Halogenated 7-deazapurine nucleosides: Stereoselective synthesis and conformation of 2′ -deoxy-2′ -fluoro-β-D-arabinonucleosides

Article abstract of DOI:10.1039/b409648g

The stereoselective syntheses of 5-halogenated 7-(2-deoxy-2-fluoro-β- D-arabinofuranosyl)-7 H-pyrrolo[2,3-d]-pyrimidine nucleosides 3b-d, 4a-c as well as 7-deaza-2′-deoxyisoguanosine 2 are described. Nucleobase anion glycosylation of 2-amino-4-chloro-7 H-pyrrolo[2,3-d]pyrimidine (5) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-α-D-arabino-furanosyl bromide (6) exclusively gave the β-D-anomer 7, which was deblocked (→ 8), aminated at C(4) (→ 3a) and selectively deaminated at C(2) to yield 2′-deoxy-2′-fluoro-β-D-arabinofuranosyl 7-deazaisoguanine (2). Condensation of the 5-halogenated 4-chloro-2-pivaloylamino-7 H-pyrrolo[2,3-d]pyrimidines 9a c with 6 furnished the N⁷-nucleosides 10a-c together with N², N⁷-bisglycosylated compounds 11a-c. The former was converted to the corresponding 2,4-diamino-compounds 3b-d, and the latter was deblocked by NaOMe/MeOH to yield the 4-methoxy-nucleosides 4a-c. Conformational analysis of the sugar moiety of the nucleosides 2 and 3a-d was performed on the basis of vicinal [¹H,¹H] coupling constants. The fluorine atom in the sugar moiety shifts the sugar conformation from S towards N by about 10%, while the halogen substituants in the base moiety increase the hydrophobicity and polarizability of the nucleobases.

Full text of DOI:10.1039/b409648g

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A R T I C L E
Halogenated 7-deazapurine nucleosides: stereoselective synthesis and
conformation of 2-deoxy-2-fluoro-b-D-arabinonucleosides
Xiaohua Penga,b and Frank Seela*a,b
a
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie,
Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de; Fax: +49(541)9692370
Laboratory of Bioorganic and Biophysical Chemistry, Center for Nanotechnology,
b
Gievenbecker Weg 11, 48149 Münster, Germany
Received 25th June 2004, Accepted 9th August 2004
First published as an Advance Article on the web 9th September 2004
The stereoselective syntheses of 5-halogenated 7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine nucleosides 3b–d, 4a–c as well as 7-deaza-2-deoxyisoguanosine 2 are described. Nucleobase anion glyco-
sylation of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (5) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-a-D-arabino-
furanosyl bromide (6) exclusively gave the b-D-anomer 7, which was deblocked (→ 8), aminated at C(4) (→ 3a)
and selectively deaminated at C(2) to yield 2-deoxy-2-fluoro-b-D-arabinofuranosyl 7-deazaisoguanine (2). Con-
densation of the 5-halogenated 4-chloro-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidines 9a–c with 6 furnished the
N7-nucleosides 10a–c together with N2,N7-bisglycosylated compounds 11a–c. The former was converted to the cor-
responding 2,4-diamino-compounds 3b–d, and the latter was deblocked by NaOMe/MeOH to yield the 4-methoxy-
nucleosides 4a–c. Conformational analysis of the sugar moiety of the nucleosides 2 and 3a–d was performed on the
basis of vicinal [1H,1H] coupling constants. The fluorine atom in the sugar moiety shifts the sugar conformation
from S towards N by about 10%, while the halogen substituents in the base moiety increase the hydrophobicity and
polarizability of the nucleobases.
atom strongly influences the N/S-conformational equilibrium
of the pentofuranose moiety. The electronegative character of
the fluorine substituent shifts the conformation of the sugar
moiety from S to N. This phenomenon is observed for substitu-
ents introduced near the anomeric centre, e.g. in the 7-position
of the nucleobase as well as the 2-position of the sugar moiety.
This was confirmed by a recent observation made on 7-fluoro-
2-deoxytubercidin 1c synthesized in our laboratory as well as on
Introduction
Halogenated nucleosides are widely used in biochemistry,
medicinal chemistry, and pharmacology.1–3 Much effort has been
devoted to the synthesis of various halogenated nucleoside and
nucleotide analogues,3,4 which are widely employed as experi-
mental antitumor and antiviral agents.5–11 They are also useful
for probing the structure of protein–RNA, protein–DNA and
DNA–RNA complexes in crosslinking experiments.12 Beyond
this, halogenated 7-deazapurine (pyrrolo[2,3-d]pyrimidine)
nucleosides have gained extensive attention since some of
them, such as 7-iodotubercidin 1a,13 2-deoxy-2-fluoroarabino-
tubercidin 1b14 and 2-amino-2-deoxy-2-fluoroarabino-
tubercidin 3a,15 exhibit a broad spectrum of biological activity
(purine numbering is used throughout the general section).
Furthermore, 7-halogenated 7-deazapurine nucleosides can
stabilize the DNA duplex structure16–20 and are useful for
antisense purposes.20
other 2-deoxy-2-fluoroarabinonucleosides.22,23a Steric as well as
stereoelectronic effects are responsible for this behaviour.
Previously, several 7-deazapurine nucleosides containing
fluoro-sugar moieties have been synthesized by glycosylation
of 6-chloro-7-deazapurines with 3,5-di-O-benzoyl-2-deoxy-
2-fluoro-b-D-arabinofuranosyl bromide (6) using sodium
hydride for the generation of the nucleobase anion.14,15 How-
ever, 7-halogenated 7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-
7-deazapurine nucleosides are still unknown. In continuation of
our studies performed on 7-deazapurine nucleosides16–19,24–28
and on fluoronucleosides,22,23 we report herein the synthesis and
conformational properties of the halogenated 7-deazapurine
nucleosides 2, 3a–d, 4a–c with a F substituent in the 2-arabino-
orientation. They are the key intermediates for later studies on
oligonucleotides.
Recently, it has been shown that sugar modifications, in
particular the addition of a fluorine atom ‘up’ in the 2-position
can enhance the biological activity, while increasing chemical
stability.9,10,21 Apart from the effects of the fluorine substitu-
ent on the biological activity, the introduction of a fluorine
Fig. 1 Structures of nucleosides 1–4.
2 8 3 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6
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 4

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di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride.28 It was
Results and discussion
also not observed in the glycosylation reaction of the 2-amino-
unprotected base 5 with the fluorosugar 6. The most likely
explanation for the formation of 11a–c is that on one hand, the
pivaloyl group increases the acidity of N-H(2), which is easily
deprotonated by KOH, affording a nitrogen anion, on the other
hand the presence of the F-atom in the 2 position and the Br-
atom in the 1 position of compound 6 make it more reactive
than deoxyribose. Compounds 11a–c were assigned as N2,N9-
The synthesis of 2-deoxy-2-fluoro-b-D-arabinofuranosyl 7-
deazaisoguanine (2) was accomplished by nucleobase-anion
glycosylation25,29 of 2-amino-6-chloro-7-deazapurine (5)26b
with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-a-D-arabinofuranosyl
bromide (6)30 (Scheme 1). The reaction of 5 with bromide 6 in
MeCN in the presence of powdered KOH and TDA-1 {tris[2-
(2-methoxyethoxy)ethyl]amine} exclusively gave the b-D-nucleo-
side 7 (59%). The protected nucleoside 7 was deblocked with
methanolic ammonia (saturated at 0 °C) at room temperature
affording compound 8, which on amination with 25% aqueous
ammonia at 90 °C gave the 2-amino-7-deazaadenosine 3a. Selec-
tive deamination of compound 3a with sodium nitrite in AcOH/
H2O (v/v, 1:5) furnished compound 2 in 69% yield.
1
bisglycosylated compounds on the basis of H- and 13C-NMR
1
as well as elemental analysis studies. Both H- and 13C-NMR
spectra of 11a–c show two sets of the sugar signals, while only
one set of the base moiety appears. In addition, the proton signal
of the 2-amino group in the base moiety has disappeared from
the 1H-NMR spectra. The sugar moiety attached to the 2-amino
group is very labile and was lost when being kept in MeOH at
room temperature for several days.
The removal of the benzoyl protecting groups and displace-
ment of the 6-chloro group of 10a–c were performed by treat-
ment with 25% aqueous ammonia in a steel bomb at 90 °C
without affecting the 7-halogen substituents, furnishing com-
pounds 3b–d. The displacement of the 6-chloro group as well as
the deprotection of the benzoyl and pivaloyl groups of N2,N9-
bisglycosylated compounds 11a–c was accomplished by reflux-
ing in 0.5 N NaOMe/MeOH, in the meantime the sugar moiety
attached to the 2-amino group was removed, which afforded the
corresponding nucleosides 4a–c. Without separation, the mix-
tures of compounds 10a–c and 11a–c can directly be converted
to the corresponding 3b–d or 4a–c by the procedures mentioned
above.
All new compounds were characterized by 1H- and 13C-NMR
spectroscopy (Tables 1 and 2) as well as by elemental analysis.
Compounds 2, 3a–d and 4a–c are assigned as having b-D-
configuration from the 1H- and 13C-NMR spectra, referring to
reference 23a. The assignments of the carbon resonances of the
bases are made according to reference 28.
Scheme 1 The synthesis of nucleoside 2 by nucleobase-anion glyco-
sylation.
Conformation of sugar moiety in solution
The synthesis of the 2-deoxy-2-fluoro-b-D-arabinofuranosyl
7-halogenated 7-deazapurine nucleosides 3b–d, 4a–c was accom-
plished according to Scheme 2. In order to introduce 7-halogen
atoms, the 7-halogenated bases 9a–c28 were employed for the
glycosylation reaction because of their good solubility. The
condensation of 9a–c with the halogenose 6 was performed
in MeCN in the presence of powdered KOH and TDA-1. This
reaction resulted in the formation of the desired b-D-nucleo-
sides 10a–c (44–45%) along with the N2,N9-bisglycosylated
compounds 11a–c (11–12%), which was not found in the closely
related glycosylation of the same bases 9a–c with 2-deoxy-3,5-
A conformational analysis of the sugar moiety in solution was
performed with the aid of the PSEUROT (version 6.3) program
employing updated values for the substituent electronegativity
constants31a,b (Due to the lack of accurate electronegativity data
of the modified bases, the default electronegativity values for
the normal purine bases were used). In the PSEUROT program
a minimization of the differences between the experimental
and calculated couplings is accomplished by a nonlinear
Newton–Raphson minimization. This procedure presupposes
the existence of a two-state N/S equilibrium (Fig. 2).31c The
Scheme 2 The synthesis of 7-halogenated nucleosides.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6
2 8 3 9

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2 8 4 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6

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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6
2 8 4 1

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Table 3 Pseudorotational parameters of compounds 2 and 3a–da
Pseudorotational parameters
Compd
%N
%S
PN/deg
WN b/deg
PS/deg
WS b/deg
rms/Hz
2
35
34
36
37
36
65
66
64
63
64
−1.9
10.4
−3.6
−2.1
5.1
41.0
41.0
41.0
41.0
41.0
129.0
130.2
130.8
131.0
129.8
41.0
41.0
41.0
41.0
41.0
0.116
0.082
0.012
0.016
0.154
3a
3b
3c
3d
a Measured in D2O. b Kept fixed during the final minimization.
Table 4 Sugar conformations of the fluoro-nucleosides 2, 3a–d, 1422
and the 2-deoxyribonucleosides 12,28 13a–d,28 1533 (Scheme 3)
3
3
program calculates the best fits of three JH,H and two JH,F
experimental coupling constants to the five conformational
parameters: the phase angles (PS and PN) and puckering ampli-
tudes (WS and WN) of the S- and N-conformers, and the popula-
tion of the S-type conformer (XS; XS + XN = 1).
Conformation
Conformation
2
35% N
34% N
36% N
37% N
36% N
100% N
12
27% N
26% N
28% N
29% N
28% N
37% N
3a
3b
3c
3d
14
13a
13b
13c
13d
15
Fig. 2 N and S conformations of sugar rings of fluoroarabino-
nucleosides.
The input contained the following coupling constants:
3J(H1,H2), J(H2,H3), J(H3,H4), J(H1,F), J(H3,F). In
our cases, the PSEUROT calculation was started with a ‘free
trial’ run without fixing any parameters, which shows that the
N conformer is the minor one. In following runs, the puckering
amplitude of the minor form N was fixed and the fixed value
was varied stepwise (27, 30, 33,...45 degrees) in order to meet
two targets: a) the lowest rms; b) the puckering amplitude of the
two forms should not differ too much. It was found that fixing
the puckering amplitude at 41 degrees resulted in the lowest rms
value. Therefore, the value of 41 degrees was chosen for the
puckering amplitude of both conformers (WS and WN) and fixed
in the final PSEUROT calculation. For detailed procedures of
the PSEUROT calculation refer to references 31a, 32a. The
calculated pseudorotational parameters are shown in Table 3.
From the data given in Table 4 it can be seen that the presence
of the fluorine atom in the sugar moiety drives the N  S equili-
brium of 7-deaza-2-deoxy-2-fluoroisoguanosine 2 towards the
N-conformation (35% N) in comparison with the corresponding
2-deoxyribonucleoside 12 (27% N).28 The same observation can
be also made in the case of 7-deazapurin-2,6-diamine 2-deoxy-
2-fluoroarabinonucleosides 3a–d (34–37% N) and their corres-
ponding 2-deoxyribonucleosides 13a–d (26–29% N).28 This
means that a fluorine atom ‘up’ in the 2-position enhances the
population of the N conformers by 8% in the cases of both the
7-deazaisoguanine nucleoside and the 2-amino-7-deazaadenine
nucleosides (similar results for adenosine analogs were reported
by Marquez and co-workers32b). This enhancement is more
pronounced in the case of 8-aza-7-deazapurine nucleosides.
The N-conformer population of 3-bromo-1-(2-deoxy-2-fluoro-
b-arabinofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4,6-diamine
(systematic numbering) (14)22 increases to about 100% which
is 63% higher than that of the corresponding 2-deoxyribo-
nucleoside 15 (37% N).33 This is due to the replacement of
the 2-ara-proton by an electronegative fluorine atom creates
the repulsive Coulomb interactions between the N8 and the
F2driving compound 14 to adopt an N conformation, while
the corresponding effect does not exist in the 2-deoxynucleoside
15 and 7-deazapurine 2-arabinofluoronucleosides.
Scheme 3 The structures of nucleosides 12–15.
of the nucleosides 3b–d with 7-halogen substituents are shifted
only a little towards the N-population (36–37% N).34,35
Normally, the N/S pseudorotational equilibrium in the
nucleosides is controlled by steric interactions and stereo-
electronic factors known as the anomeric effect (AE) and
gauche effect (GE).31c,32a Due to the AE the heterocyclic base
at C1 is driven to adopt the pseudoaxial position (gauche
C4–O4–C1–B). Preference for a pseudoaxial position at C1
always results in a drive towards the N form in the b-D-series
nucleosides. Therefore, the difference of the N  S equilibrium
between 2-deoxy-2-fluoroarabinonucleosides and 2-deoxy-
ribonucleosides is primarily induced by GE and steric effects.
The term GE means that a vicinal fragment such as X–C–C–Y
prefers to adopt a gauche conformation along the central C–C
bond in cases where X and Y represent highly electronegative li-
gands (or electron pairs). For 2-deoxy-2-fluoroarabinonucleo-
sides, let us look along the C(1)–C(2) and C(2)–C(3) bonds of
the sugar ring, concentrating on the effect involving the C(2)–F
bond (Fig. 2). In the N-form the strongly polar C(2)–F bond is
gauche oriented with respect to C(3)–O(3) and C(1)–N, simul-
taneously, itis trans toC(1)–O(4)andtoC(3)–C(4). Therefore,
compared to 2-deoxyribonucleosides, fluoronucleosides show a
larger N conformer population because of the preferred gauche
orientation of the strongly polar C(2)–F bond to C(3)–O(3)
and C(1)–N. This is also found in the case of 8-aza-7-deaza-
purine22 and 5-aza-7-deazaguanine nucleosides.23a
pKa Values, hydrophobicity and polarizability of
fluoronucleosides
Similar to the 2-deoxyribonucleosides,28 the 7-halogen sub-
stituents have a minor influence on the N  S equilibrium of the
7-halogenated nucleosides 3b–d. The non-halogenated nucleo-
side 3a shows a population of 34% N, while the conformations
Although the introduction of halogen substituents on the base
moiety can not change the N  S equilibrium of nucleosides
2 8 4 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6

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so evidently, they greatly influence other properties of nucleo-
sides, such as pKa values, hydrophobicity and polarizability.
This might lead to significant changes of the biological prop-
erties of oligonucleotides containing these nucleosides.36 The
halogen substituents alter the mobility of the nucleosides on a
reversed-phase HPLC column with the iodinated nucleoside 3d
as the slowest migrating compound (Fig. 3), which shows that
the hydrophobic properties of the 7-halogenated nucleosides
are enhanced. The introduction of 7-halogen substituents also
increases the polarizabilities (am/10−24 cm3) of the nucleobases
(15.77 for 3a, 17.70 for 3b, 18.40 for 3c and 20.80 for 3d calcu-
lated by Hyperchem 7.0). These data suggest that the stacking
interactions of 3b–d can be strengthened compared to that of
non-halogenated nucleoside 3a, therefore, it is reasonable that
the incorporation of compounds 3b–d into oligonucleotides will
stabilize the DNA duplex.36
Furthermore, the pKa values of the halogenated compounds
3b–d were measured by spectrophotometric titration37 (pH
1.5–12.5) at 220–350 nm and compared to that of 3a. Com-
pounds 3b–d show very similar pKa values (4.72 for 3b, 4.77
for 3c and 4.82 for 3d), which are lower than that of 3a (5.67)
(Fig. 4). Obviously, the electron-withdrawing halogen sub-
stituents reduce the basicity of the 7-deazaadenine moieties;
in the meantime, the 6-amino group can become a better
proton donor. With regard to these properties, 3b–d might form
more stable base pairs with thymine than compound 3a. Also,
7-deazaisoguanosine 2 was found to give two pKa values (4.47
and 10.38) (Fig. 4(d)); the lower one corresponds to proton-
ation, the higher one to deprotonation of the base.
Finally, the fluoronucleosides 2, 3a–d and 4b were evaluated in
vitro for their cytotoxity and activity against five human viruses,
namely human immunodeficiency virus-1 (HIV-1), bovine viral
diarrhea virus (BVDV), yellow fever virus (YFV), dengue virus
2 (DENV-2) and west Nile virus (WNV).38 Compound 3a shows
activity against HIV-1 and nucleoside 3d is active against all
viruses, but develops also cytotoxicity.
Conclusions
It was reported that nucleobase anion glycosylation25,29 of
2-amino-6-chloro-7-deazapurine (5) or its 7-halogenated
derivatives 9a–c with the fluoro-sugar bromide 6 proceeds in a
stereoselective way with almost exclusive formation of the b-D-
anomers leading to the 2-deoxy-2-fluoroarabinonucleosides 2,
3a–d and 4a–c. From the conformational analyses of the sugar
moieties of these nucleosides, it is obvious that compared
to the 2-deoxynucleosides,
a 2-fluoro substituent in
arabino configuration shifts the N  S equilibrium of the
7-deazapurine 2-deoxy-2-fluoronucleosides towards N. The
7-halogenated nucleosides 3b–d show higher polarizabilities and
hydrophobicities and lower pKa values compared to the non-
halogenated compound 3a. These properties would contribute
to the stabilization of oligoribonucleotide duplexes. Currently,
Fig. 3 HPLC profiles of the nucleosides 3a–d (the nucleoside mixtures
were analyzed by reversed-phase HPLC at 260 nm on a RP-18 column
(200 × 10 cm). Gradient: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 90:10,
flow rate 1.0 mL min−1).
Fig. 4 a) UV-spectra of compound 3b in phosphate buffer solution (7.8 g NaH2PO4·H2O in 500 mL H2O) from pH 1.5 to 12.5. b) Absorbance
of compound 3b as a function of pH values measured at 286 nm. c) Absorbance of compound 3a as a function of pH values measured at 280 nm.
d) Absorbance of compound 2 as a function of pH values measured at 255 nm.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6
2 8 4 3

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biochemical and biophysical experiments of the incorporation
7-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-7H-pyrrolo[2,3-
of nucleosides 2 and 3a–d into nucleic acids are in progress.
d]pyrimidin-2,4-diamine 3a
A suspension of 8 (260 mg, 0.86 mmol) in a mixture of 25%
aqueous ammonia (40 mL) and dioxane (20 mL) was stirred in a
sealed vessel for 24 h at 90 °C. The clear solution was evaporated
under reduced pressure to leave a yellow oil, which was purified
by column chromatography on silica gel using CH2Cl2–MeOH
(95:5) as eluent to give 3a as a white solid (220 mg, 90%).
Crystallization from MeOH yielded colorless crystals (Found:
C, 46.45; H, 5.01; N, 24.53%. C11H14FN5O3 requires C, 46.64;
H, 4.98; N 24.72%); mp 188 °C (from MeOH); TLC (silica gel,
CH2Cl2–MeOH, 9:1) Rf 0.21; kmax (MeOH)/nm 220 (e/dm3 mol−1
cm−1 31 900), 263 (12 100) and 284sh (9 300); dF (250 MHz;
Experimental
General
All chemicals were purchased from Sigma-Aldrich (Sigma-Al-
drich Chemie GmbH, Taufkirchen, Germany). 1,3,5-Tri-O-ben-
zoyl-2-deoxy-2-fluoro-a-D-arabinofuranose was a commercial
product. Solvents were of laboratory grade. Thin-layer chro-
matography (TLC): aluminium sheets, silica gel 60 F254, 0.2
mm layer (VWR International, Darmstadt, Germany). Column
flash chromatography (FC): silica gel 60 (VWR International,
Darmstadt, Germany) at 0.4 bar. Solvent systems of FC and
TLC: CH2Cl2 (A), CH2Cl2/MeOH 98:2 (B), CH2Cl2/MeOH 9:1
(C), CH2Cl2/MeOH 4:1 (D); Sample collection with a Multi-
Rac fractions collector (LKB Instruments Sweden). UV-Spectra
were recorded on a U-3200 spectrophotometer (Hitachi, Japan),
kmax in nm, e in dm3 mol−1 cm−1. NMR spectra were measured
on an Avance DPX 250 or an AMX-500 spectrometer (Bruker,
Rheinstetten, Germany); chemical shifts (d) are in ppm down-
field from internal TMS (1H, 13C). The J-values are given in Hz.
Melting points were determined with a Linström apparatus and
are not corrected. Elemental analyses were performed by the
Mikroanalytisches Laboratorium Beller, Göttingen, Germany.
2
3
[d6]DMSO; Me4Si) −198.69 (dt, JF, H2 = 52.3, JF, H3 = 19.3,
3JF, H1 = 17.2 Hz).
4-Amino-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-one 2
To a solution of 3a (200 mg, 0.71 mmol) in 15% AcOH–H2O
(v/v 1:5; 5.0 mL), a solution of NaNO2 (150 mg) in H2O (3 mL)
was added dropwise at room temperature under stirring. The
stirring was continued for 2.5 h, and the pH of the dark solution
was adjusted to 5.5 with 25% aqueous ammonia. The mixture
was purified by column chromatography on Serdolit AD04
(resin 0.1–0.2; Serva, Germany) using H2O–iPrOH (100:0 →
95:5) as eluent. Compound 2 was directly crystallized from
the solvent as yellowish crystals (138 mg, 69%) (Found: C,
46.70; H, 4.59; N, 19.42%. C11H13FN4O4 requires C, 46.48;
H, 4.61; N 19.71%); mp 197 °C (from H2O); TLC [silica gel,
NH3 (25%)–iPrOH–H2O, 1:7:2] Rf 0.90; kmax (MeOH)/nm
225 (e/dm3 mol−1 cm−1 26 300), 256 (7 900) and 304 (6 900);
1-Bromo-2-deoxy-2-fluoro-3,5-di-O-benzoyl-a-D-
arabinofuranose 630
Into a solution of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluro-a-D-
arabinofuranose (1.0 g, 2.15 mmol) in CH2Cl2 (6.0 mL), a 30%
solution of HBr in acetic acid (1.2 mL) was added, and the
mixture was stirred at room temperature for 16 h and evaporated
to dryness. The syrup was redissolved in CH2Cl2 (20.0 mL), then
washed with an aqueous saturated NaHCO3 solution (10.0 mL)
and water (10.0 mL), dried over anhydrous Na2SO4, and
concentrated to a viscous syrup which was further dried under
high vacuum for 24 h. The colorless syrup 6 (839 mg, 1.98 mmol,
92%) was used in the next step without purification.
2
dF (250 MHz; [d6]DMSO; Me4Si) −198.67 (dt, JF, H2 = 52.7,
3JF, H3 = 19.5, 3JF, H1 = 17.0 Hz).
7-(2-Deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-arabinofuranosyl)-
4,5-dichloro-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine 10a
and 4,5-dichloro-2,7-di-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-
arabinofuranosyl)-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine
11a
2-Amino-4-chloro-7-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-
arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine 7
To a stirred suspension of powdered KOH (528 mg, 85%,
8.0 mmol) in MeCN (40 mL), TDA-1 (0.1 mL, 0.3 mmol)
was added at room temperature. After 10 min of stirring, 4,5-
dichloro-2-pivaloylamino-7H–pyrrolo[2,3-d]pyrimidine28 (9a:
568 mg, 1.98 mmol) was added, and the stirring was continued
for another 10 min. Then, the solution of 6 (839 mg, 1.98 mmol)
in MeCN (5.0 mL) was added in two portions. The reaction was
continued for 30 min, after which it was filtered, condensed,
and loaded onto a silica gel column. Flash chromatography
(FC) with CH2Cl2–MeOH (99:1) gave two compounds in the
following order.
To a stirred suspension of powdered KOH (528 mg, 85%,
8.0 mmol) in MeCN (35 mL), TDA-1 (0.1 mL, 0.3 mmol)
was added at room temperature. After 10 min of stirring,
compound 5 (334 mg, 1.98 mmol) was added, and the
stirring was continued for another 10 min. Then, the solution
of 6 (839 mg, 1.98 mmol) in MeCN (5.0 mL) was added in two
portions. The reaction was continued for 30 min, after which it
was filtered, condensed, and loaded onto a silica gel column.
Flash chromatography (FC) with CH2Cl2–MeOH (99:1) gave
compound 7 as a colorless foam (596 mg, 59%) (Found: C,
58.99; H, 4.01; N, 11.01%. C25H20ClFN4O5 requires C, 58.77; H,
3.95; N, 10.97%); TLC (silical ge, CH2Cl2–MeOH, 98:2) Rf 0.20;
kmax (MeOH)/nm 232 (e/dm3 mol−1 cm−1 55 900), 316 (7 200);
10a. Slower migrating zone (562 mg, 45%) (Found: C, 57.30;
H, 4.35; N, 9.00%. C30H27Cl2FN4O6 requires C, 57.24; H, 4.32;
N, 8.90%); TLC (silica gel, CH2Cl2–MeOH, 98:2) Rf 0.31; kmax
(MeOH)/nm 232 (e/dm3 mol−1 cm−1 34 900), 252 (35 900) and
341 (2 100); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.23 (9 H, s,
3 × Me), 4.51–4.77 (3 H, m, 4-H, 5-H), 5.80 (1 H, dm, J2,F 52.0,
2-H), 5.98 (1 H, dm, J3,F 19.9, 3-H), 6.78 (1 H, dd, J1,F 16.5,
J1,2 4.2, 1-H), 7.82 (1 H, d, J6,F 2.4, 6-H), 7.47–8.25 (10 H, m,
2 × Ph), 10.40 (1 H, s, NH).
2
dF (250 MHz; [d6]DMSO; Me4Si) −199.31 (dt, JF, H2 = 51.9,
3JF, H3 = 19.0, 3JF, H1 = 20.9 Hz).
4-Chloro-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidine 8
A suspension of 7 (556 mg, 1.09 mmol) in ammonia-saturated
MeOH (20.0 mL) was stirred in a sealed vessel for 18 h at
room temperature. Concentration and purification by FC with
CH2Cl2–MeOH (95:5) as eluent furnished compound 8 as a
colorless foam, which was crystallized from MeOH, yielding
colorless crystals (284 mg, 86%) (Found: C, 43.39; H, 4.02; N,
18.33%. C11H12ClFN4O3 requires C, 43.65; H, 4.00; N 18.51%);
mp 164 °C (from MeOH); TLC (silica gel, CH2Cl2–MeOH, 95:
5) Rf 0.35; kmax (MeOH)/nm 234 (e/dm3 mol−1 cm−1 45 800), 262
(7 400) and 318 (8 900).
11a. Faster migrating zone (206 mg, 11%) (Found: C, 60.92;
H, 4.36; N, 5.85%. C49H42Cl2F2N4O11 requires C, 60.56; H, 4.36;
N, 5.77%); TLC (silica gel, CH2Cl2–MeOH, 98:2) Rf 0.61; kmax
(MeOH)/nm 233 (e/dm3 mol−1 cm−1 64 200), 310 (4 900) and
342 (2 100); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.05 (9 H, s,
3 × Me), 4.56–4.76 (6 H, m, 4-H, 5-H), 5.60–5.86 (4 H, m,
2-H, 3-H), 6.60–6.62 (1 H, m, 1-H), 6.76 (1 H, dd, J1,F 16.6,
J1,2 4.1, 1-H), 7.70 (1 H, s, 6-H), 7.45–8.09 (20 H, m, 4 × Ph).
2 8 4 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 8 3 8 – 2 8 4 6

View Article Online
5-Bromo-4-chloro-7-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-
arabinofuranosyl)-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine
10b and 5-bromo-4-chloro-2,7-di-(2-deoxy-2-fluoro-3,5-di-O-
benzoyl-b-D-arabinofuranosyl)-2-pivaloylamino-7H-pyrrolo[2,3-
d]pyrimidine 11b
90%) (Found: C 36.69, H 3.61, N 19.25%. C11H13BrFN5O3
requires C 36.48, H 3.62, N 19.34%); mp 197 °C (from MeOH);
TLC (silica gel, CH2Cl2–MeOH, 9:1) Rf 0.25; kmax (MeOH)/nm
227 (e/dm3 mol−1 cm−1 27 800), 267 (9 600) and 287 (7 200);
2
dF (250 MHz; [d6]DMSO; Me4Si) −198.89 (dt, JF, H2 = 52.4,
3JF, H3 = 19.0, 3JF, H1 = 17.1 Hz).
As described for 10a and 11a, with 9b (656 mg, 1.98 mmol),
KOH (528 mg, 85%, 8.0 mmol), TDA-1 (0.1 mL, 0.3 mmol),
MeCN (40 mL) and 6 (839 mg, 1.98 mmol).
7-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodo-7H-
pyrrolo[2,3-d]pyrimidin-2,4-diamine 3d
As described for 3a, with 10c (400 mg, 0.55 mmol), dioxane
(20 mL) and 25% aqueous ammonia (40 mL). Compound 3d
(196 mg, 86%), colorless crystals (Found: C, 32.26; H, 3.24; N,
17.30%. C11H13FIN5O3 requires C, 32.29; H, 3.20; N, 17.12%);
mp 198 °C (from MeOH); TLC (silica gel, CH2Cl2–MeOH, 9:1)
Rf 0.25; kmax (MeOH)/nm 230 (e/dm3 mol−1 cm−1 26 800), 269 (8
700), 286sh (7 000); dF (250 MHz; [d6]DMSO; Me4Si) −198.91
(dt, 2JF, H2 = 52.4, 3JF, H3 = 19.0, 3JF, H1 = 16.7 Hz).
10b. Slower migrating zone (600 mg, 45%) TLC (silical gel,
CH2Cl2–MeOH, 98:2) Rf 0.31. Directly used for the next step
without further analysis.
11b. Faster migrating zone (203 mg, 10%) (Found: C, 57.58; H,
4.25; N, 5.36%. C49H42BrClF2N4O11 requires C, 57.91; H, 4.17;
N, 5.51%); TLC (silica gel, CH2Cl2–MeOH, 98:2) Rf 0.61; kmax
(MeOH)/nm 233 (e/dm3 mol−1 cm−1 64 300), 310 (5 000) and 343
(2 100); dH (250 MHz; [d6]DMSO; Me4Si) 1.06 (9 H, s, 3 × Me);
4.56–4.74 (6 H, m, 4-H, 5-H); 5.60–5.82 (4 H, m, 2-H, 3-H);
6.60–6.62 (1 H, m, 1-H); 6.78 (1 H, dd, J1,F 16.5, J1,2 4.2, 1-H);
7.71 (1 H, s, 6-H); 7.48–8.07 (20 H, m, 4 × Ph).
2-Amino-5-chloro-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-4-
methoxy-7H-pyrrolo[2,3-d]pyrimidine 4a
A solution of compound 11a (180 mg, 0.19 mmol) in 0.5 M
MeONa/MeOH (20 mL) was heated under reflux for 3 h.
The solution was neutralized with glacial acetic acid, concen-
trated and adsorbed on silica gel for FC with CH2Cl2–MeOH
(95:5) to give compound 4a as a colorless solid (55 mg, 89%)
(Found: C, 43.20; H, 4.22; N, 16.80%. C12H14ClFN4O4 requires
C, 43.32; H, 4.24; N, 16.84%); mp 160 °C (from MeOH); TLC
(silica gel, CH2Cl2–MeOH, 9:1) Rf 0.38; kmax (MeOH)/nm 228
(e/dm3 mol−1 cm−1 27 100), 263 (9 700) and 287 (7 200).
4-Chloro-7-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-arabino-
furanosyl)-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine
10c and 4-chloro-2,7-di-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-b-
D-arabinofuranosyl)-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]-
pyrimidine 11c
As described for 10a and 11a, with 9c (750 mg, 1.98 mmol),
KOH (528 mg, 85%, 8.0 mmol), TDA-1 (0.1 mL, 0.3 mmol),
MeCN (40 mL) and 6 (839 mg, 1.98 mmol).
2-Amino-5-bromo-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-4-
methoxy-7H-pyrrolo[2,3-d]pyrimidine 4b
10c. Slower migrating zone (628 mg, 44%) (Found: C, 50.11;
H, 3.83; N, 7.71%. C30H27ClFIN4O6 requires C, 49.98; H, 3.77;
N, 7.77%); TLC (silica gel, CH2Cl2–MeOH, 98:2) Rf 0.31; kmax
(MeOH)/nm 232 (e/dm3 mol−1 cm−1 34 800), 252 (35 900) and 341
(2 000); dH (500 MHz; [d6]DMSO; Me4Si) 1.24 (s, 9 H, 3 × CH3);
4.66–4.81 (3 H, m, 4-H, 5- H); 5.78 (1 H, dm, J2,F 51.2, 2-H);
5.99 (1 H, dm, J3,F 19.6, 3-H); 6.76 (1 H, dd, J1,F 16.5, J1,2 4.3,
H-1); 7.83 (1 H, d, J6,F 2.2, 6-H); 7.52–8.10 (10 H, m, 2 × Ph);
10.34 (1 H, s, NH).
As described for 4a, with 11b (180 mg, 0.18 mmol) and 0.5 M
MeONa/MeOH (20 mL). FC gave 4b as colorless solid (60 mg,
90%) (Found: C, 38.42; H, 3.75; N, 15.05%. C12H14BrFN4O4
requires C, 38.21; H, 3.74; N, 14.85%); mp 159 °C (from MeOH);
TLC (silica gel, CH2Cl2–MeOH, 9:1) Rf 0.38; kmax (MeOH)/nm
229 (e/dm3 mol−1 cm−1 27 000), 264 (8 900) and 287 (7 000).
2-Amino-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodo-4-
methoxy-7H-pyrrolo[2,3-d]pyrimidine 4c
11c. Faster migrating zone (245 mg, 12%) (Found: C, 55.10;
H, 3.92; N, 5.34%. C49H42ClF2IN4O11 requires C, 55.35; H, 3.98;
N, 5.27%); TLC (silical gel, CH2Cl2–MeOH, 98:2) Rf 0.61; kmax
(MeOH)/nm 232 (e/dm3 mol−1 cm−1 64 500), 309 (5 000) and 341
(2 200); dH (250 MHz; [d6]DMSO; Me4Si) 1.05 (9 H, s, 3 × Me);
4.50–4.76 (6 H, 3 m, 4-H, 5-H); 5.61–5.89 (4 H, m, 2-H, 3-H);
6.60–6.62 (1 H, m, 1-H); 6.75 (1 H, dd, J1,F 16.6, J1,2 4.1, 1-H);
7.73 (1 H, d, J6,F 2.1, 6-H); 7.45–8.07 (20 H, m, 4 × Ph).
As described for 4a, with 11c (220 mg, 0.21 mmol) and 0.5 M
MeONa/MeOH (20 mL). FC gave 4c as colorless solid (72 mg,
82%) (Found: C, 33.80; H, 3.30; N, 13.11%. C12H14FIN4O4
requires C, 33.98; H, 3.33; N, 13.21%); mp 157 °C (from MeOH);
TLC (silica gel, CH2Cl2–MeOH, 9:1); Rf 0.38; kmax (MeOH)/nm
227 (e/dm3 mol−1 cm−1 26 100), 262 (8 800) and 287 (7 000).
Acknowledgements
5-Chloro-7-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-7H-
pyrrolo[2,3-d]pyrimidin-2,4-diamine 3b
We thank Prof. Paolo La Colla, University of Cagliari, Italy, for
providing us with the antiviral test data, Dr. Helmut Rosemeyer
and Dr. Yang He for the measurement of NMR spectra, and Dr.
Hong Li for the calculation of pseudorotational parameters. We
also thank Monika Dubiel and Elisabeth Michalek for their kind
help in the preparation of this manuscript. Financial support by
the European Community (grant no.: OLK3-CT-2001-00506,
“Flavitherapeutics”) is gratefully acknowledged.
As described for 3a, with 10a (400 mg, 0.64 mmol), dioxane
(20 mL) and 25% aqueous ammonia (40 mL). FC with
CH2Cl2–MeOH (95:5) furnished compound 3b as white solid
(176 mg, 87%), which was crystallized from MeOH, yield-
ing colorless crystals (Found: C, 41.53; H, 4.07; N, 21.92%.
C11H13ClFN5O3 requires C, 41.59; H, 4.12; N, 22.04%); mp
198 °C (from MeOH); TLC (silica gel, CH2Cl2–MeOH, 9:1) Rf
0.25; kmax (MeOH)/nm 226 (e/dm3 mol−1 cm−1 26 100), 267 (10
100) and 287 (7 100); dF (250 MHz; [d6]DMSO; Me4Si) −198.70
(dt, 2JF, H2 = 52.4, 3JF, H3 = 18.9, 3JF, H1 = 16.9 Hz).
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