"Molecular chameleons". Design and synthesis of a second series of flexible nucleosides

Article abstract of DOI:10.1021/jo048218h

(Chemical Equation Presented) The second series of flexible shape-modified nucleosides is introduced. The "fleximers" feature the purine ring systems split into their individual imidazole and pyrimidine components. This structural modification serves to introduce flexibility to the nucleoside while still retaining the elements essential for recognition. As a consequence, these structurally innovative nucleosides can more readily adapt to their environment and should find use as bioprobes for investigating enzyme-coenzyme binding sites as well as nucleic acid and protein interactions. Their design and synthesis is described.

Full text of DOI:10.1021/jo048218h

“Molecular Chameleons”. Design and Synthesis of a Second Series
of Flexible Nucleosides
Katherine L. Seley,* Samer Salim, Liang Zhang, and Peter I. O’Daniel
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County,
Baltimore, Maryland 21250
kseley@umbc.edu
Received October 8, 2004
The second series of flexible shape-modified nucleosides is introduced. The “fleximers” feature the
purine ring systems split into their individual imidazole and pyrimidine components. This structural
modification serves to introduce flexibility to the nucleoside while still retaining the elements
essential for recognition. As a consequence, these structurally innovative nucleosides can more
readily adapt to their environment and should find use as bioprobes for investigating enzyme-
coenzyme binding sites as well as nucleic acid and protein interactions. Their design and synthesis
is described.
Introduction
exhibits a significant difference between the “open” and
“closed” conformations.8 As a possible means to explore
this phenomenon, we have designed a series of structur-
ally innovative nucleosides that possess a heteroaromatic
purine ring split into its two components (for example,
an imidazole and pyrimidine ring), thereby conferring
additional degrees of conformational freedom and tor-
sional flexibility to the ligand. As a result, these “molec-
ular chameleons” can readily adapt to the environment
of the flexible binding site in order to maximize comple-
mentary structural interactions, without losing the in-
tegrity of the basic scaffold required for the enzyme’s
mechanism of action.
One focus for our research has involved the design and
synthesis of novel shape-modified nucleosides to explore
fundamental aspects of nucleic acid structure, function,
and stability, as well as to investigate enzyme binding
site parameters. With the increasing number of crystal
structures for various enzyme-substrate complexes, it
has become apparent that many binding sites are more
flexible than previously thought and can therefore adjust
to fit a wide range of substrates.1-3 This phenomenon
causes inaccuracies when employing structure-based
drug design of a potential enzyme-substrate complex
from a crystallographic basis; although the fit may
appear to be good, in reality the “best guess” drug often
proves to be a poor inhibitor.
(4) Tuske, S.; Sarafianos, S. G.; Clark, A. D., Jr.; Ding, J.; Naeger,
L. K.; White, K. L.; Miller, M. D.; Gibbs, C. S.; Boyer, P. L.; Clark, P.;
Wang, G.; Gaffney, B. L.; Jones, R. A.; Jerina, D. M.; Hughes, S. H.;
Arnold, E. Nat. Struct. Mol. Biol. 2004, 11, 469-474.
(5) Das, K.; Clark, A. D., Jr.; Lewi, P. J.; Heeres, J.; de Jonge, M.
R.; Koymans, L. M. H.; Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.;
Kukla, M. J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye, H.;
Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de Bethune, M.-P.; Boyer,
P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A. J.; Arnold, E. J. Med.
Chem. 2004, 47, 2550-2560.
(6) Lewis, P. J.; de Jonge, M.; Frits, D.; Koymans, L.; Vinkers, M.;
Heeres, J.; Janssen, P. A. J.; Arnold, E.; Das, K.; Clark, A. D., Jr;
Huges, S. H.; Boyer, P. L.; de Bethune, M.-P.; Pauwels, R.; Andries,
K.; Kukla, M.; Ludovici, D.; De Corte, B.; Kavash, R.; Ho, C. J.
Comput.-Aided Mol. Des. 2003, 17, 129-134.
(7) Henry, C. M. Chem. Eng. News 2004, 82, 40-41.
(8) Yin, D.; Yang, X.; Hu, Y.; Kuczera, K.; Schowen, R. L.; Borchardt,
R. T.; Squier, T. C. Biochemistry 2000, 39, 9811-9818.
Significant to this observation, recent reports have
shown that (i) flexible inhibitors can overcome drug
resistance mutations in viral HIV4-7 and (ii) the binding
site of S-adenosylhomocysteine hydrolase (SAHase), an
enzyme critical in the replication mechanism of viruses,
* To whom correspondence should be addressed. Phone: 410-455-
8684. Fax: 410-455-2608.
(1) Bugg, C. E.; Carson, W. M.; Montgomery, J. A. Sci. Am. 1993,
269, 92-98.
(2) Carlson, H. A.; McCammon, J. A. Mol. Pharmacol. 2000, 57,
213-218.
(3) Gane, P. J.; Dean, P. M. Curr. Opin. Struct. Biol. 2000, 10, 401-
404.
10.1021/jo048218h CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/03/2005
1612
J. Org. Chem. 2005, 70, 1612-1619

“Molecular Chameleons”
FIGURE 3. Twisted G and isoG fleximers.
FIGURE 1. C-5- and C-4-substituted guanosine fleximers.
formation than the C-5 analogues, so we were anxious
to explore this experimentally. We also wanted to explore
the arrangement of the substituents, since the “twisted”
guanosine fleximer closely resembles the isoguanosine
(isoG) ring system (Figure 3). As a result, it occurred to
us that if we “prearranged” the substituents in the
conformation they appeared to prefer, it might result in
a more favorable binding energy, since theoretically the
pyrimidine would not have to expend any energy to attain
the desired conformation.
FIGURE 2. Weisz’s C-4-substituted analogues.
Our initial calculations in SAHase had shown that the
binding energy for the C-5 guanosine fleximer was
-24.88 kcal/mol, but these had been carried out on a
lower level of theory, using a class I force field (CVFF),
so in an effort to increase our accuracy, we moved to a
class II force field16,17 (CFF) and repeated the calcula-
tions. The new results all showed lower binding energies
than for the previous force field with the new energy for
the C-5 guanosine being used as the reference in this
study. The corresponding C-4 guanosine fleximer was
5.36 kcal/mol lower in energy that the C-5 guansosine;
however, as we predicted, both the C-5- and C-4-
substituted isoG’s exhibited much more favorable binding
energies. Relative to the C-5 guanosine fleximer, the C-5
and C-4 isoG’s were more favorable by -20.59 and
-21.97 kcal/mol, respectively.
The lead provided by the unusual inhibitory activity
exhibited by our guanosine C-5 substituted imidazole
fleximer 1 (Figure 1) against S-adenosylhomocysteine
hydrolase (SAHase), an enzyme that recognizes adeno-
sine analogues, prompted us to expand our initial efforts
with these structurally unique analogues and this bio-
logically significant enzyme. In contrast to our previously
reported9-11 C-5-substituted imidazole fleximers, the C-4
series is connected at the C-4 of the imidazole and the
C-5 of the pyrimidine (as in 2, Figure 1), rather than the
C-5 of the imidazole and the C-6 of the pyrimidine (as in
1).
To our knowledge, the only other example in the
literature of a split nucleoside such as these was recently
introduced by Weisz et al.;12,13 however, as shown in
Figure 2, the connectivity of the C-4 of the imidazole is
substituted with either a pyridine or a benzene ring
attached at the C-3 of the benzene ring (or the C-2 of the
pyridine ring), rather than C-5 or C-6 of the pyrimidine
ring, thereby differing from the standard purine motif.
As a result, our analogues are more faithful to a classic
purine design, and as such, they will provide a unique
perspective on enzyme/ligand interactions. Their synthe-
sis is described herein.
Analogous to isoguanosine,18 the isoG fleximers each
have two tautomeric forms, so it was important for us to
determine which would be preferred, since it would affect
the hydrogen bonding and recognition.19,20 Our calcula-
tions showed that when bound in the active site of
SAHase, the C-5-substituted isoG fleximer preferred to
exist as the N-3-H tautomer (shown in Figure 4), with a
difference in energy between the two tautomers of 18.09
kcal/mol, while the C-4 isoG fleximer preferred the N-1-H
tautomeric form (Figure 5) with a relative binding energy
of -23.08 kcal/mol lower than the N-3-H tautomer.
Figure 6 depicts the C-5- and C-4-substituted imidazole
G’s, as well as the C-5- and C-4-substituted imidazole
isoG’s aligned in the binding site of SAHase by the
residues they interact with. As mentioned above, the C-5
and C-4 isoG fleximers exhibit the most favorable binding
energy values, but it is clear that they are both capable
Results and Discussion
The aforementioned unusual activity exhibited11 by our
C-5-substituted imidazole guanosine fleximer (1) against
SAHase, as well as the interesting conformation it
adopted in our modeling studies when bound into the
SAHase active site, prompted us to investigate the
connectivity for the fleximer bond. Our modeling stud-
ies,10,11,14 as well as other computational studies15 involv-
ing our fleximers, had suggested that the C-4-substituted
imidazole fleximers would adopt quite a different con-
(14) Polak, M.; Seley, K. L.; Plavec, J. J. Am. Chem. Soc. 2004, 126,
8159-8166.
(15) Bardon, A. B.; Wetmore, S. D. J. Phys. Chem. A 2005, 106, 262-
272.
(9) Seley, K. L.; Zhang, L.; Hagos, A. Org. Lett. 2001, 3, 3209-3210.
(10) Seley, K. L.; Zhang, L.; Hagos, A.; Quirk, S. J. Org. Chem. 2002,
67, 3365-3373.
(16) Hwang, M.-J.; Ni, X.; Waldman, M.; Ewig, C. S.; Hagler, A. T.
Biopolymers 1998, 45, 435-468.
(17) Maple, J. R.; Hwang, M.-J.; Jalkanen, K. J.; Stockfisch, T. P.;
Hagler, A. T. J. Comput. Chem. 1998, 19, 430-458.
(18) Blas, J. R.; Luque, F. J.; Orozco, M. J. Am. Chem. Soc. 2004,
126, 154-164.
(11) Seley, K. L.; Quirk, S.; Salim, S.; Zhang, L.; Hagos, A. Bioorg.
Med. Chem. Lett. 2003, 13, 1985-1988.
(12) Wang, W.; Purwanto, M. G. M.; Weisz, K. Org. Biomol. Chem.
2004, 2, 1194-1198.
(19) Benner, S. A. Acc. Chem. Res. 2004, 37, 784-797.
(20) Martinot, T. A.; Benner, S. A. J. Org. Chem. 2004, 69, 3972-
3975.
(13) Lengeler, D.; Weisz, K. Nucleosides Nucleotides 1999, 18, 1657-
1658.
J. Org. Chem, Vol. 70, No. 5, 2005 1613

Seley et al.
FIGURE 6. Relative energies of C-4- and C-5-substituted G
and IsoG fleximers in SAHase: orange, C-5 guanosine fleximer,
∆G ) reference; purple, C-4 guanosine fleximer, ∆G ) -5.36
kcal/mol; green, C-5 isoguanosine fleximer, ∆G ) -20.59 kcal/
mol; pink: C-4 isoguanosine fleximer, ∆G ) -21.97 kcal/mol.
FIGURE 4. N3(H) tautomer for the C-5 IsoG fleximer in
SAHase.
FIGURE 7. C-4-substituted fleximer targets.
SCHEME 1. Cross-Coupling Approaches: Stille,
Negishi, Kumada, Grignard, and Suzuki
FIGURE 5. N1(H) tautomer for the C-4 IsoG fleximer in
SAHase.
of adopting a significantly different conformation from
each other, while still maintaining critical contacts
necessary for recognition.
The initial approach to realize the C-4-substituted
fleximer scaffold was envisioned from traditional orga-
nometallic coupling methods to attach the imidazole and
pyrimidine rings (as depicted above in Scheme 1). The
plethora of available catalysts and coupling methods,
including Stille, Kumada, Negishi, Grignard, and Suzuki,
made this effort more attractive and straightforward
than the nontrivial and tedious linear synthesis that had
been required to attain the C-5-substituted fleximers.
While there were examples of both the imidazole and the
pyrimidine components serving as either the electrophilic
or nucleophilic coupling partner, we first chose to attempt
In light of this, we focused our initial efforts on the
synthesis of the C-4-substituted guanosine (2) and isoG
(3) analogues. While 2 and 3 could not be realized directly
due to complications with the exocyclic amine group, they
could be obtained by manipulation of either the diamino
(4) or xanthosine (5) fleximers (Figure 7), which were of
interest to us as well as they would allow a complete
comparison of the effect of the amino and carbonyl
groups.
1614 J. Org. Chem., Vol. 70, No. 5, 2005

“Molecular Chameleons”
SCHEME 2a
conditions surprised us, since Negishi coupling had been
reported26 to work well with C-4 iodo-substituted imida-
zoles and was more tolerant of sensitive functional groups
than traditional Grignard and organolithium approaches.
We speculated that it could have been due to the
π-electron-deficient nature of the dimethyluracil since we
were successful in forming the nucleophilic imidazole
moiety, both in situ and as an isolated intermediate. As
a result, it appeared that the pyrimidine system was
simply insufficiently activated to participate in the cross-
coupling.
a Reaction conditions: (a) KCl, ICl; (b) (i) â-D-ribofuranose
1,2,3,5-O-tetraacetate, BSA, (ii) TMSOTf; (c) NH4OH, EtOH, 18
h; (d) (i) NaH, THF, 0 °C, 3 h; (ii) BnBr, TBAl; (e) EtMgBr, Et2O,
3 h.
We turned next to the Stille27-31 and Suzuki32,33
coupling methods, as there were examples in the litera-
ture that had been obtained in reasonable yields. In some
respects, Stille coupling has many advantages that made
it an attractive choice: the conditions are tolerant of
many functional groups, and the stannanes are readily
prepared and purified and are known to be fairly
stable.27,28,33 Unfortunately, all efforts with Stille or
Suzuki conditions failed; however, use of n-BuLi and
EtMgBr provided the dehalogenated imidazole nucleoside
14 in a 90% yield (Scheme 4 on the next page).
At this point, we reversed our strategy to employ the
pyrimidine as the nucleophilic component in an effort to
obtain the critical xanthosine intermediate. Once again
using traditional coupling approaches, we attempted to
couple 11 to a variety of organometallic pyrimidine
species, including the pyrimidinylstannane 15,34,35 as well
as the corresponding Kumada and Negishi metalated
pyrimidines (16 and 17, respectively, Scheme 5 on the
next page).24,25 Pyrimidinylstannane 15 was isolated in
reasonable yield (66%) but failed to undergo coupling
with the usual palladium reagents. A procedure34 em-
ploying copper(I)thiophene carboxylate and 1-methyl-2-
pyrrolidinone (NMP) was also tried, unfortunately, to the
same end so this route was abandoned as well.
We speculated that the inherent electron-deficient
nature of the pyrimidine ring system was the cause for
the significant lack of reactivity noted to date. If this was
indeed true, it was clear that to be successful, the
electronics of the pyrimidine ring system had to be
enhanced; therefore, electron-donating substituents such
as ethers or thioethers might prove to be a possible
solution. Suzuki coupling with 5-bromo-2,4-bis(meth-
ylthio)pyrimidine was tried,37 but NMR and MS analysis
of the product proved it to be a mixture of dimerized
pyrimidines, which were the result of self-cross coupling
at C-5/C-5′ and C-5/C-6′, a fate apparently not uncommon
for boronic acid pyrimidines.38
coupling with the imidazole moiety as the nucleophilic
species, since we had ready access to the necessary
starting materials and many of the examples using this
approach had produced results in reasonably good yields.
Since many of the routes appeared to have a bias for
the C-4 iodo-substituted imidazole rather than with the
analogous 4-bromoimidazole for coupling procedures, we
adapted our previously reported route,9,10 which had
featured a dibromoimidazole ribofuranosyl intermediate.
This proved to be advantageous, since the workup and
purification steps for the diiodoimidazole analogue were
much less tedious than for the dibromoimidazole ana-
logue.
Synthesis began with replacement of the C-4 and C-5
hydrogens with iodine using a facile literature proce-
dure21 to give 7 in 93% yield (Scheme 2). Next, coupling
diiodoimidazole 7 with commercially available tetra-
acetate-protected ribofuranose using bis(trimethylsilyl)-
acetamide (BSA) and trimethylsilyltriflate (TMSOTf)
gave 8.9,10 It was then necessary to replace the acetate
groups with a more robust protecting group, due to the
various reaction conditions that would be encountered
in subsequent steps that would not be tolerated by either
an alcohol or ester functionality. Standard base-promoted
deprotection afforded 9 in a 75% overall yield for the two
steps. Reprotection of the three hydroxyls with the in
situ22 formation of benzyliodide, a method we have found
to be superior to other traditionally used routes when
attempting to benzylate all three hydroxyls, was then
accomplished to give 10 (80% yield), followed by displace-
ment of the C-5 iodide using Grignard conditions,23 which
yielded 11 also in an 80% yield.
All of the desired targets were available from the
xanthosine fleximer. As a result, the Kumada and
Negishi were the first cross-coupling methods tried as
shown in Scheme 3 on the next page.24,25 Unfortunately,
all efforts to construct the C-4-substituted scaffold in this
manner proved fruitless; altering the nature of the
halouracil derivative, the choice of solvent, the reaction
temperature, or the palladium catalyst failed to yield the
desired intermediate, from which all four of the target
fleximers could be realized. The failure of the Negishi
(26) Negishi, E.-I.; Luo, F.-I.; Frisbee, R.; Matsushita, H. Heterocycles
1982, 18, 117.
(27) Stille, J. K. In Modern Synthetic Methods; Scheffold, R., Ed.;
Springer: Berlin, 1983; Vol. 3, p 2.
(28) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508.
(29) Cliff, M. D.; Pyne, S. G. Tetrahedron 1996, 52, 13703-13712.
(30) Jetter, M. C.; Reitz, A. B. Synthesis 1998, 829-831.
(31) Wang, J.; Scott, A. I. Tetrahedron Lett. 1996, 37, 3247-3250.
(32) Hassan, J.; Sevignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M.
Chem. Rev. 2002, 102, 1359-1470.
(33) Li, J. J.; Gribble, G. W. Palladium in Heterocyclic Chemistry;
Pergamon: Amsterdam, 2000.
(21) Iddon, B. Tetrahedron Lett. 1986, 27, 1635-1638.
(22) Czernecki, S.; Georgoulis, C.; Provelenghiou, C. Tetrahedron
Lett. 1976, 17, 3535-3536.
(34) Allred, G. D.; Liebeskind, L. S. J. Am. Chem. Soc. 1996, 118,
2748-2749.
(23) Katritzky, A. R. J. Chem. Soc., Perkin Trans. 1 1989, 1139-
1145.
(35) Santagostino, M.; Palmisano, G. Tetrahedron 1993, 49, 2533-
2542.
(24) Hirota, K.; Watanabe, K. A.; Fox, J. J. J. Org. Chem. 1978, 43,
1193-1197.
(36) Bumagin, N. A.; Luzikova, E. V. J. Organomet. Chem. 1997,
532, 271-273.
(25) Erdik, E. Tetrahedron 1992, 48, 9577-9648.
(37) Strekowski, L. Rocziniki Chem. 1975, 49, 1693-1705.
J. Org. Chem, Vol. 70, No. 5, 2005 1615

Seley et al.
SCHEME 3a
a Reaction conditions: (a) Me2SO4, NaOH, H2O; (b) (i) 11 with EtMgBr or ZnCl2 in Et2O, then (ii) 13 with (c) PdCl2(dppf); (e) Pd(PPh3)4;
or (f) Pd2(dba)3.
SCHEME 4a
SCHEME 6a
a Reaction conditions: n-BuLi, EtMgBr.
SCHEME 5a
a Reaction conditions: (a) NaH, BnOH; (b) (i) B(OiPr)3, THF/
toluene (1:4), (ii) n-BuLi, THF, -78 °C, (iii) aq HCl; (c) Pd(PPh3)4,
1,2-dimethoxyethane (DME), NaHCO3, reflux; (d) 10% NH3,
BuOH, heat.
a Reaction conditions: for 15: (i) 13, (Bu3Sn)2, 66%; then (ii)
11, Pd(PPh3)4, CuI, DMF, or DME, 80 °C, NR; or (ii) 11,
(PPh3)2PdCl2, CH3CN, reflux, NR; or (ii) 11, copper(l)thiophen-
ecarboxylate, NMP, 0 °C to rt; for 16 or 17: (i) 13, EtMgBr, Et2O,
ZnCl2, then (ii) 11, (dppf)PdCl2, DMF or (ii) 11, Pd(PPh3)4, Cul,
DMF, NR.
With the first two C-4-substituted fleximers finally in
hand, the remaining task was to manipulate the diamino
fleximer to give us the guanosine and isoguanosine
fleximers we had initially set our sights on. The C-4-
substituted isoguanosine fleximer 3 was realized follow-
ing selective conversion of the C-2 amino group of the
protected diamino fleximer 22 to the desired carbonyl.
As shown in Scheme 7, formation of the diazonium using
standard conditions, followed by hydrolysis, and subse-
quent deblocking of the benzyl protecting groups gave 3
in a 41% yield (two steps).10,41,42 Finally, the diamino
fleximer 4 was converted directly to 2 using aqueous
sodium bisulfite and heat in 88% yield.37
In summary, a series of traditional cross-coupling
methods using a variety of catalysts and conditions were
explored to realize the synthesis of the second series of
fleximers. It is clear from the efforts outlined herein that
the imidazole and pyrimidine ring systems are recalci-
trant at best and that work remains to fully understand
the ideal conditions to manipulate these heterocycles
more efficiently. We are, however, encouraged by the
reasonably good results finally obtained with the Suzuki
system, and we will continue to optimize this promising
route.
Attention then turned to the analogous dibenzyloxy-
ether pyrimidine system. Beginning with 5-bromo-2,4-
dichloropyrimidine (18) that is commercially available,
but is also an intermediate in the synthesis36 of 5-bromo-
2,4-bis(thiomethyl)pyrimidine, conversion to the diben-
zyloxy analogue 19 (Scheme 6) was accomplished in
excellent yield (91%) using standard benzylation condi-
tions, followed by conversion to the boronic acid 20, which
was also obtained in excellent yield (95%).39 Coupling was
then successfully carried out with tetrakis (triphenylphos-
phine)palladium (0) to give 21 (67%).40 Removal of all five
of the benzyl groups was accomplished in 88% yield to
give the xanthosine fleximer 5 directly.41 Alternatively,
treatment of 21 with ammonia in butanol in a steel Parr
bomb afforded 22 in moderate yield (40%), which, fol-
lowed by deprotection,10 gave the desired diamino fleximer
4 (90%).42
(38) Rouhi, A. M. Chem. Eng. News 2004, 82, 59-61.
(39) Schinazi, R. F.; Prusoff, W. H. J. Org. Chem. 1985, 50, 841-
847.
(40) Wellmar, U.; Hornfeldt, A.-B.; Gronowitz, S. J. Heterocycl.
Chem. 1995, 32, 1159-1163.
(41) Koshkin, A. A.; Fensholdt, J.; Pfundheller, H. M.; Lomholt, C.
J. Org. Chem. 2001, 66, 8504-8512.
(42) Ogilvie, K. K.; Nghe, N.-B.; Gillen, M. F.; Radatus, B. K.;
Cheriyan, U. O.; Smith, K. O.; Galloway, K. S. Can. J. Chem. 1984,
62, 241-252.
1616 J. Org. Chem., Vol. 70, No. 5, 2005

“Molecular Chameleons”
SCHEME 7a
solved. The clear solution was then acidified to pH 9.50 by
dropwise addition of concentrated HCl. The resulting product
was filtered to afford an off-white powder, which, following
recrystallization in EtOH, gave 7 as a white crystalline solid
1
(131.2 g, 93%): mp 188-191 °C; H NMR (DMSO-d6) δ 7.76
(s, 1 H); 13C NMR (DMSO-d6) δ 141.1, 143.2.
2,3-Diacetoxy-5-acetoxymethyl-1-(4,5-diiodoimidazol-
3-yl)-â-D-ribofuranose (8).9,10 N,O-Bis(trimethylsilyl)acet-
amide (BSA) (73.9 mL, 0.30 mol) was added to a stirred
solution of 7 (50.0 g, 156 mmol) and 1,2,3,5-tetra-O-acetate-
â-D-ribofuranose (50.0 g, 157 mmol) in anhydrous acetonitrile
(500 mL). The mixture was stirred for an additional 4 h at rt
under Ar. The solution was then cooled to 0 °C, at which point
trimethylsilyl trifluromethylsulfonate (TMSOTf) (31 mL, 172
mmol) was added dropwise. The mixture was heated at 60 °C
under Ar for 18 h. The solvent was removed under reduced
pressure, and the resulting residue was cooled to 0 °C, followed
by portionwise addition of aqueous NaHCO3 until production
of gas ceased. The mixture was extracted with CH2Cl2 (3 ×
400 mL); the organic extracts were combined, washed with
brine (2 × 300 mL), and dried over MgSO4; and the solvent
was removed under reduced pressure to give 8 as a light brown
syrup (67.6 g), which was used without further purification.
(4,5-Diiodoimidazol-3-yl)-1-â-D-ribofuranose (9). A mix-
ture of crude 8 (67.6 g, 0.117 mol) and concentrated ammonium
hydroxide (28%, 500 mL) in EtOH (300 mL) was stirred at rt
for 18 h. The resulting precipitate was filtered and washed
with cold H2O to afford 9 as a white crystalline solid (52.8 g,
75% for two steps): 1H NMR (DMSO-d6) δ 3.49 (dd, 3.9 Hz,
12.0 Hz, 1 H), 3.57 (dd, 3.9 Hz, 12.0 Hz, 1 H), 3.88 (q, 3.6 Hz,
1 H), 4.01 (t, 3.6 Hz, 1 H), 4.24 (t, 5.0 Hz, 1 H), 5.19 (t, 5.1 Hz,
1 H), 5.33 (d, 4.8 Hz, 1 H), 5.46 (d, 5.1 Hz, 1 H), 5.60 (d, 6.3
Hz, 1 H), 8.15 (s, 1 H); 13C NMR (DMSO-d6) δ 61.1, 70.2, 75.0,
85.6, 92.4, 97.3, 130.5, 140.8.
2,3-Dibenzyloxy-5-benzyloxymethyl-1-(4,5-diiodoimi-
dazol-3-yl)-1-â-D-ribofuranose (10). To a stirred solution of
9 (21.0 g, 45.9 mmol) in anhydrous THF (300 mL) at 0 °C was
added NaH (95% dry, 4.06 g, 161 mmol) in small portions over
a period of 10 min. The resulting mixture was stirred at rt for
3 h, at which point tetrabutylammonium iodide (3.4 g, 9.20
mmol) was added, followed by dropwise addition of benzyl
bromide (19.1 mL, 161 mmol).22 The new mixture was stirred
at rt for 18 h. The solvent was then removed under reduced
pressure, and saturated aqueous NH4Cl solution (200 mL) was
added to the residue. The mixture was extracted with CH2Cl2
(3 × 250 mL); the organic extracts were combined, washed
with brine (2 × 300 mL), and dried over MgSO4; and the
solvent was removed under reduced pressure to give a yellow
syrup. Column chromatography eluting with hexane/EtOAc
(3:1) gave 10 as a white crystalline solid (26.5 g, 80% yield):
mp 86-88 °C; 1H NMR (CDCl3) δ 3.53 (dd, 2.5 Hz, 10.8 Hz, 1
H), 3.75 (dd, 2.4 Hz, 10.8 Hz, 1 H), 4.08-4.18 (m, 2 H), 4.31-
4.34 (m, 1 H), 4.44-4.70 (m, 6 H), 5.82 (d, 3.6 Hz, 1 H), 7.21-
7.37 (m, 15 H), 7.99 (s, 1 H); 13C NMR (CDCl3) δ 66.3, 72.6,
72.9, 73.5, 75.7, 79.8, 80.9, 81.9, 92.0, 127.8, 127.9, 128.2, 128.5,
128.6, 136.9, 137.2, 140.3. Anal. Calcd for C29H28I2N2O4: C,
48.22; H, 3.91; N, 3.88; I, 35.14. Found: C, 48.48; H, 3.91; N,
3.86; I, 35.41.
2,3-Dibenzyloxy-5-benzyloxymethyl-1-(4-iodoimidazol-
3-yl)-1-â-D-ribofuranose (11). To a solution of 10 (34.7 g, 48.0
mmol) in anhydrous ether (500 mL) at rt was added EtMgBr
(3.0 M, 19 mL, 57 mmol) in a dropwise manner.23 The mixture
was stirred at rt for 3 h and then quenched with anhydrous
EtOH (50 mL). The solvent was removed under reduced
pressure, saturated aqueous NH4Cl solution (300 mL) added,
and the mixture extracted with CH2Cl2 (3 × 200 mL). The
organic layers were combined, washed with brine (2 × 400
mL), and dried over MgSO4, and the solvent was removed
under reduced pressure. Column chromatography of the
residue, eluting with hexane/EtOAc (3:1), gave 11 as a colorless
syrup (23 g, 80%): 1H NMR (CDCl3) δ 3.51 (dd, 2.4 Hz, 10.8
Hz, 1 H), 3.67 (dd, 3.0 Hz, 10.8 Hz, 1 H), 4.04-4.16 (m, 3 H),
a Reaction conditions: (a) NaNO2, AcOH, H2O/THF (1:1); (b) Pd/
C, HCO2NH4, EtOH, reflux; (c) NaHSO3, H2O.
With the first four C-4 substituted fleximers in hand,
we will be able to continue our investigations into
exploring the confines of biologically significant enzymes,
and by comparing the results already obtained with the
C-5 substituted fleximers to see if altering the position
of the fleximer bond has a significant effect on recognition
with SAHase and the other enzymes we are studying.
In the meantime, efforts are also underway to realize the
C-4-substituted adenosine and inosine fleximers from a
different route, since those are of interest to us as well.
Results of those efforts and the results of the broad screen
antiviral, anticancer, and antiparasitic testing presently
underway will be reported in the future.
Experimental Section
Modeling. All potential inhibitors were docked into the
SAHase crystal structure (PDB 1A7A) and calculations per-
formed on an SGI Octane2 using Accelrys InsightII/Discover
with a CFF force field, which is derived from ab initio
calculations on the Hartree-Fock level of theory using the
6-31G* basis set. The inhibitors were optimized using steepest
descent calculations. Then, using Monte Carlo techniques, up
to 20 spatial conformers were generated for each inhibitor. The
10 lowest energy conformations were then minimized using
simulated annealing techniques. The final structures were
analyzed for the best ligand structure with the lowest energy
value. The overlapped images depicted in Figure 6 are the best
final structures docked into the SAHase binding site. The
nonessential hydrogens have been removed for clarity.
1
General Procedures. Melting points are uncorrected. H
and 13C spectra were operated at 300 and 75 MHz, respec-
tively, all referenced to internal tetramethylsilane (TMS) at
0.0 ppm. The spin multiplicities are indicated by the symbols
s (singlet), d (doublet), t (triplet), q(quartet), m (multiplet), and
b (broad). Reactions were monitored by thin-layer chromatog-
raphy (TLC) using 0.25 mm Whatman Diamond silica gel 60-
F254 precoated plates. Column chromatography was performed
on Whatman silica, 200-400 mesh, 60 and elution with the
indicated solvent system. Yields refer to chromatographically
and spectroscopically (1H and 13C NMR) homogeneous materi-
als.
4,5-Diiodoimidazole (7).21 An aqueous solution (500 mL)
of imidazole (30.0 g, 0.441 mol) was added dropwise to a
solution of KICl2 (2.0 M, 550 mL, 1.1 mol, prepared from
dissolving ICl (79.9 g, 1.03 mol) in an aqueous solution of KCl
(131.5 g, 1.77 mol in 550 mL of H2O) at rt. The mixture was
stirred for an additional 10 h, followed by slow addition of 2
M NaOH solution until the suspension was completely dis-
J. Org. Chem, Vol. 70, No. 5, 2005 1617

Seley et al.
4.30-4.66 (m, 6 H), 5.69 (d, 6.0 Hz, 1 H), 7.03 (d, 1.2 Hz, 1 H),
7.11 (dd, 2.0 Hz, 7.8 Hz, 2 H), 7.21-7.42 (m, 13 H), 7.46 (d,
1.2 Hz, 1 H); 13C NMR (CDCl3) δ 60.3, 69.7, 72.3, 72.6, 73.7,
76.5, 81.6, 82.6, 88.8, 121.9, 127.8, 127.9, 128.0, 128.2, 128.5,
128.6, 136.6, 137.1, 137.2, 137.5. Anal. Calcd for C29H29-
IN2O4: C, 58.40; H, 4.90; N, 4.70; I, 21.28. Found: C, 58.29;
H, 4.93; N, 4.61; I, 21.06.
5-Iodo-1,3-dimethyluracil (13). Dimethyl sulfate (13.0
mL, 137 mmol) was added dropwise to a stirring slurry of
5-iodoracil 12 (14.9 g, 62.6 mmol) and NaOH (7.4 g, 156 mmol)
in H2O (100 mL) at 0 °C.34 The mixture was heated under
reflux for 2 h, cooled to rt, and extracted with CH2Cl2 (3 ×
200 mL). The organic layers were combined, washed with brine
(300 mL), and dried over MgSO4. The solvent was removed
under reduced pressure to give a white solid, which, following
recrystallization in hot EtOH, gave 13 as a white crystalline
solid (10.6 g, 63%): mp 224 °C (Aldrich, 225 °C); 1H NMR
(CDCl3) δ 3.40 (s, 3 H), 3.41 (s, 3 H), 7.62 (s, 1 H); 13C NMR
(CDCl3) 29.5, 37.3, 67.1, 147.2, 151.2, 160.2.
2,3-Dibenzyloxy-5-benzyloxymethyl-1-(imidazol-3-yl)-
1-â-D-ribofuranose (14). EtMgBr (3.0 M in ethyl ether, 0.62
mL, 1.84 mmol) was added dropwise to a solution of 11 (1.0 g,
1.67 mmol) in anhydrous CH2Cl2 (15 mL) at rt under Ar. The
resulting solution was stirred at rt for 2 h, followed by dropwise
addition of trimethyltin chloride (1.0 M in CH2Cl2, 2.0 mL, 2.0
mmol) at rt.35 The mixture was allowed to stir at rt for 18 h
and then quenched with water. The organic layer was sepa-
rated and the aqueous layer washed with CH2Cl2 (2 × 20 mL).
The organic extracts were combined and washed sequentially
with saturated KF solution (30 mL) to form insoluble organotin
fluoride and then brine (50 mL). The organic layer was dried
over MgSO4 and the solvent removed to afford 14 as a colorless
syrup (714 mg, 91%): 1H NMR (CDCl3) δ 3.48 (dd, 2.4 Hz, 10.8
Hz, 1 H), 3.62 (dd, 3.0 Hz, 10.8 Hz, 1 H), 4.03 (dd, 2.8 Hz, 4.8
Hz, 1 H), 4.10 (br t, 5.4 Hz, 1 H), 4.29 (dd, 2.8 Hz, 5.4 Hz, 1
H), 4.36-4.61 (m, 6 H), 5.74 (d, 6.4 Hz, 1 H), 6.98 (d, 6.8 Hz,
2 H), 7.10-7.32 (m, 15 H), 7.59 (s, 1 H); 13C NMR (CDCl3) δ
69.5, 72.0, 72.3, 73.3, 76.4, 81.4, 82.1, 88.5, 116.1, 127.3, 127.4,-
127.5, 127.6, 127.7, 128.1, 129.3, 135.7, 136.5, 137.0, 137.1.
5-(Dihydroxyboryl)-2,4-bis(benzyloxy)pyrimidine (20).39
N-Butyllithium (1.3 M in hexane, 2.5 mL, 3.2 mmol) was
dropwise added over a 1 h period to a solution of 19 (1.0 g, 2.7
mmol) and B(OiPr)3 (1.0 mL, 5.3 mmol) in a mixture of
anhydrous THF and toluene (1:4 volume ratio, 50 mL total)
at -78 °C under Ar.39 The mixture was stirred for an
additional 18 h at rt, followed by addition of dilute HCl (1 M).
The reaction mixture was evaporated under reduced pressure.
The residue was then treated with H2O (50 mL) and extracted
with CH2Cl2 (3 × 30 mL). The organic extracts were combined,
washed with brine (50 mL), and dried over MgSO4, and the
solvent was removed under reduced pressure to give 20 as a
white powder (8.8 g, 95%). Spectral data are in agreement with
the literature:39 1H NMR (CDCl3) δ 5.46 (s, 4 H), 7.25-7.48
(m, 10 H), 8.72 (s, 1H); 13C NMR (CDCl3) δ 118.5, 160.1, 161.4,
165.9.
2,3-Dibenzyloxy-5-benzyloxymethyl-1-[4-(2,4-dibenzyl-
oxy-5-pyrimidinyl)imidazol-1-yl]-â-D-ribofuranose (21). A
mixture of 11 (1.7 g, 2.9 mmol) and Pd(PPh3)4 (185 mg, 0.16
mmol) in 40 mL of 1,2-dimethoxyethane (DME) was stirred
at rt under Ar for 10 min. To this mixture was added
5-(dihydroxyboryl)-2,4-bis(benzyloxy)pyrimidine (20) (3.2 mmol
in 20 mL of DME).40 Saturated aqueous NaHCO3 (40 mL) was
added and the mixture refluxed under Ar for 4 h. The solution
was cooled to rt and the DME layer separated and set aside.
The aqueous layer was then extracted with EtOAc (3 × 50
mL), and the organic extracts were combined with the DME
layer, washed with brine (100 mL), and dried over MgSO4. The
solvent was removed to give a pale brown syrup. Column
chromatography eluting with 2% EtOH in CH2Cl2 gave 21 as
a colorless syrup (1.54 g, 70%): 1H NMR (CDCl3) δ 3.45 (dd,
3.0 Hz, 10.6 Hz, 1 H), 3.56 (dd, 3.3 Hz, 10.6 Hz, 1 H), 4.01-
4.48 (m, 1 H), 4.11 (br t, 5.5 Hz, 1 H), 4.31-4.62 (m, 7 H),
5.42-5.52 (m, 4 H), 5.78 (d, 6.0 Hz, 1 H), 7.15-7.54 (m, 26
H), 7.73 (s, 1 H), 9.15 (s, 1 H); 13C NMR (CDCl3) δ 68.3, 68.9,
69.4, 72.1, 72.5, 73.1, 76.5, 81.5, 82.1, 88.7, 109.5, 115.6, 127.3,
127.6, 127.8, 127.9, 128.3, 134.0, 135.6, 136.1, 136.5, 136.7,
137.2, 137.3, 155.9, 162.7, 166.0. Anal. Calcd for C47H44N4O6‚
1H2O: C, 72.48; H, 5.95; N, 7.19. Found: C, 72.82; H, 5.75; N,
7.03.
5-Tri-n-butylstannyl-1,3-dimethyluracil (15). A mixture
of 5-iodo-1,3-dimethyluracil (13) (2.66 g, 10.0 mmol), bis-
(tributyltin) (10.1 mL, 20.0 mmol), and PdCl2(PPh3)4 (280 mg,
0.40 mmol) in anhydrous toluene (300 mL) was heated under
Ar at 90 °C for 4 h.35 The mixture was filtered through a pad
of alumina, the solvent removed under reduced pressure, and
the residue purified by column chromatography (silica gel
pretreated with 5% triethylamine in hexanes), eluting with
2% triethylamine in hexanes, to afford 5-tri-n-butylstannyl-
1,3-dimethyluracil (15) as a colorless oil (2.7 g, 62%): 1H NMR
(CDCl3) δ 0.88 (t, 7.2 Hz, 9 H), 1.03 (t, 8.4 Hz, 6 H), 1.31 (q,
7.6 Hz, 6 H), 1.50 (q, 8.4 Hz, 6 H), 3.32 (s, 3 H), 3.36 (s, 3 H),
6.87 (s, 1 H); 13C NMR (CDCl3) δ 9.9, 13.8, 27.4, 27.7, 29.1,
36.8, 111.0, 146.7, 152.4, 166.5.
2,4-Dibenzyloxy-5-bromopyrimidine (19).39 A stirred
solution of benzyl alcohol (13.4 mL, 129 mmol) in anhydrous
toluene (140 mL) was treated with NaH (60% in mineral oil,
4.84 g, 121 mmol) under Ar. The mixture was warmed to 50
°C to facilitate the formation of the sodium salt and stirred
until all signs of gas evolution had subsided.39 The suspension
was cooled and 5-bromo-2,4-dichloropyrimidine36 (9.1 g, 40
mmol) added dropwise, while maintaining the temperature
below 25 °C. After being stirred for 18 h at rt, the reaction
mixture was filtered to remove precipitated NaCl and thor-
oughly washed with toluene. The filtrate was then evaporated
under reduced pressure to give an oil which solidified upon
cooling. The crude solid was then recrystallized in EtOH to
afford 19 as a white crystalline solid (16 g, 91%): mp 88-90
°C (lit.39 mp 89-91 °C); 1H NMR (CDCl3) δ 5.40 (s, 2 H), 5.48
(s, 2 H), 7.32-8.48 (m, 10 H), 8.35 (s, 1 H); 13C NMR (CDCl3)
δ 69.1, 69.7, 98.4, 127.7, 128.0, 128.1, 128.2, 128.5, 128.6, 135.5,
136.1, 159.4, 159.5, 163.5, 166.2.
1-[4-(Uracil-5-yl)imidazol-1-yl]-1-â-D-ribofuranose-2,3,5-
triol (5). A mixture of 21 (200 mg, 0.26 mmol), palladium (10%
Pd/C, 200 mg), and ammonium formate (300 mg) in EtOH (50
mL) was heated under reflux for 2 h.10 The solvent was then
removed under reduced pressure and the residue purified by
column chromatography eluting with EtOAc/acetone/EtOH/
H2O (7:1:1:0.5) to give 5 as a white crystalline solid (71 mg,
88%): mp 239-242 °C; 1H NMR (DMSO-d6) δ 3.49 (dd, 4.5
Hz, 12.3 Hz, 1 H), 3.55 (dd, 5.0 Hz, 12.3 Hz, 1 H), 3.86 (br d,
3.3 Hz, 1 H), 4.0 (dd, 4.0 Hz, 6.0 Hz, 1 H), 4.11 (br t, 5.1 Hz,
1 H), 5.00 (br t, 4.8 Hz, 1 H), 5.14 (br d, 3.9 Hz, 1 H), 5.36 (br
d, 5.7 Hz, 1 H), 5.52 (d, 6.0 Hz, 1 H), 7.67 (s, 1 H), 7.81 (s, 1
H), 7.86 (s, 1 H). 11.0 (s, 1 H), 112.2 (s, 1 H); 13C NMR (DMSO-
d6) δ 61.3, 70.3, 75.1, 85.2, 89.3, 107.3, 114.5, 133.5, 135.7,
136.3, 150.5, 162.2. Anal. Calcd for C12H14N4O6‚0.6H2O: C,
44.88; H, 4.80; N, 17.38. Found: C, 45.14; H, 4.60; N, 17.03.
1-[4-(2,4-Diaminopyrimidin-5-yl)imidazol-1-yl]-1-â-D-ri-
bofuranose-2,3,5-triol (4). In a Parr bomb, anhydrous am-
monia gas was bubbled in an anhydrous methanolic solution
of 21 (1.0 g, 1.3 mmol in 50 mL of MeOH) at -78 °C for 5 min.
The bomb was sealed and heated at 170 °C for 96 h. After the
mixture was cooled to 0 °C, the solvent was removed under
reduced pressure. The residue was purified by column chro-
matography eluting with 8% EtOH in CH2Cl2 to give 22 (398
mg, 52%) as a colorless syrup, which was used directly in the
next step: 1H NMR (CDCl3) δ 3.54 (dd, 2.4 Hz, 10.4 Hz, 1 H),
3.70 (dd, 2.8 Hz, 10.4 Hz, 1 H), 4.09 (dd, 2.5 Hz, 4.5 Hz, 1 H),
4.16 (br t, 4.8 Hz, 1 H), 4.33-4.36 (m, 1 H), 4.39 (d, 12.0, 1 H),
4.47-4.68 (m, 6 H), 5.06 (br s, 2 H), 5.66 (br s, 2 H), 5.74 (d,
6.4 Hz, 1 H), 7.01 (s, 1 H), 7.10-7.35 (m, 15 H), 7.60 (s, 1 H),
7.78 (s, 1 H); 13C NMR (CDCl3) δ 70.2, 72.7, 73.0, 74.0, 76.9,
81.7, 82.9, 89.2, 102.6, 110.4, 127.3, 127.8, 128.0, 128.1, 128.2,
1618 J. Org. Chem., Vol. 70, No. 5, 2005

“Molecular Chameleons”
128.3, 128.4, 128.5, 128.7, 128.8, 129.0, 135.3, 136.9, 137.4,
137.5, 138.9, 152.3, 161.2, 161.4.
ammonium formate and subsequently purified by column
chromatography eluting with EtOAc/acetone/EtOH/H2O (4:1:
1:0.5) to give 3 as a white powder (42 mg, 41% for two steps):
1H NMR (D2O) δ 3.61 (dd, 3.6 Hz, 12.0 Hz, 1 H), 3.71 (dd, 3.2
Hz, 12.0 Hz, 1 H), 4.06 (q, 3.2 Hz, 1 H), 4.19, (dd, 3.6 Hz, 4.2
Hz, 1 H), 4.33 (dd, 1 H), 5.68 (d, 3.9 Hz, 1 H), 7.39 (s, 1 H),
7.56 (s, 1 H), 7.89 (s, 1 H), 8.23 (s, 2H); 13C NMR (D2O) δ 61.7,
70.3, 75.0, 85.1, 89.5, 101.1, 115.1, 134.2, 137.7, 158.5, 165.1,
171.1.
1-[4-(2-Aminopyrimidin-5-yl-4-one)imidazol-1-yl]-1-â-
D-ribofuranose-2,3,5-triol (2). A solution of 4 (14 mg, 0.05
mmol) and saturated aqueous NaHSO3 (5 mL) was heated at
60 °C for 10 h.37 The solution was evaporated under reduced
pressure to afford the crude product, which was purified by
preparative TLC eluting with EtOAc/acetone/EtOH/H2O (4:1:
1:1) to obtain 2 as a white powder (12.4 mg, 88%): 1H NMR
(D2O) δ 3.65 (dd, 3.6 Hz, 11.2 Hz, 1 H), 3.76 (dd, 3.3 Hz, 12.1
Hz, 1 H), 4.05-4.08 (m, 1 H), 4.13, (dd, 3.6 Hz, 4.2 Hz, 1 H),
4.31 (dd, 1 H), 5.99 (d, 1 H), 6.61 (s, 1 H), 7.46 (s, 1 H), 8.05 (s,
1 H); 13C NMR (D2O) δ 60.6, 69.3, 75.5, 85.4, 90.7, 103.1, 121.1,
132.9, 136.6, 153.9, 163.5, 168.4.
In a similar fashion as was used to obtain 5, the desired
triol 4 was obtained in 88% yield (156 mg) as a white powder
following deprotection of 22 with Pd/C and ammonium for-
mate: mp 268-272 °C. 1H NMR (DMSO-d6) δ 3.72 (dd, 3.6
Hz, 12.0 Hz, 1 H), 3.80 (dd, 3.2 Hz, 12.0 Hz, 1 H), 4.05 (q, 3.2
Hz, 1 H), 4.20, (dd, 3.6 Hz, 4.2 Hz, 1 H), 4.27 (br t, 4.2 Hz, 1
H), 5.64 (d, 5.6 Hz, 1 H), 7.56 (s, 1 H), 7.91 (s, 1 H), 7.96 (s, 1
H); 13C NMR (DMSO-d6) δ 61.4, 70.4, 75.1, 85.4, 89.4, 100.6,
111.0, 135.5, 137.5, 150.4, 160.3, 160.5. Anal. Calcd for
C12H16N6O4‚1H2O: C, 45.42; H, 5.40; N, 26.48. Found: C,
45.71; H, 5.22; N, 26.26.
1-[4-(4-Aminopyrimidin-5-yl-2-one)imidazol-1-yl]-1-â-
D-ribofuranose-2,3,5-triol (3). To a solution of 22 (850 mg,
1.46 mmol) in a 1:1 mixture of THF and H2O (30 mL) was
added sodium nitrite (486 mg, 7.03 mmol), followed by glacial
acetic acid (0.62 mL, 10.69 mmol). The mixture was heated to
60 °C and stirred for 2 h, at which point the TLC showed no
traces of starting material. The solution was cooled to rt,
neutralized with concentrated NH4OH (1 mL), and evaporated
under reduced pressure. The crude product was used directly
in the next step.
In a similar fashion as was used for the deprotection of 21
to get 5, the benzyl groups were deblocked with Pd/C and
JO048218H
J. Org. Chem, Vol. 70, No. 5, 2005 1619