz, 2H, CH2 ), 2.97–2.91 (m, H, 300H), 2.72–
2.62 (m, 2H, 20,200H), 2.43 (s, 3H, Ar-CH3), 2.39 (s,
3H, Ar-CH3), 1.40 (t, J = 6.9 Hz, 3H, CH3); 13C NMR
(CDCl3, 75 MHz) d 165.4, 165.2, 159.4, 144.2, 143.8,
137.2, 129.2, 129.0, 128.8, 128.7, 125.8, 125.4, 108.9,
106.4, 86.2, 83.4, 73.9, 63.0, 61.3, 39.3, 21.2, 21.1. Mass
(FAB) m/z 518.20 (MH+). Anal. Calcd for C28H27N3O7:
C, 64.98; H, 5.26; N, 8.12. Found: C, 64.82; H, 5.28; N,
8.08.
1723, 1541, 1437, 1207 cmꢀ1 1H NMR (DMSO-d6,
;
400 MHz), d 8.55 (s, 1H, H-2), 6.50 (t, J = 5.52 Hz,
1H, 10H), 5.32–5.31 (d, J = 4.56 Hz, 1H, OH), 5.12 (t,
J = 5.04 Hz, 1H, OH), 4.30–4.25 (m, 1H, CH), 3.88 (s,
3H, CH3), 3.86–3.84 (d, 1H, CH), 3.68–3.55 (m, 1H,
20-H), 2.44–2.27 (m, 2H, CH2); 13C NMR (DMSO-d6,
100 MHz) d 158.2, 141.2, 140.9, 114.7, 88.5, 88.1, 60.7,
53.0, 40.4. Mass (FAB) m/z 268.20 (MH+). Anal. Calcd
for C11H13N3O5: C, 49.44; H, 4.90; N, 15.72. Found: C,
49.07; H, 4.92; N, 15.34.
4.4. Compound 9
4.7. 7-Amino-1-[(20-deoxy)-b-D-erythropento-furanosyl]-
1H-imidazol[4,5-d]pyridazin-4(5H)-one (1) and 4-amino-
1-[(20-deoxy)-b-D-erythropento-furanosyl]-3H-imi-
dazol[4,5-d]pyridazin-7(6H)-one (2)
Yield 5.92 g, 61%; mp 128 ꢀC; Rf 0.41 (2:1 hexane/ethyl
acetate); IR 2241, 1720, 1611, 1265, 1203, 1176, 1086,
1018, 756 cmꢀ1; H NMR (CDCl3, 300 MHz), d 8.11
1
(s, 1H, H-2), 7.93 (d, J = 8.1 Hz, 2H, ArH), 7.80 (d,
J = 7.8 Hz, 2H, ArH), 7.23 (dd, J = 7.8 Hz, 4H, ArH),
6.72 (t, J = 6.6 Hz, 1H, 10H), 5.60–5.58 (m, 1H, 30H),
4.71–4.67 (m, 3H, 40, 50 and 500H), 4.42 (q, J = 7.2 Hz,
2H, CH2), 3.08–3.05 (m, 2H, 20,200H), 2.42 (s, 3H, Ar-
CH3), 2.39 (s, 3H, Ar-CH3), 1.43 (t, J = 7.5 Hz, 3H,
CH3); 13C NMR (CDCl3, 75 MHz) d 165.5, 165.3,
157.3, 144.0, 143.9, 138.2, 129.2, 129.0, 128.8, 128.7,
126.7, 125.7, 125.6, 120.6, 112.81, 87.92, 83.3, 73.8,
63.1, 61.8, 40.4, 21.2, 21.1. Mass (FAB) m/z 518.20
(MH+). Anal. Calcd for C28H27N3O7: C, 64.98; H,
5.26; N, 8.12. Found: C, 64.95; H, 5.39; N, 8.11.
To a solution of 10 or 11 (0.534 g, 0.002 mol) in anhy-
drous methanol (15 mL), hydrazine hydrate (2 mL)
was added. This reaction mixture was stirred at ice-cold
temperature for 30 min. Then methanol and unreacted
hydrazine hydrate were removed under vacuum. The
residue was refluxed in ethanolic solution of sodium eth-
oxide (2 mL, 0.35 mmol). After 8 h reflux, the reaction
mixture was brought to room temperature and the
separated solid was filtered and washed with ethanol.
4.8. Compound 1
4.5. Methyl 5-cyano-1-[(20-deoxy)-b-D-erythropento-
furanosyl]imidazole-4-carboxylate (10)
Recrystallized from ethanol. Yield 0.43 g, 76%;
mp > 300 ꢀC; Rf 0.20 (chloroform/methanol/ammonium
hydroxide, 2:1:0.25); IR 3432, 3339, 3216, 3106, 1657,
1
Compound 8 (3.23 g, 0.0063 mol) was placed in dry
methanol (35 mL) and tert-butylamine (2.28 g,
0.0312 mol) was added, and the reaction mixture was
stirred at room temperature for 48 h, when a TLC (chlo-
1628, 1594, 1566, 1461, 1221 cmꢀ1; H NMR (DMSO-
d6, 400 MHz) d 11.61 (s, 1H, CONH), 8.48 (s, 1H,
H-2), 6.43 (t, J = 5.96 Hz, 1H, 10-H), 5.49 (s, 2H,
NH2), 5.35 (s, 1H, OH), 5.00 (s, 1H, OH), 4.33–4.35

6366
R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
(m, 1H, 40-H), 3.90–3.89 (m, 1H, 30-H), 3.56–3.44 (m,
1H, 50,500H), 2.65–2.38 (m, 2H, 20,200H); 13C NMR
(DMSO-d6, 100 MHz) d 157.5, 142.0, 140.3, 138.3,
126.7, 88.6, 85.8, 70.2, 61.2, 40.8. Mass (FAB) m/z
268.20 (MH+). Anal. Calcd for C10H13N5O4: C, 44.94;
H, 4.90; N, 26.21. Found: C, 44.70; H, 4.94; N, 26.11.
85% and 95% cell killing at 6 days post-infection.
TCID50 calculations by endpoint titration in CEM-SS
cells indicated that the multiplicity of infection of these
assays was approximately 0.01.
4.11.3. Assessment of IC50 and TC50 values. Samples
were evaluated for antiviral efficacy with triplicate mea-
surements using 6 concentrations at half-log dilutions in
order to determine the IC50 values and with duplicate
measurements to determine cellular cytotoxicity
(TC50). At assay termination, the assay plates were
stained with the soluble tetrazolium-based dye MTS
(CellTiter 96 Reagent, Promega) to determine cell viabil-
ity and quantify compound toxicity. At termination of
the assay, 20–25 lL MTS reagent was added per well,
and the microtiter plates were then incubated for 4–6 h
at 37 ꢀC, 5% CO2 to assess cell viability. The plate was
read spectrophotometrically at 490/650 nm with a
Molecular Devices Vmax plate reader. Using an in-
house computer program, % CPE reduction, % cell
4.9. Compound 2
Recrystallized from methanol. Yield 0.46 g, 81%;
mp > 300 ꢀC; Rf 0.44 (chloroform/methanol/ammonium
hydroxide, 2:1:0.25); IR, 3415, 3308, 3332, 3158, 1662,
1636, 1584, 1469, 1200 cmꢀ1 1H NMR (DMSO-d6,
;
400 MHz) d 11.55 (s, 1H, CONH), 8.57 (s, 1H, H-2),
6.78–6.75 (t, J = 6.4 Hz, 1H, 10H), 5.78 (s, 2H, NH2),
5.29 (s, 1H, OH), 5.02 (s, 1H, OH), 4.31–4.32 (s, 1H,
40H), 3.85–3.84 (m, 1H, 30H), 3.60–3.51 (m, 1H,
50,500H), 2.46–2.31 (m, 2H, 20,200H); 13C NMR
(DMSO-d6, 100 MHz) d 154.7, 145.5, 142.2, 135.9,
125.1, 88.6, 85.9, 70.7, 61.7, 41.7. Mass (FAB) m/z
268.20 (MH+). Anal. Calcd for C10H13N5O4: C, 44.94;
H, 4.90; N, 26.21, Found, C, 44.79; H, 4.93; N, 26.21.
viability, and IC50 and TC
values were calculated.
50
AZT was evaluated in parallel as a positive control
in the anti-HIV assay. The computed values for 2 are:
IC50 = 89.2 lM and TC50 > 200 lM, while those for
AZT are: IC50 = 15.6 nM and TC50 > 500 nM.
4.10. Molecular modeling studies
Molecular modeling studies were performed on a PS/2
workstation, employing the Biosym/InsightII software
(Accelrys, San Diego), coupled with Discover algorithm
for energy minimization. Nucleosides 1 and 2 were sep-
arately built and energy-minimized before association
into a complex and further energy minimization. The
complex was minimized to convergence using the steep-
est descent and conjugate gradient forcefield protocols
of the software program.
Acknowledgments
This paper is dedicated to Professor Nelson J. Leonard
on the occasion of his 90th birthday. The research was
supported by a Grant (#9 RO1 AI55452) from the
National Institute of Allergy and Infectious Diseases
(NIAID) of the National Institutes of Health (NIH).
The antiviral screenings were performed with funds
from NIAID, NIH, Department of Health and Human
Services under Contract No. NO1-AI-25478. We are
highly obliged to Dr. Steven R. Turk, Program Officer,
NIAID, Division of AIDS, for making arrangements
for the anti-HIV screening studies through NIAID
contract.
4.11. Antiviral screening
Antiviral assays were performed at Southern Research
Institute (SRI), Frederick, Maryland.
4.11.1. Cell preparation. CEM-SS cells were passaged in
T-75 flasks prior to use in the antiviral assay. On the day
preceding the assay, the cells were split 1:2 to assure they
were in an exponential growth phase at the time of infec-
tion. Total cell and viability quantification was per-
formed using a hemacytometer and Trypan blue
exclusion. Cell viability was greater than 95% for the
cells to be utilized in the assay. The cells were resuspend-
ed at 5 · 104 cells/mL in tissue culture medium and
added to the drug-containing microtiter plates in a
volume of 50 lL.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.05.043. Description of the general experi-
mental procedure and Tables I–IX listing concentration
1
and temperature-dependent H NMR chemical shifts of
target nucleosides 1 and 2, either alone or as a mixture
(seven pages), can be found in the supplementary data
associated with the online version of this journal.
4.11.2. Virus preparation. The lymphocytropic virus
strain HIV-1RF was obtained from the NIH AIDS Re-
search and Reference Reagent Program, and was
grown in CEM-SS cells for the production of stock
virus pools. For each assay, a pre-titered aliquot of
virus was removed from the freezer (ꢀ80 ꢀC) and
allowed to thaw slowly to room temperature in a bio-
logical safety cabinet. The virus was resuspended and
diluted into tissue culture medium such that the
amount of virus added to each well in a volume of
50 lL was the amount determined to give between
References and notes
1. Henry, A. A.; Yu, C.; Romesberg, F. E. J. Am. Chem. Soc.
2003, 125, 9638–9646.
2. Kool, E. T. Annu. Rev. Biochem. 2002, 71, 191–219.
3. Matsuda, S.; Henry, A. A.; Schultz, P. G.; Romesberg, F.
E. J. Am. Chem. Soc. 2003, 125, 6134–6139.

R. K. Ujjinamatada et al. / Bioorg. Med. Chem. 14 (2006) 6359–6367
6367
4. Paul, N.; Nashine, V. C.; Hoops, G.; Zhang, P.; Zhou, J.;
Bergstrom, D. E.; Davisson, V. J. Chem. Biol. 2003, 10,
815–825.
5. Adelfinskaya, O.; Nashine, V. C.; Bergstrom, D. E.;
Davisson, V. J. J. Am. Chem. Soc. 2005, 127, 16000–16001.
6. Romesberg, F. E. Polymer Prep. 2005, 46, 137–138.
7. Henry, A. A.; Olsen, A. G.; Matsuda, S.; Yu, C.;
Geierstanger, B. H.; Romesberg, F. E. J. Am. Chem.
Soc. 2004, 126, 6923–6931.
23. Tor, Y.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115,
4461–4467.
24. Roberts, C.; Bandaru, R.; Switzer, C. J. Am. Chem. Soc.
1997, 119, 4640–4649.
25. Piccirilli, J. A.; Moroney, S. E.; Benner, S. A. Biochemistry
1991, 30, 10350–10356.
26. Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem.
Soc. 1989, 111, 8322–8323.
27. Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Biochemistry
1993, 32, 10489–10496.
28. Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A.
Nature 1990, 343, 33–37.
29. Minakawa, N.; Kojima, N.; Hikishima, S.; Sasaki, T.;
Kiyosue, A.; Atsumi, N.; Ueno, Y.; Matsuda, A. J. Am.
Chem. Soc. 2003, 125, 9970–9982.
30. Seela, F.; Peng, X.; Li, H. J. Am. Chem. Soc. 2005, 127,
7739–7751.
8. Henry, A. A.; Romesberg, F. E. Curr. Opin. Chem. Biol.
2003, 7, 727–733.
9. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F.
E. J. Am. Chem. Soc. 2001, 123, 7439–7440.
10. Berger, M.; Ogawa, A. K.; McMinn, D. L.; Wu, Y.;
Schultz, P. G.; Romesberg, F. E. Angew. Chem., Int. Ed.
Engl. 2000, 39, 2940–2942.
11. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P.
G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122,
3274–3287.
12. McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P.
G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121,
11585–11586.
13. Kim, T. W.; Kool, E. T. J. Org. Chem. 2005, 70, 2048–2053.
14. Kool, E. T. Acc. Chem. Res. 2002, 35, 936–943.
15. Lee, A. H.; Kool, E. T. J. Am. Chem. Soc. 2005, 127,
3332–3338.
31. Berry, D. A.; Wotring, L. L.; Drach, J. C.; Townsend, L.
B. Nucleosides Nucleotides 1994, 13, 2001–2011.
32. Subrayan, R. P.; Thurber, E. L.; Rasmussen, P. G.
Tetrahedron 1994, 50, 2641–2656.
33. Bhat, C. C. 2-Deoxy-3,5-di-O-p-toluoyl-D-erythropento-
syl chloride. Preparation of a crystalline O-acyl-2-
deoxypentofuranosyl halide. In Synthetic Procedures in
Nucleic Acid Chemistry; John Wiley and sons: New
York, 1968; pp 521–522.
16. Lee, A. H.; Kool, E. T. J. Org. Chem. 2005, 70, 132–140.
17. Liu, H.; Gao, J.; Lynch, S. R.; Saito, Y. D.; Maynard, L.;
Kool, E. T. Science 2003, 302, 868–871.
18. Liu, H.; Gao, J.; Maynard, L.; Saito, Y. D.; Kool, E. T.
J. Am. Chem. Soc. 2004, 126, 1102–1109.
19. Liu,H.;Gao,J.;Kool,E.T.J.Org.Chem.2005,70,639–647.
20. Liu, H.; Gao, J.; Kool, E. T. J. Am. Chem. Soc. 2005, 127,
1396–1402.
34. Zhang, N.; Chen, H.-M.; Koch, V.; Schmitz, H.; Minczuk,
M.; Stepien, P.; Fattom, A. I.; Naso, R. B.; Kalicharran,
K.; Borowski, P.; Hosmane, R. S. J. Med. Chem. 2003, 46,
4776–4789.
35. Revankar, G. R.; Robins, R. K. Nucleosides Nucleotides
1989, 8, 709–724.
36. Shoup, R. R.; Miles, H. T.; Becker, E. D. Biochem.
Biophys. Res. Commun. 1966, 23, 194–201.
21. Mitsui, T.; Kimoto, M.; Harada, Y.; Yokoyama,
S.; Hirao, I. J. Am. Chem. Soc. 2005, 127, 8652–
8658.
22. O’Neill, B. M.; Ratto, J. E.; Good, K. L.; Tahmassebi, D.
C.; Helquist, S. A.; Morales, J. C.; Kool, E. T. J. Org.
Chem. 2002, 67, 5869–5875.
37. Mumper, M. W.; Aurenge, C.; Hosmane, R. S. J. Biomol.
Struct. Dyn. 1994, 11, 1107–1131.
38. Janke, E. M. B.; Dunger, A.; Limbach, H.-H.; Weisz, K.
Magn. Reson. Chem. 2001, 39, S177–S182.
39. Raszka, M.; Kaplan, N. O. Proc. Natl. Acad. Sci. U.S.A.
1972, 69, 2025–2029.