1,N6-Etheno-7-deaza-2,8-diazaadenosine: Syntheses, properties and conversion to 7-deaza-2,8-diazaadenosine

Article abstract of DOI:10.1039/b418849g

1,N⁶-Etheno-7-deaza-2,8-diazaadenosine (4) was synthesized from 8-aza-7-deazaadenosine (6) in 64% overall yield. The starting material 6 was obtained by the direct glycosylation of 8-aza-7-deazaadenine (7) with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-

Full text of DOI:10.1039/b418849g

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A R T I C L E
1,N6-Etheno-7-deaza-2,8-diazaadenosine: syntheses, properties and
conversion to 7-deaza-2,8-diazaadenosine†
Wenqing Lin,a,b Hong Li,a,b Xin Minga,b and Frank Seela*a,b
a Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t
Osnabru¨ck, Barbarastr. 7, 49069, Osnabru¨ck, Germany
b Center for Nanotechnology (CeNTech), Gievenbecker Weg 11, 48149, Mu¨nster, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; Fax: +49-541-9692370;
Tel: +49-541-969-2791
Received 16th December 2004, Accepted 10th March 2005
First published as an Advance Article on the web 30th March 2005
1,N6-Etheno-7-deaza-2,8-diazaadenosine (4) was synthesized from 8-aza-7-deazaadenosine (6) in 64% overall yield.
The starting material 6 was obtained by the direct glycosylation of 8-aza-7-deazaadenine (7) with
1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (8) (NO2 CH3, BF3·Et2O; 77% yield). Compound 4 was transformed
into 7-deaza-2,8-diazaadenosine (5). The fluorescence of compound 4 shows an emission maximum at 531 nm
(phosphate buffer; pH 7.0), which is bathochromically shifted compared to 1,N6-etheno-2-azaadenosine (3a) (495
nm). A conformational analysis was performed in the solid state and in solution.
Introduction
The etheno derivatives of adenosine and cytidine, as well as their
2ꢀ-deoxy congeners, are strongly fluorescent.1 This behavior is
different from the canonical nucleosides which do not show
appreciable fluorescence in aqueous solution. The physical
properties of etheno nucleosides2 enable them to be used as
structural and functional probes in DNA and RNA. Thus, they
are applicable in real-time PCR and have the potential to be
used for the detection of nucleic acids in various RNA and DNA
formats. The etheno adducts of the 2ꢀ-deoxyadenosine and the
2ꢀ-deoxycytidine cause mutagenic lesions in DNA.3 This refers to
an unusual base pairing of 1,N6-etheno-2ꢀ-deoxyadenosine (e-2ꢀ-
deoxyadenosine, 1a) and 1,N6-etheno-adenosine (e-adenosine,
1b) (Scheme 1) with guanosine or its 2ꢀ-deoxy derivative.
Moreover, these nucleosides are rather unstable in acidic and
alkaline solution thereby cause problems during oligonucleotide
synthesis.1b This problem is circumvented by the use of 1,N6-
etheno-7-deaza-2ꢀ-deoxyadenosine (2) (purine numbering is
used in the results and discussion sections, except otherwise
stated) being stable under acidic and alkaline conditions due to
the replacement of the purine by the pyrrolo[2,3-d]pyrimidine
system.4
Among the various etheno nucleosides, 2-azapurine deriva-
tives, such as 1,N6-etheno-2-azaadenosine (2-aza-e-adenosine,
3a)5a,b as well as its 2ꢀ-deoxy derivative 3b5c have been incor-
porated into RNA and DNA chemically or enzymatically.1b,1c,6
Scheme 1 Structures of nucleosides 1–6.
From fluorescence studies performed on those nucleosides it
compounds 4 and 5 was 8-aza-7-deazaadenosine (6). As this
nucleoside is not readily accessible,10 a more efficient synthesis
was developed. Apart from the synthetic studies, investigations
on the fluorescence and the conformational properties of the
nucleosides are performed.
became obvious that the fluorescence emission maximum of
compound 3a (495 nm) is bathochromically shifted compared
to 1b (415 nm)—both measured at pH 7.0 in aq. buffer solution.7
Nevertheless, the quantum yield of the 2-aza nucleoside 3a
is low compared to that of 1b.7 This manuscript reports
on the synthesis and properties of 1,N6-etheno-7-deaza-2,8-
diazaadenosine (4) being related to the previously published 7-
deaza-2,8-diaza-2ꢀ-deoxyadenosine.8 In contrast to compound
1a,b and 3a,b, compound 4 cannot form Watson–Crick or
Hoogsteen base pairs. Thus, it can act as an universal nucleoside
when incorporated in duplex RNA.
Results and discussion
Syntheses of nucleosides
Compound 6 was the starting material for the etheno nu-
cleoside 4 as well as for the 2-aza nucleoside 5. The first
chemical synthesis of 6 was performed by condensation of
the chloromercury salt of N6-benzoyl-8-aza-7-deazaadenine (4-
benzamidopyrazolo[3,4-d]pyrimidine) with 2,3,5-tri-O-acetyl-
D-ribofuranosyl chloride in boiling toluene giving com-
pound 6 in only 10–20% yield.10a Montgomery applied
During our work, it became apparent that compound 4
can be converted efficiently to 7-deaza-2,8-diazaadenosine (5)
which is otherwise difficult to synthesize.9 Starting material for
† In memory of the late Professor John A. Montgomery.
1 7 1 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 1 4 – 1 7 1 8
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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the acid-catalysed fusion procedure employing N6-benzoyl-
8-aza-7-deazaadenine and tetra-O-acetyl-b-D-ribofuranose in
the melt to furnish 6 in 12.5% yield.10b Later, Townsend
and Revankar synthesized 6 via the glycosylation of sily-
lated 4-chloropyrazolo[3,4-d]pyrimidine with 2,3,5-tri-O-acetyl-
D-ribofuranosyl bromide in the presence of NaI followed by
deprotection and ammonolysis in 30% yield.10c A more efficient
procedure was reported by Korbukh et al.10d using the fusion
of 4-methylthiopyrazolo[3,4-d]pyrimidine with 1,2,3,5-tetra-O-
acetyl-D-ribofuranose at 180 ◦C (70% yield), which required
the additional conversion of the 4-methylthio group into the
4-amino group. Herein, the direct high-temperature glycosy-
lation of 8-aza-7-deazaadenine (7)11 with 1-O-acetyl-2,3,5-tri-
O-benzoyl-b-D-ribofuranose (8) in MeNO2 with BF3·Et2O as
catalyst is performed.12 The glycosylation product, 2ꢀ,3ꢀ,5ꢀ-
tribenzoylated 8-aza-7-deazaadenosine (9) is isolated in 79%
yield. Deblocking with MeONa–MeOH furnished 6 almost
quantitatively (Scheme 2). The 1H NMR data of 6 were in
accordance with those of Montgomery.10b An unambiguous
assignment of the glycosylation position was performed on the
basis of 13C NMR chemical shifts. According to previous work
on 8-aza-7-deazapurines,13 the 13C NMR signal of C7 is located
around 132 ppm when the 8-aza-7-deazapurine is glycosylated
at nitrogen-9, while on glycosylation at nitrogen-8, a 10 ppm
shift of C-7 towards higher field (122 ppm), is observed.
Scheme 3 The synthetic route for the formation of fluorescent nucleo-
side 4.
yield increase by pH change14 we studied this in detail. However,
the reaction yield was also not improved when the pH was
altered. An increase of the NBS amount or its replacement
by ammonium persulfate (phosphonate buffer, pH 7.2) was
also unsuccessful.16 Finally, we observed that the treatment of
compound 4 with NBS in DMF–H2O (1 : 1)7 furnished the
nucleoside 5 in 51% yield (Scheme 4).
Scheme 2 The route for the synthesis of nucleoside 6.
Treatment of compound 6 with 50% aqueous chloroacetalde-
hyde in 1 M aq. sodium acetate buffer at pH 4.5–5.0 (room
temperature) gave 1,N6-etheno-8-aza-7-deazaadenosine (10) in
77% yield.14 Stirring of compound 10 with 0.5 M aqueous NaOH
at ambient temperature overnight furnished the ring-opened
compound 11. It was cyclized afterwards by diazotization with
NaNO2 in 80% aqueous AcOH to give 1,N6-etheno-7-deaza -
2,8-diazaadenosine (4) in 83% yield (based on 10) (Scheme 3).
With compound 4 in hand, we became interested to remove
the 1,N6-etheno group selectively, thereby transforming com-
pound 4 into the 2-aza nucleoside 5. Although compound 5
has been already synthesized from 6 via its N-oxide in a 5
step synthesis (9.8% overall yield),9 little information was given
regarding the analytical data. Treatment of 4 with NBS in
sodium acetate buffer (pH 4–4.5) formed only trace amounts
of 5 as monitored by TLC.15 As Yamaji et al. reported on a
Scheme 4 Removal of the etheno residue.
The structures of the nucleosides 4,5 and the intermediates
were confirmed by 1H- and 13C-NMR spectroscopy (see Table 1
and Experimental section). The 13C NMR chemical shifts were
assigned with the help of gated-decoupled spectra (Table 2).
As shown in Table 1, the replacement of C2 by N2 drives
the chemical shift of C6 of both etheno and non etheno
nucleosides to higher field by 8.5 ppm (6: 158.0 ppm, 5: 149.5
ppm) and 8.3 ppm (10: 139.3 ppm, 4: 131.0 ppm) which is in
accordance with findings on related molecules.5c,8 Smaller effects
are observed on the chemical shift of carbon-4. The introduction
of nitrogen-2 affects also the chemical shift of C5 of the non
etheno nucleosides (6: 100.4 ppm, 5: 95.9 ppm).
Table 1 13C NMR data of nucleosidesa
Compound C(2)b C(6)c C(4)b C(7a)c C(5)b C(3a)c C(6)b C(4)c C(7)b C(3)c C(10)b,e C(11)b,e C(1ꢀ) C(2ꢀ) C(3ꢀ) C(4ꢀ) C(5ꢀ)
4
—
—
156.0
156.5
140.3
—
143.1
153.1
153.9
154.2
145.4
146.1
104.3
95.9
100.4
100.3
103.2
94.5
131.0
149.5
158.0
158.0
139.3
142.3
131.6
132.9
133.2
133.4
131.9
135.6
132.9
—
—
—
132.3
126.9
115.5
—
—
—
112.8
114.1
89.8
89.2
88.4
85.9
88.6
89.1
73.7
73.2
73.0
74.0
73.4
73.1
70.7
70.7
70.8
71.0
70.8
70.8
85.7
85.4
85.0
78.6
85.3
84.7
61.9
62.1
62.3
63.2
62.2
62.3
5
6
9d
10
11
a Measured in (D6) DMSO at 303 K. b Purine numbering. c Systematic numbering. d 165.3, 164.6, 164.5 for three carbonyl in benzoyl group; 134.5,
133.9, 133.8, 129.3, 129.2, 128.8, 128.7, 128.6, 128.5, 128.3 for three phenyl in benzoyl group. e Tentative.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 1 4 – 1 7 1 8
1 7 1 5

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Table 2 J(H,C) coupling constants (Hz) of nucleosidesa
4
5
6
9
10
1J(C(2), H–C(2))b
2J(C(5), H–C(7))b
1J(C(7), H–C(7))b
1J(C(10), H–C(10))b,c
2J(C(10), H–C(11))b,c
1J(C(11), H–C(11))b,c
2J(C(11), H–C(10))b,c
1J(C(1ꢀ), H–C(1ꢀ))
1J(C(2ꢀ), H–C(2ꢀ))
1J(C(3ꢀ), H–C(3ꢀ))
1J(C(4ꢀ), H–C(4ꢀ))
1J(C(5ꢀ), H–C(5ꢀ))
—
10.9
199.5
193.0
9.4
200.2
16.1
166.3
149.6
150.1
147.8
140.3
—
—
196.6
—
—
—
—
163.7
148.6
147.9
148.2
139.6
198.8
10.8
193.3
—
—
—
199.3
12.0
192.3
—
—
—
215.6
10.3
194.8
190.5
10.3
197.8
16.4
164.4
148.6
146.6
147.6
140.2
—
—
163.1
145.9
143.8
146.8
139.3
168.9
163.5
157.8
149.7
150.0
a Measured in (D6) DMSO at 303 K. b Purine numbering. c Tentative.
Fig. 1 The fluorescence emission spectra of compound 4 and related
nucleosides measured in MeOH at room temperature. Intensities are
normalized.
Conformation of compounds 4 and 5 in solid and in solution‡
The conformation of compounds 4 and 5 was determined in
the solid state and in solution. The crystal structures of both
compounds 417 and 5 are shown in Fig. 2. In the solid state,
both compounds 4 and 5 adopt the high-anti conformation with
torsion angles v being at 83.0(3) and −103.5(3)◦, respectively. A
similar high-anti conformation was also observed for compound
6 (v −77.6◦).18 The ribofuranose moieties of compounds 4–
6 adopts the C2ꢀ-endo conformation (S-type sugar). Usually
the exocyclic C4ꢀ-C5ꢀ bond of several purine and 8-azapurine
nucleosides prefers to adopt the trans conformation, e.g. in
compound 6, which has been correlated with the C2ꢀ-endo
ribose pucker and the high-anti glycosylic bond conformation.18
However, in the case of the N2-modified nucleosides, the
conformation of this side chain –CH2OH group changed to the
syn (gauche–gauche, + sc) conformation (compounds 4,5 and
2-azaadenosine). The etheno group does not significantly alter
the conformational properties of the nucleosides.
In solution, the conformational analysis of the sugar moi-
ety of compounds 4–6 was performed with the aid of the
PSEUROT program (version 6.3).19 A minimization of the
differences between the experimental and calculated couplings is
accomplished by a nonlinear Newton–Raphson minimization;
the quality of the fit is expressed by the root-mean-square (rms)
difference. This procedure presupposes the existence of a two
state N/S equilibrium. The input contained the following cou-
pling constants: J(H1ꢀ,H2ꢀ), J(H2ꢀ,H3ꢀ), J(H3ꢀ,H4ꢀ), J(H4ꢀ,H5ꢀ),
J(H4ꢀ,H5ꢀꢀ). During the iterations either the puckering param-
eters (P, Wmax) of the minor conformer (N) or the puckering
amplitudes of both conformers were constrained. The coupling
constants and the pseudo rotational parameters are shown in
Table 3.
Both compounds, 4 and 5, measured in D2O show the pre-
ferred gauche–gauche conformation of 51 and 46% population,
respectively, similar to that in the crystalline state. In solution,
compound 6 exhibits a gauche–gauche conformer population of
51%, which is different from that in the crystal.
UV and fluorescence properties of the etheno nucleoside 4
As observed for the 13C NMR spectra, the replacement of
carbon-2 by nitrogen-2 also affects the UV-spectra and the pKa
values. The UV maxima of compounds with nitrogen at position
2 show a strong red shift compared to those with a carbon at
that position: 6 (kmax = 260 nm) to 5 (kmax = 309 nm); 10 (kmax
=
278 nm) to 4 (kmax = 295 nm), 1a (kmax = 275 nm) to 3a (kmax
=
282 nm), and 2ꢀ-deoxyadenosine (kmax = 260 nm) to 2-aza-2ꢀ-
deoxyadenosine (kmax = 293 nm). The presence of nitrogen-2
also lowers the pKa values of protonation, which results from
the higher electronegativity of nitrogen compared to carbon: 6
(pKa = 4.0) to 5 (pKa = 1.7), 10 (pKa = 3.7) to 4 (pKa = 2.2)
and 2ꢀ-deoxyadenosine (pKa = 3.8) to 2-aza-2ꢀ-deoxyadenosine
(pKa = 1.9).
The fluorescence emission spectra of compound 4 and the
related nucleosides (Scheme 5) are shown in Fig. 2, and the
corresponding data are summarized in Table 4. Compound 4
shows an emission maximum at 511 nm in MeOH (Fig. 2 and
Table 4) which is further shifted to 531 nm in aq. phosphate
buffer (pH 7.0). This relative long wavelengths emission is rather
unusual for the etheno nucleosides. The data of this nucleoside
Compound 5 shows a nearly equal population of N- and S-
conformers (51 vs. 49%), while compound 4 and 6 are biased
toward S (63 and 64% S) (Table 3). The conformation of the
exocyclic C(4ꢀ)–C(5ꢀ) bond of compounds 4–6 were calculated
based on J(4ꢀ,5ꢀ) and J(4ꢀ,5ꢀꢀ) according to Westhof et al.20
‡ CCDC reference numbers 262698. See http://www.rsc.org/suppdata/
ob/b4/b418849g/ for crystallographic data in CIF format.
Scheme 5 Structures of the fluorescent nucleosides 12–14.
Table 3 1H NMR coupling constants (3J(H, H)) and conformation of the nucleosides 4–6a
3J(H,H)/Hz
Pseudorotational parameters
PN wm(N) PS wm(S)
Conformations
J(1ꢀ,2ꢀ)
J(2ꢀ,3ꢀ)
J(3ꢀ,4ꢀ)
J(4ꢀ,5ꢀ)
J(4ꢀ,5ꢀꢀ)
Rms
%N
%S
c g+
c t
c g−
4
5
6
4.34
4.69
5.20
4.87
4.88
4.79
5.05
4.57
4.50
3.33
3.20
3.38
5.53
5.25
5.08
−1.6 32.0
10.4 32.0
−1.2 32
193.7 35.0
163.3 35.0
170.6 35.0
0.256 36
0.033 51
0.193 37
64
49
63
0.46
0.51
0.51
0.14
0.13
0.15
0.39
0.36
0.34
a Measured in D2O at 303 K.
1 7 1 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 1 4 – 1 7 1 8

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Table 4 Fluorescence data of compound 4 and related nucleosides measured in MeOH at room temperaturea
Excitation
max/nm
Emission
max/nm
Quantum
yieldb
Excitation
max/nm
Emission
max/nm
Quantum
yieldb
Compound
Compound
4
367
300
365
303
511
441
514
438
0.01
0.14
0.003
0.04
3a
1b
2
349
298
297
301
481
410
409
381
0.05
0.2
0.14
0.08
10
12
13
14
a The concentration of the nucleosides were in about 1 × 10−5 M. b Fluorescence quantum yield were determined using quinine sulfate dihydrate in
0.1 M HClO4 (φs = 0.59)23 according to Onidas et al.24
high-anti conformation in the crystalline state, with the ribose in
the unusual S-conformation. In solution, the population of the
S-conformers population is 63% for 4 and 49% for 5. Compound
4 possesses unique fluorescent properties with regard to the
emission at 511 nm (MeOH) and 531 nm (sodium phosphate
buffer; pH 7.0). This relatively long wavelength emission suggests
that compound 4 will broaden the application of etheno
nucleosides to be used as a fluorescent probes. As compound
4 is not expected to form base pairs with canonical DNA
constituents it can be considered as an universal nucleoside.
Experimental
General
Solvents: technical grade, distilled before use. Flash chro-
matography (FC): 0.4 bar, silica gel 60 H (VWR, Darm-
stadt, Germany). TLC: Aluminium sheet, silica gel 60 F254
(0.2 mm, VWR, Germany). UV spectra were recorded on a
U3200 spectrophotometer (Hitachi, Japan). NMR Spectra were
measured on an Avance-DPX-250 spectrometer or AMX-500
spectrometer (Bruker, Rheinstetten, Germany), at 250.13 MHz
and 500 MHz for 1H and 62.90 and 125.13 MHz for 13C, d values
are in ppm relative to internal SiMe4 (1H, 13C). Fluorescence
spectra were determined on Fluorescence Spectrophotometer F-
4500 (Hitachi, Japan). Microanalyses were performed by Mikro-
analytisches Labor Beller (Go¨ttingen, Germany). Chemicals
were commercial products of ACROS, Fluka or Sigma-Aldrich
(Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
4-Amino-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-1H-pyra-
zolo[3,4-d]pyrimidine (9). 4-Aminopyrazolo[3,4-d]pyrimidine
Fig. 2 Single crystal X-ray structures of compounds 417a and 5.17b
711 (1.5 g, 11.1 mmol) and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-
ribofuranose 8 (8.1 g, 16.1 mmol) were suspended in MeNO2
are compared with that of the related etheno nucleosides in Fig. 1
and Table 3. From the comparison of the fluorescence data, one
can conclude that, (i) the replacement of C(2)-H by nitrogen-2
(150 mL). The mixture was heated under reflux. BF3·Et2O
(2 mL) was added in one portion resulting in a clear solution.
After refluxing for 2 h, the reaction mixture was cooled to room
temperature and evaporated in vacuo. The residue was applied
shifts the emission maxima bathochromically from 441 nm for
10 to 511 nm for 4, from 438 nm for 1321 to 514 nm for 1221
to flash chromatography (FC) (silica gel, EtOAc–petroleum
and, 410 nm for 1b to 481 nm for 3a, (ii) the shift of nitrogen-7
ether, 2 : 1) to give 9 (5.1 g, 79%) as a colourless foam (found:
in the imidazole to the position-8 in the pyrazole moiety also
C, 64.23; H, 4.40; N, 12.03%. C31H25N5O7 requires C, 64.24; H,
causes a bathochromic shift, albeit to a smaller degree than that
of nitrogen-2 (see Table 4), (iii) the displacement of nitrogen-7
within the imidazole moiety by C(7)-H in the pyrrole system
does not change the emission maxima as seen for compound
4.35; N, 12.08%); TLC (silica gel, CH2Cl2–MeOH, 20 : 1): Rf
0.15; dH (250.13 MHz; [d6]DMSO; Me4Si) 4.51–4.67 (2H, m,
5ꢀ-H), 4.87 (1H, m, 4ꢀ-H), 6.20–6.31 (2H, m, 3ꢀ-H, 2ꢀ-H), 6.70
(1H, d, J 2.13, 1ꢀ-H), 7.42–8.02 (17H, m, NH2, 3 × Ph), 8.22,
=
1b and 2, (iv) the replacement of the HC CH moiety of the
etheno group by an N N residues causes an hypsochromic shift
8.27 (2H, 2s, 3-H, 6-H).
=
(compound 2 and 1422), (v) the bromination has a small effect
on the emission maxima (compound 10 and 13, compound 4
and 12), (vi) both the presence of a nitrogen in the 2-position
and the bromo sustituent at position-7 lower the quantum yield
strongly.
4-Amino-1-(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine
(6). To a solution of compound 9 (5.1 g, 8.8 mmol) in MeOH
(250 mL), MeONa (0.6 g, 11.1 mmol) was added until the pH
reached 11.0. The mixture was stirred at room temperature for
1 h, and then heated at 60 ◦C for an additional 1 h. The reaction
volume was reduced to ca. 150 mL in vacuo, and THF (100 mL)
was added. The solution was stored in a refrigerator for 1 h. The
precipitate was filtered off and washed with MeOH–THF (1 :
Conclusions
An efficient synthesis of 1,N6-etheno-7-deaza-2,8-diazaadeno-
sine (4) is described. The synthesis of 7-deaza-2,8-diazaadeno-
sine (5) and the precursor molecule 8-aza-7-deazaadenosine (6)
were significantly improved. Both compounds 4 and 5 show a
1.5, 3 × 5 mL). The solid material was dried in vacuo at 70 ◦C
◦
to give 6 (1.5 g) as colourless crystals; mp 250–252 C (lit10c
:
255 ◦C). The mother liquid was evaporated and purified by FC
(silica gel, CH2Cl2–MeOH, 6 : 1→4 : 1) to give another 0.81 g
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 1 4 – 1 7 1 8
1 7 1 7

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of 6. In total, 2.31 g (98%) of 6 was obtained; TLC (silica gel,
CH2Cl2–MeOH, 4 : 1): Rf 0.38; dH (250.13 MHz; [d6]DMSO;
Me4Si) 3.44, 3.57 (2H, 2m, 5ꢀ-H), 3.88 (1H, m, 4ꢀ-H), 4.21 (1H,
m, 3ꢀ-H), 4.58 (1H, m, 2ꢀ-H), 4.89 (1H, t, J 6.0, 5ꢀ-OH), 5.14
(1H, d, J 5.5, 3ꢀ-OH), 5.36 (1H, d, J 5.9, 2ꢀ-OH), 6.08 (1H, d, J
4.7, 1ꢀ-H), 7.77 (2H, bs, NH2), 8.16, 8.19 (2H, 2s, 3-H, 6-H).
Acknowledgements
We are grateful to Dr Helmut Rosemeyer and Dr Yang He
for the measurement of NMR spectra. Financial support
by the European Community (grant no. QLRT-2001-00506,
“Flavitherapeutics”) is gratefully acknowledged.
7-(b-D-Ribofuranosyl)-imidazo[1,2-c]-7H-pyrazolo[4,3-e]pyri-
midine (10). Compound 6 (0.76 g, 2.8 mmol) was dissolved in
aqueous AcONa (1 M, pH 4.5–5.0, 43 mL) at 40–50 ◦C. After
addition of chloroacetaldehyde (50% aqueous solution, 9 mL),
the solution was stirred at room temperature for 24 h. The
reaction mixture was evaporated, and the residue was applied
to FC (silica gel, CH2Cl2–MeOH, 6 : 1) to give 10 (0.64 g, 77%)
References
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◦
as colourless needles; mp 211–213 C from methanol (lit14:
179–181 ◦C); TLC (silica gel, CH2Cl2–MeOH, 4 : 1): Rf 0.74;
dH (250.13 MHz; [d6]DMSO; Me4Si) 3.45, 3.59 (2H, 2m, 5ꢀ-H),
3.94 (1H, m, 4ꢀ-H), 4.26 (1H, m, 3ꢀ-H), 4.64 (1H, m, 2ꢀ-H),
4.78(1H, s, 5ꢀ-OH), 5.21 (1H, m, 3ꢀ-OH), 5.47 (1H, m, 2ꢀ-OH),
6.26 (1H, s, H-1ꢀ), 7.53 (1H, s, 2-H (tentative)), 8.07(1H, s, 3-H
(tentative)), 8.43 (1H, s, 9-H), 9.33 (1H, s, 2-H).
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5-Amino-1-(b-D-ribofuranosyl)-4-(imidazol-2-yl)pyrazole (11).
Compound 10 (0.79 g, 2.7 mmol) was stirred in aqueous NaOH
(0.5 M, 19 mL) overnight at room temperature. The solution
was neutralized with aq. HCl (2 M), and concentrated in vacuo
to afford 11 as a syrup. This was applied directly to the next
reaction step. For analytical purposes, the syrup was purified by
FC (silica gel, CH2Cl2–MeOH, 6 : 1) to give a yellowish foam
(Found: C, 46.85; H, 5.42; N, 24.80%. C11H15N5O4 requires C,
46.97; H, 5.38; N, 24.90%); TLC (silica gel, CH2Cl2–MeOH, 6 :
1): Rf 0.41; kmax(MeOH)/nm 319 (e/dm3 mol−1 cm−1 6 900) and
256 (5 700); dH (250.13 MHz; [d6]DMSO; Me4Si) 3.42, 3.55 (2H,
2m, 5ꢀ-H), 3.86 (1H, m, 4ꢀ-H), 4.15 (1H, t, J 4.81, 3ꢀ-H), 4.47
(1H, t, J 4.41, 2ꢀ-H), 4.94–5.23 (3H, 3br, 2ꢀ-OH), 3ꢀ-OH, 5ꢀ-OH),
5.67 (1H, d, J 2.01, 1ꢀ-H), 6.39 (2H, s, 3ꢀ-H, 4ꢀ-H in imidazole),
6.91 (2H, br, NH2), 7.65 (1H, s, 3-H), 11.92 (1H, s, NH).
9 J. A. Montgomery and H. J. Thomas, J. Med. Chem., 1972, 15, 182–
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10 (a) J. Davoll and K. A. Kerridge, J. Chem. Soc., 1961, 2589–2591;
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Townsend, J. Chem. Soc., Perkin Trans. I, 1971, 2440–2442; (d) I. A.
Korbukh, N. G. Yakunina and M. N. Preobrazhenskaya, Bioorg.
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11 R. K. Robins, J. Am. Chem. Soc., 1956, 78, 784–790.
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13 F. Seela and H. Steker, Helv. Chim. Acta, 1985, 68, 563–570.
14 G. A. Bhat and L. B. Townsend, J. Chem. Soc., Perkin Trans. I, 1981,
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17 (a) W. Lin, F. Seela, H. Eickmeier and H. Reuter, Acta Crystallogr.,
Sect. C, 2004, 60, 566–568; (b) W. Lin, X. Ming, H. Eickmeier and
F. Seela, Acta Crystallogr., 2005, in preparation.
18 S. Sprang, R. Scheller, D. Rohrer and M. Sundaralingam, J. Am.
Chem. Soc., 1978, 100, 2867–2872.
19 L. van Wijk, C. A. G. Haasnoot, F. A. A. M. de Leeuw, B. D.
Huckriede, A. J. A. Westra Hoekzema and C. Altona, PSEUROT 6.3,
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1999.
20 E. Westhof, O. Ro¨der, I. Croneiss and H.-D. Lu¨demann, Z. Natur-
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21 W. Lin and F. Seela, 2005 submitted.
22 M. Zulauf, PhD Thesis, University of Osnabru¨ck, 1999.
23 R. A. Verapoldi and K. D. Mielenz, A fluorescence standard reference
material: quinine sulfate dihydrate, NBS special publication SP 260–
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24 D. Onidas, D. Markovitsi, S. Marguet, A. Sharonov and T. Gustavs-
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7-(b-D-Ribofuranosyl)-imidazo[1,2-c]-7H-pyrazolo[4,3-e]-
[1,2,3]triazine (4). Compound 11 (prepared from 10 (0.79 g,
2.7 mmol)) was dissolved in 80% aqueous HOAc (30 mL)
and treated with NaNO2 (0.19 g, 2.8 mmol) for 0.5 h at room
temperature. The reaction mixture was evaporated. The residue
was applied to FC (silica gel, CH2Cl2–MeOH, 15 : 1) to give
4 (0.66 g, 83% based on 10) as yellow needles from methanol
(Found: C, 45.25; H, 4.04; N, 28.56%. C11H12N6O4 requires
C, 45.21; H, 4.14; N, 28.76%); mp 209–210 ◦C; TLC (silica
gel, CH2Cl2–MeOH, 15 : 1): Rf 0.26; kmax(MeOH)/nm 294
(e/dm3 mol−1 cm−1 5 300) and 232 (34 300); dH (250.13 MHz;
[d6]DMSO; Me4Si) 3.48, 3.62 (2H, 2m, 5ꢀ-H), 4.01 (1H, m,
4ꢀ-H), 4.31 (1H, m, 3ꢀ-H), 4.75 (2H, m, 2ꢀ-H, 5ꢀ-OH), 5.33 (1H,
d, J 5.7, 3ꢀ-OH), 5.62 (1H, d, J 5.78, 2ꢀ-OH), 6.53 (1H, d, J
4.36, 1ꢀ-H), 7.82 (1H, s, 3-H (tentative)), 8.76 (1H, s, 9-H), 8.80
(1H, d, J 1.57, 2-H (tentative)).
4-Amino-7-(b-D-ribofuranosyl)-7H-pyrazolo[3,4-d][1,2,3]tri-
azine (7-deaza-2,8-diazaadenosine) (5). To
a solution of
compound 4 (200 mg, 0.7 mmol) in DMF (20 mL)–H2O
(20 mL) was added N-bromosuccinimide (1.3 g, 7.3 mmol).
The mixture was stirred at room temperature overnight, diluted
with H2O (50 mL) and purified by Dowex 1 × 2(OH−) to give
0.13 g of a yellowish solid, which was crystallized from H2O to
yield 5 (93 mg, 51%) as colourless crystals; mp 208–209 ◦C (lit9:
207–208 ◦C); TLC (silica gel, CH2Cl2–MeOH, 4 : 1): Rf 0.43;
kmax(MeOH)/nm 309 (e/dm3 mol−1 cm−1 9 500) and 238 (6 000);
dH (250.13 MHz; [d6]DMSO; Me4Si) 3.44, 3.57 (2H, 2m, 5ꢀ-H),
3.96 (1H, m, 4ꢀ-H), 4.25(1H, m, 3ꢀ-H), 4.67(1H, m, 2ꢀ-H), 4.82
(s1H, 5ꢀ-OH), 5.23 (1H, d, J 4.8, 3ꢀ-OH), 5.48 (1H, d, J 5.5,
2ꢀ-OH), 6.30 (1H, d, J 3.8, 1ꢀ-H), 8.31 (3H, s, NH2 and 5-H).
1 7 1 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 7 1 4 – 1 7 1 8