Synthesis and biological evaluation of new pyrazole- and tetrazole-related C-nucleosides with modified sugar moieties

Article abstract of DOI:10.1016/S0040-4020(01)01126-7

3(5)-Carboxamido-4-(β-D-ribofuranosyl)pyrazoles bearing 2′-benzamido (15) and 3′-mesyloxy (29) isosteric groups, as well as the tetrazole C-nucleosides with 2-benzamido-2-deoxy-β-D-ribofuranose (19) and 3-azido-3-deoxy-β-D-xylofuranose (36) as sugar segments, have been synthesized starting from D-glucose, by utilizing the 2,5-anhydro-D-glucose ethylene acetal derivatives 1 and 20 as divergent intermediates. The C-nucleosides 15 and 36 were shown to be moderate inhibitors of the in vitro growth of both N2a and BHK 21 tumour cell lines, whereas 29 showed a selective, although not potent cytotoxic activity against N2a cells. Compound 29 also showed a moderate in vitro antiviral activity towards the rabies virus.

Full text of DOI:10.1016/S0040-4020(01)01126-7

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 569±580
Synthesis and biological evaluation of new pyrazole- and
tetrazole-related C-nucleosides with modi®ed sugar moieties
a,p
a
a
b
c
Â
Ï
Â
Ï
Âd
Mirjana Popsavin, Ljilja Torovic, Sasa Spaic, Srdjan Stankov, Agnes Kapor, Zoran Tomic
and Velimir Popsavina
aInstitute of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 3, 21000 Novi Sad, Yugoslavia
bPasteur Institute, Hajduk Veljkova 1, 21000 Novi Sad, Yugoslavia
cInstitute of Physics, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 4, 21000 Novi Sad, Yugoslavia
dInstitute of Nuclear Sciences ªVincaº, P.O. Box 522, 11001 Belgrade, Yugoslavia
Received 17 September 2001; accepted 15 November 2001
AbstractÐ3(5)-Carboxamido-4-(b-d-ribofuranosyl)pyrazoles bearing 20-benzamido (15) and 30-mesyloxy (29) isosteric groups, as well as
the tetrazole C-nucleosides with 2-benzamido-2-deoxy-b-d-ribofuranose (19) and 3-azido-3-deoxy-b-d-xylofuranose (36) as sugar
segments, have been synthesized starting from d-glucose, by utilizing the 2,5-anhydro-d-glucose ethylene acetal derivatives 1 and 20 as
divergent intermediates. The C-nucleosides 15 and 36 were shown to be moderate inhibitors of the in vitro growth of both N2a and BHK 21
tumour cell lines, whereas 29 showed a selective, although not potent cytotoxic activity against N2a cells. Compound 29 also showed a
moderate in vitro antiviral activity towards the rabies virus. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results and discussion
A number of nucleoside analogues have been found to show
a broad spectrum of biological activity and have stimulated
considerable interest as potential antitumour and/or antiviral
agents.1 Signi®cant antitumour and antiviral activity of
some pyrazole-related nucleosides2 prompted us to prepare
the corresponding C-nucleoside analogues. A number of
4-(b-d-ribofuranosyl)pyrazoles,3 along with several 4- and
5-(d-lyxofuranosyl)pyrazoles4 and a pyrazole homo-C-
nucleoside,5 have been already synthesized. Conversely,
the synthesis of C-nucleosides containing the pyrazole-5-
carboxamide moiety and a modi®ed sugar segment has
not been described in the literature so far. Some C-(glyco-
pyranosyl)tetrazoles6,7 were also synthesized as potential
inhibitors of early steps in the shikimate pathway,7 whereas
certain 5-(glycofuranosyl)-tetrazoles8,9 were designed as
possible bioisosteres of the well known antiviral agent
ribavirin.9 However, the data related to their antiviral and/
or cytotoxic activities were not reported so far. In this paper
we describe the synthesis of the pyrazole-related C-nucleo-
sides 15 and 29,10 as well as the tetrazole-related C-nucleo-
sides 19 and 36, along with their preliminary biological
evaluations, including their in vitro cytotoxic and antiviral
activities.
2.1. Chemistry
The crucial problem in the synthesis of all targets 15, 19, 29
and 36 was the establishment of the requisite b-con®gura-
tion at the anomeric position. In a previous paper we have
described11 the conversion of d-glucose to the 2,5-anhydro-
d-glucose derivative 1 (Scheme 1), a compound already
containing the desired b-d-glycosidic bond, as well as the
functionalities suitable for further introduction of diversity
into the nucleoside carbohydrate segment.
The 2,5-anhydro-d-glucose derivative 1 reacted with benzyl
bromide in ether, in the presence of silver(I)-oxide as a
catalyst, to afford the expected 4-O-benzyl derivative 2 in
87% yield. Reaction of 2 with potassium benzoate in N,N-
dimethylformamide gave the corresponding 6-O-benzoyl
derivative 3 in 83% yield. Compound 3 readily reacted
with sodium azide in dimethyl sulfoxide to afford the
desired 3-azido-3-deoxy derivative 5 as a major reaction
product (77%) accompanied with a small amount of the
corresponding 2,3-unsaturated derivative 4 (10%). Catalytic
reduction of 5 over 10% Pd/C according to the procedure
developed by Secrist and Logue,12 gave the amine hydro-
chloride 6 as a product of sequential azide reduction/benzyl
ether hydrogenolysis process. Treatment of crude 6 with
benzoyl chloride in pyridine gave the fully benzoylated
product 7 (71% from 5). O-Debenzoylation of 7 with
sodium methoxide in methanol afforded the diol 8 in 86%
Keywords: C-nucleosides; pyrazoles; tetrazoles; cytotoxic activity; anti-
viral activity; 2,5-Anhydro sugars; X-ray crystallography.
p
Corresponding author. Fax: 138-121-54-065;
e-mail: popsavin@ih.ns.ac.yu
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)01126-7

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M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
Scheme 1. (a) BnBr, Ag2O, Et2O, re¯ux, 22 h, 87%; (b) KOBz, DMF, 1008C, 48 h, 83%; (c) NaN3, DMSO, 1208C, 47 h, 10% of 4, 77% of 5; (d) H2, Pd/C,
CHCl3 (cat.), EtOH, rt, 120 h; (e) BzCl, Py, rt, 96 h, 71% from 5; (f) NaOMe, MeOH, rt, 2 h for 7, 86% of 8, 1.5 h for 13, 45% of 14 from 10; (g) 4:1
CF3CO2H±6 M HCl, 148C, 96 h; (h) Ph3P:CHCO2Me, CH2Cl2, rt, 19 h, 85% from 7; (i) CH2N2, Et2O, 08C, 2 h for 10, 45 h for 11; (j) Cl2/CCl4, CH2Cl2, rt,
2.5 h, 97% from 10, 98% from 11; (k) NH3, MeOH, rt, 7 days, 86%.
Scheme 2. (a) 4:1CF 3CO2H±6 M HCl, 148C, 120 h; (b) NH2OH´HCl, NaOAc, EtOH, rt, 2 h; (c) MsCl, Py, 2158C!rt, 2.5 h, 63% from 7; (d) NaN3, NH4Cl,
DMF, 1008C, 4 h, 55%; (e) NaOMe, MeOH, rt, 3.5 h, 72%.

M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
571
Scheme 3. (a) DMF, H2O (5%), CaCO3, 155±1608C, 20 h, 86%; (b) MsCl, Py, 148C, 48 h, for 23: 41% from 20; (c) 4:1CF 3CO2H±6 M HCl, 148C, 24 h;
(d) Ph3P:CHCO2Me, CH2Cl2, rt, 24 h, 48% from 23; (e) CH2N2, Et2O, 08C, 5 h; (f) Cl2/CCl4, CH2Cl2, rt, 3 h, 91% from 26; (g) NH3, MeOH, rt, 7 days, 86%.
yield. Hydrolytic removal of the dioxolane protective group
in 7 was achieved with 4:1mixture of tri¯uoroacetic acid
and 6 M hydrochloric acid at 148C, whereupon the corre-
sponding aldehyde 9 was obtained. Due to its instability the
crude 9 was immediately treated with (carbomethoxy-
methylene)-triphenylphosphorane in dichloromethane to
afford a 2:1mixture of the corresponding Z and E
unsaturated esters 10 and 11 in 85% combined yield. The
unsaturated esters 10 and 11 were readily separated by
column chromatography and converted to the C-nucleoside
15 according to the methodology developed by Moffatt et
al.13 Both 10 and 11 readily underwent 1,3-dipolar cyclo-
addition with an excess of diazomethane in ether at 08C to
yield the 2-pyrazoline 12 as the only reaction product. The
transformation of 10 to 12 was complete in 2 h, while
the conversion of 11 to 12 required 45 h for completion.
The enhanced reactivity of the Z-isomer 10 is presumably
due to the activation of its a,b-unsaturated system by the
intramolecular hydrogen bond between H (amide) and O
(ester carbonyl). Indeed, the 1H NMR spectrum of 10
showed a signi®cant down®eld shift of the amide proton
signal (dNH 7.67) in contrast to the corresponding signal in
the spectrum of 11, which appeared at a high®eld position
(dNH 6.78). This result is consistent with the presence of an
intramolecular hydrogen bond in 10. Without puri®cation or
further characterization the intermediate 12 was treated with
a saturated solution of chlorine in carbon tetrachloride to
give the pyrazole derivative 13. Quite unexpectedly, pure 13
was isolated as highly hygroscopic syrup. However, the
corresponding di-O-deprotected product 14, prepared by
treatment of 13 with sodium methoxide in methanol, was
a highly crystalline stable compound, which was fully char-
acterized by corresponding spectral and analytical data.
Finally, treatment of 13 with methanolic ammonia afforded
the O-deprotected C-nucleoside 15, ready for biological
testing. Under these reaction conditions both intermediates
10 and 11 were converted to the target 15 in 97 and 85%
overall yields, respectively.
2,5-Anhydro derivative 7 has been conveniently used as a
divergent intermediate for preparation of the corresponding
tetrazole C-nucleoside 19 (Scheme 2).
Hydrolytic removal of the dioxolane protective group from
7, followed by subsequent treatment of the liberated alde-
hyde 9 with hydroxylamine hydrochloride, in the presence
Scheme 4. (a) NaN3, DMSO, 1208C, 144 h, 62% of 30, 9% of 31; (b) 4:1CF 3CO2H±6 M HCl, 148C, 96 h; (c) NH2OH´HCl, NaOAc, EtOH, rt, 2 h; (d) MsCl,
Py, 2158C!rt, 2.5 h, 22% from 30; (e) NaN3, NH4Cl, DMF, 1008C, 2.5 h, 58%; (f) NaOMe, MeOH, rt, 2.5 h, 91%.

572
M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
of sodium acetate in ethanol, gave an inseparable mixture of
the corresponding E- and Z-oxime 16. As the mixture 16
appeared as a single spot in TLC, it was not puri®ed further,
but was immediately treated with mesyl chloride in pyridine
to afford the corresponding nitrile 17 in an overall yield of
63% with respect to 7. The intermediate 17 was then
reacted with an excess of sodium azide in the presence of
ammonium chloride, under the Buchanan reaction con-
ditions,7 to give the corresponding tetrazole derivative 18
in good yield. Moreover, an action of sodium methoxide
onto 18 furnished the O-deprotected C-nucleoside 19 in
72% yield.
Synthesis of the pyrazole C-nucleoside 29 bearing the 3-O-
mesyl functionality as an isostere is outlined in Scheme 3.
Solvolysis of 2011 in wet N,N-dimethylformamide (5% of
water), in the presence of calcium carbonate as a proton
acceptor, gave a mixture of 3,6- and 4,6-di-O-benzoyl
derivatives 21 and 22 in 86% combined yield. The regio-
isomers 21 and 22 could not be separated by column
chromatography, presumably due to their rapid inter-
conversion caused by silica gel. However, direct crystalliza-
tion of the crude reaction mixture from benzene±hexane
afforded pure 21, which was subsequently mesylated to
give the key intermediate 23. The addition of mesyl chloride
in pyridine to the mother liquor, which remained after
crystallization of 21 afforded an additional quantity of 23.
The total yield of 23 was 41% with respect to 20.
Figure 1. ORTEP drawing of 3(5)-carboxamido-4-(2-benzamido-2-deoxy-
b-d-ribofuranosyl)pyrazole (15´H2O) with non-H labelling scheme. The
displacement ellipsoids were drawn at 30% probability.
and consistent with a strong intramolecular hydrogen bond
between them.
Two tautomeric forms with hydrogen atoms at N1and N2
are randomly present in a three dimensional lattice. The
®nal re®nement gave the bond lengths of N1±
Ê
Ê
C5ˆ1.338(7) A and N2±C3ˆ1.330(8) A, as well as the
population of N1±H and N2±H tautomers in an
approximate ratio of 3:7, respectively. Both tautomers
and a water molecule are built into the three-dimensional
crystal lattice by intermolecular hydrogen bonds
whereby the water molecule is included0as a proton donor
The intermediate 23 was converted to the target molecule 29
by using the same ®ve-step sequence already applied for the
conversion of 7 into the C-nucleoside 15 (Scheme 1). In this
way the 4-O-mesyl derivative 23 has been transformed into
the target 29 in 38% overall yield.
0
Ê
of two hydrogen bonds (O ±HA´´´O3 ˆ2.802 A, HA´´´
O3 ˆ1.703 A, /O0 ±HA´´´O30ˆ166.88; O0-HB´´´O50ˆ
0
Ê
0
0
0
Ê
Ê
2.782 A, HB´´´O5 ˆ2.030 A, /O ±HB´´´O5 ˆ165.48), as
well as a proton acceptor of one hydrogen bond (N1±
0
0
H1´´´O ˆ3.164 A, H1´´´O ˆ2.340 A, /N1±H1´´´O 0ˆ
160.88). There are no signi®cant deviations from the
expected bond lengths and angles.
Ê
Ê
Finally, the intermediate 23 was converted into the tetrazole
C-nucleoside 36 as shown in Scheme 4. The sequence
started with nucleophilic displacement of the C-4 mesyloxy
group with azide anion, whereupon the 4-azido-4-deoxy
derivative 30 was obtained as a major reaction product
(62%) accompanied with small amount of the 6-O-
debenzoylated derivative 31 (9%). The intermediate 30
was then converted to the tetrazole 36 by using the same
®ve-step sequence already applied for the conversion of 7
into the C-nucleoside 19 (Scheme 2).
2.3. Biological evaluation
The biological activities of compounds 8, 15, 29 and 36
were evaluated as follows: a preliminary in vitro evaluation
for cytotoxic activity against mouse neuroblastoma (N2a)
and baby hamster kidney (BHK 21) continuous cell lines, as
well as for antiviral activity against rabies virus.
2.2. X-Ray analysis
As shown in Table 1, the C-glycoside 8 was found to be
inactive against both N2a and BHK 21cells. Among the
pyrazole-related C-nucleosides, compound 15 showed a
An X-ray diffraction analysis of compound 15 (Fig. 1)
unambiguously con®rmed its structure providing a proof
that all intermediates generated by the multistep sequence
7!15 retained the required b-con®guration at the anomeric
position. The values of torsion angles C30 ±C20 ±C10 ±
C4ˆ162.9(58) and C10 ±O±C40 ±C50ˆ147.6(58) are con-
sistent with the b-d-con®guration of 15. The torsion angle
of O±C10 ±C4±C5 is 2145.8(6)8 that indicates that
pyrazole-5-carboxamide moiety in 15 has the anti orienta-
tion. The ®ve membered furanose ring adopts an envelope
conformation [wˆ41.2(9)8], with C10 below the best plane
Table 1. Cytotoxic activity of synthesized compounds 8, 15, 29 and 36
a
IC50 (mM)
Compounds
N2a
BHK 21
8
3500
220
440
430
.5000
170
1300
630
15
29
36
Ê
[0.261(4) A] of the ring. The distance between the H-atom
Ê
from N7 (amide) and O6 (carbonyl) of 2.176 A is consider-
a
IC50 values represent the compound concentration required to inhibit 50%
of the cells growth.
14
Ê
ably less then the sum of their van der Waals radii (2.72 A)

M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
Table 2. Antiviral activity of synthesized compounds 8, 15, 29 and 36
573
Final conc. (mM).
GM (%)
0.5% Tween 80 (%)
8 (%)
15 (%)
29 (%)
36 (%)
5000
2500
1250
625
70
70
70
70
70a
70a
70a
70a
70
70
70
70
NCPb
70a
70a
70
,1a
1a
70a
70a
70a
70
10a
50
31
2
70
70
70
70
70
70
a
Signi®cant cytotoxic effect.
NCP-no cells present.
b
moderate cytotoxic effect against both N2a and BHK 21
cells, whereas compound 29 showed a selective, although
not potent cytotoxic activity against the N2a cells. More-
over, the tetrazole C-nucleoside 36 showed a weak cyto-
toxic activity towards both N2a and BHK 21cell lines.
were taken on a Micromass LCT KA111 spectrometer.
TLC was performed on DC Alufolien Kieselgel 60 F254
(E. Merck). Column chromatography was carried out
using Kieselgel 60 (under 0.063 mm; E. Merck). All organic
extracts were dried with anhydrous Na2SO4. Organic
solutions were concentrated in a rotary evaporator under
diminished pressure at a bath temperature below 358C.
The results related to antiviral activities of 8, 15, 29 and 36,
are shown in Table 2.
4.1.1.
2,5-Anhydro-4-O-benzyl-3,6-di-O-methanesul-
Obviously, only the pyrazole related C-nucleoside 29
showed a slight antiviral activity towards the rabies virus,
while other compounds were devoid of any antiviral
activity. Each compound in higher concentrations exhibited
a notable cytotoxicity as evidenced by cell rounding of a
signi®cant proportion of cells (.5%). Due to the cytotoxic
effect of 29, its virus-inhibitory activity is of negligible
practical value.
fonyl-d-glucose ethylene acetal (2). Compound 111
(9.31g, 25.72 mmol) was dissolved in dry ether (620 mL),
and then benzyl bromide (32.12 mL, 268.95 mmol) and
Ag2O (59.30 g, 255.78 mmol) were added. The reaction
mixture was stirred at re¯ux temperature for 22 h, then
®ltered and the precipitate was washed successively with
toluene (100 mL) and Me2CO (250 mL). The organic solu-
tions were evaporated and the oily residue was submitted to
column chromatography (toluene; 4:1!7:3 toluene±
EtOAc) to furnish pure 2 (10.13 g, 87%) as a colorless
1
syrup: [a]Dˆ195 (c, 1.23 in CHCl3). H NMR (CDCl3):
3. Conclusions
d 2.98 and 3.09 (2£s, 3H each, 2£MeSO2), 3.89 (m, 1H,
J4,5ˆ4.5 Hz, H-5), 3.9±4.1(m, 4H, 2 £CH2±dioxolane),
4.20±4.27 (m, 4H, H-2, H-4 and 2£H-6), 4.69 (2£d, 2H,
Jgemˆ11.8 Hz, PhCH2), 5.09 (d, 1H, J2,3ˆ3.2 Hz, H-3), 5.12
(d, 1H, J1,2ˆ6.8 Hz, H-1), 7.3±7.4 (m, 5H, Ph); 13C NMR
(CDCl3): d 37.51and 38.35 (2 £MeSO2), 65.27 and 65.36
(2£CH2±dioxolane), 67.75 (C-6), 72.31(Ph CH2), 80.77
(C-2), 82.57 (C-3), 82.81 and 83.54 (C-4 and C-5), 101.35
(C-1), 127.94, 128.2, 128.56 and 136.73 (Ph); FAB-MS: m/z
453 (M111).
In summary, we have synthesized novel pyrazole- and tetra-
zole-related C-nucleosides bearing the 20-benzamido (15
and 19), 30-mesyloxy (29) and 30-azido group (36) as
isosteres. The C-nucleosides 15 and 36 were shown to be
moderate inhibitors of the in vitro growth of both N2a and
BHK 21tumour cell lines, whereas 29 showed a moderate
cytotoxic activity only against N2a cells. Compound 29 also
showed a moderate in vitro antiviral activity towards the
rabies virus. Molecules of type 15 or 19 might be of wider
medicinal interest because they are partly related to certain
20-benzamido-20-deoxy adenosines, the potential agents for
treatment of sleeping sickness.15 In addition the protected
5-(glycofuranosyl)tetrazoles 18 and 35 might be used as
convenient synthons for preparation of a range C-nucleo-
sides bearing the 4,5-substituted pyrazoles or 1,3,4-oxa-
diazoles as heterocyclic aglycons.16
4.1.2. 2,5-Anhydro-6-O-benzoyl-4-O-benzyl-3-O-methane-
sulfonyl-d-glucose ethylene acetal (3). To a solution of 2
(10.13 g, 22.41 mmol) in DMF (143 mL) was added potas-
sium benzoate (15.11 g, 94.39 mmol). The mixture was stir-
red for 48 h at 1008C, then poured into cold water (300 mL)
and the emulsion was extracted with CH2Cl2 (4£200 mL).
The combined extracts were washed successively with
water (200 mL) and satd aq. NaHCO3 (200 mL), dried and
the solvents were evaporated. The oily residue solidi®ed
after triturating with EtOH. Recrystallization from EtOH
gave pure 3 (8.90 g, 83%) as colorless crystals: mp 88±
4. Experimental
4.1. General methods
1
898C; [a]Dˆ170.9 (c, 1.54 in CHCl3); H NMR (CDCl3):
d 3.08 (s, 3H, MeSO2), 3.82±4.04 (m, 4H, 2£CH2±dioxo-
lane), 4.02 (m, 1H, J2,3ˆ4 Hz, H-2), 4.30 (m, 1H,
J5,6ˆ4.5 Hz, H-5), 4.31(bs, 1H, J3,4,1Hz, H-4), 4.43 (m,
2H, 2£H-6), 4.70 (2£d, 2H, Jgemˆ12 Hz, PhCH2), 5.15 (d,
1H, H-3), 5.17 (d, 1H, J1,2ˆ7.3 Hz, H-1), 7.25±8.05 (m,
10H, 2£Ph); 13C NMR (CDCl3): d 38.17 (MeSO2), 63.69
(C-6), 65.21and 65.79 (2 £CH2±dioxolane), 72.16
(PhCH2), 80.51(C-2), 83.05 and 84.41(C-4 and C-5),
83.18 (C-3), 101.4 (C-1), 127.71, 127.82, 128.20, 128.56,
Melting points were determined on a BuÈchi 510 apparatus
and were not corrected. Optical rotations were measured on
a Perkin Elmer 141 MC polarimeter. IR spectra were
recorded with a Specord 75IR spectrophotometer. NMR
spectra were recorded on a Bruker AC 250 E instrument
and chemical shifts are expressed in ppm down®eld from
tetramethylsilane. Low resolution mass spectra were
recorded on Finnigan-MAT 8230 (CI) and VG AutoSpec
(FAB) mass spectrometers. High resolution mass spectra

574
M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
129.84, 133.01 and 136.82 (2£Ph), 166.01 (PhCO). Anal.
Calcd for C23H26O9S: C 57.74, H 5.48, S 6.68. Found: C
57.33, H 5.72, S 6.04.
(C-2), 80.46 (C-5), 102.62 (C-1), 127.57, 127.71, 127.91,
128.35, 128.47, 128.82, 129.02, 129.31, 129.51, 129.56,
129.61, 129.72, 131.12, 131.68, 133.08, 133.77, 133.88
and 134.23 (3£Ph), 165.28, 165.86 and 167.2 (2£PhCOO
and PhCONH). Anal. Calcd for C29H27NO8: C 67.30, H
5.26, N 2.71. Found: C 67.54, H 5.33, N 2.60.
4.1.3. 2,5-Anhydro-3-azido-6-O-benzoyl-4-O-benzyl-3-
deoxy-d-allose ethylene acetal (5). A mixture of 3
(1.35 g, 2.82 mmol) and sodium azide (1.8464 g,
28.41 mmol) in DMSO (48 mL) was stirred at 115±1208C
for 47 h. The mixture was poured into cold water and the
resulting emulsion was extracted with 1:1 benzene±hexane
(4£80 mL). The combined extracts were washed with water
(2£100 mL), dried and evaporated to yellow oil. Column
chromatography (toluene; 97:3 toluene±EtOAc) of the
residue gave pure 4 (0.1095 g, 10%) as a colorless syrup:
4.1.5. 2,5-Anhydro-3-benzamido-3-deoxy-d-allose ethyl-
ene acetal (8). To a solution of 7 (0.1029 g, 0.2 mmol) in
anhydrous MeOH (10 mL) was added a solution of 5%
NaOMe in MeOH (0.05 mL). The mixture was stirred for
2 h at room temperature and then neutralized by stirring
with Amberlite IR-120[H1] resin at room temperature
for 30 min. The mixture was ®ltered and the resin was
washed with MeOH. The combined organic solutions
were evaporated to an oil, which solidi®ed upon triturating
with 3:2 toluene±EtOAc (5 mL). Recrystallization from
CH2Cl2±hexane afforded pure 8 (0.0526 g, 86%) as white
crystals: mp 157±157.58C; [a]Dˆ264.9 (c, 1.28 in MeOH);
1
[a]Dˆ1477.6 (c, 1.45 in CHCl3); H NMR (CDCl3): d
3.90±4.06 (m, 4H, 2£CH2±dioxolane), 4.38 (m, 2H,
J5,6ˆ5.3 Hz, 2£H-6), 4.54 (s, 2H, PhCH2), 4.74 (dd, 1H,
J3,4ˆ2.3 Hz, J4,5ˆ3.2 Hz, H-4), 4.88 (m, 1H, H-5), 5.35
(dd, 1H, J1,3ˆ0.8 Hz, H-3), 5.55 (s, 1H, H-1), 7.25±8.06
(m, 10H, 2£Ph); 13C NMR (CDCl3) d 64.30 (C-6), 65.10
and 65.20 (2£CH2±dioxolane), 69.82 (PhCH2), 82.61(C-4),
84.69 (C-5), 97.39 (C-1), 98.69 (C-3), 127.73, 128.35,
128.41, 129.68, 133.16 and 137.77 (2£Ph), 159.40 (C-2),
166.18 (PhCO). Further elution gave 5 (0.92 g, 77%) as a
colorless oil: [a]Dˆ178 (c, 1.11 in CHCl3); 1H NMR
(CDCl3): d 3.82±4.00 (m, 5H, J2,3ˆ4.3 Hz, J3,4ˆ5.5 Hz,
H-3 and 2£CH2±dioxolane), 4.14 (dd, 1H, J1,2ˆ2.8 Hz,
1H NMR (acetone-d6):d 3.55 (dd, 1H, J5,6aˆ4.9 Hz, J6a,6b
ˆ
11.9 Hz, H-6a), 3.62 (dd, 1H, J5,6bˆ4.8 Hz, H-6b), 3.80±
4.00 (m, 6H, 2£CH2±dioxolane, OH and H-5), 4.06 (dd,
1H, J1,2ˆ2.9 Hz, J2,3ˆ8.3 Hz, H-2), 4.26 (dd, 1H, J3,4ˆ
5.9 Hz, J4,5ˆ3 Hz, H-4), 4.61(m, 1H, J3,NHˆ6.1Hz, H-3),
5.04 (d, 1H, H-1), 7.52±8.00 (m, 6H, Ph and NH); 13C NMR
(acetone-d6): d 53.87 (C-3), 63.55 (C-6), 65.93 and 65.98
(2£CH2±dioxolane), 72.64 (C-4), 81.91 (C-2), 87.38 (C-5),
104.27 (C-1), 128.09, 129.02, 131.96 and 135.59 (Ph),
167.51 (PhCONH). Anal. Calcd for C15H19NO6£0.5MeOH:
C 57.17, H 6.51, N 4.30. Found: C 57.04, H 6.57, N 4.08.
H-2), 4.18 (dd, 1H, J4,5ˆ6.1Hz, H-4), 4.30 (m, 1H, J5,6a
ˆ
4.5 Hz, J5,6bˆ3.4 Hz, H-5), 4.34 (dd, 1H, J6a,6bˆ11.5 Hz,
H-6a), 4.50 (m, 1H, H-6b), 4.68 (2£d, 2H, Jgemˆ11.7 Hz,
PhCH2), 4.96 (d, 1H, H-1), 7.25±8.04 (m, 10H, 2£Ph); 13
C
NMR (CDCl3) d 60.48 (C-3), 63.50 (C-6), 65.10 and
65.26 (2£CH2±dioxolane), 72.79 (PhCH2), 78.93 (C-4),
79.11 (C-5), 81.85 (C-2), 102.29 (C-1), 127.79, 127.91,
128.08, 128.27, 129.45, 129.57, 132.84 and 135.78
(2£Ph), 165.89 (PhCO); FAB-MS: m/z 448 (M11Na),
426 (M11H).
4.1.6. Methyl Z-4,7-anhydro-5-benzamido-6,8-di-O-
benzoyl-2,3,5-trideoxy-d-allo-oct-2-enoate (10) and
methyl E-4,7-anhydro-5-benzamido-6,8-di-O-benzoyl-
2,3,5-trideoxy-d-allo-oct-2-enoate (11). A solution of 7
(3 g, 5.8 mmol) in a mixture of CF3CO2H (18 mL) and
6 M HCl (4.5 mL) was kept at 148C for 96 h. The mixture
was concentrated to 5±6 mL and poured into satd aq.
NaHCO3 (15 mL). The aqueous solution was rendered
alkaline with solid NaHCO3 to pH 8±9 and extracted with
CH2Cl2 (4£80 mL). The combined extracts were washed
successively with satd aq. NaHCO3 (50 mL) and water,
dried and evaporated to an unstable oil. The crude aldehyde
9 was immediately dissolved in anhydrous CH2Cl2 (55 mL)
and treated with (carbomethoxymethylene)triphenyl-
phosphorane (4.87 g, 14.6 mmol) in dry CH2Cl2 (16 mL).
After 19 h at room temperature the solvent was evaporated
and the residue was chromatographed on a column of silica
gel (20:1!10:1!5:1toluene±EtOAc). Pure Z-isomer 10
(1.4 g, 56%) was isolated as a white crystalline solid.
Recrystallization from CH2Cl2±hexane gave an analytical
sample 10: mp 114±1158C; [a]Dˆ254 (c, 0.85 in CHCl3);
1H NMR (CDCl3): d 3.76 (s, 3H, OCH3), 4.52 (td, 1H,
J6,7ˆ1.5 Hz, J7,8aˆ4.1Hz, J7,8bˆ3.6 Hz, H-7), 4.61(m,
1H, J4,5ˆ10.1 Hz, J5,NHˆ6.7 Hz, J5,6ˆ5.5 Hz, H-5), 4.63
(dd, 1H, J8a,8bˆ11.9 Hz, H-8a), 4.75 (dd, 1H, H-8b), 5.77
(ddd, 1H, J2,4ˆ1.3 Hz, J3,4ˆ7.8 Hz, H-4), 5.84 (dd, 1H, H-
6), 6.02 (dd, 1H, J2,3ˆ11.7 Hz, H-2), 6.35 (dd, 1H, H-3),
7.67 (d, 1H, NH), 7.33±8.21 (m, 15H, 3£Ph); 13C NMR
(CDCl3) d 52.07 (OCH3), 57.51(C-5), 64.54 (C-8), 74.54
(C-4), 75.67 (C-6), 83.18 (C-7), 122.40 (C-2), 127.03,
128.45, 128.56, 129.48, 129.63, 129.81, 131.63, 133.16,
133.36 and 133.40 (ArC), 146.92 (C-3), 165.57, 166.28,
4.1.4.
2,5-Anhydro-3-benzamido-4,6-di-O-benzoyl-3-
deoxy-d-allose ethylene acetal (7). A solution of 5 (5.4 g,
12.7 mmol) in a mixture of dry EtOH (185 mL) and CHCl3
(9.2 mL) was hydrogenated over 10% Pd/C (12 g) for 120 h
at room temperature. The mixture was ®ltered and the
catalyst washed with EtOH. The ®ltrate and washings
were combined and evaporated to give crude 6 as a colorless
solid. The crude product 6 was dissolved in anhydrous
pyridine (165 mL) and benzyl chloride (13.6 mL,
116.84 mmol) was added to the solution. The mixture was
left at room temperature for 96 h, then poured onto ice and
acidi®ed with 6 M HCl (to pH 3±4) whereupon a white
precipitate was formed. The precipitate was collected by
®ltration, washed with water and dried. Recrystallization
from MeOH gave pure 7 (4.67 g, 71%) as white crystals:
mp 177±177.58C; [a]Dˆ2110.1 (c, 1.02 in CHCl3); nmax
(KBr): 3350, 1730, 1640, 1600 cm21; 1H NMR (DMSO-d6):
d 3.80±3.98 (m, 4H, 2£CH2±dioxolane), 4.34 (dd, 1H,
J1,2ˆ2.9 Hz, J2,3ˆ8.1Hz, H-2), 4.43 (dd, 1H, J5,6a
ˆ
3.8 Hz, J6a,6bˆ11.0 Hz, H-6a), 4.49 (m, 1H, J4,5ˆ3.3 Hz,
J5,6bˆ3.6 Hz, H-5), 4.56 (dd, 1H, H-6b), 4.87 (m, 1H,
J3,4ˆ6.2 Hz, J3,NHˆ8.5 Hz, H-3), 5.00 (d, 1H, H-1), 5.56
(dd, 1H, H-4), 7.72±8.09 (m, 15H, 3£Ph), 8.76 (d, 1H,
NH); 13C NMR (DMSO-d6) d 51.68 (C-3), 64.47 (C-6),
65.11 and 65.28 (2£CH2±dioxolane), 73.9 (C-4), 80.24

M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
575
167.18 and 167.66 (4£CvO). Anal. Calcd for C30H27NO8:
C 68.04, H 5.14, N 2.65. Found: C 67.55, H 5.03, N 3.21.
Further elution with 5:1toluene±EtOAc gave pure E-isomer
11 (0.74 g, 29%) which crystallized from CH2Cl2±hexane to
afford colorless needless mp 1658C; [a]Dˆ2256.6 (c, 0.96
in CHCl3); 1H NMR (CDCl3): d 3.67 (s, 3H, OCH3), 4.52±
4.70 (m, 3H, J6,7ˆ2.2 Hz, J7,8aˆ3.7 Hz, J7,8bˆ4.8 Hz,
COOMe). Anal. Calcd for C17H19N3O6: C 56.51, H 5.30,
N 11.63. Found: C 56.40, H 5.35, N 11.54.
4.1.8. 3(5)-Carboxamido-4-(2-benzamido-2-deoxy-b-d-
ribofuranosyl)pyrazole (15). Procedure A: A solution of
compound 13 (0.2072 g, 0.47 mmol) in saturated methano-
lic solution of NH3 (60 mL) was stored at room temperature
for 6 days. The volatiles were evaporated and the remaining
residue was puri®ed by column chromatography (4:1
toluene±EtOAc, 4:1EtOAc±MeOH) whereupon pure 15
(0.1594 g, 97%) was obtained as a white solid. Recrystal-
lization from EtOH±MeOH gave an analytical sample 15 as
colorless crystals.
J8a,8bˆ12.4 Hz, 2£H-8 and H-7), 4.71(m, H1,
J2,4ˆ
1.5 Hz, J3,4ˆ5.0 Hz, J4,5ˆ9.2 Hz, H-4), 4.92 (m, 1H,
J5,NHˆ8.9 Hz, J5,6ˆ 5.9 Hz, H-5), 5.66 (dd, 1H, H-6), 6.21
(dd, 1H, J2,3ˆ15.7 Hz, H-2), 6.78 (bs, 1H, NH), 7.04 (dd,
1H, H-3), 7.29±8.15 (m, 15H, 3£Ph); 13C NMR (CDCl3) d
51.57 (OCH3), 55.39 (C-5), 64.20 (C-8), 74.82 (C-6), 80.05
(C-4), 81.99 (C-7), 122.28 (C-2), 126.88, 128.44, 128.53,
128.58, 128.89, 129.08, 129.34, 129.48, 129.67 131.84,
133.16, 133.50 and 133.70 (ArC), 143.50 (C-3), 165.22,
166.11, 166.21 and 167.39 (4£CvO). Anal. Calcd for
C30H27NO8: C 68.04, H 5.14, N 2.65. Found: C 67.98, H
5.07, N 2.59.
Procedure B: Compound 11 (0.5798 g, 1.1 mmol) was
dissolved in a mixture of anhydrous ether (7 mL), freshly
distilled dioxane (17 mL) and dry CH2Cl2 (1mL). To the
cooled (08C) and stirred mixture was added a solution of
diazomethane (generated from 3.325 g, 22.6 mmol, of
N-methyl-N0-nitro-N-nitrosoguanidine) in dry ether
(75 mL). The mixture was stirred on an ice bath for 45 h.
After workup as described above (preparation of 14) pure 13
(0.6111 g; 98%) was obtained which was immediately
treated with saturated methanolic solution of NH3 (50 mL)
for 7 days at room temperature. The usual workup (pro-
cedure A) gave pure 15 (0.3206 g, 85% from 11) as color-
4.1.7. 3(5)-Carbomethoxy-4-(2-benzamido-2-deoxy-b-d-
ribofuranosyl)pyrazole (14). To a stirred and cooled
(08C) solution of 10 (0.2512 g, 0.48 mmol) in anhydrous
ether (4.5 mL) was added a solution of diazomethane
(generated from 1.729 g, 11.75 mmol of N-methyl-N0-
nitro-N-nitrosoguanidine) in Et2O (37.5 mL). After 2 h at
08C the volatiles were evaporated whereupon the pyrazoline
12 remained as a white foam. The crude 12 was dissolved in
dry CH2Cl2 (15 mL) and oxidized with a freshly prepared
saturated solution of Cl2 in CCl4 (55 mL). The mixture was
stirred at room temperature for 2.5 h and then evaporated.
Column chromatography (7:3 toluene±EtOAc) of the
residue gave pure pyrazole 13 (0.2072 g, ,100%) as a
less needless: mp 1338C; [a]Dˆ2116.6 (c, 1.08MeOH). 1H
0
0
0
NMR (DMSO-d6): d 3.54 (d, 2H, J4 ,5 ˆ4.6 Hz, 2£H-5 ),
0
0
0
0
0
0
3.82±3.95 (m, 2H, J1 ,2 ˆ10.4 Hz, J2 ,3 ˆ5.2 Hz, J3 ,4
ˆ
1.5 Hz, H-40 and H-20), 3.40±4.60 (bs, 2H, 2£OH), 4.25
(dd, 1H, H-30), 5.39 (d, 1H, H-10), 7.38±7.84 (m, 5H, Ph),
7.60 and 7.90 (2£bs, 1H each, CONH2), 7.85 (s, 1H, H-3),
8.54 (d, 1H, J2 ,NHˆ6.1Hz, NH); 13C NMR (DMSO-d6): d
0
1
colorless hygroscopic syrup: H NMR (CDCl3): d 3.88 (s,
61.17 (C-20), 62.06 (C-50), 70.77 (C-30), 72.34 (C-10), 86.85
(C-40), 121.30 (C-3), 127.11, 128.41, 131.46 and 133.83
(ArC), 130.20 and 142.82 (C-4 and C-5), 164.97
(CONH2), 166.24 (PhCONH).; HRMS (EI): Calcd for
C16H18N4O5: 346.1277. Found: m/z 346.1307 (M1). Anal.
Calcd for C16H18N4O5: C 55.49, H 5.24, N 16.18; Found: C
55.78, H 5.53, N 16.23.
0
0
0
0
0
3H, OMe), 4.50±4.63 (m, 2H, J1 ,2 ˆ10.4 Hz, J2 ,3 ˆ5.6 Hz,
0
0
0
0
0
0
0
J3 ,4 ˆ1.5 Hz, J4 ,5a ˆ4.1Hz, J4 ,5b ˆ3.2 Hz, H-2 and H-4 ),
0
0
0
0
4.73 (dd, 1H, J5a ,5b ˆ11.9 Hz, H-5a ), 5.79 (d, 1H, H-1 ),
5.92 (dd, 1H, H-30), 7.34±8.18 (m, 17H, 3£Ph, H-3 and
N3 ±H), 12.96 (bs, 1H, N1±H); 13C NMR (CDCl3): d
0
52.36 (OMe), 60.43 (C-20), 64.50 (C-50), 73.78 (C-10),
74.77 (C-30), 82.76 (C-40), 123.43 and 138.43 (C-4 and
C-5), 127.01, 128.15, 128.40, 128.50, 128.54, 128.96,
129.41, 129.50, 129.59, 129.65, 131.68, 133.19, 133.26
and 133.40 (3£Ph), 163.92, 165.66, 166.39 and 167.32
(2£PhCOO, PhCONH and COOMe). To a solution of 13
in dry MeOH (10 mL) was added a solution of 5% NaOMe
in MeOH (0.3 mL). The mixture was stirred at room
temperature for 1.5 h, then neutralized with Amberlite IR-
120 [H1] resin, and ®ltered. The resin was washed with
MeOH and the combined ®ltrate and washings were evapo-
rated to dryness. Trituration of the residue with benzene
(2£10 mL) gave crude 14 as a white solid. Recrystallization
from MeOH afforded pure 14 (0.0766 g, 45% from 10) as
4.1.9.
2,5-Anhydro-3-benzamido-4,6-di-O-benzoyl-3-
deoxy-d-allononitrile (17). A solution of 7 (1.2575 g,
2.43 mmol) in a mixture of TFA (9 mL) and 6 M HCl
(2.7 mL) was left at 148C for 120 h. The workup as
described above (preparation of 10 and 11) gave crude alde-
hyde 9 which was immediately dissolved in a mixture of
EtOH (2 mL) and CH2Cl2 (6 mL) and treated with
NH2OH£HCl (0.3724 g, 5.36 mmol) and NaOAc
(0.6487 g, 7.91mmol) while stirring at room temperature
for 2 h. The mixture was evaporated and the residue
extracted with CH2Cl2 (4£40 mL). The extract was dried
and evaporated to afford crude oxime 16 (1.12 g) as a
mixture of the corresponding E- and Z-isomers. To a cooled
(2158C) and stirred solution of crude 16 (1.12 g), in
anhydrous pyridine (4.4 mL) was added dropwise during
30 min a cold solution of MsCl (0.96 mL, 12.46 mmol) in
dry pyridine (2.65 mL). The mixture was warmed to room
temperature, stirred for the next 2 h, then poured into a 1:1
mixture of ice and conc. HCl (pH,2). The emulsion was
extracted with CH2Cl2 (4£80 mL), the combined extracts
were washed with water (50 mL), satd aq. NaHCO3
(50 mL) and again with water (50 mL). The extract was
colorless crystals: mp 2388C; [a]Dˆ276.4 (c, 1.02 in
1
MeOH); H NMR (methanol-d4): d 3.74 (dd, 1H, J4 ,5a
0
0
ˆ
0
0
0
4.6 Hz, J5a ,5b ˆ11.9 Hz, H-5a ), 3.80 (s, 3H, OMe), 3.81
0
0
0
0
0
(dd, 1H, J4 ,5b ˆ3 Hz, H-5b ), 4.05 (m, 1H, J3 ,4 ˆ2.6 Hz,
H-40), 4.35 (dd, 1H, J2 ,3 ˆ5.8 Hz, H-3 ), 4.44 (dd, 1H,
0
0
0
0
0
0
0
J1 ,2 ˆ9.4 Hz, H-2 ), 5.49 (d, 1H, H-1 ), 7.40±7.84 (m, 5H,
Ph), 7.95 (s, 1H, H-3); 13C NMR0 (methanol-d4) d 52.44
(OMe), 61.26 (C-20), 63.61(C-5 ), 72.74 (C-30), 75.04
(C-10), 88.27 (C-40), 124.83 (C-3), 128.31, 129.54, 132.86
and 135.22 (Ph), 169.86 and 169.97 (PhCONH and

576
M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
dried and evaporated, and the residue was puri®ed on a
column of silica gel (4:1cyclohexane±Me CO). Pure 17
132.70 and 135.32 (Ph), 161.58 (C-5), 170.22 (PhCONH).
FAB-MS: m/z 350 (M112Na±H), 328 (M11Na). HR-MS:
Calcd for C13H15N5NaO4: 328.1022. Found: m/z 328.1031.
Anal. Calcd for C13H15N5O4£2.5H2O: C 44.57, H 5.71, N
20.00. Found: C 44.23, H 5.12, N 20.41.
2
(0.7171 g, 63%) was obtained as a white solid. Recrystal-
lization from MeOH±EtOAc gave an analytical sample 17:
mp 2048C; [a]Dˆ282.4 (c, 1.1 in Me2CO). 1H NMR
(acetone-d6): d 4.67 (dd, 1H, J5,6aˆ3.8 Hz, J6a,6bˆ12.2 Hz,
H-6a), 4.75 (dd, 1H, J5,6bˆ3.4 Hz, H-6b), 4.81(m, H1 ,
J4,5ˆ2.3 Hz, H-5), 5.23 (d, 1H, J2,3ˆ9.5 Hz, H-2), 5.58
(m, 1H, J3,NHˆ8.5 Hz, J3,4ˆ5.8 Hz, H-3), 5.89 (dd, 1H,
H-4), 7.33±8.27 (m, 15H, 3£Ph), 8.42 (d, 1H, NH); 13C
NMR (acetone-d6): d 56.71(C-3), 64.92 (C-6), 68.65
(C-2), 74.25 (C-4), 84.05 (C-5), 118.12 (CN), 128.19,
129.17, 129.39, 129.50, 130.14, 130.45, 130.56, 132.57,
134.19, 134.45 and 134.54 (3£Ph), 165.90, 166.51 and
168.08 (2£PhCOO and PhCONH). Anal. Calcd for
C27H22N2O6: C 68.93, H 4.71, N 5.95. Found: C 68.52, H
5.11, N 5.55.
4.1.12. 2,5-Anhydro-3,6-di-O-benzoyl-d-allose ethylene
acetal (21). To a solution of 2011 (3.55 g, 7.21mmol) in
95% aq. DMF (73 mL) was added CaCO3 (1.44 g,
14.0 mmol). The mixture was stirred for 20 h at 155±
1608C and then ®ltered. The ®ltrate was diluted with
water (300 mL) and extracted with 1:1 mixture of benzene±
hexane (3£200 mL). The combined extracts were washed
with water (2£50 mL) dried and ®ltered. The organic solu-
tion was left at room temperature for 10 h whereupon pure
21 was obtained as a white solid. Recrystallization from
CH2Cl2±hexane gave an analytical sample 21 (0.72 g,
24%) as colorless crystals: mp 1358C; [a]Dˆ22.8 (c, 0.6
in CHCl3). 1H NMR (CDCl3): d 2.45 (d, 1H, JOH,4ˆ5.8 Hz,
OH-4), 3.79±4.10 (m, 4H, 2£CH2±dioxolane), 4.28 (ddd,
1H, J4,5ˆ10.9 Hz, J5,6aˆ5.1Hz, J5,6bˆ2.9 Hz, H-5), 4.37 (t,
1H, J1,2ˆJ2,3ˆ2.3 Hz, H-2), 4.45±4.56 (m, 2H, J3,4ˆ5.4 Hz,
J6a,6bˆ12.2 Hz, H-4 and H-6a), 4.69 (dd, 1H, H-6b), 5.09 (d,
1H, H-1), 5.51 (dd, 1H, H-3), 7.39±8.16 (m, 10H, 2£Ph);
13C NMR (CDCl3): d 64.14 (C-6), 65.50 and 65.62
(2£CH2±dioxolane), 71.65 (C-4), 74.12 (C-3), 80.46
(C-5), 82.69 (C-2), 102.35 (C-1), 128.32, 128.50, 129.29,
129.79, 133.08 and 133.51 (2£Ph), 166.22 and 166.53
(2£PhCOO). Anal. Calcd for C22H22O8: C 63.76, H 5.35.
Found: C 63.96, H 5.50. Mother liquor was evaporated to
afford an inseparable mixture of 21 and 22 (1.86 g, 62%) as
colorless syrup, which was used for further work without
additional puri®cation.
4.1.10. 5-(2-Benzamido-3,5-di-O-benzoyl-2-deoxy-b-d-
ribofuranosyl)tetrazole (18). A mixture of 17 (0.5574 g,
1.18 mmol), NaN3 (0.1534 g, 2.36 mmol) and NH4Cl
(0.1203 g, 2.25 mmol) in DMF (4.3 mL) was stirred at
1008C for 2.5 h. An additional amount of NaN3 (0.0394 g,
0.61mmol) and NH 4Cl (0.0298 g, 0.56 mmol) was added to
the reaction mixture, the stirring was continued at 1008C for
the next 1.5 h, and the solvent was evaporated. The residue
was extracted with CH2Cl2, and the extract was ®ltered and
evaporated. Column chromatography (4:1EtOAc±MeOH)
of the residue gave pure 18 (0.3339 g, 55%) as a pale yellow
solid. Recrystallization from MeCN±MeOH gave an analy-
tical sample 18 as colorless crystals: mp 262±2638C;
1
[a]Dˆ2196.2 (c, 0.98 in pyridine). H NMR (acetone-d6):
0
0
0
d
4.73 (d, 2H, J4 ,5 ˆ4 Hz, 2£H-5 ), 4.86 (m, 1H,
J3 ,4 ˆ2.2 Hz, H-40), 5.46 (ddd, 1H, J1 ,2 ˆ8.8 Hz,
0
0
0
0
0
0
0
0
0
J2 ,NHˆ8.4 Hz, J2 ,3 ˆ5.7 Hz, H-2 ), 5.84 (d, 1H, H-1 ),
5.94 (dd, 1H, H-30), 7.32±8.24 (m, 16H, 3£Ph and
NH-tetrazole), 8.49 (d, 1H, PhCONH); 13 C NMR (acetone-
d6): d 57.36 (C-20), 65.54 (C-50), 73.99 (C-10), 75.18 (C-30),
83.72 (C-40), 127.85, 128.23, 128.31, 128.98, 129.06,
129.38, 129.44, 130.46, 130.66, 130.72, 132.31, 134.07,
134.36 and 134.95 (3£Ph), 166.14, 166.73 and 168.08
(2£PhCOO and PhCONH). HR-MS: Calcd for
C27H23N5O6: 513.1648. Found: m/z 513.1661.
4.1.13. 2,5-Anhydro-3,6-di-O-benzoyl-4-O-methanesul-
fonyl-d-allose ethylene acetal (23). Procedure A: To a stir-
red and cooled (08C) solution of 21 (2.0122 g, 4.86 mmol)
in dry pyridine (50 mL) was added MsCl (1.25 mL,
16.08 mmol). The mixture was left at 148C for 48 h, then
poured onto ice, acidi®ed with conc. HCl (to pH 2) and the
resulting solution was extracted with CH2Cl2 (4£100 mL).
The combined extracts were washed with water (100 mL),
satd aq. NaHCO3 (100 mL) and again with water (100 mL),
dried and evaporated to give crude 23 as a pale yellow
crystalline mass. Recrystallization from EtOH gave pure
23 (2.27 g, 95%) as colorless crystals: mp 1078C;
[a]Dˆ115.15 (c, 1.2 in CHCl3). 1H NMR (CDCl3): d
2.98 (s, 3H, MeSO2), 3.78±4.05 (m, 4H, 2£CH2±dioxo-
lane), 4.40 (dd, 1H, J1,2ˆ2.0 Hz, J2,3ˆ4.0 Hz, H-2), 4.48
(dd, 1H, J5,6aˆ4.1Hz, J6a,6bˆ12.1 Hz, H-6a), 4.57 (m, 1H,
H-5), 4.75 (dd, 1H, J5,6bˆ3 Hz, H-6b), 5.10 (d, 1H, H-1),
5.37 (dd, 1H, J3,4ˆ5.5 Hz, J4,5ˆ6.5 Hz, H-4), 5.65 (dd, 1H,
H-3), 7.40±8.20 (m, 10H, 2£Ph); 13C NMR (CDCl3): d
38.11 (MeSO2), 62.71(C-6), 65.50 and 65.62 (2 £CH2±
dioxolane), 71.35 (C-3), 76.62 (C-4), 79.36 (C-5), 81.97
(C-2), 101.85 (C-1), 128.35, 128.57, 129.71, 129.75,
133.12 and 133.67 (2£Ph), 165.40 and 166.00
(2£PhCOO). Anal. Calcd for C23H24O10S: C 56.10, H
4.87, S 6.50. Found: C 56.26, H 4.75, S 6.46.
4.1.11.
5-(2-Benzamido-2-deoxy-b-d-ribofuranosyl)-
tetrazole (19). To a solution of 18 (0.2392 g, 0.47 mmol)
in dry MeOH (20 mL) was added 5% solution of MeONa in
MeOH (0.2 mL) and the mixture was stirred at room
temperature for 2.5 h. An additional amount of the base
(0.2 mL) was added to the mixture and stirring at room
temperature was continued for the next 1h. The workup
as described above (preparation of 8) gave crude 19 as a
yellow oil. Silica gel column chromatography (EtOAc, 4:1,
7:3 EtOAc±MeOH) gave pure 19 (0.1029 g, 72%) as a
white solid. Recrystallization from MeOH±EtOAc gave
an analytical sample 19 as colorless crystals: mp 1888C;
1
[a]Dˆ2118.8 (c, 0.99 in MeOH). H NMR (methanol-d4):
0
0
0
0
0
d 3.75 (dd, 1H, J4 ,5a ˆ4.3 Hz, J5a ,5b ˆ12.3 Hz, H-5a ), 3.87
0
0
0
0
0
(dd, 1H, J4 ,5b ˆ3.3 Hz, H-5b ), 4.14 (m, 1H, J3 ,4 ˆ2.8 Hz,
H-40), 4.42 (dd, 1H, J2 ,3 ˆ5.9 Hz, H-3 ), 4.75 (dd, 1H,
0
0
0
0
0
0
0
J1 ,2 ˆ9.2 Hz, H-2 ), 5.33 (d, 1H, H-1 ), 7.32±7.82 (m, 5H,
Ph); 13C NMR (methanol-d4): d 59.76 (C-20), 63.44 (C-50),
72.30 (C-30), 75.78 (C-10), 88.79 (C-40), 128.48, 129.39,
Procedure B: To a stirred and cooled (08C) solution of crude
mixture 21 and 22 (mother liquor, 1.42 g, 3.43 mmol) in dry
pyridine (50 mL) was added MsCl (0.81mL, 10.41mmol).

M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
577
The solution was stored at 148C for 48 h. After workup as
described above (procedure A) the crude mixture was
obtained which was puri®ed on a column of silica gel (5:1
toluene±EtOAc). Physical and spectral data of thus obtained
sample 23 (0.56 g, 33%) were in full agreement with the
corresponding values reported above (procedure A). Further
elution gave pure 24 (0.21g, 12.5%) as a colorless oil:
[a]Dˆ152.9 (c, 1.12 in CHCl3). 1H NMR (CDCl3): d
2.97 (s, 3H, MeSO2), 3.92±4.05 (m, 4H, 2£CH2±dioxo-
lane), 4.46 (t, 1H, J1,2ˆ2.3 Hz, J2,3ˆ2.8 Hz, H-2), 4.49
(dd, 1H, J5,6aˆ4.5 Hz, J6a,6bˆ11.8 Hz, H-6a), 4.58 (m, 1H,
J4,5ˆ7.5 Hz, J5,6bˆ3.5 Hz, H-5), 4.70 (dd, 1H, H-6b), 5.09
(d, 1H, H-1), 5.38 (dd, 1H, J3,4ˆ5.5 Hz, H-3), 5.53 (dd, 1H,
H-4), 7.32±8.11 (m, 10H, 2£Ph); 13C NMR (CDCl3): d
38.34 (MeSO2), 63.49 (C-6), 65.53 and 65.64 (2£CH2±
dioxolane), 71.93 (C-4), 77.54 (C-3), 78.27 (C-5), 83.07
(C-2), 101.59 (C-1), 128.28, 128.54, 129.70, 129.83,
133.08 and 133.66 (2£Ph), 165.53 and 166.07
(2£PhCOO). LRMS (EI): m/z 492 (M1).
(4:1, 7:3, 3:2 toluene±EtOAc). Pure product 28 (0.41g,
48% from 23) was isolated as colorless syrup: [a]Dˆ19.4
(c, 0.91in CHCl 3). 1H NMR (CDCl3): d 2.99 (s, 3H,
MeSO2), 3.73 (s, 3H, OMe), 0 4.59±4.720 (m, 2H,
0
0
0
0
J4 ,5a ˆ3.8 Hz, J5a ,5b ˆ14.5 Hz, H-4 and H-5a ), 4.85 (m,
1H, H-5b0), 5.43 (t, 1H, J2 3 ˆ5.4 Hz, J3 ,4 ˆ5.6 Hz, H-3 ),
0
0
0
0
0
0
0
0
0
5.61(t, 1H, J1 ,2 ˆ5.2 Hz, H-2 ), 5.75 (d, 1H, H-1 ), 7.35±
8.17 (m, 10H, 2£Ph), 7.85 (s, 1H, H-3), 13.42 (bs, 1H, NH);
13C NMR (CDCl3): d 38.23 (MeSO2), 52.09 (OMe), 62.85
(C-50), 75.77 (C-10), 76.22 (C-20), 76.38 (C-30), 79.53
(C-40), 121.57 (C-4), 128.53, 128.65, 128.75, 128.99,
129.40, 129.67, 129.86, 133.33, 133.82 (2£Ph), 130.24
(C-3), 138.55 (C-5), 161.82 (COOMe) 165.49 and 166.15
(2£PhCOO).
4.1.15. 3(5)-Carboxamido-4-(3-O-methanesulfonyl-b-d-
ribofuranosyl)pyrazole (29). A solution of 28 (0.5304 g,
0.97 mmol) in saturated methanolic solution of NH3
(50 mL) was left at room temperature for 7 days, and then
evaporated. The remaining crude mixture was triturated
with benzene. The benzene solution was decanted, and the
remaining crude oil was puri®ed by preparative TLC (7:3
CHCl3±MeOH). Pure 29 (0.313 g, 86%) was thus obtained
as colorless oil: [a]Dˆ126.4 (c, 1.03 in MeOH). Crystal-
lization from MeOH±iPr2O gave an analytical sample 29 as
colorless crystals: mp 110±1118C. 1H NMR (DMSO-d6): d
4.1.14.
methanesulfonyl-b-d-ribofuranosyl)pyrazole (28).
3(5)-Carbomethoxy-4-(2,5-di-O-benzoyl-3-O-
A
solution of 23 (2.8261g, 5.74 mmol) in a mixture of TFA
(28 mL) and 6 M HCl (7 mL) was kept at 148C for 24 h.
The workup as described earlier (preparation of 10 and 11)
gave crude aldehyde 25 which was immediately dissolved in
dry CH2Cl2 (50 mL) and allowed to react with a solution of
(carbomethoxymethylene)triphenylphosphorane (4.7916 g,
14.37 mmol) in dry CH2Cl2 (15 mL) for 24 h at room
temperature. The mixture was evaporated and puri®ed by
column chromatography (10:1 toluene±EtOAc) whereupon
the oily unsaturated ester 26 (1.1346 g, 48%) was obtained
as a 4:1mixture of E- and Z-isomers: [a]Dˆ261.2 (c, 1.11
in CHCl3). 1H NMR (CDCl3): d 2.95 (s, MeSO2, E-isomer),
3.00 (s, MeSO2, Z-isomer), 3.69 (s, OMe, Z-isomer), 3.70 (s,
OMe, E-isomer), 4.55 (dd, J7,8aˆ3.2 Hz, J8a,8bˆ12 Hz,
H-8a, E-isomer), 4.67 (m, J6,7ˆ4.5 Hz, J7,8bˆ3.1Hz, H-7,
E-isomer), 4.74 (dd, H-8b, E-isomer), 4.85 (m, J2,4ˆ1.5 Hz,
J3,4ˆ4.8 Hz, J4,5ˆ6.5 Hz, H-4, E-isomer), 5.21(t,
J5,6ˆ5.6 Hz, H-5, E-isomer), 5.37 (t, H-6, E-isomer), 5.97
(dd, J2,3ˆ11.6 Hz, J2,4ˆ1.2 Hz, H-2, Z-isomer), 6.17 (dd,
J3,4ˆ7.9 Hz, H-3, Z-isomer), 6.24 (dd, J2,3ˆ15.7 Hz, H-2,
E-isomer), 7.00 (dd, H-3, E-isomer), 7.40±8.18 (m, 10H,
2£Ph); 13C NMR (CDCl3): d 38.08 (MeSO2, E-isomer),
38.22 (MeSO2, Z-isomer), 51.55 (OMe, Z-isomer), 51.69
(OMe, E-isomer), 62.80 (C-8, E-isomer), 63.04 (C-8,
Z-isomer), 74.59 (C-5, E-isomer), 76.84 (C-6, E-isomer),
79.14 (C-4, E-isomer), 80.85 (C-7, E-isomer), 122.97
(C-2, E-isomer), 123.48 (C-2, Z-isomer), 125.21, 128.14,
128.39, 128.50, 128.68, 128.94, 129.27, 129.62, 129.66,
129.79, 129.90, 133.30, 133.69 and 133.95 (2£Ph), 142.36
(C-3, E-isomer), 144.11 (C-3, Z-isomer), 165.51, 165.94
and 165.98 (2£PhCOO and COOMe). To a stirred and
cooled (08C) solution of 26 (0.419 g, 0.83 mmol) in dry
Et2O (4 mL) was added a solution of diazomethane
(generated from 1.596 g, 10.85 mmol, of N-methyl-N0-
nitro-N-nitrosoguanidine) in Et2O (36 mL). The mixture
was stirred at 08C for 5 h and then evaporated. The remain-
ing crude 2-pyrazoline 27 was dissolved in a mixture of dry
CCl4 (10 mL) and CH2Cl2 (4 mL) and treated with a freshly
prepared saturated solution of Cl2 in CCl4 (15 mL) for 3 h at
room temperature. The mixture was evaporated and the
remaining crude 28 was puri®ed on a column of silica gel
0
0
3.21(s, 3H, MeSO 2), 3.51(dd, H1,
0
J5a ,5b ˆ12.1 Hz,
0
0
0
0
0
J4 ,5a ˆ4.1Hz, H-5a ), 3.56 (dd, 1H, J4 ,5b ˆ4.0 Hz, H-5b ),
4.05 (m, 1H, J3 ,4 ˆ3.2 Hz, 0H-40), 4.13 (dd, 1H,
0
0
0
0
0
0
0
J1 ,2 ˆ7.7 Hz, J2 ,3 ˆ5.2 Hz, H-2 ), 4.88 (dd, 1H, H-3 ),
5.11 (d, 1H, H-10), 3.42 and 5.84 (2£bs, 1H each, 2£OH),
7.38 and 7.64 (2£bs, 1H each, CONH2), 7.86 (s, 1H, H-3),
13.28 (bs, 1H, NH); 13C NMR (DMSO-d6): d 38.21
(MeSO2), 60.92 (C-50), 74.96 (C-20), 75.44 (C-10), 81.21
(C-30), 82.51(C-4 0), 120.44 (C-3), 130.15 and 143.05
(C-4 and C-5), 164.47 (CONH2). FAB-MS: m/z 344
(M11Na),
322
(M11H).
Anal.
Calcd
for
C10H15N3O7S£0.5H2O: C 36.36, H 4.88, N 12.72. Found:
C 36.20, H 5.00, N 12.38.
4.1.16. 2,5-Anhydro-4-azido-3,6-di-O-benzoyl-4-deoxy-
d-gulose ethylene acetal (30). To a solution of 23 (3.02 g,
6.14 mmol) in DMSO (60 mL) was added NaN3 (4.24 g,
65.23 mmol). The resulting suspension was stirred at 118±
1208C for 144 h and then poured into cold water (100 mL)
and extracted with 1:1 benzene±hexane (4£50 mL). The
extract was washed with water (2£20 mL), dried and evapo-
rated. The remaining crude mixture was puri®ed by column
chromatography (99:1, 11:4 toluene±EtOAc) to afford pure
30 (1.68 g, 62%) as a colorless oil: [a]Dˆ26.4 (c, 1.59 in
CHCl3). nmax (®lm): 2100 cm21; 1H NMR (CDCl3): d 3.86±
4.15 (m, 4H, 2£CH2±dioxolane), 4.18 (dd, 1H, J1,2ˆ4.7 Hz,
J2,3ˆ3 Hz, H-2), 4.30 (dd, 1H, J3,4ˆ1.1 Hz, J4,5ˆ4.1Hz,
H-4), 4.46 (m, 1H, J5,6aˆ5.7 Hz, J5,6bˆ5.9 Hz, H-5), 4.55±
4.69 (m, 2H, 2£H-6), 5.17 (d, 1H, H-1), 5.58 (dd, 1H, H-3),
7.40±8.13 (m, 10H, 2£Ph); 13C NMR (CDCl3): d 62.64
(C-6), 65.35 and 65.68 (2£CH2±dioxolane), 66.70 (C-4),
78.14 (C-3), 78.30 (C-5), 83.76 (C-2), 102.25 (C-1),
127.96, 128.14, 128.53, 128.57, 129.26, 129.31, 132.76
and 133.28 (2£Ph). Further eluting gave pure 31 (0.18 g,
9%) as a colorless syrup: 1H NMR (CDCl3): d 2.72 (bs, 1H,
exchangeable with D2O, OH), 3.75 (t, 1H, J1,2ˆ5.1Hz,
J2,3ˆ5.3 Hz, H-2), 3.81±4.08 (m, 4H, 2£CH2±dioxolane),

578
M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
0
0
0
4.18 (dd, 1H, J3,4ˆ4.2 Hz, J4,5ˆ5.3 Hz, H-4), 4.29±4.60 (m,
4H, H-3, H-5 and 2£H-6), 5.00 (d, 1H, H-1), 7.30±8.13 (m,
5H, Ph); 13C NMR (CDCl3): d 63.21(C-6), 65.21and 65.35
(2£CH2±dioxolane), 68.10 (C-4), 76.42 (C-3), 77.50 (C-5),
84.63 (C-2), 102.97 (C-1), 128.29, 128.45, 129.53, 129.59
and 133.09 (Ph), 166.25 (PhCOO).
nol-d4): d 3.83 (d, 2H, J4 ,5 ˆ5.8 Hz, 2£H-5 ), 4.17 (dd, 1H,
0 0
0 0 0 0
J3 ,4 ˆ4.6 Hz, J2 ,3 ˆ2.6 Hz, H-3 ), 4.40 (m, 1H, H-4 ), 4.58
(t, 1H, J1 ,2 ˆ3 Hz, H-2 ), 5.14 (d, 1H, H-1 ); 13C NMR
(methanol-d4): d 61.37 (C-50), 69.39 (C-30), 79.57 (C-10),
81.19 (C-20), 82.72 (C-40), 157.69 (C-5). LR-MS (CI): m/z
257 (M11C4H10±N2), 228 (M11H).
0
0
0
0
4.1.17. 2,5-Anhydro-4-azido-3,6-di-O-benzoyl-4-deoxy-
d-gulononitrile (34). A solution of 30 (1.68 g, 3.83 mmol)
in a mixture of TFA (8.5 mL) and 6 M HCl (2.1mL) was
left at 148C for 96 h. The workup as described earlier
(preparation of 10 and 11) gave crude aldehyde 32,which
was immediately dissolved in a mixture of EtOH (13 mL)
and CH2Cl2 (2 mL) and allowed to react with NH2OH£HCl
(0.6 g, 8.63 mmol) and NaOAc (0.96 g, 11.58 mmol) by
stirring at room temperature for 2 h. After work up as
described above (preparation of 17), crude oxime 33 was
obtained as a mixture of E- and Z-isomers. To a cooled
(2158C) and stirred solution of crude 33 (1.1 g) in
anhydrous pyridine (5 mL) was added dropwise precooled
(08C) solution of MsCl (1.29 mL,16.55 mmol) in dry
pyridine (3.5 mL). The mixture was warmed to room
temperature and stirred for the next 2 h. The usual workup
(preparation of 17) gave crude 34 as brown oil. Chromato-
graphic puri®cation on a column of silica gel (4:1cyclo-
4.2. X-Ray analysis17
Colorless, transparent single crystal of 15 was mountered on
a Enraf±Nonius CAD-4 diffractometer equipped with
graphite-monochromated Mo Ka radiation. Cell parameters
(Table 3) were determined by least-squares re®nement of
diffractometer angles for 24 re¯ections collected in the
range 7.0#u#1.08. Three standard re¯ections (21 1 26),
(2116) and ( 21 1 29) were monitored every 60 min with
intensity variations Rintˆ0.00057, R(s)ˆ0.0931. Re¯ec-
tions were recorded with v±2u scan with Miller indices
hminˆ21, hmaxˆ8, kminˆ0, kmaxˆ15, lminˆ29, lmaxˆ22
and merged with Rˆ0.0057 to 1806 unique re¯ections.
Data were corrected for Lorentz and polarization effects.
The structure was solved by a direct method using the
SHELXS86 program.18 The E-map computed from
the phase set with best combined ®gure of merit revealed
the positions of all non-hydrogen atoms along with an
oxygen atom (O0) from the independent water molecule
that was also included into the crystal lattice. Full-matrix
least squares re®nement of the fractional coordinates of the
non-hydrogen atoms with anisotropic atomic displacement
parameters was performed with SHELXL97.19 Positions of
hydrogen atoms were generated from the assumed
geometry, checked in difference Fourier map and re®ned
isotropically with common displacement parameters
hexane±Me2CO) gave pure 34 (0.324 g, 22% from 30) as
1
colorless syrup: [a]Dˆ232.7 (c, 2.61in CHCl ). H NMR
3
(CDCl3): d 4.51(dd, 1H, J4,5ˆ3.5 Hz, J3,4ˆ1.4 Hz, H-4),
4.56±4.76
(m,
3H,
J6a,6bˆ11.7 Hz,
J5,6aˆ6 Hz,
J5,6bˆ5.2 Hz, H-5 and 2£H-6), 4.84 (d, 1H, J2,3ˆ1.6 Hz,
H-2), 5.74 (t, 1H, H-3), 7.40±8.12 (m, 10H, 2£Ph); 13C
NMR (CDCl3): d 62.29 (C-6), 65.70 (C-4), 70.22 (C-5),
79.86 (C-2), 80.50 (C-3), 115.40 (CN), 127.72, 128.44,
129.18, 129.68, 129.84, 133.39 and 134.30 (2£Ph), 164.88
and 165.99 (2£PhCOO).
Ê 2
Ê 2
U1ˆ0.053(7) A and U2ˆ0.093(12) A . For solvated water
hydrogens isotropic displacement parameters were ®xed to
4.1.18. 5-(3-Azido-2,5-di-O-benzoyl-3-deoxy-b-d-xylo-
furanosyl) tetrazole (35). A suspension of compound 34
(0.12 g, 0.3 mmol), NaN3 (0.04, 0.55) and NH4Cl (0.03,
0.53 mmol) in DMF (1mL) was stirred at 100 8C for 2.5 h.
The workup as described earlier (preparation of 18) gave
crude 35, which was puri®ed by preparative TLC (80:23
CHCl3±MeOH) to afford pure 35 (0.08 g, 58%) as a color-
Table 3. Crystallographic data and structure re®nement of 15
Crystallographic parameter
Empirical formula
Formula weight
Temperature (K)
C16H18N4O5£H2O
364.36
293(2)
Ê
Wavelength (A)
0.71070
Orthorhombic
P212121
less syrup: [a]Dˆ229.4 (c, 1.85 in CHCl3). 1H NMR
Crystal system
Space group
Unit cell dimensions
0
0
0
(acetone-d6): d 4.63 (d, 2H, J4 ,5 ˆ5.5 Hz, 2£H-5 ), 4.81
Ê
aˆ7.122(2) A, aˆ908
0
Ê
0
0
0
0
(m, 1H, J3 ,4 ˆ4.3 Hz, H-4 ), 4.87 (dd, 1H, J2 ,3 ˆ1.5 Hz,
bˆ13.049(2) A, bˆ908
H-30), 5.62 (d, 1H, J1 ,2 ˆ3.3 Hz, H-1 ), 5.96 (dd, 1H,
H-20), 7.43±8.18 (m, 10H, 2£Ph); 13C NMR (acetone-d6):
d 63.72 (C-50), 67.47 (C-30), 77.43 (C-10), 80.02 (C-40),
82.53 (C-20), 129.38, 129.46, 130.02, 130.31, 130.55,
130.65, 134.10 and 134.53 (2£Ph), 157.29 (C-5), 165.96
and 166.52 (2£PhCOO). LR-MS (CI): m/z 493
(M11C4H10), 436 (M11H).
0
Ê
cˆ19.229(2) A, gˆ908
0
0
Ê 3
Volume (A )
Z
1787.0(6)
4
1.354
Density (calculated) (mg m23
)
)
Absorption coef®cient (mm21
F(000)
0.105
768
Crystal size (mm3)
0.35£0.25£0.20
Q range for data collection (8)
Index ranges
Re¯ections collected
Independent re¯ections
Re®nement method
Data/restraints/parameters
Goodness-of-®t on F2
Final R indices [I.2s(I)]
R indices (all data)
Absolute structure parameter
Extinction coef®cient
2.1±24.95
21#h#8, 0#k#15, 29#l#22
1913
4.1.19. 5-(3-Azido-3-deoxy-b-d-xylofuranosyl)tetrazole
(36). To a solution of 35 (0.13 g, 0.29 mmol) in dry
MeOH (6 mL) was added 5% solution of MeONa in
MeOH (0.35 mL) and the mixture was stirred at room
temperature for 2.5 h. The workup as described earlier
(preparation of 19) yielded crude 36, which was separated
from methyl benzoate after triturating with benzene. Pure
product 36 (0.06 g, 91%) was thus obtained as a colorless
syrup: [a]Dˆ2122.6 (c, 0.99 in MeOH). 1H NMR (metha-
1806 [R(int)ˆ0.0057]
Full-matrix least-squares on F2
1806/0/245
0.947
R1ˆ0.0661, wR2ˆ0.1520
R1ˆ0.1128, wR2ˆ0.1692
3(4)
0.012(4)
0.321and 20.290
Largest difference peak and
Ê 23
hole (e A
)

M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
579
1.3Ueq of the parent atom O0. The uncertainty of the
Acknowledgements
hydrogen position within the pyrazole ring indicated the
presence of two tautomeric forms.
This work was supported by a research grant from the
Ministry of Science, Technologies and Development of
the Republic of Serbia. The authors are grateful to Mrs T.
Marinko-Covell (University of Leeds, UK) and to Mr D.
Djokovic (Faculty of Chemistry, University of Belgrade,
YU), for recording the mass spectra, as well as to Mr M.
Huscroft (University of Leeds, UK) for elemental micro-
analyses.
The re®nement of the occupational factors of hydrogens H1
and H2 bonded to N1and N2 atoms lead to the ®nal value of
0.28(8) and 0.72(8), respectively. Final R-factor was
Rˆ0.0661, for 245 parameters using the F2 values of 1806
(I.2s(I)) re¯ections. The highest and the lowest peaks in
Ê 23
the ®nal difference map were 0.321and 0.290 e A
Scattering factors were taken from SHELXL97.
.
References
4.3. Cytotoxic activity
1. For reviews, see: De Clercq, E. In Design of Anti-AIDS Drugs,
De Clercq, E., Ed.; Elsevier: Amsterdam, 1990; pp 1±95.
Huryn, D. M.; Okabe, M. Chem. Rev. 1992, 92, 1745±1768.
Perigaud, C.; Gosselin, G.; Imbach, J.-L. Nucleosides Nucleo-
tides 1992, 11, 903±945.
Cytotoxic activity was determined for mouse neuro-
blastoma (N2a) and baby hamster kidney (BHK 21) cells
suspended at a density of 1024 cells/0.1mL. Stock solutions
of the tested compounds (cˆ0.1mol/L) in growth medium
(GM) were prepared as follows: Compound 15 was
dissolved in Eagle Minimal Essential Medium (EMEM)
supplemented with 10% calf serum. Due to their poor solu-
bilities compounds 8, 29 and 36 were dissolved in GM
containing 0.5% of Tween 80. Eight serial twofold diluted
solutions were prepared on a microplate for cell culture
(0.1mL volume per well), starting with 0.01M concentra-
tion for each tested substance. In parallel, GM with and
without Tween 80 were included as controls (GM with
0.5% Tween 80 was serially diluted as above). N2a and
BHK 21cell suspensions (0.1mL) were added separately
to each tested solution and controls. The ®nal concentrations
of tested compounds were: 5000, 2500, 1250, 625, 312, 156,
78 and 39 mM/L. The microplate was then incubated for
24 h at 378C in the atmosphere with 5% of CO2. At the
end of the incubation period, the microplate was examined
under an inverted optical microscope (magni®cation 40£)
and the percentage of rounded cells recorded for each well.
The concentration that resulted in 50% inhibition of growth
(IC50) was determined by linear regression technique
(Table 1).
2. Manfredini, S.; Bazzanini, R.; Baraldi, P. G.; Simoni, D.;
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4.4. Antiviral activity
Compounds 8, 15, 29 and 36 were evaluated for possible
inhibition of rabies virus growth in mouse neuroblastoma
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tested compounds were prepared as described above
(0.05 mL per well) starting with 0.02 M solution for each
substance. In parallel, GM with and without Tween 80 were
included as controls. Rabies virus strain CVS was added to
each well (0.05 mL) in concentrations which form
speci®c nucleocapsid inclusions in 70% of cells after 24 h
incubation in GM control wells. N2a cell suspension (104
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atmosphere with 5% CO2. The cells were ®xed with 80%
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compound 15 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication

580
M. Popsavin et al. / Tetrahedron 58 (2002) 569±580
È È
Crystal Structures, University of Gottingen: Gottingen,
no. CCDC 167534. Copies of the data can be obtained, free of
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@ccdc.cam.ac.uk).
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