Imidazole nucleosides, V. Synthesis of 3′-fluoro-3′-deoxy-ribofuranosides of 4(5)-amino-imidazole-5(4)carboxamide

Article abstract of DOI:10.1515/znb-1999-0814

The synthesis of the 3′-fluoro-derivatives of 5-amino-1-(β-D-ribofuranosyl)imidazole-4-carboxamide (AICA-riboside) and the isomeric 4-amino-1(β-D-ribofuranosyl)imidazole-5-carboxamide (iso-AICA-riboside) are described. Structures were confirmed by element

Full text of DOI:10.1515/znb-1999-0814

Imidazole Nucleosides, V.
Synthesis of 3'-Fluoro-3'-deoxy-ribofuranosides of
4(5)-Amino-imidazoIe-5(4)carboxamide
H ans G uglielm i3 *, M arkus D achtlerb, and K laus A lb ertb
a Physiologisch-Chemisches Institut der Universität Tübingen, Hoppe-Seyler-Str. 4,
D-72076 Tübingen
b Institut für Organische Chemie der Universität Tübingen, Auf der Morgenstelle 18,
D-72076 Tübingen
* Reprint requests to Prof. Dr. Hans Guglielmi. Fax: (+49) 7071-295360.
E-mail: brigitte.pfeiffer@uni-tübingen.de
Z. Naturforsch. 54b, 1055-1060 (1999); received June 5, 1999
Fluorinated Imidazole Nucleosides, Structure, UV Data, NMR-Data, Biological Evaluation
The synthesis of the 3'-fluoro-derivatives of 5-amino-l-(/?-D-ribofuranosyl)imidazole-4-
carboxamide (AICA-riboside) and the isomeric 4-amino-l(/?-D-ribofuranosyl)imidazole-5-
carboxamide (wo-AICA-riboside) are described. Structures were confirmed by elemental
analysis, UV and 'H NMR spectroscopy. The anti-viral and anti-cancer activities of these
imidazole nucleosides were tested.
Introduction
4-carboxam ide (A IC A -riboside) and the isom eric
4-am ino-l-(/3-D -ribofuranosyl)im idazole-5-car-
boxam ide (/so-A IC A -riboside). The route is il-
lustrated in Fig. 1. The silver salt of m ethyl-4(5)-
nitroim idazole-5(4)-carboxylate 1 was used as a
starting m aterial and prepared by modifying a p ro-
cedure described earlier [4].
Im idazole nucleosides have been extensively
studied as naturally occuring interm ediates of the
de novo purine nucleotide biosynthesis and as en -
zyme inhibitors of this im portant anabolic p ath -
way. Therefore, there has been great interest in the
synthesis and evaluation of analogues of A IC A R
(5-am ino-l-(/3-D -ribofuranosyl)im idazole-4-car-
boxam ide-5'-phosphate) which is one of the in ter-
m ediates of this process [1]. F urtherm ore, im idaz-
ole nucleosides serve as useful synthetic interm e-
diates for the preparation of related purine
nucleosides of biological interest. A recent survey
was given by Shaw [2],
For the synthesis of 3'-fluoro-analogues we uti-
lized
l-0-acetyl-2,5-di-0-benzoyl-3-fluoro-3-
deoxy-a,/?-D -ribofuranose (2) which is available by
a twelve step process [5]. It was converted to the
unknow n 1-chloro derivative 3 which was not fur-
ther characterized. C ondensation of 3 with an ex-
cess of 1 in boiling toluene resulted in a m ixture of
the expected N -l and N-3 substituted im idazoles 4
and 5, respectively. They w ere separated by silica
gel colum n chrom atography. M ost rem arkably, the
5-nitro-4-carboxylic acid derivative (5) is the
m ayor product (23% yield) and not the 4-nitro-5-
carboxylic acid derivative (4) (13% yield). This
was never observed in a series of sim ilar syntheses
[6,8]. C om pounds 4 and 5 w ere deblocked and
converted to the carboxam ides 6 and 7 by trea t-
m ent w ith m ethanolic am m onia. H ow ever, 5-nitro-
l-(3'-fluoro-3'-deoxy-/3-D -ribofuranosyl)im id-
azole-4-carboxam ide (7) did not crystallize and
darkened w ithin a few days because of decom posi-
tion. A m ethanolic solution of the foregoing 4-nit-
roim idazole (6) was shaken with a platinium cata-
The synthesis of im idazole nucleosides can be
achieved by condensation of a m etal salt of an im -
idazole derivative with acylated glycosyl halides
which leads to the isom eric N -l and N-3 substi-
tuted imidazoles. Furtherm ore, syntheses have
been reported applying the glycosylam ine m ethod
[2] or alkaline treatm ent of N -l substituted purine
nucleosides [3], Im idazole nucleosides w ith a fluo-
rine atom in the carbohydrate m oiety are u n -
known.
Results
We describe the synthesis of 3'-fluoro deriva-
tives of 5-am ino-(l-/3-D -ribofuranosyl)im idazole-
0932-0776/99/0800-1055 $06.00 © 1999 Verlag der Zeitschrift für Naturforschung, Tübingen ■www.znaturforsch.com
D
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1056
H. Guglielmi et al. • Imidazole Nucleosides, V
N ^N O O
i I
o
N-^COOCH,
lyst in an atm osphere of hydrogen for
5
h
to
m ore, the position of the am ino group can easily
be determ ined with the Pauly reaction [7]. As
found earlier, all nucleosides of 4-amino-im idaz-
ole-5-carboxam ide led to a yellow or brown-yel-
low colour and nucleosides of 5-am ino-im idazole-
4-carboxam ide show ed a violet colour [6]. 8 pro-
duced a yellow colour, and therefore it is a 4-
am ino-im idazole. In contrast, we obtained a violet
colour with 9. thus confirm ing a 5-amino-im idaz-
ole nucleoside.
The structures of the synthesized com pounds are
confirm ed by ‘H N M R data, which are listed in
Table I. Fig. 3 shows the ’H NM R spectrum of
com pound 4 including peak picking and integ-
ration. The m ultiplet at 4.67 ppm arises from the
two diastereotopic protons H -5' and FI-5". B e-
cause of their m agnetical non-equivalency an
A B C -spectrum was found and the chemical shift
produce the 4-am inoim idazole (8) as described [6].
In a sim ilar m anner, the 5-nitroim idazole (7) was
reduced to the 5-am inoim idazole (9). The o rien ta-
tion of the isom eric nucleosides 8 and 9 was estab -
lished by U V spectra. They w ere com pared to
those of the corresponding nucleosides w ithout
fluorine atom [4], O ne of the isom ers has the sam e
absorption m axim um as 5-am ino-l-(/?-D -ribofura-
nosyl)-im idazole-4-carboxam ide (266 nm ) which
could be converted to inosine, identical with the
naturally occuring product. Therefore, this isom er
is 5-am ino-l-(3'-fluoro-3'-deoxy-/3-D -ribofurano-
syl)im idazole-4-carboxam ide (9) (268 nm ). L ike-
wise, the isom eric nucleoside 8 has the sam e ab-
sorption m axim um (275 nm ) as 4-am ino-l-(/?-D -
ribofuranosyl)im idazole-5-carboxam ide (272 nm ),
which could be converted to isoinosine. F u rth er-
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1057
H. Guglielmi et al. • Imidazole Nucleosides, V
Table I.
NMR spectral data of compounds 4-6, 8, 9.
No. H-2
H-l'
H-4'
others
H-2'
H-3'
H-5'
H-5”
4
5
6
8.37 s 6.52 d
5.91 ddd
6.03 ddd
5.72 ddd
5.71 ddd
5.05 ddd
4.87 dddd
4.88 ddt
4.27 dddd
4.66 dd
4.71 d
3.33 ddd
4.70 dd
4.71 d
3.37 ddd
3.79 s (OCH3), 8.06-7.53 m (2 Bz)
3.84 s (OCH3), 8.07-7.51 m ( 2 Bz)
6.09 d (OH-2'), 5.27 dd (OH-5'),
8.32 s (CO-NH,)
8.37 s
8.23 s
6.61 d
5.65 d 4.65 dddd
8
9
7.75 s
6.04 d (OH-2'), 5.12 dd (OH-5'),
6.91 s (CO-NH,), 5.41 s (NH,)
6.00 d 4.45 dddd
4.96 ddd
4.90 ddd
4.18 dddd
4.20 dddd
~ 3.56 ddd
3.55 ddd
~ 3.56 ddd
7.32 s 5.54 d
4.50 dddd
3.65 ddd 5.90 d (OH-2'), 5.44 dd (OH-5'),
6.74 s (CO-NH,), 6.01 s (NH,)
r
.......................... -;■■, ,
........................ , — ,
, =g=j ppm Fig. 2. ’H, XH COSY spectrum of com-
ppm
6.5
6.0
5.5
5.0
4.5
pound 4.
values as well as the coupling constants w ere sim u-
lated with the B R U K E R W inDaisy program . The
results are listed in Table I and II. The CO SY
spectrum in Fig. 2 depicts all crosspeaks of the ali-
phatic protons. Proton FI-5' couples with the p ro -
ton FI-5" with the typical gem inal coupling con-
stant of 12.2 Hz and with the vicinal proton H -4'
( 3/ H -5\ h -4 ' 5.4 Hz). H -4' has also a crosspeak with
proton H -3'. So the doublet at 5.72 ppm belongs
to the proton H -3'. The typical coupling constant
of 52.4 H z indicates the coupling to a fluor atom .
The coupling constants to the /3-H-atoms prove
also the existence of the fluor atom ( 3/ h - 2 \ f 14.8
Hz; 3/ H -4\
f
20.7 Hz). The coupling constants 7H f
are collected in Table III. The COSY spectrum in
Fig. 2 shows also clearly the crosspeaks H -37H -2'
and H -27H -1'. The singulet at 8.37 ppm is caused
by the proton H-2. The m ultiplets betw een
8.06 ppm and 7.53 ppm arise from the 10 protons
of the two benzoyl groups. All other structures can
be proved in an analogous way by N M R spectros-
copy. Table I to III show the results of all com -
pounds. O f interest are the accidental isochronous
protons H -5' and H-5" of com pound 5. Therefore,
the spectrum exhibits only a doublet of an A B-
spectrum at 4.71 ppm.
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1058
H. Guglielmi et al. ■Imidazole Nucleosides, V
Table II. Coupling constants 7HH of compounds 4-6, 8, 9.
No. H -l\ H-2' H-2', H-3' H-3', H-4'
H-4'. H-5'
H-4', H-5"
H-2', OH-2'
H-5'. H-5"
H-5'. OH-5'
H-5", OH-5"
_
_
5.4
4.9
4.0
4.4
4.4
4
5
6
8
9
5.4
4.5
7.6
8.0
8.0
3.6
4.5
4.0
4.4
4.4
5.4
4.0
4.0
3.5
4.0
4.0
4.0
4.0
3.5
4.4
12.2
-
-
-
_
12.1
~ 12
11.9
6.7
~ 6.2
6.2
5.4
~ 4.8
4.9
5.4
~ 4.8
4.9
■—r~-ao o -*»-u->c''Joocnu3
OCTIlDCNI—■—C'vJLOCD-—
^-ooc
U3U3
CMCVi
POmrOrOmKIKIfOrOCNCNCM
r^CNirMOslCslCNJCMCsjrviCNJCVICsl
(ppm)
Fig. 3. *H NMR spectrum of compound 4.
Table III. Coupling constants 7HF of compounds 4-6, 8,
9.
Biological evaluation. N ucleosides 6, 8 and
9
have been evaluated for in vitro anti-H IV activity
[9]. N one of the com pounds show ed significant in-
hibitory effects. The anti-cancer activity of these
com pounds was tested with an in vitro m odel of
hum an tum or cell lines [10]. A s prim ary anti-can-
cer screen a three-cell line panel was used, con-
sisting of M C F 7 (B reast), N C I-H 460 (Lung) and
SF-268 (CNS). All three substances were inactive.
No.
H-2', F
H-3', F
H-4', F
4
5
6
8
9
14.8
12.1
24.7
23.4
26.1
52.1
51.6
54.3
54.8
54.8
22.9
20.6
26.5
27.4
28.8
The /^-configuration of the carbohydrate m oiety
was also assigned by N M R spectroscopy. The val-
ues of the coupling constant V H-r. h-2' in Table II
indicate no evidence for the form ation of a-nucle-
osides. D ue to the electron-w ithdraw ing effect of
the benzoyl group in position 2', the coupling con-
stants of com pound 4 and 5 decrease.
E xperim ental
N M R experim ents were recorded with
a
B R U K E R A R X 400 spectrom eter. All com -
pounds were dissolved in D M SO -d6. The tem per-
ature was set to 300 K. 32 transients with a time
dom ain of 32 k per FID were coadded using a
pulse length of 7.7 //s and a sweep w idth of 7352.9
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H. Guglielmi et al. • Imidazole Nucleosides, V
1059
flux for 1 h with the exclusion of m oisture. The
cooled m ixture was filtered and evaporated to dry-
ness. The residual sirup was fractionated by silica
gel colum n chrom atography (A).
Hz. The CO SY spectra w ere recorded with a tim e
dom ain of 2 k per F ID and 256 experim ents in F I
dim ension. D ata processing was perform ed with
X W IN N M R
and
ID
W IN N M R
softw are
C om pound
(200 mg).
M.p. 122 °C. R f = 0,63 (A ), [a]578 - 26°, [a]546 -
28°, c 1 in CHC13. U V (A,f): 275 nm (m ax) 10608,
258 nm (m in) 4795 in ethanol.
4
was obtained in 13% yield
(B R U K E R ). Zero-filling to 32 k d ata points and
exponential m ultiplication with a line broadening
of 0.3 H z was applied to the FID prior to F ourier
transform ation in the F2 dim ension.
M elting points w ere determ ined on
a Biichi
m elting point instrum ent and are uncorrected. O p -
tical rotations w ere determ ined in a 5 cm cell with
a light electric Zeiss precision polarim eter L E P
A2 at 22 °C. U ltraviolet absorption spectra w ere
m easured on a C ontron U vicon 930 spectrom eter
using solutions in phosphate buffer (pH 7) unless
otherw ise stated. Thin-layer chrom atography was
perform ed on precoated silica sheets (layer thick-
ness 0,25 mm, IC N B iom edicals) w ith fluorescent
indicator 254/366 nm. C om pounds w ere detected
on t.l.c. plates by U V absorbance and the Pauly
reaction [7]. C olum n chrom atography (45 x 3 cm)
was perform ed on silica gel 60 M erck 7734. The
following solvent system s w ere used (v/v): Light
petroleum (5 0 -7 0 °C) - ethyl acetate 2:1 (A ) and
n-butanol saturated w ith w ater (B).
C24H 20N3O9F (513,45)
Calcd
C 56.14 H 3.90 N 8.19 F 3.69% ,
Found C 56.24 H 3.96 N 8.26 F 3.58% .
C om pound
(350 mg).
5
was obtained in 23% yield
M.p. 125 °C. R f = 0,38 (A ), [a],78 - 25°, [a]546 -
23°, c 1 in CHCI3, U V (A,e): 275 nm (m ax) 8538,
258 nm (m in) 6597 in ethanol.
C24H 20N 3O 9F (513,45)
Calcd
C 56.14 H 3.90 N 8.19 F 3.69% ,
Found C 56.38 H 4.14 N 8.21 F 3.56% .
E lem ental analyses w ere perform ed by M ikroa-
nalytisches L abor Pascher, D-53424 R em agen-
B andorf (G erm any).
4-N itro-l-(3 '-fluoro-3'-deoxy-ß-D -ribofuranosyl) -
im idazole-5-carboxam ide (6)
450 mg (0,88 m m ol) 4 w ere treated with m etha-
nolic am m onia (100 ml) at 0 °C for 50 h. The solu-
tion was evaporated, the residue dissolved in 30 ml
of w ater and extracted two tim es with 30 ml of
ether. The aqueous solution was evaporated. The
residue cristallized from ethanol, yield 220 mg
(87% ). M.p. 225 °C.
M ethyl-4-nitro-l -(2 '-5 '-di-O -benzoyl-3'-fluoro-3'-
deoxy-ß-D -ribofuranosyl)im idazole-5-carboxylate
(4) and m ethyl-5-nitro-1 -(2' -5' -di-O -benzoyl-3' -
flu o ro -3 '-deoxy-ß-D -ribofuranosyl)im idazole-4-
carboxylate (5)
U V (A,f): 294 nm (m ax) 5777, 257 nm (m in)
2854. Rf = 0,55 (B).
1
g (6 m m ol) m ethyl-4(5)-nitroim idazole-5(4)-
carboxylate was dissolved in 150 ml of hot m eth a-
nol and m ixed with the solution of 1 g (6 m m ol)
silver nitrate in 150 m l of hot m ethanol. The sus-
pension was treated w ith 10 ml pyridine, cooled
and centrifugated. The precipitate was stirred w ith
m ethanol, centrifugated again, the precipitate sus-
pended in 400 ml of toluene and dried azeotropi-
cally by distillation of 150 ml of the solvent. 1,2 g
(3 m m ol) l-0-acetyl-2,5-di-0-benzoyl-3-fluoro-3-
deoxy-a,/3-D -ribofuranose (2) w ere dissolved in
100 ml of ether and saturated w ith dry hydrogen
chloride gas at 0 °C. A fter eight days the clear so-
lution was evaporated. The residual sirupy 1-
chloro-2,5-di-0-benzoyl-3-fluoro-3-deoxy-a,/?-D -
ribofuranose (3) was dissolved in toluene and re -
evaporated with toluene (2x50 m l). The resulting
sirup was dissolved in 50 ml anhydrous toluene
and added to the suspension of the silver salt. The
reaction m ixture was stirred and heated under re -
C9H n N40 6F (290,21)
Calcd
C 37.24 H 3.82 N 19.30 F 6.54%
Found C 37.56 H 3.99 N 19.39 F 6.29% .
5-N itro-l-(3 '-fluoro-3'-deoxy-ß-D -ribofuranosyl)-
im idazole-4-carboxam ide (7)
510 m g (1 m m ol) 5 w ere treated w ith m etha-
nolic am m onia (100 ml) at 0 °C for 50 h. The reac-
tion m ixture was processed as described for 6. The
aqueous solution was evaporated to give a gum
which did not crystallize and darkened w ithin a
few days. Yield 250 mg (86% ).
U V (A,e): 298 nm (m ax) 3373, 265 nm (m in)
2752. Rf = 0,51 (B).
C9H „ N 40 6F (290,21)
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1060
H. Guglielmi et al. ■Imidazole Nucleosides, V
4 -A m in o -l-(3 '-fluoro-3'-deoxy-ß-D -ribo-
furanosyl)im idazole-5-carboxam ide (8)
tallisation from w ater. Yield 165 m g (91% ). M.p.
260 °C (dec.), R f= 0,50 (B).
U V (A, e): 268 nm (m ax) 9860, 217 nm (m in)
3113.
250 mg (0,86 m ol) 6 w ere dissolved in 60 ml of
freshly distilled m ethanol and hydrogenated with
a platinium catalyst. The catalyst was rem oved by
filtration and the filtrate evaporated to a sirup
which crystallized from ethanol. Recrystallization
from w ater. Yield 210 mg (94% ), M.p. 197 °C, R f =
0,40 (B).
C9H n N 40 4F (260,11)
Calcd
C 41.55 H 5.03 N 21.54 F 7.30% ,
Found C 41.50 H 5.04 N 21.60 F 7.45% .
U V (2,e): 275 nm (m ax) 8117, 241 nm (m in)
2860.
A cknow ledgem ents
C9H n N 40 4F (260,11)
Calcd
C 41.55 H 5.03 N 21.54 F 7.30% ,
Found C 41.50 H 5.06 N 21.80 F 7.32% .
We thank the N ational Institutes of H ealth , the
N ational C ancer Institute, B ethesda, M aryland,
U. S. A ., for perform ance of the anti-viral and anti-
cancer assays.
5-Am ino-1 -(3'-fluoro- 3' -deoxy-ß-D-ribofiiranosyl)-
imidazole-4-carboxamide (9)
200 mg (0,7 m ol) 7 w ere hydrogenated as d e-
scribed for 8. C rystallization from ethanol. R ecrys-
The authors express sincere thanks to Mrs. U l-
rike W öll for expert assistance.
[5b] H. S. El Khadem, T. D. Audichaya, M. J. Withee,
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339 (1967).
[5d] J. A. Wright, N. F. Taylor, Carbohydr. Res. 13,
297-300 (1970).
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ima, A. Matsuda, Chem. Pharm. Bull. 44(2), 288-
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[4] J. Baddiley, J. G. Buchanan, F. E. Hardy, J. Stewart,
J. Chem. Soc. 1959, 2893.
[7] H. Pauly, Hoppe-Seyler's Z. Physiol. Chem. 42,
508 (1904).
[8] H. Guglielmi, Liebigs Ann. Chem. 1973, 1286-
1293.
[5] I. A. Mikhailopulo. N. E. Poopeiko. T. I. Pricota.
G. G. Sivets, E. I. Kvayuk, J. Balzarini, E. De
Clerk. J. Med. Chem. 34, 2195-2202 (1991).
[5a] B. R. Baker. R. E. Schaub. J. P. Joseph. J. H. Wil-
liams, J. Am. Chem. Soc. 77, 7-12 and 12-15
(1955).
[9] O. W. Weisloh, R. Kiser, D. Fine, J.Bader,
R. H.Shoemaker. M. R.Boyd, J. Natl. Cancer Inst.
81. 577-586 (1989).
[10] J. Natl. Cancer Inst. 83. 757-766 (1991).
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