Design and synthesis of new C-nucleosides as potential adenosine deaminase inhibitors

Article abstract of DOI:10.1016/j.tet.2010.10.005

A number of new pyrazolo[3,4-c] and [4,3-b]pyridine C-nucleosides, which can be viewed as 4- or 6-deazaformycin analogues were synthesized and examined as potential adenosine deaminase (ADA) inhibitors. The compounds were prepared through the condensation

Full text of DOI:10.1016/j.tet.2010.10.005

Tetrahedron 66 (2010) 9620e9628
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Tetrahedron
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Design and synthesis of new C-nucleosides as potential adenosine deaminase
inhibitors
Tony Tite a, Nikolaos Lougiakis a, Vassilios Myrianthopoulos a, Panagiotis Marakos a, Emmanuel Mikros a,
,
b
b
b
Nicole Pouli a , Roxane Tenta , Elisabeth Fragopoulou , Tzortzis Nomikos
*
a Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens, Panepistimiopolis, 15771 Zografou, Greece
b Department of Science of Nutrition-Dietetics, Harokopio University,70 El. Venizelou Street, Athens 17671, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 June 2010
Received in revised form 10 September 2010
Accepted 4 October 2010
Available online 4 November 2010
A number of new pyrazolo[3,4-c] and [4,3-b]pyridine C-nucleosides, which can be viewed as 4- or
6-deazaformycin analogues were synthesized and examined as potential adenosine deaminase (ADA)
inhibitors. The compounds were prepared through the condensation of a suitably substituted, lithiated
2- or 4-methylpyridine with tri-O-benzyl-D-ribonolactone, followed by borohydride reduction of the
resulting hemiacetals, intramolecular Mitsunobu cyclisation of the derived diols, formation of the pyr-
azolopyridine ring system and subsequent removal of the protecting groups. These derivatives were
designed on the structural basis provided by docking simulations performed within the enzyme catalytic
site, however they demonstrated weak ADA inhibitory activity. Theoretical calculations assisted in the
interpretation of the obtained biological data, thus providing guidance for rational structural modifica-
tions within this molecular scaffold.
Keywords:
Pyrazolopyridine
Nucleosides
Mitsunobu
Enthalpy of hydration
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
ADA is ubiquitous to almost all human tissues, regulating both
intra- and extra-cellular adenosine concentrations, and conse-
Adenosine deaminase (ADA), a monomeric, 41KDa zinc metal-
loenzyme, plays a key role in purine metabolism and catalyzes
the irreversible hydrolytic degradation of both adenosine and
20-deoxyadenosine to the corresponding inosine nucleosides and
ammonia, probably via a tetrahedral high energy complex in-
termediate (Scheme 1).1 The rate of the deamination reaction is
accelerated by approximately 12 orders of magnitude, in the
presence of the enzyme.2
quently, a decrease or increase in its levels triggers several patho-
logical conditions. For instance, genetic deficiency of ADA results in
a severe combined immunodeficiency disease (SCID) caused by the
accumulation of lymphotoxic adenosine and 20-deoxyadenosine in
B- and T-cells.3 On the other hand, ADA is an essential enzyme for
vital homeostasis, as adenosine plays a crucial role in the differ-
entiation and maturation of the immune system and, additionally it
is considered as an important factor in the attenuation of in-
O
NH2
N
H2N OH
N
N
N
N
HN
N
HN
H O (activated by zinc)
2
N
N
N
N
+
NH
3
O
O
O
HO
HO
HO
H
H
H
R (H, OH)
HO
R (H, OH)
HO
R (H, OH)
HO
Scheme 1. Deamination of heteroaromatic compounds via an unstable hydrated intermediate.
flammation, signalled through the A2a adenosine receptor.4
Therefore, immunosuppressive ADA inhibitors have already been
proposed for the treatment of lymphoproliferative malignancies,
they demonstrate therapeutic potential for the treatment of
* Corresponding author. Tel.: þ30 210 7274185; fax: þ30 210 7274747; e-mail
address: pouli@pharm.uoa.gr (N. Pouli).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.10.005

T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
9621
cerebral and myocardial ischemia,5 and could also serve as novel
anti-inflammatory drugs, presenting limited side effects.6 Fur-
thermore, it is known that ADA inhibitors can prevent the meta-
bolic breakdown, and thus exhibit synergism with adenosine-based
antiviral and antitumour drugs.7 A number of ADA inhibitors with
various degrees of potency have been reported.8 They are generally
grouped into two main classes designated as ‘transition-state in-
hibitors’, and ‘ground-state inhibitors’. Current ‘transition-state
inhibitors’, including pentostatin, the only ADA inhibitor in clinical
use, suffer from poor pharmacokinetics, and moreover they bind to
the enzyme so tightly that their activity is nearly irreversible, giving
rise to many toxic effects.9 On the other hand, the usually rapid
metabolism of ‘ground-state inhibitors’ allows a fast recovery of the
ADA catalytic activity, which eventually leads to poor therapeutic
results.10 In recent years research efforts are mainly focused on the
better understanding of ADA-ligand interactions11 and the design
of new derivatives presenting improved metabolic and pharma-
cokinetic profiles.8b,10
promising ADA inhibitors, endowed with a fairly good inhibitory
activity (IC50 M), demonstrating an 18-fold affinity improvement
5
m
for the enzyme, compared to the structurally related analogue
nebularine.13
In this respect and in conjunction with molecular modelling
studies, we have planned the replacement of the pyrimidine het-
erocyclic core of deaminoformycin by pyridine, in order to evaluate
the chemical and biological properties of the resulting new pyr-
azolo[3,4-c] or [4,3-b]pyridine C-nucleoside derivatives. The in-
troduction of a small 5-amino substitution was also explored, in
accordance with previous observations concerning the molecular
recognition pattern of the enzyme.14
2. Results and discussion
2.1. Molecular modelling
As a first step, docking calculations of the novel nucleosides 16,
17 (Scheme 3), 33 and 34 (Scheme 4) within the ADA catalytic site
were carried out. Results strongly suggested that the designed
chemical modifications would possibly enhance stabilizing in-
teractions between the novel analogues and specific residues be-
longing to the enzyme active site. The theoretical lowest energy
As a continuation of an ongoing medicinal chemistry project
towards the design and synthesis of new metabolically stable
C-nucleoside antibiotics,12 we have focused our efforts on the
exploitation of the deazaformycin nucleoside scaffold for the de-
velopment of potential ADA inhibitors, using deaminoformycin as
lead compound (Scheme 2). This C-nucleoside is one of the most
structures revealed
a
favourable binding mode for all four
7
1
7a
NH
N
N
6 N
N
N
2
N
NH
H
5
N
N
3a
3
4 N
N
O
O
O
HO
HO
HO
H
H
OH
OH
OH
nebularine
HO
HO
HO
deaminoformycin
Scheme 2. Structures of deaminoformycin and nebularine.
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
a
N
N
N
N
b
c
R
R
R
R
CH3
O
O
OH
BnO
BnO
BnO
OH
OH
H
OBn
OBn
OBn
BnO
BnO
BnO
1 R= H
2 R= NHBoc
4 R= H
5 R= NHBoc
6 R= H
7 R= NHBoc
8 R= H
9 R= NHBoc
d
R
Ac
N
N
NH
N
NH
N
N
N
N
f
e
N
H
R
R
R
O
BnO
O
O
O
BnO
HO
BnO
H
H
H
NH
N
OBn
BnO
OBn
OH
OBn
BnO
HO
BnO
16 R= H
17 R= NH
12 R= H
14 R= NHBoc
13 R= H
15 R= NHBoc
10 R= H
11 R= NHBoc
2
Scheme 3. Reagents and conditions:(a) (i) n-BuLi, THF, ꢀ78 ꢁC. (ii) 2,3,5-tri-O-benzyl-D-ribonolactone (3), THF, ꢀ30 ꢁC, 43% (for 4), 25% (for 5); (b) NaBH4, MeOH, rt, 2 h, 93% (for 6),
80% (for 7); (c) PPh3, DEAD, reflux 90 min, 40% (for 8), 38% (for 9); (d) AcOK, Ac2O, isoamyl nitrite, toluene dry, reflux, 15 h; (e) NH3, dry CH3OH, rt, 12 h, 40e70% (two steps); (f) BCl3,
CH2Cl2, ꢀ78 ꢁC, 2 h 60e70%.

9622
T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
NHR'
NH2
NHCOCF3
NHCOCF3
a
d
b
R
N
R
N
R
N
O
R
N
CH3
OH
O
BnO
BnO
BnO
OH
OH
H
OBn
BnO
OBn
BnO
OBn
BnO
22 R= R'= H
23 R= NHBoc, R'= COCF3 (25%)
24 R= NHBoc, R'= H (65%)
18 R= H
19 R= NHBoc
20 R= H
21 R= NHBoc
25 R= H
26 R= NHBoc
c
e
Ac
N
NHCOCH3
NH
NH
N
N
N
g
h
f
R
N
R
N
R
N
R
N
O
O
O
BnO
BnO
O
HO
H
H
H
BnO
H
OBn
OBn
BnO
BnO
OH
HO
OBn
BnO
33 R= H
34 R= NH2
31 R= H
32 R= NHBoc
29 R= H
30 R= NHBoc
27 R= H
28 R= NHBoc
Scheme 4. Reagents and conditions: (a) (i) n-BuLi, THF, ꢀ78 ꢁC, (ii) 2,3,5-tri-O-benzyl-D-ribonolactone (3), THF, ꢀ30 ꢁC 44e55%; (b) NaBH4, MeOH, rt, 2 h 80%; (c) K2CO3, MeOH,
H2O, 60 ꢁC, 15 h, 65%; (d) PPh3, DEAD, reflux 90 min; 77e89%; (e) Ac2O, CH2Cl2, 15 h, rt 90%; (f) AcOK, Ac2O, isoamyl nitrite, toluene dry, reflux; (g) NH3, dry CH3OH, rt; 85% (two
steps); (h) BCl3, CH2Cl2, ꢀ78 ꢁC 79e96%.
compounds, indicating potential interaction partners within ADA
binding pocket and thus supporting the proposed modifications
(Fig. 1).
The coupling of the lithiated derivative of 1 or 2 with 2,3,5-tri-O-
benzyl- anomeric mix-
-ribonolactone (3)18 provided a 6:1,
D
a/b-D
ture of the hemiacetals 4 (43%) and 5 (25%), respectively. A small
amount of a side product is also produced, resulting from the attack
of the anion formed on the acetamide methyl upon the lactone 3.
The unambiguous structural elucidation of compounds 4 and 5 was
made through the examination of the corresponding HMBC spec-
troscopic data, where the protons of the acetamide methyl group
correlate with the carbonyl and at the same time, the methylene
group possesses a strong correlation peak with both the anomeric
carbon and the aromatic carbons 4 (2J coupling) and 5 (3J coupling).
The hemiacetals were then reduced with sodium borohydride into
their corresponding S/R diols 6 (93%) and 7 (80%). Very good dia-
stereoselectivity was observed in this reduction step, with a ster-
eofacial preference for the R epimer (ratio S/R: 1/7 for 6 and 1/6 for
7, respectively, as indicated by 1H NMR data). Intramolecular Mit-
sunobu cyclisation of the diols 6 and 7 provided an inseparable
The N(1)H, which appears in the predominant tautomeric form
of the molecule, is positioned in close proximity to the side chain
carboxylate of Asp296, forming a strong hydrogen bond with it.
Conversely, according to calculations, the N(2)H tautomer is not
expected to form a similar interaction, since its orientation is not
optimal for the formation of a hydrogen bond with the aforemen-
tioned aspartate residue. This observation is in full agreement with
the experimental binding mode of a transition state purine ana-
logue as it appears in the crystal structure of murine ADA.15 Fur-
thermore, the introduction of an amino group at position 5 was
strongly encouraged by calculations. Position 5 of the nucleoside is
oriented towards the side chain of Glu217 and thus, as modelling
suggested, the amine moiety of the analogues could form a hydro-
gen bond with the glutamate carboxylate. This modification was
also in accordance with existing SAR data indicating that the in-
troduction of an amine at this particular position favours affinity,
however in an inversely proportional fashion to the bulk of the
amine substituent.14 Finally, the conversion of the heterocyclic ni-
trogen of position 4 into carbon was justified by calculations as
potentially favourable, as this part of the molecule is oriented to-
wards a rather hydrophobic sub-site of the binding pocket. This part
of the pocket is comprised by the side chains of Leu62, Gly184 and
Leu58 and, as a result, a hydrophobic carbon atom could possibly be
accommodated in such an environment under more favourable
terms than the corresponding polar nitrogen. The permutation of
the carbon atom between positions 4 and 6 was finally performed in
order to fully elucidate the biological functionality of N(6).
mixture of the
a
,
b-C-glycosides 8 (ratio
a/b 1/7) and 9 (ratio a/b
1/2.5) in moderate yield (40%). The
a,b
anomeric mixture of each
derivative was treated with isoamyl nitrite in toluene at reflux,19 in
the presence of acetic anhydride and potassium acetate, to give the
rearranged intermediate N-nitroso compounds, giving a mixture of
1- and 2-acetylpyrazolopyridines (10 and 11, respectively). Sub-
sequent methanolic ammonia deacetylation of 10 and 11, resulted
in the
b
-pyrazolopyridine C-nucleosides 12 and 14, which were
isomeric counterparts 13 and
separated from the corresponding
a
15 by column chromatography (50e70% isolated yield). The ste-
reochemistry of the compounds was unambiguously determined
on the basis of NOE data, where a strong correlation peak between
the H-10 and H-40 was indicative of the
b anomers. Concomitant
removal of the protective groups with boron trichloride in
a dichloromethane solution yielded the desired 4-deazaformycin
analogue 16, as well as the 5-amino derivative 17.
2.2. Synthesis
For the synthesis of the pyrazolo[3,4-c]pyridine analogues, we
used as starting material the previously prepared 3-acetamido-4-
methylpyridine (1)16 and the corresponding 6-tert-butyl carbamate
217 (Scheme 3).
In order to get access to the pyrazolo[4,3-b]pyridine analogues
the 3-trifluoroacetamido-2-methyl pyridine (18) as well as the 6-
carbamate derivative 19 were used as starting materials (Scheme 4).
The trifluoracetamide 18 was prepared upon diazotisation of the

T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
9623
R-aminodiols 24 (65% yield) together with the trifluoroacetamide
derivative 23 (25% yield). This latter can be easily and selectively
deprotected to 24 in mild basic media. The aminodiols 22 and 24
were subjected to intramolecular Mitsunobu cyclisation and were
converted successively into the corresponding
b-C-glycoside de-
rivatives 25 (77% yield) and 26, respectively and then to the acetates
27 and 28 (89% yield two steps). The unambiguous NMR character-
ization of these analogues (25e28) proved to be more convenient
when performed on the acetates.
The acetates 27 and 28 were refluxed in toluene with isoamyl
nitrite in the presence of acetic anhydride and potassium acetate, as
already described, to give the b-1-acetylpyrazolopyridines 29 and
30, respectively. The acetyl groups were easily cleaved upon
treatment of 29 or 30 with methanolic ammonia, and the protective
groups were then removed with boron trichloride in a dichloro-
methane solution to yield the target b-C-nucleosides 33 and 34.
2.3. Assessment of the bioactivity
The compounds were tested regarding the inhibition of calf
spleen ADA and the results are presented in Table 1. Several con-
centrations were tested ranging from 0.01 to 1 mM. The com-
pounds proved to be very weak inhibitors of ADA, even in high
concentrations and therefore Ki determinations were not per-
formed. Nevertheless, for each pair of isomeric pyrazolopyridines, it
is the 5-aminosubstituted analogue that appears to be the more
potent inhibitor of the enzyme.
Table 1
Compounds inhibitory activities against calf spleen ADA
Compound
C (
mM)
% Inhibition
16
17
33
34
319
237
295
237
8.2
18
19
25
Fig. 1. (a) Superposition of the theoretical binding mode of compound 17 and the ex-
perimental binding geometry of 6-hydroxyl-1,6-dihydropurine ribonucleoside (yellow-
pdb id: 2ADA) within the catalytic site of murine adenosine deaminase. The coordinated
zinc cation is visible as a sphere between His15, His17, His214 and Asp295. The protein is
depicted in a ribbon representation. The nearly-identical interaction mode of the two
molecules is fairly reasonable since both compounds share a high degree of structural
similarity. (b) The proposed binding mode of compound 17, as determined by docking
calculations within the catalytic site of murine ADA. Two hydrogen bonds (yellow
dashed lines) stabilize the pyrazolopyridine ring of the nucleoside inside the protein.
Those bonds are formed between the 5-amino group and the side chain of Glu217 and
between the N1(H), which appears in the dominant tautomeric form of 17 and the side
chain of Asp296. Two additional bonds are formed between the sugar moiety and the
residues Asp19 and Ser103. The hydrophobic sub-site of the pocket, which is formed by
the side chains of Leu58, Leu62 and Gly184 (not visible) is oriented towards position 4 of
the heteroaromatic system and hydrophobic interactions are depicted in green dashed
lines.
According to results obtained by initial docking calculations
(Section 2.1), it was anticipated that the new C-nucleoside de-
rivatives could ultimately present enhanced binding affinity to-
wards ADA. However, the biological data (Table 1) did not confirm
this hypothesis. These rather unexpected results prompted us to
investigate alternative factors that could possibly influence the
biological activity of the new compounds. Some structurally related
triazolotriazine nucleoside derivatives possessing strong ADA in-
hibitory activity were previously studied by the use of theoretical
calculations.14 Reported results suggested that the biological ac-
tivity of nebularine-related derivatives could be partially attributed
to the relative stability of their corresponding covalent hydrated
transition state analogues based on calculated values for stabilities.
However, it is still unclear whether binding of the nucleoside
framework or the tendency of these molecules to undergo the
hydration reaction is predominant for the activity. Consequently,
the newly synthesized nucleosides were evaluated with respect to
their potential to form stable hydrates. A theoretical study (Section
4.2) was undertaken using high-level quantum mechanical calcu-
lations. A full vibrational and thermochemical analysis was per-
formed for the nucleosides and their corresponding hydrates, as
well as for the molecule of water at the restricted HF level of theory
using the triple zeta LACV3P basis set, augmented with full polar-
isation and diffusion functions. The enthalpy of hydration was
calculated for compounds 16, 17, 33 and 34 as well as for nebularine
and deaminoformycin at the HF/LACV3P**þþ level, in accordance
with the formalism followed in previous studies,14 as the difference
of heat of formation between reactants and products for the re-
action: nucleosideþwater/hydrated nucleoside.
known 6-amino-3-nitro-2-picoline,20 followed by chlorination,21
reduction and trifluoracetylation.22 Analogously, trifluoracetylation
of tert-butyl-N-(3-amino-2-methylpyridin-6-yl)carbamate17 pro-
vided 19.23 As already reported, concerning the lithiation of ami-
nosubstituted 2-picoline, it is preferable to use the trifluoracetamide
instead of the corresponding acetamide in order to avoid the for-
mation of a substantial amount of side-products.24 The lithiation of
the trifluoroacetamides 18 and 19 was accomplished according to
the above mentioned methodology to result in a 3:1 and a 8:1 a/b-D
anomeric mixture of the hemiacetals 20 (55%) and 21 (44%),
respectively. The borohydride reduction of the hemiacetals 20
afforded the corresponding R- and S-aminodiols 22 in 80% yield
(ratio R/S 1/3), whereas the reduction of 21 provided a mixture of the

9624
T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
In all cases the calculated enthalpy of hydration values were
J¼13.65 Hz), 3.07 (d, 1H, 4-CH2, J¼13.65 Hz), 3.34 (dd, 1H, H-50,
0
0
0
0
0
0
0
positive, ranging from 19.9 to 26.8 kcal/mol. The corresponding
values for nebularine and deaminoformycin were found to be
negative, suggesting that hydration of pyrazolopyridines is less
favoured.
These results could provide a strong indication that hydration is
a critical factor, concerning the ADA inhibitory profile of nucleoside
derivatives with an aglycone resembling to 6-unsubstituted pu-
rines, and cannot be effectively counterbalanced by moderate en-
hancement of intermolecular interactions between the enzyme and
the potential inhibitor.
J5 e4 ¼3.75 Hz, J5 e5 ¼10.59 Hz),0 3.39 (dd, 1H, H-5 , J5 e4 ¼3.75 Hz,
0
0
0
0
J5 e5 ¼10.59 Hz), 3.76 (d,1H, H-2 , J2 e3 ¼4.78 Hz), 3.90e3.95 (m, 1H,
H-30), 4.33e4.64 (m, 7H, 3ꢃCH2ePh, H-40), 6.77 (d, 1H, H-5,
J5e6¼5.12 Hz), 7.17e7.40 (m, 15H, 3ꢃC6H5), 8.15 (d, 1H, H-6,
J6,5¼5.12 Hz), 8.92 (s, 1H, NH, D2O exch.), 9.03 (s, 1H, H-2); 13C NMR
(50 MHz, CDCl3)
d
23.91 (CH3), 39.45 (CH2), 69.81 (C-50), 72.34,
72.43, 73.37 (CH2ePh), 76.52 (C-30), 78.24 (C-20), 80.72 (C-40),
104.54 (C-10), 126.44 (C-5), 127.60, 127.70, 127.86, 127.94, 128.13,
128.22, 128.46, 128.52 [CH(Ph)], 134.14 (C-3), 136.25, 136.56, 136.68,
137.25 [C(Ph), C-4], 144.69 (C-6), 145.46 (C-2), 168.59 (CO). Anal.
Calcd for C34H36N2O6: C, 71.81; H, 6.38; N, 4.93. Found: C, 71.57; H,
6.73; N, 5.14.
3. Conclusion
In conclusion, we have performed the synthesis of four new
C-nucleosides through the lithiation of suitable aminopicolines,
reduction, application of the Mitsunobu cyclodehydration and
elaboration of the pyrazolopyridine ring system. The compounds
were evaluated as ADA inhibitors and they proved to be very weak
inhibitors of ADA, in spite of the favourable ADA binding affinity
predicted by docking studies. This result strongly supports the
hypothesis of poor covalent hydrate stability observed in theoret-
ical studies, and argues in favour of the importance of the hydration
of the heterocyclic ring system, which seems to be the critical factor
for effective ADA inhibition, concerning these 7-unsubstituted
nucleoside derivatives.
4.1.2. tert-Butyl-N-[5-acetamido-4-(2,3,5-tri-O-benzyl-1-hydroxy-
a,
b-
D
-ribofuranosylmethyl)-pyridin-2-yl]carbamate (5). This com-
pound was prepared by a procedure analogous to that of 4, starting
from217 to resultin the hemiacetals 5 (yellowoil, 25%,
ratio 6:1, as
determined by 1H NMR). Data for the
-anomer: IR (film) nmax
3365e3290 (br), 1719, 1686, 1543, 1403, 1159 cmꢀ1 1H NMR
(400MHz, CDCl3) 1.55(s, 9H,(CH3)3), 2.08(s, 3H,CH3), 3.04(d,1H,4-
a/b
a
;
d
CH2, J¼13.74 Hz), 3.11 (d,1H, 4-CH2, J¼13.74 Hz), 3.32e3.42(m, 2H, H-
50), 3.83e3.89 (m, 2H, H-20, H-30), 4.33e4.38 (m,1H, H-40), 4.38e4.59
(m, 6H, 3ꢃCH2ePh), 7.20e7.41 (m, 15H, 3ꢃC6H5), 7.93 (s, 1H, H-3),
8.70 (s,1H, H-6), 8.78 (s,1H, NHAc, D2O exch.), 9.08(s,1H, NHBoc, D2O
exch.); 13C NMR (50 MHz, CDCl3)
d 24.01 (CH3), 28.39 [(CH3)3], 40.22
(CH2), 69.84 (C-50), 72.61, 72.95, 73.56 (CH2ePh), 76.88 (C-30), 78.75
(C-20), 80.67(C-40), 80.81[(CH3)3C],104.84(C-10),114.89(C-3),127.70,
128.00, 128.31, 128.53, 128.60, 128.66 [CH(Ph)], 129.39 (C-5), 136.84,
137.07, 137.51 [C(Ph)], 139.37 (C-4), 143.83 (C-6), 148.77 (C-2), 152.72
(OCONH), 168.72 (COCH3). Anal. Calcd for C39H45N3O8: C, 68.50; H,
6.63; N, 6.15. Found: C, 68.79; H, 6.44; N, 5.78.
4. Experimental
4.1. Chemistry
€
Melting points were determined on a Buchi apparatus and are
uncorrected. Optical rotations were obtained on a PerkineElmer
341 Polarimeter. FT-IR spectra were recorded on a PerkineElmer RX
1 FT-IR spectrometer. 1H NMR spectra and 2-D spectra were
recorded on a Bruker Avanche 400 instrument, whereas 13C NMR
spectra were recorded on a Bruker AC 200 spectrometer in deu-
4.1.3. N-[4-[(2RS,5S)-3,4,6-Tri-O-benzyl-2,5-dihydroxyhexane]pyr-
idin-3-yl]acetamide (6). To a solution of the hemiacetals 4 (510 mg,
0.89 mmol) in dry MeOH (15 mL) was added cautiously sodium
borohydride (84 mg, 2.24 mmol) and the mixture was stirred at
room temperature for 90 min. The reaction was quenched with ice-
water, neutralized with a 0.1 M HCl solution and extracted with
CH2Cl2 (3ꢃ50 mL) and AcOEt (2ꢃ50 mL). The combined organic
extracts were dried (Na2SO4) and concentrated to dryness. The res-
idue was purified by flash chromatography (silica gel) using a mix-
ture of CH2Cl2/MeOH 100/3 as the eluent to result in a mixture of the
diols 6 (480 mg, 93%) as a white foam. IR (film) nmax 3465e3170 (br),
1678, 1568, 1525, 1453, 1417, 1216 cmꢀ1; 1H NMR (400 MHz, CDCl3)
terated solvents and were referenced toTMS (d scale). The signals of
1H and 13C spectra were unambiguously assigned by using 2D NMR
techniques: 1H1H COSY, NOESY HMQC and HMBC. Flash chroma-
tography was performed on Merck silica gel 60 (0.040e0.063 mm).
Analytical thin layer chromatography (TLC) was carried out on
precoated (0.25 mm) Merck silica gel F-254 plates. Elemental
analyses were within ꢂ0.4% of the theoretical values.
4.1.1. N-[4-(2,3,5-Tri-O-benzyl-1-hydroxy-
a
,
b
-
D
-ribofur-
d
2.05 (s, 3H, CH3), 2.48 (m, 1H, H-10), 3.03 (dd, 1H, H-10,
0
0
0
0
0
0
0
anosylmethyl)pyridin-3-yl]acetamide (4). To a solution of 4-meth-
ylpyridine 116 (910 mg, 6.06 mmol) in dry THF (70 mL) at ꢀ78 ꢁC
was added under argon n-BuLi (9.30 mL, 15.16 mmol, 1.6 M solution
in hexanes), the resulting solution was stirred at ꢀ78 ꢁC for 15 min
and the temperature then raised to ꢀ25 ꢁC for 90 min. The solution
J1 e1 ¼13.54 Hz, J1 e2 ¼9.88 Hz), 3.65 (dd, 1H, H-6 , J6 e5 ¼4.76 Hz,
0
0
0
0
0
0
0
J6 e6 ¼9.51 Hz), 3.70 (dd, 1H, H-6 , J6 e5 ¼2.93 Hz, J6 e6 ¼9.51 Hz),
3.77e3.81 (m, 1H, H-30), 3.92 (dd, 1H, H-40, J4 e3 ¼1.46 Hz,
0
0
0
0
0
0
J4 e5 ¼8.40 Hz), 3.96e4.01 (m, 1H, H-5 ), 4.10e4.14 (m, 1H, H-2 ),
4.46e4.85 (m, 6H, 3ꢃCH2ePh), 6.95 (br s, 1H, H-5), 7.20e7.43 (m,
15H, 3ꢃC6H5), 8.15 (br s,1H, H-6), 8.99 (s, 1H, H-2), 9.29 (s, 1H, NH,
was cooled to ꢀ78 ꢁC and a solution of
D
-ribonolactone 318 (3.03 g,
7.28 mmol) in dry THF (20 mL) was added dropwise. The resulting
mixture was stirred at ꢀ78 ꢁC for 10 min and then at ꢀ20 ꢁC for 3 h.
A saturated ammonium chloride solution was then added to the
yellow solution to quench the excess n-BuLi. The solvent was vac-
uum-evaporated, water was added to the residue and then
extracted with CH2Cl2 (4ꢃ50 mL). The organic extracts were dried
(Na2SO4) and the solvent was evaporated under reduced pressure
to give an orange oil. Flash chromatography purification (silica gel)
using a mixture of CH2Cl2/MeOH 50/1 as the eluent afforded a light
yellow oil consisting of a mixture of the hemiacetals 4 (1.5 g, 43.5%),
D2O exch.); 13C NMR (50 MHz, CDCl3) 24.04 (CH3), 36.97 (C-10),
d
69.65 (C-50), 70.95 (C-60), 73.21 (C-20), 73.07, 73.58, 74.54 (CH2ePh),
79.99 (C-30), 80.58 (C-40), 125.51 (C-5), 128.08, 128.17, 128.23, 128.57,
128.60 [CH(Ph)], 134.38 (C-3), 137.34, 137.67, 137.74 [C(Ph)], 140.38
(C-4), 145.03 (C-2, C-6), 168.90 (CO). Anal. Calcd for C34H38N2O6: C,
71.56; H, 6.71; N, 4.91. Found: C, 71.35; H, 6.57; N, 4.79.
4.1.4. tert-Butyl-N-[5-acetamido-4-[(2RS,5S)-3,4,6-tri-O-benzyl-2,5-
dihydroxyhexane]pyridin-2-yl]carbamate (7). Following an analo-
gous procedure to that described for 6, the mixture of the diols 7
was obtained as transparent oil in 80% yield (chromatographic
purification was effected using a mixture of CH2Cl2/MeOH 40/1 as
the eluent). IR (film) nmax 3410e3147 (br), 1723, 1678, 1552, 1404,
with a clear predominance of the
termined by 1H NMR). Data for the
3400e3270 (br), 1694, 1568, 1518, 1416, 1116 cmꢀ1
(400 MHz, CDCl3) 2.06 (s, 3H, CH3), 2.90 (d, 1H, 4-CH2,
a
-anomer (
-anomer: IR (film) nmax
1H NMR
a/b ratio 6:1, as de-
a
;
d
1160 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d 1.53 (s, 9H, (CH3)3), 2.07 (s,

T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
9625
3H, CH3), 2.52e2.59 (m, 1H, H-10), 3.10 (dd, 1H, H-10, J¼13.70,
isoamyl nitrite (155
m
L, 1.13 mmol) was then added dropwise and
9.39 Hz), 3.66 (dd, 1H, H-60, J6 e5 ¼5.48 Hz, J6 e6 ¼9.78 Hz), 3.72 (d0d,
the resulting mixture was heated at 100 ꢁC for 16 h. The insoluble
material was filtered off, washed with hot toluene and the com-
bined filtrates were evaporated to dryness to afford an orange oil
corresponding to a mixture of the acetamides 10. A saturated
methanolic ammonia solution (30 mL) was added to the above
mixture and the resulting solution was stirred at room temperature
overnight. The solvent was removed in vacuo and the residue was
purified by flash chromatography (silica gel) using a mixture of
0
0
0
0
1H, H-60, J6 e5 ¼2.74 Hz, J6 e6 ¼9.78 Hz), 3.77e3.84 (m, 1H, H-3 ),
0
0
0
0
3.87 (dd, 1H, H-40, J4 e3 ¼1.56 Hz, J4 e5 ¼8.60 Hz), 4.02e4.10 (m, 1H,
H-50), 4.16e4.19 (m, 1H, H-20), 4.50e4.86 (m, 6H, 3ꢃCH2ePh),
7.25e7.39 (m, 15H, 3ꢃC6H5), 7.87 (s, 1H, H-3), 8.71 (s, 1H, H-6), 9.18
(s, 1H, NHAc, D2O exch.), 9.23 (s, 1H, NHBoc, D2O exch.); 13C NMR
0
0
0
0
(50 MHz, CDCl3) d
23.86 (CH3), 28.31 [(CH3)3], 37.46 (C-10), 69.31 (C-
50), 71.00 (C-60), 73.18 (CH2ePh), 73.34 (C-20), 73.41, 74.31
(CH2ePh), 79.98 (C-30), 80.76 [(CH3)3eC], 81.09 (C-40), 113.64 (C-3),
127.97, 128.07, 128.12, 128.49, 129.37 [CH(Ph), C-5], 137.34, 137.58,
137.66 [C(Ph)], 143.06 (C-4), 143.18 (C-6), 148.77 (C-2), 152.90
(OCONH), 168.78 (COCH3). Anal. Calcd for C39H47N3O8: C, 68.30; H,
6.91; N, 6.13. Found: C, 68.04; H, 7.17; N, 6.20.
cyclohexane/AcOEt 2/1 as the eluent to give the
(170 mg, 72%) and the -anomer 13 (24 mg, 10%) as light yellow oils.
b-anomer 12
a
4.1.8. Data for 3-(2,3,5-Tri-O-benzyl-b-D-ribofuranosyl)pyrazolo[3,4-
c]pyridine (12). [
a
;
]
22 ꢀ55 (c 0.41, MeOH); IR (film) nmax 3286, 1454,
D
1360, 1268, cmꢀ1
1H NMR (400 MHz, CDCl3)
d
3.606 (dd, 1H, H-50,
0
0
0
0
0 0
4.1.5. N-[4-(2,3,5-Tri-O-benzyl-
a,
b
-
D
-ribofuranosylmethyl)pyridin-3-
J5 e4 ¼3.75 Hz, J5 e5 ¼10.58 Hz), 3.79 (dd, 01H, H-5 , J5 4 ¼3.41 Hz,
0
0
0
yl]acetamide (8). Triphenylphosphine (470 mg, 1.78 mmol) was
added under argon to a solution of the diols 6 (510 mg, 0.894 mmol)
in dry THF (15 mL) and the resulting mixture was heated at reflux.
Diethylazodicarboxylate (0.27 mL, 1.78 mmol) was then added
dropwise at this temperature and heating was continued for 90 min.
The solvent was evaporated and the resulting oil was purified by
flash chromatography (silica gel) using a mixture of CH2Cl2/AcOEt 5/
2 as the eluent to give 8 (200 mg, 40%) as an oil. An adequate quantity
J5 e5 ¼10.58 Hz), 4.18e4.22 (m, 1H, H-3 ), 4.39 (dd, 1H, H-2 ,
0
0
0
0
0
J2 e1 ¼6.82 Hz, J2 e3 ¼5.47 Hz), 4.42e4.48 (m, 1H, H-4 ), 4.50e4.79
(m, 6H, 3ꢃCH2ePh), 5.57 (d, 1H, H-10, J1 e2 ¼6.82 Hz), 7.08e7.41 (m,
0
0
15H, 3ꢃC6H5), 7.74 (d, 1H, H-4, J4e5¼5.80 Hz), 8.06 (d, 1H, H-5,
J5e4¼5.80), 9.00 (s, 1H, H-7); 13C NMR (50 MHz, CDCl3)
d 70.33 (C-
50), 72.31, 72.41, 73.68 (CH2ePh), 77.51 (C-30), 78.43 (C-10), 81.10 (C-
20), 82.43 (C-40), 115.47 (C-4), 125.04 (C-3
[CH(Ph)], 134.85 (C-7), 137.41, 137.89 [C(Ph)], 138.00 (C-7
a
), 128.02, 128.28, 128.56
), 138.16
a
of pure
cation. Data for the
1415, 1275 cmꢀ1; 1H NMR (400 MHz, CDCl3)
b
-anomer could be isolated from the mixture for identifi-
-anomer: oil; IR (film) nmax 3318, 1693, 1519,
2.01 (s, 3H, CH3), 2.70
(C-5), 138.35 [C(Ph)], 144.38 (C-3). Anal. Calcd for C32H31N3O4: C,
73.68; H, 5.99; N, 8.06. Found: C, 73.81; H, 5.82; N, 7.89.
b
d
(dd, 1H, CH2, J¼6.14, 14.34 Hz), 3.03 (dd, 1H, CH2, J¼2.73, 14.34 Hz),
4.1.9. Data for 3-(2,3,5- tri-O-benzyl-
a
-
D
-ribofuranosyl)pyrazolo[3,4-
22
0
3.29 (dd,1H, H-50, J5 e4 ¼4.78 Hz, J5 e5 ¼10.24 Hz), 3.41 (dd,1H, H-5 ,
c]pyridine (13). [
a
]
ꢀ3 (c 0.25, MeOH); IR (film) nmax 3271, 1458,
0
0
0
0
D
J5 e4 ¼3.75 Hz, J5 e5 ¼10.24 Hz), 3.51 (dd, 1H, H-2 , J2 e1 ¼8.19 Hz,
1361,1264,1113 cmꢀ1; 1H NMR (400 MHz, CDCl3) 3.98 (dd,1H, H-50,
d
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
J2 e3 ¼5.46 Hz), 3.72 (dd, 1H, H-3 , J3 e2 ¼5.46 Hz, J3 e4 ¼3.07 Hz),
4.19e4.24 (m, 1H, H-40), 4.26e4.33 (m, 1H, H-10), 4.34e4.75 (m, 6H,
3ꢃCH2ePh), 6.79 (br s,1H, H-5), 7.17e7.45 (m,15H, 3ꢃC6H5), 8.21 (br
s, 1H, H-6), 8.80 (s, 1H, NH, D2O exch.), 8.98 (s, 1H, H-2); 13C NMR
J5 e4 ¼4.09 Hz, J5 e5 ¼10.24 Hz), 4.05 (dd, 1H, H-5 , J5 e4 ¼6.83 Hz,
0
0
J5 e5 ¼10.24 Hz), 4.09 (d, 1H, CH2ePh, J¼11.95 Hz), 4.26 (d, 1H,
CH2ePh, J¼11.95 Hz), 4.33e4.43 (m, 3H, H-20, H-30, H-40), 4.51e4.73
(m, 4H, 2ꢃCH2ePh), 5.49 (d, 1H, H-10, J1 e2 ¼4.77 Hz), 6.76e7.42 (m,
15H, 3ꢃC6H5), 7.98 (d, 1H, H-4, J4e5¼5.37 Hz), 8.05 (d, 1H, H-5,
0
0
(50 MHz, CDCl3) d
23.89 (CH3), 34.23 (CH2), 70.11 (C-50), 72.06, 72.42,
73.49 (CH2ePh), 76.39 (C-30), 78.91 (C-20), 81.21 (C-10), 82.22 (C-40),
125.71 (C-5), 128.11, 128.17, 128.30, 128.36, 128.44, 128.59, 128.61,
128.70 [CH(Ph)], 134.24 (C-3), 137.32 (C-4), 137.40, 137.91 [C(Ph)],
145.53 (C-6),146.24 (C-2),168.65 (CO). Anal. Calcd for C34H36N2O5: C,
73.89; H, 6.57; N, 5.07. Found: C, 73.62; H, 6.44; N, 5.26.
J5e4¼5.37 Hz), 8.96 (s,1H, H-7); 13C NMR (50 MHz, CDCl3)
d 69.92 (C-
50), 73.19, 73.60, 73.66 (CH2ePh), 77.67 (C-10), 78.61, 79.41, 80.47 (C-30,
C-20, C-40), 117.45 (C-4), 127.50, 127.62, 127.81, 127.96, 128.02, 128.05,
128.30 [CH(Ph)],128.53 (C-3
5), 137.86, 138.26 [C(Ph)], 138.71 (C-7
C32H31N3O4:C,73.68;H, 5.99;N, 8.06. Found:C,73.92;H, 5.78;N,7.96.
a),134.78 (C-7),137.57 [C(Ph)],137.66 (C-
a
), 142.91 (C-3). Anal. Calcd for
4.1.6. tert-Butyl-N-[5-acetamido-4-(2,3,5,tri-O-benzyl-a,b-D-ribofur-
anosylmethyl)pyridin-2-yl]carbamate (9). The anomeric mixture of 9
was prepared by a procedure analogous to that described for 8 in 38%
4.1.10. tert-Butyl-N-[3-(2,3,5-tri-O-benzyl-b-D-ribofuranosyl)pyr-
azolo[3,4-c]pyridin-5-yl]carbamate (14). Both anomers 14 and 15
were prepared by a procedure analogous to that of the corre-
sponding compounds 12 and 13, starting from the acetamides 9.
yield. Pure
b-anomer could be isolated from the mixture. Data for the
b
-anomer: oil; IR (film) nmax 3325, 1717, 1671, 1540, 1408, 1160 cmꢀ1
;
1H NMR (400 MHz, CDCl3)
d
1.56 (s, 9H, (CH3)3),1.99 (s, 3H, CH3), 2.74
(dd, 1H, CH2, J¼6.65, 14.08 Hz), 3.06 (dd, 1H, CH2, J¼3.13, 14.08 Hz),
4.1.11. Data for tert-butyl-N-[3-(2,3,5-tri-O-benzyl-
b-D-ribofuranosyl)
3.30 (dd,1H, H-50, J5 e4 ¼3.08 Hz, J5 e5 ¼10.57 Hz), 30.45 (dd,1H, H-5 ,
pyrazolo[3,4-c]pyridin-5-yl]carbamate (14). Oil; Yield: 63%; [
a]
D
22 ꢀ13
0
0
0
0
0
J5 e4 ¼3.53 Hz, J5 e5 ¼10.57 Hz), 3.61 (dd, 1H, H-2 , J2 e1 ¼5.09 Hz,
(c 0.14, MeOH); IR (film) nmax 3248, 1718, 1450, 1272, 1117 cmꢀ1; 1H
0
0
0
0
0
0
0
J2 e3 ¼7.44 Hz), 3.67 (dd, 1H, H-3 , J3 e2 ¼5.09 Hz, J3 e4 ¼3.52 Hz),
NMR (400 MHz, CDCl3) d
1.53 (s, 9H, (CH3)3), 3.62e3.68 (m,1H, H-50),
0
0
0
0
0
0
4.16e4.23 (m, 1H, H-40), 4.33 (dd, 1H, H-10, J1 e2 ¼5.09, 7.40 Hz),
4.37e4.67 (m, 6H, 3ꢃCH2ePh), 7.18e7.45 (m, 15H, 3ꢃC6H5), 7.88 (s,
1H, H-3), 8.37 (s, 1H, H-6), 8.61 (s,1H, NHBoc, D2O exch.), 8.66 (s,1H,
3.73e3.78 (m,1H, H-50), 4.14e4.17 (m,1H, H-30), 4.37e4.42 (m,1H, H-
40), 4.50e4.67 (m, 7H, 3ꢃCH2ePh, H-20), 5.51 (d, 1H, H-10,
0
0
0
0
J1 e2 ¼6.26 Hz), 7.10e7.34 (m,15H, 3ꢃC6H5), 8.28 (s,1H, H-4), 8.52 (br
NHAc, D2O exch.); 13C NMR (50 MHz, CDCl3)
d
23.78 (CH3), 28.41
s, 1H, NHBoc, D2O exch.), 8.58 (s, 1H, H-7); 13C NMR (50 MHz, CDCl3)
[(CH3)3], 34.97 (CH2), 70.12 (C-50), 72.06, 72.72, 73.50 (CH2ePh),
76.55 (C-30), 79.42 (C-20), 80.97 [(CH3)3eC], 81.59 (C-10), 81.86 (C-40),
114.02 (C-3), 127.73, 127.99, 128.19, 128.29, 128.61 [CH(Ph)], 137.40,
137.57, 137.65, 137.73 [C(Ph), C-5], 141.36 (C-4), 144.24 (C-6), 148.84
(C-2), 152.60 (OCONH), 168.78 (COCH3). Anal. Calcd for C39H45N3O7:
C, 70.14; H, 6.79; N, 6.29. Found: C, 70.31; H, 6.57; N, 6.46.
d
28.43 [(CH3)3], 69.79 (C-50), 72.15, 72.33, 73.36 (CH2ePh), 77.71 (C-
10), 80.25(C-30), 80.34(C-40), 80.44 [(CH3)3eC], 81.81 (C-20),101.09(C-
4), 127.65, 127.69, 127.80, 127.94, 127.98, 128.16, 128.18, 128.38 [CH
(Ph)], 132.64 (C-7), 135.58 (C-7a), 137.69 (C-3a), 137.91 (C-3), 143.08,
143.62, 143.85 [C(Ph)], 152.73 (C-5), 153.65 (CO). Anal. Calcd for
C37H40N4O6:C, 69.79;H, 6.33;N,8.80. Found:C, 69.93;H, 6.18;N, 8.61.
4.1.7. 3-(2,3,5-Tri-O-benzyl-
dine (12). To a suspension of 8 (250 mg, 0.452 mmol) in dry toluene
b
-
D
-ribofuranosyl)pyrazolo[3,4-c]pyri-
4.1.12. Data for tert-butyl-N-[3-(2,3,5-tri-O-benzyl-
pyrazolo[3,4-c]pyridin-5-yl]carbamate (15). Oil; Yield: 12%; [
ꢀ24 (c 0.44, MeOH); IR (film) nmax 3263, 1722, 1458, 1264, 1152,
1117 cmꢀ1;1HNMR(400MHz, CDCl3)
1.55(s, 9H,(CH3)3), 4.00e4.08
a-D-ribofuranosyl)
22
a]
D
(30 mL) were added under argon AcOK (46 mg, 0.292 mmol) and
Ac2O (133
m
L, 1.35 mmol). The reaction mixture was heated at 80 ꢁC,
d

9626
T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
(m, 2H, H-50), 4.19e4.25 (m, 1H, H-30), 4.35e4.38 (m, 1H, H-40),
4.40e4.44 (m, 1H, H-20), 4.57e4.78 (m, 6H, 3ꢃCH2ePh), 5.53 (d, 1H,
3.75e3.78 (m, 1H, H-30), 3.92 (d, 1H, H-20, J3 e2 ¼4.78 Hz),
4.29e4.34 (m, 1H, H-40), 4.34e4.62 (m, 6H, 3ꢃCH2ePh), 4.94 (s, 1H,
OH, D2O exch.), 7.04 (br s,1H, NHBoc, D2O exch.), 7.07e7.40 (m,15H,
3ꢃC6H5), 7.77 (d, 1H, H-5, J5e4¼8.88 Hz), 7.89 (d, 1H, H-4,
J4e5¼8.88 Hz), 10.04 (s, 1H, NHCOCF3, D2O exch.); 13C NMR (50
0
0
H-10, J1 e2 ¼5.47 Hz), 6.90e7.40 (m, 15H, 3ꢃC6H5), 8.41 (s, 1H, H-4),
0
0
8.85 (br s,1H, NHBoc, D2O exch.), 8.89 (s,1H, H-7); 13C NMR (50 MHz,
CDCl3)
d
28.49 [(CH3)3], 69.76 (C-50), 73.16, 73.65, 73.75 (CH2ePh),
75.37 (C-10), 78.88 (C-30), 78.96 (C-40), 79.04 (C-20), 80.07 [(CH3)3eC],
MHZ, CDCl3) d
28.38 [(CH3)3], 42.85 (CH2), 69.43 (C-50), 72.74, 72.88,
102.22 (C-4), 127.72, 127.94, 128.11, 128.28, 128.45, 128.61 [CH(Ph)],
131.31 (C-7),132.20 (C-7a),133.32 (C-3a),137.44 (C-3),137.75,137.86,
138.14 [C(Ph)],152.36 (C-5),152.71 (CO). Anal. Calcd for C37H40N4O6:
C, 69.79; H, 6.33; N, 8.80. Found: C, 69.65; H, 6.06; N, 8.99.
73.54 (CH2ePh), 76.89 (C-30), 77.90 (C-20), 80.62 (C-40), 81.29
[(CH3)3eC], 104.80 (C-10), 107.38, 113.20, 118.93, 124.66 (CF3), 110.66
(C-5), 126.86 (C-3), 127.73, 127.81, 127.99, 128.26, 128.54, 128.64 [CH
(Ph)], 133.72 (C-4), 136.99, 137.21, 137.54 [C(Ph)], 146.85 (C-2),
148.78 (C-6), 152.03 (OCONH), 154.32, 155.06, 155.80, 156.55
(COCF3). Anal. Calcd for C39H42F3N3O8: C, 63.49; H, 5.74; N, 5.70.
Found: C, 63.18; H, 5.90; N, 5.51.
4.1.13. 3-(b-D-Ribofuranosyl)pyrazolo[3,4-c]pyridine (16). A solution
of BCl3 (1.61 mL,1 M in hexane) was added dropwise under argon at
ꢀ78 ꢁC to a solution of 12 (70 mg, 0.134 mmol) in dry CH2Cl2
(10 mL). The reaction mixture was stirred at ꢀ78 ꢁC for 90 min and
then quenched with a cold solution of CH2Cl2/MeOH (1/2). The
solvents were evaporated and the residue was purified by flash
chromatography (silica gel), using a mixture of CH2Cl2/MeOH 8.5/
4.1.17. 2-[(2RS,5S)-2,5-Dihydroxy-3,4,6-tri-O-benzylhexane]pyridin-
3-yl-amine (22). This compound was prepared by a procedure
analogous to that of 6, starting from 20. Yield 80%; oil; IR (film) nmax
3490e3186 (br), 1454, 1308, 1093 cmꢀ1; 1H NMR (400 MHz, CDCl30)
1.5 as the eluent to give pure 16 (25 mg, 74%) as a clear oil. [
a
]
22 ꢀ11
d
2.89 (dd,1H, H-10, J1 e2 ¼4.30 Hz, J1 e1 ¼14.87 Hz), 3.02 (dd,1H, H-1 ,
0
0
0
0
D
0
0
0
0
0
(c 0.09, MeOH); IR (film) nmax 3457e3170 (br), 1458, 1377,
J1 e2 ¼7.04 Hz, J1 e1 ¼14.87 Hz), 3.68e3.80 (m, 2H, H-6 ), 3.94e4.18
(br s, 2H, NH2, D2O exch.), 3.96e4.00 (m,1H, H-30), 4.01e4.08 (m,1H,
H-40), 4.11e4.18 (m, 1H, H-50), 4.51e4.88 (m, 7H, 3ꢃCH2ePh, H-20),
6.85e7.01 (m, 2H, H-5, H-4), 7.25e7.45 (m, 15H, 3ꢃC6H5), 7.86e7.91
1264 cmꢀ1 1H NMR (400 MHz, CD3OD) 3.76 (dd, 1H, H-50,
; d
0
0
0
0
0
0
0
J5 e4 ¼4.70 Hz, J5 e5 ¼11.74 Hz), 3.84 (dd, 1H, H-5 , J5 e4 ¼03.52 Hz,
0
0
0
J5 e5 ¼11.74 Hz), 4.05e4.09 (m, 1H, H-4 ), 4.20 (dd, 1H, H-3 , J¼4.30,
5.48 Hz), 4.38 (et, 1H, H-20, J¼6.26 Hz), 5.21 (d, 1H, H-10,
(m, 1H, H-6); 13C NMR (50 MHz, CDCl3)
d
36.64 (C-10), 70.11 (C-50),
J1 e2 ¼7.04 Hz), 8.02 (d, 1H, H-4, J4e5¼5.87 Hz), 8.19 (d, 1H, H-5,
70.60 (C-20), 71.52 (C-60), 73.01, 73.44, 74.13 (CH2ePh), 79.99 (C-30),
80.29 (C-40),122.28 (C-5),122.49 (C-4),125.85,127.32,127.65,127.74,
127.97, 128.16, 128.37 [CH(Ph)], 138.17 (C-6), 138.36, 138.58 [C(Ph)],
142.16 (C-3), 144.90 (C-2). Anal. Calcd for C32H36N2O5: C, 72.70; H,
6.86; N, 5.30. Found: C, 72.94; H, 7.07; N, 5.13.
0
0
J5e4¼5.87 Hz), 8.97 (s, 1H, H-7); 13C NMR (50 MHz, CD3OD)
d 63.53
(C-50), 72.90 (C-30), 76.89 (C-20), 80.48 (C-10), 87.04 (C-40), 116.82 (C-
4), 126.13 (C-3a), 135.70 (C-7), 135.90 (C-7a), 138.49 (C-5), 146.90
(C-3). Anal. Calcd for C11H13N3O4: C, 52.59; H, 5.22; N, 16.72. Found:
C, 52.36; H, 5.08; N, 16.49.
4.1.18. tert-Butyl-N-[3-amino-2-[(2RS,5S)-2,5-dihydroxy-3,4,6-tri-O-
benzylhexane]pyridin-6-yl]carbamate (24). This compound was
prepared by a procedure analogous to that of 6, starting from 21.
Chromatographic purification was made using a mixture of AcOEt/
CH2Cl2 1/4 as the eluent and provided first the trifluoroacetamide
diols 23 (25%) then the aminodiols 24 (65%) as light yellow oils.
4.1.14. 5-Amino-3-(b-D-ribofuranosyl)pyrazolo[3,4-c]pyridine
(17). This compound was prepared by a procedure analogous to that
of 16, starting from 14. Yield: 60%; oil; [
a
]
22 ꢀ21 (c 0.15, MeOH); IR
D
(film) nmax 3534e3093 (br), 1442, 1261, 1106 cmꢀ1
;
1H NMR
0
0
(400 MHz, CD3OD)
d
3.77 (dd, 1H, H-50, J5 e4 ¼4.69 Hz,
0
0
0
0
0
0
0
J5 e5 ¼12.13 Hz), 3.83 (dd, 1H, H-5 , J5 e4 ¼3.52 Hz, J5 e5 ¼12.13 Hz),
4.04e4.09 (m, 1H, H-40), 4.18 (dd, 1H, H-30, J3 e2 ¼5.48 Hz,
4.1.19. Data for tert-butyl-N-[3-trifluoroacetamido-2-[(2RS,5S)-2,5-
dihydroxy-3,4,6-tri-O-benzylhexane]pyridin-6-yl]carbamate (23). IR
0
0
0
0
0
0
0
0
0
J3 e4 ¼3.92 Hz), 4.37 (dd,1H, H-2 , J2 e3 ¼5.48 Hz, J2 e1 ¼7.04 Hz), 5.13
(d, 1H, H-10, J1 e2 ¼7.04 Hz), 7.56 (s, 1H, H-4), 8.66 (s, 1H, H-7); 13C
(film) nmax 3457e3178 (br), 1731, 1525, 1396, 1271, 1154 cmꢀ1 1H
;
0
0
NMR (50 MHz, CD3OD)
d
63.51 (C-50), 72.95 (C-30), 76.41 (C-20), 80.15
NMR (400 MHz, CDCl3) d
1.51 (s, 9H, (CH3)3), 2.75e2.86 (m,1H, H-10),
0
(C-10), 87.26 (C-40), 102.27 (C-4), 124.06 (C-7), 133.66 (C-7
(C-3 ), 144.99 (C-3), 148.36 (C-5). Anal. Calcd for C11H14N4O4: C,
49.62; H, 5.30; N, 21.04. Found: C, 49.78; H, 5.53; N, 20.96.
a), 134.40
3.16 (dd, 1H, H-10, J1 e1 ¼14.00 Hz, J1 e2 ¼8.20 Hz), 3.64 (dd, 1H, H-6 ,
0
0
0
0
0
0
0
0
0
0
0
a
J6 e5 ¼4.10 Hz, J6 e6 ¼9.56 Hz), 3.71 (0dd, 1H, H-6 , J6 e5 ¼2.050 Hz,
0
0
J6 e6 ¼9.56 Hz), 3.76e3.85 (m, 1H, H-3 ), 3.90e3.99 (m, 2H, H-4 , H-
50), 4.34e4.41 (m, 1H, H-20), 4.42e4.84 (m, 6H, 3ꢃCH2ePh),
7.18e7.40 (m, 16H, 3ꢃC6H5, NHBoc), 7.81 (d, 1H, H-5, J5e4¼8.87 Hz),
8.13 (d,1H, H-4, J4e5¼8.87 Hz),10.65 (br s,1H, NHCOCF3, D2O exch.);
4.1.15. N-[2-(2,3,5-Tri-O-benzyl-1-hydroxy-a,b-D-ribofuranosyl)
methylpyridin-3-yl]trifluoroacetamide (20). This compound was
prepared by a procedure analogous to that of 4, starting from 18.
13C NMR (50 MHz, CDCl3) 28.37 [(CH3)3], 39.41 (C-10), 69.73 (C-50),
d
Yield: 55%; Yellow syrup. Data for the predominant
a
-anomer: IR
;
70.94 (C-60), 72.23 (C-20), 73.05, 73.67, 74.81(CH2ePh), 79.48 (C-30),
80.31 (C-40), 81.32 [(CH3)3eC], 110.62 (C-5), 106.55, 112.60, 118.70,
124.79 (CF3), 127.03 (C-3), 127.83, 128.09, 128.18, 128.24, 128.30,
128.64, 128.69 [CH(Ph)], 133.40 (C-4), 137.40, 137.70 [C(Ph)], 148.73
(C-6), 149.53 (C-2), 152.16 (OCONH), 154.40, 155.14, 155.88, 156.62
(COCF3). Anal. Calcd for C39H44F3N3O8: C, 63.32; H, 5.99; N, 5.68.
Found: C, 63.05; H, 6.27; N, 5.79.
(film) nmax 3403e3240 (br), 1749, 1455, 1202, 1130 cmꢀ1
1H NMR
(400 MHz, CDCl3)
d
3.35e3.53 (m, 3H, 2ꢃCH2, H-50), 3.54e3.71 (m,
1H, H-50), 3.80e3.86 (m, 1H, H-30), 4.09 (d, 1H, H-20, J2 e3 ¼4.69 Hz),
4.35e4.40 (m, 1H, H-40), 4.41e4.82 (m, 6H, CH2ePh), 6.11 (s, 1H, OH,
D2O exch.), 7.15e7.46 (m, 16H, 3ꢃC6H5, H-5), 8.06 (d, 1H, H-4,
J4e5¼8.21 Hz), 8.36 (d, 1H, H-6, J6e5¼4.69 Hz), 10.50 (s, 1H, NH, D2O
exch.). Anal. Calcd for C34H33F3N2O6: C, 65.59; H, 5.34; N, 4.50.
Found: C, 65.78; H, 5.51; N, 4.36.
0
0
4.1.20. Data for tert-Butyl-N-[3-amino-2-[(2RS,5S)-2,5-dihydroxy-
3,4,6-tri-O-benzylhexane]pyridin-6-yl]carbamate (24). IR (film) nmax
4.1.16. tert-Butyl-N-[3-trifluoroacetamido-2-(2,3,5-tri-O-benzyl-1-hy-
3527e3178 (br), 1722, 1514, 1470, 1282, 1160 cmꢀ1 1H NMR
;
droxy-a,b-D-ribofuranosylmethyl)pyridin-6-yl] carbamate (21). This
(400 MHz, CDCl3) d
1.50 (s, 9H, (CH3)3), 2.75 (dd, 1H, H-10,
0
0
0
0
0
0
0
compound was prepared by a procedure analogous to that of 4,
starting from 19. Yield: 44%; oil. Data for the predominant
J1 e1 ¼14.68 Hz, J1 e2 ¼3.75 Hz), 2.91 (dd, 1H, H-1 , J1 e1 ¼14.68 Hz,
0
0
0
0
0
0
0
J1 e2 ¼7.51 Hz), 3.67 (dd, 1H, H-6 , J6 e5 ¼5.12 Hz, J6 e6 ¼9.90 Hz),
3.73 (dd, 1H, H-60, J6 e5 ¼2.73 Hz, J6 e6 ¼9.90 Hz), 3.85e3.91 (m, 1H,
H-30), 3.95 (dd, 1H, H-40, J¼2.05, 8.19 Hz), 4.05e4.14 (m, 1H, H-50),
4.12e4.35 (br s, 2H, NH2, D2O exch.), 4.39e4.45 (m, 1H, H-20),
4.48e4.84 (m, 6H, CH2ePh), 6.96 (d, 1H, H-5, J5e4¼8.53 Hz), 7.18 (br
0
0
0
0
a
-anomer: IR (film) nmax 3380e3217 (br), 1735, 1595, 1514, 1396,
1270, 1153 cmꢀ1 1H NMR (400 MHz, CDCl3)
; d 1.57 (s, 9H, (CH3)3),
3.15e3.25 (m, 2H, CH2), 3.37 0(dd, 1H, H-50, J5 e4 ¼3.42 Hz,
0
0
0
0
0
0
0
0
J5 e5 ¼10.58 Hz), 3.43 (dd, 1H, H-5 , J5 e4 ¼3.74 Hz, J5 e5 ¼10.58 Hz),

T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
9627
s, 1H, NH, D2O exch.), 7.22e7.39 (m, 15H, 3ꢃC6H5), 7.59 (d, 1H, H-4,
76.67 (C-30), 78.44 (C-20), 81.01 [(CH3)3eC], 81.30 (C-40), 82.36 (C-10),
110.55 (C-5), 127.73, 128.05, 128.25, 128.33, 128.53 [CH(Ph)], 129.17
(C-3), 134.51 (C-4), 137.48, 137.64,137.97 [C(Ph)], 147.68 (C-2), 148.09
(C-6), 152.24 (OCONH), 168.75 (CO). Anal. Calcd for C39H45N3O7: C,
70.14; H, 6.79; N, 6.29. Found: C, 70.37; H, 6.53; N, 6.41.
J4e5¼8.53 Hz); 13C NMR (50 MHz, CDCl3)
d 28.41 [(CH3)3], 36.69 (C-
10), 70.19 (C-50), 70.88 (C-20), 71.37 (C-60), 73.36, 73.56, 74.12
(CH2ePh), 80.17 (C-30), 80.50 (C-40), 80.56 [(CH3)3eC], 111.47 (C-5),
126.07 (C-4), 127.87, 128.05, 128.31, 128.50 [CH(Ph)], 137.59, 138.10
[C(Ph)], 138.10 (C-3), 142.52 (C-2), 143.05 (C-6), 152.59(OCONH).
Anal. Calcd for C37H45N3O7: C, 69.03; H, 7.05; N, 6.53. Found: C,
68.76; H, 6.91; N, 6.36.
4.1.24. 3-(2,3,5-Tri-O-benzyl-
b-D-ribofuranosyl)pyrazolo-[4.3-b]pyri-
dine (31). This compound was prepared by a procedure analogous
22
to that of 12 starting from 27. Yield 85%; oil; [
a
]
ꢀ27 (c 0.39,
D
4.1.21. 2-[(2,3,5-Tri-O-benzyl-
b
-
D-ribofuranosyl)methyl]pyridin-3-yl-
MeOH); IR (film) nmax 3286, 1481, 1458, 1233, 1079 cmꢀ1 1H NMR
;
3.73 (dd, 1H, H-50, J5 e4 ¼4.78 Hz,
0
0
amine (25). This compound was prepared by a procedure analo-
(400 MHz, CDCl3) d
22 þ24 (c 0.15,
J5 e5 ¼10.58 Hz), 3.80 (dd, 1H, H-5 , J5 e4 ¼4.19 Hz, J5 e5 ¼10.58 Hz),
0
0
0
0
0
0
0
gous to that of 8, starting from 22. Yield. 77%; oil; [a]
D
MeOH); IR (film) nmax 3426, 3348, 1582, 1452, 1268, 1094 cmꢀ1; 1H
4.36 (et, 1H, H-30, J¼5.46 Hz), 4.45e4.72 (m, 7H, 3ꢃCH2ePh, H-40),
4.82 (et, 1H, H-20, J¼4.78 Hz), 5.74 (d,1H, H-10, J1 e2 ¼4.78 Hz), 7.15
(dd, 1H, H-6, J6e5¼4.10 Hz, J6e7¼8.53 Hz), 7.17e7.40 (m, 15H,
3ꢃC6H5), 7.77 (d, 1H, H-7, J7e6¼8.53 Hz), 8.47 (d, 1H, H-5,
J5e6¼4.10 Hz), 11.00e11.60 (v br s, 1H, NH, D2O exch.); 13C NMR
0
0
NMR (400 MHz, CDCl3)
d
2.93 (dd, 1H, CH2, J¼7.50, 14.00 Hz), 3.10
(dd, 1H, CH2, J¼3.75, 14.00 Hz), 3.42 (dd, 1H, H-50, J5 e4 ¼4.10 Hz,
0
0
0
0
0
0
0
0
0
J5 e5 ¼10.59 Hz), 3.57 (dd, 1H, H-5 , J5 e4 ¼3.07 Hz, J5 e5 ¼10.59 Hz),
3.71 (dd, 1H, H-30, J¼5.46, 6.49 Hz), 3.88e4.03 (br s, 2H, NH2, D2O
exch.), 3.93 (et, 1H, H-20, J¼4.43 Hz), 4.15e4.21 (m, 1H, H-40),
4.35e4.70 (m, 7H, 3ꢃCH2ePh, H-10), 6.79 (dd,1H, H-4, J4e5¼7.85 Hz,
J4e6¼1.36 Hz), 6.94 (dd, 1H, H-5, J5e4¼7.85 Hz, J5e6¼4.78 Hz),
7.22e7.41 (m, 15H, 3ꢃC6H5), 7.95 (dd, 1H, H-6, J6e5¼4.78 Hz,
(50 MHz, CDCl3) d
70.38 (C-50), 72.20, 72.25, 73.48 (CH2ePh), 77.29
(C-10), 78.20 (C-30), 79.78 (C-20), 81.44 (C-40), 119.02 (C-7), 121.12 (C-
6), 127.66, 127.80, 127.86, 128.10, 128.16, 128.26, 128.42 [CH(Ph)],
134.55 (C-7a), 138.07, 138.22, 138.25 [C(Ph)], 138.60 (C-3a), 143.49
(C-3), 145.53 (C-5). Anal. Calcd for C32H31N3O4: C, 73.68; H, 5.99; N,
8.06. Found: C, 73.56; H, 5.72; N, 8.15.
J6e4¼1.36 Hz); 13C NMR (50 MHz, CDCl3)
d
38.67 (CH2), 69.45 (C-50),
71.97, 72.05, 73.45 (CH2ePh), 76.95 (C-30), 79.53 (C-20), 80.38 (C-40),
82.95 (C-10),122.66 (C-5), 123.16 (C-4), 127.81, 127.95,128.14,128.20,
128.46, 128.57 [CH(Ph)], 137.92, 138.00, 138.15 [C(Ph)], 138.71 (C-6),
142.74 (C-3), 144.55 (C-2). Anal. Calcd for C32H34N2O4: C, 75.27; H,
6.71; N, 5.49. Found: C, 75.01; H, 6.59; N, 5.63.
4.1.25. tert-Butyl-N-[2-(2,3,5-tri-O-benzyl-b-D-ribofuranosyl)pyr-
azolo-[4,3-b]pyridin-5-yl]carbamate (32). This compound was pre-
pared by a procedure analogous to that of 12, starting from 28. Yield
22
84%; oil; [
a]
ꢀ17 (c 0.29, MeOH); IR (film) nmax 3294, 1725, 1497,
D
4.1.22. N-[2-(2,3,5-Tri-O-benzyl-
b
-
D
-ribofuranosyl)methylpyridin-
1253, 1156 cmꢀ1 1H NMR (400 MHz, CDCl3)
; d 1.58 (s, 9H, (CH3)3),
3.67 (dd, 1H, H-50, J5 e4 ¼4.76 Hz, J5 e5 ¼10.61 Hz), 3.73 (dd, 1H, H-
0
0
0
0
3yl]acetamide (27). To a solution of 25 (40 mg, 0.078 mmol) in dry
CH2Cl2 (15 mL) was added acetic anhydride (0.009 mL, 0.093 mmol)
and the mixture was stirred overnight. The reaction was quenched
with a saturated NaHCO3 solution, extracted with CH2Cl2 and the
organic extracts were dried (Na2SO4) and concentrated to dryness.
Column chromatography (silica gel) of the residue using a mixture of
CH2Cl2/MeOH 37.5/1 as the eluent afforded 27 (40 mg, 92%) as an oil.
50, J5 e4 ¼4.02 Hz, J5 e5 ¼10.61 Hz), 4.32 (et, 1H, H-3 , J¼5.49 Hz),
0
0
0
0
0
4.43e4.48 (m, 1H, H-40), 4.48e4.75 (m, 6H, 3ꢃCH2ePh), 4.79 (et, 1H,
H-20, J¼5.12 Hz), 5.64 (d,1H, H-10, J1 e2 ¼5.12 Hz), 7.15e7.39 (m,15H,
3ꢃC6H5), 7.49 (br s, 1H, NH, D2O exch.), 7.77 (d, 1H, H-7,
J7e6¼9.15 Hz), 8.02 (d, 1H, H-6, J6e7¼9.15 Hz), 10.70e12.10 (v br s,
0
0
1H, NH, D2O exch.); 13C NMR (50 MHz, CDCl3)
d 28.45 [(CH3)3],
[a]
22 þ9 (c 0.22, MeOH); IR (film) nmax 3325, 1682, 1589, 1436, 1192,
70.22 (C-50), 72.10, 72.14, 73.44 (CH2ePh), 77.36 (C-10), 77.87 (C-30),
79.34 (C-20), 81.09 [(CH3)3eC], 81.42 (C-40), 112.95 (C-6), 121.35 (C-
7), 127.65, 127.71, 127.87, 128.08, 128.19, 128.26, 128.40, 128.45 [CH
D
1119 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d 2.02 (s, 3H, CH3), 3.03 (dd,
1H, CH2, J¼6.48, 14.00 Hz), 3.27e3.36 (m, 2H, CH2, H-50), 3.46e3.53
(m, 2H, H-50, H-30), 3.80 (t,1H, H-20, J¼5.80 Hz), 4.14e4.20 (m,1H, H-
40), 4.33e4.70 (m, 7H, 3ꢃCH2ePh, H-10), 7.10e7.42 (m, 16H, 3ꢃC6H5,
H-5), 8.06 (dd, 1H, H-4, J4e5¼7.85 Hz, J4e6¼1.36 Hz), 8.28 (dd, 1H, H-
6, J6e5¼4.78 Hz, J6e4¼1.36 Hz), 8.97 (s, 1H, NH, D2O exch.); 13C NMR
(Ph)], 132.62 (C-7a), 135.99 (C-3a), 137.98, 138.14, 138.20 [C(Ph)],
141.62 (C-3), 148.25 (C-5), 152.82 (CO). Anal. Calcd for C37H40N4O6:
C, 69.79; H, 6.33; N, 8.80. Found: C, 69.98; H, 6.56; N, 8.57.
(50 MHz, CDCl3)
d
24.18 (CH3), 38.43 (CH2), 69.86 (C-50), 72.20, 72.61,
4.1.26. 3-(
pound was prepared by a procedure analogous to that of 16 starting
from 31. Yield 96%; oil; [a D22
ꢀ50 (c 0.26, CH3OH); IR (film) nmax
b-D-Ribofuranosyl)pyrazolo[4,3-b]pyridine (33). This com-
73.52 (CH2ePh), 76.96 (C-30), 79.33 (C-20), 81.43 (C-40), 82.67 (C-10),
122.34 (C-5), 127.76, 128.01, 128.20, 128.24, 128.52, 128.59 [CH(Ph)],
131.36 (C-4), 134.07 (C-3), 137.52, 137.71, 137.97 [C(Ph)], 144.97 (C-6),
149.66 (C-2), 168.98 (CO). Anal. Calcd for C34H36N2O5: C, 73.89; H,
6.57; N, 5.07. Found: C, 73.69; H, 6.39; N, 5.22.
]
3519e3124, 1380, 1078 cmꢀ1; 1H NMR (400 MHz, CD3OD)
d 3.96 (dd,
1H, H-50, J5 e4 ¼1.95 Hz, J5 e5 ¼11.74 Hz), 4.05 (dd, 1H, H-500,
0
0
0
0
0
0
0
0
0
J5 e4 ¼2.74 Hz, J5 e5 ¼11.74 Hz), 4.19e4.24 (m,1H, H-4 ), 4.28 (et,1H, H-3 ,
J¼4.70 Hz), 4.40 (et, 1H, H-20, J¼5.48 Hz), 5.36 (d, 1H, H-10,
0
0
4.1.23. tert-Butyl-N-[3-acetamido[2-(2,3,5-tri-O-benzyl-
b
-
D
-ribofur-
J1 e2 ¼5.87 Hz), 8.00 (dd, 1H, H-6, J6e5¼5.48 Hz, J6e7¼8.60 Hz), 8.83 (dd,
anosyl)]methylpyridin-6-yl]carbamate (28). This compound was
prepared from 24 by an analogous procedure as described for
compounds 25 and 27. The intermediate carbamate 26 was acety-
1H, H-5, J5e6¼5.48 Hz, J5e7¼0.78 Hz), 8.86 (dd, 1H, H-7, J7e6¼8.60 Hz,
J7e5¼0.78 Hz); 13C NMR (50 MHz, CD3OD)
d
62.55 (C-50), 73.12 (C-30),
77.91 (C-20), 81.64 (C-10), 86.06 (C-40),123.04 (C-6),129.03 (C-3
a),130.96
lated without further purification to provide 28. Yield (two steps)
(C-7), 138.74 (C-7
C11H13N3O4: C, 52.59; H, 5.22; N,16.72. Found: C, 52.74; H, 5.36; N,16.46.
a), 139.86 (C-5), 142.60 (C-3). Anal. Calcd for
22
89%; oil; [
a
]
þ14 (c 0.48, MeOH); IR (film) nmax 3372e3263 (br),
D
1731, 1686, 1503, 1368, 1274, 1154 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
1.55 (s, 9H, (CH3)3), 1.97 (s, 3H, CH3), 2.84 (dd, 1H, CH2, J¼14.00,
4.1.27. 5-Amino-3-(
(34). This compound was prepared by a procedure analogous to
b-D-ribofuranosyl)pyrazolo[4,3-b]pyridine
5.80 Hz), 3.16 (dd, 1H, CH2, J¼14.00, 3.07 Hz), 3.26 (dd, 1H, H-50,
22
0
0
0
0
0
0
0
J5 e4 ¼4.78 Hz, J5 e5 ¼10.58 Hz), 3.044 (dd, 1H, H-5 , J5 e4 ¼3.42 Hz,
that of 16 starting from 32. Yield 79%; oil; [
a
]
ꢀ115 (c 0.43,
D
J5 e5 ¼10.58 Hz), 3.50 (et, 1H, H-3 , J¼5.12 Hz), 3.73 (et, 1H, H-2 ,
J¼5.80 Hz), 4.13e4.18 (m, 1H, H-40), 4.31e4.69 (m, 7H, 3ꢃCH2ePh,
H-10), 6.99 (br s,1H, NH, D2O exch.), 7.12e7.42 (m,15H, 3ꢃC6H5), 7.74
(d, 1H, H-5, J5e4¼8.88 Hz), 7.90 (d, 1H, H-4, J4e5¼8.88 Hz), 8.69 (br s,
CH3OH); IR (film) nmax 3520e3070, 1373, 1110 cmꢀ1 1H NMR
;
0
0
0
3.92 (dd, 1H, H-50, J5 e4 ¼2.05 Hz,
0
0
(400 MHz, CD3OD)
d
0
0
0
0
0
J5 e5 ¼11.95 Hz), 4.01 (dd, 1H, H-5 , J5e4¼2.73 Hz, J5 e5 ¼11.95 Hz),
4.12e4.16 (m, 1H, H-40), 4.20 (et, 1H, H-30, J¼4.43 Hz), 4.26 (et, 1H, H-
1H, NH, D2O exch.); 13C NMR (50 MHz, CDCl3)
d 23.96 (CH3), 28.42
20, J¼5.46 Hz), 5.16 (d, 1H, H-10, J1 e2 ¼5.81 Hz), 7.00 (d, 1H, H-6,
J6e7¼9.56 Hz), 8.19 (d, 1H, H-7, J7e6¼9.56 Hz); 13C NMR (50 MHz,
0
0
[(CH3)3], 37.49 (CH2), 68.84 (C-50), 72.14, 72.34, 73.54 (CH2ePh),

9628
T. Tite et al. / Tetrahedron 66 (2010) 9620e9628
CD3OD)
d
62.60 (C-50), 73.12 (C-30), 77.86 (C-20), 81.21 (C-10), 85.94
), 131.41 (C-7), 132.44 (C-7 ),
References and notes
(C-40), 114.35 (C-6), 123.68 (C-3
a
a
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4.2. Molecular modelling
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Vibrational analysis and thermochemical calculations were
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sides and their hydrates using Jaguar v.4.2.25 The crystal structure
of murine ADA (pdb id:2ADA) was used as a template for docking
calculations. Docking of each analogue was performed by the use of
1000 steps of the mixed Monte Carlo/Low mode search algorithm
as it is implemented in Macromodel v.7 and the GB/SA implicit
solvent model.26 The Zn atom was attributed a total charge of 2þ
and was included in all calculations. It was restrained in its crys-
tallographic position using harmonic distance potentials of
500 kcal/mol connecting it to the N3 imidazole nitrogens of His15,
His17 and His214 as well as to the side chain carboxylate of Asp295.
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4.3. Biological assays
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2%. The reaction was performed at 25 ꢁC. The conversion of adeno-
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appropriate solution, which did not contain the extracts. Absor-
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22. Data for N-(2-methylpyridin-3-yl)trifluoroacetamide (18). Mp 120e1 ꢁC. 1H
NMR (400 MHz, CDCl3)
J5e4¼7.83 Hz), 8.02 (d, 1H, H-4, J4e5¼7.83 Hz), 8.38 (d, 1H, H-6, J6e5¼4.70 Hz), 8.
47 (br s, 1H, NH, D2O exch.). 13C NMR (50 MHz, CDCl3)
20.08 (CH3), 107.26, 112.
d
2.49 (s, 3H, CH3), 7.21 (dd, 1H, H-5, J5e6¼4.70 Hz,
d
Acknowledgements
99, 118.71, 124.44 (CF3), 122.15 (C-5), 129.82 (C-3), 132.78 (C-4), 147.26 (C-6),
152.19 (C-2), 155.05, 155.81, 156.56, 157.25 (COCF3).
This work was supported by Research Grants provided by the
Greek Ministry of Industry, Energy, and Technology (Program EN-
TER) and the Special Account for Research Grants Committee of the
University of Athens (Program Kapodistrias).
23. Data for tert-butyl-N-(3-trifluoroacetamido-2-methylpyridin-6-yl) carbamate
(19). Mp 118e9 ꢁC. 1H NMR (400 MHz, CDCl3)
d 1.50 (s, 9H, (CH3)3), 2.55 (s, 3H,
CH3), 7.97 (d, 1H, H-5, J5e4¼9.00Hz), 8.10 (d, 1H, H-4, J4e5¼9.00 Hz), 10.46 (br s,
1H, NHCOCF3, D2O exch.), 11.21 (br s, 1H, NHBoc, D2O exch.). 13C NMR (50 MHz,
CDCl3)
d 16.69 (CH3), 27.88 [(CH3)3], 84.90 [(CH3)3eC], 107.15, 112.86, 118.57,
124.29 (CF3), 113.33 (C-5), 125.92 (C-3), 143.58 (C-4), 146.65 (C-2), 148.65 (C-6),
152.20 (OCONH), 155.62, 156.40, 157.17, 157.94 (COCF3).
24. Tite, T.; Lougiakis, N.; Marakos, P.; Pouli, N. Synlett 2009, 1741e1744.
25. Jaguar; Schrodinger, LLC: New York, NY, 2002.
Supplementary data
€
26. Mohamadi, F.; Richard, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Cau-
field, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11,
440e467.
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.10.005.