The synthesis of anti-fixed 3-methyl-3-deaza-2′-deoxyadenosine and other 3h-imidazo[4,5-c]pyridine analogs

Article abstract of DOI:10.1081/NCN-120016477

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2′-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2′-deoxyadenosine is replaced by a C-CH3 group to give 7-methyl-1-β-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2′-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2′-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.

Full text of DOI:10.1081/NCN-120016477

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THE SYNTHESIS OF ANTI-FIXED 3-METHYL-3- DEAZA-2′-
DEOXYADENOSINE AND OTHER 3H-IMIDAZO[4,5-
c]PYRIDINE ANALOGS
Rostem J. Irani a b & John SantaLucia Jr. c
a Department of Chemistry, Wayne State University, Detroit, MI, 48202, U.S.A.
b Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA,
02215, U.S.A.
c Department of Chemistry, Wayne State University, Detroit, MI, 48202, U.S.A.
Version of record first published: 31 Aug 2006
To cite this article: Rostem J. Irani & John SantaLucia Jr. (2002): THE SYNTHESIS OF ANTI-FIXED 3-METHYL-3- DEAZA-2′-
DEOXYADENOSINE AND OTHER 3H-IMIDAZO[4,5-c]PYRIDINE ANALOGS, Nucleosides, Nucleotides and Nucleic Acids, 21:11-12,
737-751
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 11 & 12, pp. 737–751, 2002
THE SYNTHESIS OF ANTI-FIXED 3-METHYL-
3-DEAZA-20-DEOXYADENOSINE AND OTHER
3H-IMIDAZO[4,5-c]PYRIDINE ANALOGS
Rostem J. Irani* and John SantaLucia Jr.y
Department of Chemistry, Wayne State University,
Detroit, MI 48202
ABSTRACT
Rotation of a heterocyclic base around a glycosidic bond allows the
formation of syn and anti conformations in nucleosides. The syn con-
formation has been observed primarily in purine-purine mismatches in
DNA duplexes. Such mismatches give rise to false positive oligonucleo-
tide hybridization in DNA-based diagnostics. Here we describe the
synthesis of an analog of 20-deoxyadenosine that retains its Watson-Crick
functional groups, but cannot form the syn conformation. In this analog,
the N3 atom of 20-deoxyadenosine is replaced by a C-CH3 group to give
7-methyl-1-b-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine
or 3-methyl-3-deaza-20-deoxyadenosine (3mddA). This modification
sterically prevents the syn conformation and 3mddA becomes an anti-
fixed nucleoside analog of 20-deoxyadenosine. The synthesis and confor-
mational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-
c]pyridine skeleton are described, as well as their potential applications.
*Current address: Department of Chemistry, Boston University, 590 Commonwealth Avenue,
Boston, MA 02215.
yCorresponding author. Fax: (313) 577-8822; E-mail: jsl@chem.wayne.edu
737
DOI: 10.1081=NCN-120016477
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

738
IRANI AND SANTALUCIA
INTRODUCTION
Natural DNA nucleosides can adopt either the syn or the anti con-
formation about the glycosidic dihedral angle. Several nucleosides have been
synthesized where the nucleoside is locked in either the syn or the anti con-
formation. 8-bromoadenosine and 8-bromoguanosine adopt exclusively the
syn conformation,[1] while 3-deaza-3-methylinosine adopts exclusively the
anti conformation.[2] X-ray studies on 3-deaza-3-methylinosine indicated that
steric hindrance from the extra methyl group was adequate to hinder the
rotation of the purine analog around the glycosidic bond.[3] Nuclear Over-
hauser effect (NOE) experiments showed that 3-deaza-3-halo (iodo, bromo,
chloro) derivatives of guanosine, adenosine and inosine nucleosides also
adopt an anti-fixed conformation.[4]
In Watson-Crick base pairs (Fig. 1) the nucleosides are in an
anti conformation, but for purine-purine mismatches (Fig. 2), a variety of
base pairing arrangements are possible. For single purine-purine mismatches,
it has been observed that one of the nucleosides is often in the anti confor-
mation and the other is in the syn conformation.[5–9] To destabilize the single
purine-purine mismatch, we synthesized 3-methyl-3-deaza-20-deoxyadenosine
(3mddA; Fig. 3), which is an anti-fixed analog of 20-deoxyadenosine. This
nucleoside analog may have several applications: 1. in DNA oligonucleotide
arrays, it could be used to reduce the formation false positive results caused
by purine-purine mismatches,[10] 2. to improve the specificity and binding
constant of target hybridization by DNA primers and probes in PCR and
other hybridization-based assays, 3. to act as a probe to study protein-nucleic
acid interactions in the minor groove, 4. the nucleoside 3mddA as well as the
other adenosine analogs presented here are also candidates for antiviral
activity, and 5. 3mddA is a stable uncharged analog of 3-methyl-20-deoxy-
adenosine, which is a common alkylation product[11–15] and may be useful for
mechanistic and X-ray structural studies in DNA mismatch repair of alky-
lated bases.
Figure 1. Watson-Crick base pairs showing anti conformation in base pairing.

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
739
Figure 2. Purine-purine mismatches showing syn conformation in base pairing.
Figure 3. Schematic representation of important 1D NOE interactions for compound 11.
Solid arrows indicate strong NOEs and dashed arrows indicate weak NOEs.
In this paper, we discuss the syntheses and characterization of anti-fixed
3-methyl-3-deaza-20-deoxyadenosine and several 3H-imidazo[4,5-c]pyridine
analogs.
RESULTS AND DISCUSSION
Synthesis of 3-Methyl-3-deazaadenine
Scheme 1 shows the synthesis of the nucleobase from the starting
material 3-picoline N-oxide (1). The IUPAC numbering system of imi-
dazo[4,5-c]pyridines is different from that of purines and is shown in Sch. 1
compound 7. Regioselective nitration of compound 1 with fuming nitric acid
and concentrated sulfuric acid gave 4-nitro-3-picoline N-oxide (2), which on

740
IRANI AND SANTALUCIA
Scheme 1.

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
741
recrystallization from methanol was obtained in 70% yield, by adapting the
literature procedure for other nitropyridines.[16,17] The next step was the
reduction of compound 2 to 4-amino-3-methylpyridine (3), for which several
different reaction conditions were tried. In the presence of tin (II) chloride
and concentrated HCl, only the nitro group was reduced and the N-oxide
was unaffected. On reduction with hydrogen catalyzed by palladium (10 wt.
% on activated carbon) or platinum(IV) oxide, a mixture of partially reduced
compounds along with compound 3 was generated. The same result was
obtained when 2 was refluxed with iron powder in glacial acetic acid. The
method of choice used for reduction of 2 to 3 was Raney nickel in methanol,
under 65 psi of hydrogen gas in a Parr hydrogenator (95% yield). Nitration
of 3 with concentrated nitric acid and concentrated sulfuric acid in an ice
bath gave 4-nitramino-3-methylpyridine (4) in 84% yield, as described pre-
viously.[18,19] The nitro group migrated from the amine to the ring in the
presence of concentrated sulfuric acid overnight at room temperature, to
yield 5-nitro-4-amino-3-methylpyridine (5) with a 54% yield, as described
previuosly.[18,19] Reduction of compound 5 in the presence of tin (II) chloride
and HCl afforded 4,5-diamino-6-chloro-3-methylpyridine (6) with a yield of
29%, which is lower than the previously reported yield of 58%,[19,20] but
sufficient for preparation of gram quantities for further steps. The tin (II)
chloride reduction was a key step in the synthesis since it reduced the nitro
group to an amine and regioselectively added a chloro group, which was used
later in the synthesis to introduce the final amine into the nucleoside. Ring
closure was accomplished by reflux of 6 with triethylorthoformate giving 4-
chloro-7-methyl-1(H )-imidazo[4,5-c]pyridine (7) with a 67% yield. This is an
improved method to 7 than the previously reported two-step procedure with
formic acid ring closure and POCl3 chlorination, which proceeded in 49%
yield.[19] Compound 7 was also produced from 6 by reflux with diethox-
ymethyl acetate in dimethylformamide (DMF) at 130ꢀC, though in lower
yield (58%).
Coupling of the Base with Deoxyribose
Coupling of the nucleobase to the deoxyribose sugar was attempted by
the enzymatic[21] and the sodium salt glycosylation[22] methods. The enzy-
matic method yielded no coupling product, however, indicating that purine
nucleoside phosphorylase does not recognize the modified nucleobase as a
substrate. As shown in Sch. 2, the sodium salt glycosylation method pro-
duced the desired compound 9, in 39% yield and the undesired 8 in 61%
yield. Compound 9 was deprotected by reflux at 90ꢀC in 2 M NH3
in methanol to produce compound 10 with a yield of 94%. The exchange of
the chloride with an amino group at position 6 in purines is usually
accomplished during the deprotection step.[23] Deazapurines, however, are

742
IRANI AND SANTALUCIA
Scheme 2.
more electron-rich than purines and therefore hydrazine was used to affect
the transformation.[24] 3mddA was obtained from compound 10 in 66% yield
by reaction with hydrazine monohydrate at 120ꢀC and subsequent reduction
by reflux with Raney nickel in water.
Synthesis of Additional Nucleosides with A 3H-Imidazo[4,5-c]pyridine
Skeleton
For the synthesis of a nucleoside with a 3H-imidazo[4,5-c]pyridine
skeleton, compound 8 was used as the starting material. As shown in
Sch. 3, compound 8 was deprotected using 2 M ammonia in methanol to
give compound 12 in 68% yield. Compound 12 was converted to 13 in
two steps. In step one, the chloride group was exchanged with hydrazine
by reflux with hydrazine monohydrate at 120ꢀC. This hydrazine inter-
mediate was then reduced by reflux with Raney nickel in water to give
compound 13 with a yield of 29%. In addition, compound 12 was also
reduced by reflux with Raney nickel in water to give compound 14 in 80%
yield.

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
743
Scheme 3.
Conformational Studies Using NMR Spectroscopy
The 1D NOE data for compound 11 are shown in Table 1 and Fig. 3.
1D NOE experiments are useful to qualitatively and semiquantitavely assess
the syn=anti conformational equilibrium.[25] According to Rosemeyer et al.[25]
for the anti conformation irradiation of H8 of purines (corresponding to H2
in 3mddA) exhibits a strong NOE to H20 and a weak NOE to H10. On the
other hand, for the syn conformation irradiation of H8 of purines exhibits a
strong NOE to H10 and weaker NOEs to H20 and H30. The data in Table 1,
indicate that upon irradiation of H2 of compound 11, a strong NOE to H20
(2.4%), and a weaker NOE to H10 (2.0%) is observed as shown in Fig. 3.
Table 1. Results of 1D NOE Experiments on the 3mddA Nucleoside
Proton Irradiated
NOE (%)
H2
H6
H10 (2.0), 50-OH (0.5), H30 (0.9), H20 (2.4)
CH3-7 (2.1)
H10
H2 (0.8), H40 (0.5), H20 (0.3), CH3-7 (2.8), H200(2.3)
H2 (0.6), 30-OH (1.7), H50=H500 (0.8), H20 (1.4), H200 (0.7)
H2 (1.9), H10 (1.1), H30 (1.3), H200 (3.0)
H6 (1.4), H10 (1.4)
H30
H20
CH3-7
H200
H10 (2.7), H30 (0.6), H20 (3.8)

744
IRANI AND SANTALUCIA
These data indicate that 11 is in the anti conformation. Irradiation of H6 in
compound 11, resulted in a strong NOE (2.1%) to the methyl group and no
NOEs to sugar protons, consistent with the anti conformation. Irradiation of
the H10 of compound 11 shows the strongest NOE (2.8%) to the methyl
group indicating the proximity of the H10 to the methyl group on the base,
thus further confirming its anti conformation. The anti conformation is also
consistent with the irradiation of the methyl group which only shows NOEs
to the H6 (1.4%) and H10 (1.4%).
CONCLUSIONS
The synthesis of 3mddA and the 3H-imidazo[4,5-c]pyridine analogs
have further expanded the nucleoside alphabet. 3mddA is an anti-fixed
nucleoside analog with potential applications in several fields including
DNA microarrays, hybridization-based genotyping methods, and studies
of DNA mismatch repair and DNA-protein interactions. In the future, we
plan to study the thermodynamic and structural effects of 3mddA on
single purine-purine mismatches in duplex DNA. At present, 3mddA and
the 3H-imidazo[4,5-c]pyridine described here are being tested for their
antiviral potential.
EXPERIMENTAL SECTION
General Methods
Physical data were measured as follows: Melting points are uncor-
1
rected. H and 13C NMR data were measured at 300, 400 or 500 MHz
instruments in d6-DMSO or d6-acetone. Chemical shifts (d) are reported in
parts per million (ppm) downfield from tetramethylsilane (TMS) using
TMS or residual non-deuterated solvent as reference. Multiplicity is indi-
cated using the following abbreviations: s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), q (quartet), m (multiplet) or br (broad). The NOE
experiments were performed in d6-DMSO. Each peak was irradiated, in an
interleaved fashion to minimize errors due to spectrometer drift, for 250 ms
yielding a saturation of ꢁ95%. The recycle delay was set at 3 s and the
acquisition time was 1.9 s.
1-chloro-2-deoxy-30,50-di-O-p-toluoyl-a-D-erythro-pentofuranose was
prepared from 20-deoxyribose as described by Hoffer.[26] The starting mate-
rial 3-picoline-N-oxide (compound 1), thymidine, thymidine phosphorylase,
and purine nucleoside phosphorylase were purchased from Sigma-Aldrich.
Silica gel used for column chromatography was Merck, grade 9385, 230–400
mesh, 60 A purchased from Sigma-Aldrich.

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
745
4-Nitro-3-picoline N-oxide (2). Compound 1 (20 g, 0.183 mol) was
dissolved in 100 mL of concentrated H2SO4 and warmed to 60ꢀC. While
keeping the temperature below 80ꢀC, 40 mL of 90% fuming HNO3 was added
dropwise. After 2 h, the reaction was cooled to room temperature and poured
onto crushed ice. The cooled mixture was then titrated with concentrated
NH4OH until a yellow precipitate formed. This precipitate was filtered and
dried in a vacuum oven. Upon recrystallization from methanol, yellow crys-
tals of compound 2 were obtained with a 70% yield: mp 136–138ꢀC, 1H NMR
(d6-acetone) d 8.29 (s, 1H, H2), 8.19 (m, 1H, H6), 8.11 (d, J ¼ 12 Hz, 1H, H5),
2.59 (s, 3H, CH3); EI-MS m=z 154 (Mþ). Anal. Calcd. for C6H6N2O3:
C, 46.74; H, 3.93; N, 18.18. Found: C, 45.85; H, 4.00; N, 17.98.
4-Amino-3-methylpyridine (3). Eight grams of wet Raney Ni was
added to 100 mL of methanol and pre-reduced in a Parr hydrogenator at
60 psi for 5 min. To this mixture was added compound 2 (8 g, 0.052 mol) and
reduced at 65 psi for 4 h with vigorous shaking. The mixture was then filtered
through celite and the filtrate was immediately dried in vacuo. Note that
compound 3 is easily oxidized and therefore it was dried and stored under
argon. Compound 3 was obtained in 95% yield: mp 103–105ꢀC,1H NMR
(d6-acetone) d 7.96 (s, 1H, H2), 7.94 (d, 1H, J ¼ 4.8 Hz, H6), 6.55
(d, J ¼ 4.8 Hz, H5), 5.24 (br s, 2H, NH2), 2.08 (s, 3H, CH3); EI-MS m=z 108
(Mþ). Anal. Calcd. for C6H8N2: C, 66.62; H, 7.42; N, 25.92. Found: C, 66.70;
H, 7.47; N, 25.85.
4-Nitramino-3-methylpyridine (4). Compound 3 (6.80 g, 0.0630 mol)
was dissolved in 51 mL of concentrated H2SO4 and the resulting mixture was
cooled to 5ꢀC in a dry ice=ethanol bath. Then 21 mL of concentrated HNO3
was added slowly to maintain the reaction temperature below 10ꢀC. After
1 h, the reaction mixture was poured onto 204 g of crushed ice. Concentrated
NH4OH was then added until a creamy precipitate of compound 4 was
formed at pH 7. The mixture was filtered and the precipitate was dried in a
vacuum oven. Crystals of compound 4 (8.11 g, 84% yield) were obtained by
recrystallization from hot water: mp 204–206ꢀC,1H NMR (d6-DMSO) d 8.15
(d, 2H, Ar-H), 8.00 (d, 1H, J ¼ 6.6 Hz, Ar-H), 2.08 (s, 1H, CH3); EI-MS
m=z ¼ 153 (Mþ). Anal. Calcd. for C6H7N3O2: C, 47.04; H, 4.61; N, 27.45.
Found: C, 46.78; H, 4.55; N, 27.09.
4-Amino-3-methyl-5-nitropyridine (5). Compound 4 (2.00 g, 0.013 mol)
was slowly added to 19 mL of concentrated H2SO4 to avoid increasing the
reaction temperature. The mixture was stirred at room temperature overnight
and the reaction was quenched by pouring over 200 g of crushed ice. This
mixture was then slowly titrated with concentrated NH4OH while cooling in
an ethanol=dry ice bath. When the mixture reached pH 7, a yellow precipitate
of compound 5 was formed. The precipitate was filtered and dried yielding

746
IRANI AND SANTALUCIA
1.02 g (54%) of pure compound 5: mp 194–196ꢀC,1H NMR (d6-DMSO) d
8.87 (s, 1H, H6), 8.09 (s, 1H, H2), 7.66 (s br, 2H, NH2), 2.12 (s, 3H, CH3).
EI-MS m=z 153 (Mþ). Anal. Calcd. for C6H7N3O2: C, 47.04; H, 4.61; N,
27.45. Found: C, 47.29; H, 4.59; N, 27.40.
4,5-Diamino-6-chloro-3-methylpyridine (6). Six grams of SnCl2 was
added to 300 mL of concentrated HCl and refluxed at 120ꢀC for 45 min. 3.0 g
(0.019 mol) of compound 5 was then added and allowed to react for another
45 min. The heating was then turned off and the reaction allowed to cool to
room temperature. The cooled mixture was poured over 700 g of crushed ice
and 7 M NaOH was added until the solution turned basic. The basic solution
was extracted with ethyl acetate (3 6 100 mL). The organic layer was dried
in vacuo and the residue was silica gel column purified by elution with 7:3
chloroform:methanol. After purification 0.9 g (29%) of compound 6 was
obtained: mp 150–152ꢀC,1H NMR (d6-DMSO) d 7.20 (s, 1H, H2), 5.47 (s br,
2H, NH2), 4.63 (s br, 2H, NH2), 1.95 (s, 3H, CH3). EI-MS m=z 157 (Mþ).
Anal. Calcd. for C6H8N3Cl: C, 45.85; H, 5.13; N, 26.75; Cl, 22.27. Found: C,
45.65; H, 5.19; N, 26.70; Cl, 22.46.
4-Chloro-7-methyl-1(H)-imidazo[4,5-c]pyridine (7). Method A. Under
argon atmosphere, 25 mL of anhydrous triethylorthoformate was added to
0.7 g (0.0045 mol) of compound 6 and refluxed at 160ꢀC for 4 h. After 4 h the
heating was turned off and the reaction cooled to room temperature. The
cooled reaction mixture was then dried invacuo and silica gel column purified
by elution with 7:3 chloroform:methanol. After purification, 0.5 g (67%) of
compound 7 was obtained: mp 258–260ꢀC,1H NMR (d6-DMSO) d 8.45 (s,
1H, H2), 7.91 (s, 1H. H6), 2.44 (s, 3H, CH3). EI-MS m=z 167 (Mþ). Anal.
Calcd. for C7H6N3Cl: C, 50.29, H, 3.62, N, 25.15, Cl, 20.94. Found: C, 50.14,
H, 3.77, N, 24.39, Cl, 21.59.
Method B. To 2.03 g (0.013 mol) of compound 6 was added 120 mL of dry
N,N-dimethylformamide and then 20 mL of diethoxymethyl acetate was added
under argon. The mixture was refluxed at 130ꢀC overnight. After the reaction
was cooled to room temperature, it was dried invacuo and the residue purified
by silica gel chromatography eluting with 7:3 chloroform:methanol. After
chromatography 1.26 g (58%) of compound 7 was obtained.
30,50 -O-p-toluoyl-7-chloro-4-methyl-1-b-D-deoxyribofuranosyl-3(H)-imi-
dazo[4,5-c] pyridine (8) and 30,50 -O-p-toluoyl-4-chloro-7-methyl-1-b-D-deox-
yribofuranosyl-1(H)-imidazo[4,5-c] pyridine (9). Under argon atmosphere,
compound 7 (1.00 g, 0.006 mol) was dissolved in 100 mL of dry acetonitrile
and then 0.5 g (2.1 eq) of 60% sodium hydride was added to form the sodium
salt of the heterocyclic base.[22] Evolution of H2 gas was observed during the
formation of the sodium salt. After 30 min 5.0 g (1.1 eq) of 1-chloro-2-deoxy-
3,5-di-O-p-toluoyl-a-D-erythro-pentofuranose was added and allowed
to react for 15 h. The reaction was then filtered and dried in vacuo. The

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
747
residue was purified by silica gel column chromatography with 7:3 chlor-
oform:methanol. The reaction yielded 1.9 g (61%) of compound 8 and 1.2 g
(39%) of compound 9.
Physical data for compound 8: mp 121–123ꢀC,1H NMR (d6-acetone)
d 8.70 (s, 1H, H2), 8.00–7.90 (m, 4H, toluoyl), 7.98 (s, 1H, H5), 7.36–7.14 (m,
4H, toluoyl), 7.15 (t, 1H, J ¼ 7.5 Hz and 5.5 Hz, H10), 5.83 (m, 1H, H30), 4.77
(m, 1H, H40), 4.76–4.74 (m, 2H, H50=H500), 3.11–3.05 (m, 2H, H20=H200), 2.54
(s, 3H, CH3-toluoyl), 2.42 (s, 3H, CH3), 2.39 (s, 3H, CH3-toluoyl). ESI-MS
m=z 542 (M þ Naþ). Anal. Calcd. for C28H26N3O5Cl: C, 64.72, H, 5.05, N,
8.09, Cl, 6.74. Found: C, 64.71, H, 5.02, N, 7.99, Cl, 6.71.
Physical data for compound 9: mp 143–145ꢀC,1H NMR (d6-acetone)
d 8.63 (s, 1H, H2), 8.00–7.98 (m, 4H, toluoyl), 7.94 (s, 1H, H6), 7.39–7.28
(m, 4H, toluoyl), 6.86 (t, 1H, J ¼ 7.5 Hz and 5.5 Hz, H10), 5.87 (m, 1H, H30),
4.75 (m, 1H, H40), 4.69–4.61 (m, 2H, H50=H500), 3.32–3.28 (m, 1H, H20),
3.10–3.06 (m, 1H, H200), 2.74 (s, 3H, CH3), 2.43 (s, 3H, CH3-toluoyl),
2.40 (s, 3H, CH3-toluoyl). ESI-MS m=z 542 (M þ Naþ). Anal. Calcd. for
C28H26N3O5Cl. C, 64.72, H, 5.05, N 8.09, Cl, 6.74. Found: C, 64.66, H,
5.07, N, 7.99, Cl, 6.99.
4-Chloro-7-methyl-1-b-D-deoxyribofuranosyl-1H-imidazo[4,5-c] pyridine
(10). One gram (0.0019 mol) of compound 9 was added to 20 mL of 2 M
NH3 in methanol. The reaction mixture was refluxed for 15 h at 90ꢀC. Upon
cooling to room temperature the mixture was dried in vacuo and the residue
purified by silica gel column chromatography (8:2 chloroform:methanol).
This yielded 0.5 g (94%) of compound 10 after purification: mp 146–
148ꢀC,1H NMR (d6-DMSO) d 8.72 (s, 1H, H2), 7.92 (s, 1H, H6), 6.52 (dd,
0
1H, H10, JH2 -H1 ¼ 6.4 Hz, JH2 -H1 ¼ 5.6 Hz), 5.37 (d, 1H, 3 -OH, JH3 -3 -
0
0
00
0
0
0
¼ 4.8 Hz), 4.92 (dd, 1H, 50-OH, JH5 -5 -OH ¼ 5.6 Hz, JH5 -5 -OH ¼ 4.8 Hz),
0
0
00
0
OH
4.39 (m, 1H, H30), 3.87 (m, 1H, H40), 3.48 (m, 2H, H50=H500), 2.71 (m, 1þH,
H20), 2.61 (s, 3H, CH3), 2.40 (m, 1H, H200). ESI-MS m=z 284 (M þ H ).
Anal. Calcd. for C12H14N3O3Cl: C, 50.87, H, 4.98, N, 14.84, Cl, 12.35.
Found: C, 50.59, H, 4.95, N, 14.50, Cl, 11.97.
7-Methyl-1-b-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-yla-
mine (3mddA, 11). To 0.5 g (0.0018 mol) of compound 10 was added 15 mL
of hydrazine monohydrate and refluxed at 120ꢀC for 10 h. The reaction was
then cooled to room temperature, dried invacuo, and the residue dissolved in
20 mL of H2O. Raney Ni (3 g wet) was added to the aqueous mixture and
refluxed for 6 h. The hot reaction mixture was then filtered through celite and
the filtrate dried in vacuo. The residue was then dissolved in a minimal
volume of 30% methanol=water and purified through a Dowex 162–400
(OHꢂ form) column. After chromatography, 0.31 g (66%) of compound 11
was obtained: mp 208–210ꢀC,1H NMR (d6-DMSO) d 8.38 (s, 1H, H2), 7.41
(s, 1H, H6), 6.42 (dd, 1H, H10, JH2 -H1 ¼ 7.6 Hz, JH2 -H2 ¼ 5.6 Hz , 5.89
0
0
00
0

748
IRANI AND SANTALUCIA
(s, 2H, NH2), 5.32 (d, 1H, 30-OH, JH3 -3 -OH ¼ 4 Hz), 4.90 (dd, 1H, 50-OH,
0
0
JH5 -5 -OH ¼ 5.6 Hz, JH5 -5 -OH ¼ 4.8 Hz), 4.34 (m, 1H, H30), 3.83 (m, 1H,
H40), 3.47 (m, 2H, H50=H500), 2.61 (m, 1H, H20), 2.40 (s, 3H, CH3), 2.33 (m,
1H, H200). EI-MS m=z 265 (Mþ). Anal. Calcd. for C12H16N4O3: C, 54.52, H,
6.10, N, 21.21. Found: C, 54.43, H, 6.15, N, 20.63.
0
0
00
0
7-Chloro-4-methyl-1-b-D-deoxyribofuranosyl-3H-imidazo[4,5-c]pyr-
idine (12). Compound 8 (2.7 g, 0.0052 mol) was mixed with 20 mL of 2 M
NH3 in methanol. The reaction mixture was refluxed at 90ꢀC for 12 h. Upon
cooling to room temperature the mixture was dried in vacuo and the residue
purified by silica gel chromatography (8:2 chloroform:methanol). This yiel-
ded 1.0 g (68%) of compound 12 after purification: mp 150–152ꢀC,1H NMR
(d6-DMSO) d 8.84 (s, 1H, H2), 7.98 (s, 1H, H5), 6.98 (dd, 1H, H10,
J ¼ 5.6 Hz and 6.4 Hz), 4.62 (m, 1H, H30), 4.08 (m, 1H, H40), 3.87–3.81 (m,
2H, H50=H500), 2.66–2.63 (m, 2H, H20=H200), 2.52 (s, 3H, CH3). ESI-MS m=z
306 (M þ Naþ). Anal. Calcd. for C12H14N3O3Cl: C, 50.87, H, 4.98, N, 14.84,
Cl, 12.35. Found: C, 50.77, H, 4.97, N, 14.69, Cl, 12.67.
4-Methyl-1-b-D-deoxyribofuranosyl-3H-imidazo[4,5-c]pyridine-7-
ylamine (13). Compound 12 (0.3 g, 0.0011 mol) was added to 10 mL of
hydrazine monohydrate and refluxed at 120ꢀC for 15 h. The reaction mixture
was then dried in vacuo. The residue was then dissolved in 15 mL of water.
To this mixture was added 2 g (wet) of Raney nickel and refluxed for 2 h.
After 2 h, the hot reaction mixture was filtered through celite and the filtrate
dried in vacuo. The residue was then purified by silica gel chromatography
(7:3 chloroform:methanol). This yielded 1.0 g (29%) of compound 13 after
purification: mp 170–172ꢀC, 1H NMR (d6-DMSO) d 8.40 (s, 1H, H2), 7.52 (s,
1H, H5), 6.38 (dd, 1H, H10, J ¼ 6.4 Hz and 7.6 Hz), 5.60 (br s, 2H, NH2),
5.39 (d, 1H, 30-OH, J ¼ 4 Hz), 5.02 (br s, 1H, 50-OH), 4.37 (m, 1H, H30), 3.88
(m, 1H, H40), 3.51 (m, 2H, H50=H500), 2.61 (m, 1H, H20), 2.33 (m, 1H, H200),
2.29 (s, 3H, CH3). ESI-MS m=z 265 (M þ Hþ). Anal. Calcd. for C12H16N4O3:
C, 54.52, H, 6.10, N, 21.21. Found: C, 53.92, H, 5.90, N, 20.13.
4-Methyl-1-b-D-deoxyribofuranosyl-3H-imidazo[4,5-c]pyridine
(14).
Compound 12 (0.1 g, 0.00035 mol) was added to 40 mL of water and 3 g (wet)
Raney nickel was added to the mixture. This reaction mixture was refluxed
for 2 h. After 2 h, the hot reaction mixture was filtered through celite and the
filtrate dried invacuo. The residue was dissolved in a minimal volume of 30%
methanol=water and purified through a Dowex 162 ꢂ 400 (OHꢂ form)
column using 30% methanol=water as the eluent. After chromatography,
0.07 g (80%) of compound 14 was obtained: mp 166–168ꢀC,1H NMR (d6-
DMSO) d 8.92 (s, 1H, H2), 8.61 (s, 1H, H7), 8.18 (s, 1H, H5), 6.42 (dd, 1H,
H10, J ꢃ 6 Hz and J ꢃ 6 Hz), 5.39 (d, 1H, 30-OH, J ¼ 3 Hz), 5.02 (t, 1H, 50-
OH, J ¼ 4.5 Hz), 4.40 (m, 1H, H30), 3.88 (m, 1H, H40), 3.57 (m, 2H,

ANTI-FIXED 3-METHYL-3-DEAZA-20-DEOXYADENOSINE
749
H50=H500), 2.62–2.57 (m, 1H, H20), 2.49 (s, 3H, CH3), 2.34–2.29 (m, 1H,
H200). ESI-MS m=z 250 (M þ Hþ). Anal. Calcd. for C12H15N3O3: C, 57.80,
H, 6.07, N, 16.86. Found: C, 55.50, H, 5.99, N, 15.58.
ACKNOWLEDGMENTS
We thank Kathleen Fleming for her help in searching the chemical
abstracts registry file. This work was funded by NIH grant number
HG02020.
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Received March 27, 2002
Accepted August 5, 2002