Volume 3 no.1 January 1976
NUCieJC Acids Research
Synthesis of guanosine and its derivatives from 5-amino-l-0-D-ribofuranosyl4-imidazolecarboxamide. m. Formation of a novel cydoimidazole nudeoside
and its deavage reactions *.
Masaru Okutsu and Akihiro Yamazaki
Central Research Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki,
Japan 210
Received 24 November 1975
ABSTRACT
A new cydoimidazole nudeoside, 5- (l"-benzamido-lnhydroxymethylene)amino-2',l"-anhydro-l-0-D-ribofuranosyl-4imidazolecarboxamide (III) was synthesized by reaction of
5-amino-l-B-D-ribofuranosyl-4-imidazolecarboxamide (AICAriboside) with benzoyl isothiocyanate followed by methylation
with methyl iodide. The structure of III was elucidated on
the basis of its nmr spectra and chemical reactions.
Of special interest are reactions of III with various
nucleophiles. For example, guanosine (IX) was obtained by
amination of III with ammonia in 72% yield. Analogous
reactions of III with methylamine and dimethylamine gave
N2-methylguanosine (X) and N2-dimethylguanosine (XI),
respectively. Refluxing of III in alkaline solution afforded
xanthosine (VII). The probable mechanism of formation and
facile ring-opening of III is also discussed.
INTRODUCTION
The chemistry ~
of 5-amino-l-3-D-ribofuranosyl-4o
imidazolecarboxamide (AICA-riboside), obtained by fermentation,
has been the objective of study in our laboratories.
Of particular significance has been our interest in the
synthesis of guanosine (IX) which is an immediate precursor
for the production of 5'-guanylic acid. 9-11 In recent
communications, we described two methods for the synthesis
of the 2',3'-O-isopropylidene
of IX; one is ring closure
derivative of AICA-riboside with benzoyl isothiocyanate.to
give 2*,3'-O-isopropylideneguanosine, and the other is
reaction
of AICA-riboside with sodium methylxanthate followed
by oxidation and subsequent amination to give IX. As further
extension of the former method, we have reinvestigated the
reaction between AICA-riboside and benzoyl isothiocyanate.
The present paper deals with an additional synthesis of IX via
© Information Retrieval Limited 1 Falconberg Court London W1V 5FG England
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Nucleic Acids Research
a cyclonucleoside having a novel structure. Although a number
15
14
of pyrimidine
and purine
cycloucleosides have been
synthesized, the reported examples of cycloimidazole nucleoside
are limited to some N 3 ,5'-cycloimidazole nucleosides, 1 6 " 1 8
which are of l i t t l e practical use as synthetic intermediates.
Chort I
n
0
HjN
H2N^N
PhCONCS „
HO
OH
AICA-nboside
X, R1-CH3, Rj»H
XT, R, = R 2 =CH S
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Nucleic Acids Research
When AICA-riboside was treated with 1.1 mole of benzoyl
isothiocyanate in N,N-dimethylformamide, 5-(N'-benzoylthiocarbamoyl)amino-4-imidazolecarboxamide derivative (I) was
obtained in good yield. Its structure was confirmed by
conversion with alkali to the known 2-mercaptoinosine (VIII).
Compound I was then methylated with methyl iodide, affording
the methylthio derivative (II) which failed to crystallize.
An alkaline solution of II was allowed to stand at room
temperature to give a white crystal, C,7H._O-N_, which
was devoid of sulfur and showed a negative periodide test.'19
No other product was detected by paper chromatography. Acid
hydrolysis of the product gave 5-(N'-benzoylcarbamoyl)amino-4imidazolecarboxamide (IV) identical with the authentic sample
prepared by reaction of 5-amino-4-imidazolecarboxamide with
benzoyl isocyanate. In view of these facts, the structure
of 5-(l"-benzamido-l"-hydroxymethylene)amino-2',l"-anhydro1-3-D-ribofuranosyl-4-imidazolecarboxamide (III) is plausible
among six possible compounds corresponding to the empirical
formula (Chart II). The most compelling evidence for III is
given by the nuclear magnetic resonance (nmr) data.
Chart H
-CHjSH
NH
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Nucleic Acids Research
A 100-MHz spectra of III in DMSO-dg (Figure I) showed
at lowest field (6 11.03) a broad peak caused by the benzamide
N-H.
A proton of position-2 of imidazole appeared as a broad
peak at 6 8.61 and two multiplets at 6 7.92 and 7.5 2 were
assigned to aromatic protons. Centered at 6 6.9 7 was a broad
two proton peak of imidazolecarboxamide N-2H. A complete
assignment of ribofuranose protons was established by
spin decoupling experiments. Upon irradiation of the signal
due to water proton at 6 3.36, a doublet at 6 5.81 and a broad
peak at 6 5.06 disappeared, indicating the existence of two
hydroxy groups. Simultaneous irradiation of the signal at
6 5.90 and 5.81 caused a signal at 6 4.29 to collapse to a
triplet (J=4.0 Hz) assignable to 4'-proton which coupled with
only 5'-methylene protons and caused a signal at 6 4.8 5 to
collapse to a doublet (J=5.0 Hz) assignable to 2' or 3'-proton.
At the same time, a signal at 6 4.39 was transformed into a
doublet (J=5.0 Hz) which was also assignable to 2' or 3'-proton.
Upon irradiation of the signal at 6 4.39, a doublet at 6 5.81
collapsed to a singlet, thus confirming that the signal at 6
4.39 could be assigned to 3'-proton which coupled with not
4'-proton but with 2'-proton and also that the broad doublet
at 6 5.81 to 3'-hydroxy proton. Consequently, a signal at 6
4.85 (J=4.0 Hz) was assignable to 2'-proton. When the signals
at 6 5.06 and 4.85 were irradiated, a broad peak at 6 3.55
was changed to a doublet (J=4.0 Hz) and then assigned to
5'-methylene protons which coupled with 4'-proton. Therefore,
the broad peak at 6 5.06 was assignable to 5'-hydroxy proton.
The remaining 1'-proton which coupled with 2'-proton appeared
as a doublet at 6 5.91 (J=7.3 Hz).
Examination of molecular
model shows that 2'-hydroxy group is in the most favorable
position for the formation of the internal anhydro linkage
and that such a H-l',2' coupling constant is to be expected
for III. Moreover, the nmr spectrum of diacetyl derivative of
III indicated two sharp singlets at 6 2.13 and 1.90 due to
3', 5'-diacetyl groups. Thus lack of signal to 2'-hydroxy
proton led us to formulate such a structure as III.
Of considerable interest are the formation and the
ring-opening of III.
At over pH 12, 2'-hydroxy group of II
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Nucleic Acids Research
dissociated and cyclization could proceed by nucleophilic
attack of the 2'-hydroxy anion on the carbon atom attached to
the methylthio group with the intramolecular displacement of
the same group.
Indeed, no reaction occurred when the 2',3'-
O-isopropylidene derivative
of II was used.
Compound III was
stable to alkali at room temperature, but was quickly
transformed to xanthosine (VII) at elevated temperature.
Chart BE
H2N
H
Ph-C-N-ON
II
i
OH*
0 CH3S
HOHjC
w
HO
HOHjC
w.
HO
Og
OH
D
The formation of VII could be satisfactorily
accounted for by considering the alkaline
susceptibility of III, which, by the indicated
anhydro bond cleavage, may be converted to
the intermediate, benzoylureido or presumably
carbodiimido derivative (Chart I V ) .
In similar fashion,reflux-
ing of III in a solution of sodium hydrogen sulfide gave
compound VIII.
The conversion of III to IX is an extremely interesting
example of the ring-opening reaction; III was aminated with
ammonia in an autoclave at 100° for 3 hr to produce readily IX
in 72% yield.
Without isolating I, II, and III, compound IX
was obtained in 68% yield based on AICA-riboside.
An analogous
reaction of III with methylamine and dimethylamine provided
N -methylguanosine (X) and N -dimethylguanosine (XI),
respectively.
On brief treatment of III with ethanolic
sodium ethoxide, a product was obtained which proved to be
N -benzoylguanosine (V) by elemental analysis.
The identity
of V was confirmed by ultraviolet absorption spectra which
2
21
was similar to that of N -benzoylguanosine 5'-phosphate.
As expected, compound V was hydrolyzed with alkali to IX.
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Chort IV
HOH,C
HO
Attempted conversion of III to 2-hydrazinoinosine by
reaction with hydrazine gave a product, C._H.gO_Ng. This
reaction furnished a probable intermediate s-triazino
derivative (XVI) which cyclized under the reaction conditions
to afford a ring-closure product. Such an intermediate
would be expected to be transformed by nucleophilic attack of
triazole nitrogen on the carboxamide carbon into either
9-6-D-ribofuranosyl-9H-s-(5-phenyl)triazolo[4,3-a]purine (XVII)
or 9-0-D-ribofuranosyl-9H-s-(5-phenyl)triazolo[2,3-a]purine
(VI). Presumably, the hydrazine nitrogen far from phenyl
group would be the most nucleophilic site, giving VI.
however, no substantial evidence is available to confirm to
this viewpoint. The synthetic proof of base moiety of VI may
be required.
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Chort V
Ph
Ph
>
-CH3
P
HOH2C
V?
HO
OH
vr
°thA
>C""\
path A^-J.
/P~Ph
H0H2C -°
HO
OH
-NH3
path B
W
HO OH
7VTT
EXPERIMENTAL
All melting points are uncorrected.
General Methods.
Ultraviolet absorption spectra were taken with a Hitachi EPS-2
automatic recording spectrophotometer, the nmr spectra with a
Varian HA-100 using tetramethylsilane as internal standard,
and optical rotations with a Jasco Model DIP-SL automatic
polarimeter. All chromatographies were performed on Toyo No.51
filter paper by the ascending technique. Solvent system:
A, n-PrOH-NH3(28%)-H2O (20:12:3, v/v); n-BuOH-AcOH-HjO (4:1:1,
v/v) .
5- (l"-Benzamido-l"-hydroxymethylene)amino-2',l"-anhydro-l-3-Dribofuranosyl-4-imidazolecarboxamide (III).
To a solution of 5-amino-l-B-D-ribofuranosylMethod A.
4-imidazolecarboxamide (AICA-riboside, 2.58 g, 0.01 mole) in
50 ml of N,N-dimethylformamide was added 1.8 g (0.011 mole) of
The mixture was stirred for 2 hr
benzoyl isothiocyanate.22'
at room temperature and the solvent was removed in vacuo.
Attempts to crystallize the resulting gum, 5-(N'-benzoylthiocarbamoyl)amino-l-8-D-ribofuranosyl-4-imidazolecarboxamide (I),
were unsuccessful. Paper chromatogram of the product showed
a single spot with Rf 0.50 in solvent A. The spot was excised
and eluted with 0.1N hydrochloric acid and water. The ultrapH 1
violet absorption spectra of the extracts exhibited \max 249,
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280 run and x£^ v 7 250,275 nm. The above product (4.6 g, 0.01
mole) was dissolved in 100 ml of 0.1N sodium hydroxide, methyl
iodide (1.56 g, 0.011 mole) was added, and the mixture was
stirred vigorously at room temperature for 2 hr, giving a gummy
methylthio derivative (II) in quantitative yield; Rf 0.66
1
* 1 249 ran. After II (2.2 g) was dissolved
(solvent A); UV Amax
in 20 ml of 0.5N sodium hydroxide, the solution was stirred at
room temperature for 5 hr to give a crystalline product. An
analytically pure sample was obtained by recrystallization of
the product from a large amount of water: yield 0.8 g (41%
based on II); mp 236° (dec); [a]p° +77.0°(c=l, DMSO); Rf 0.36
(solvent A ) , 0.56 (solvent B); UV X**1 nm (e): 243 (16,500),
_TI
297 (13,700);
i -j
p n XJ
max
nm (e): 244 (13,900), 338 (10,300).
TCLciX.
Anal. Calcd for C 17 H 17 OgN 5 : C, 52.69; H, 4.43; N, 18.08.'
Found: C, 52.64; H, 4.74; N, 18.01.
Method B.
AICA-riboside (2.58 g, 0.01 mole) was treated
with benzoyl isothiocyanate (1.8 g, 0.011 mole) as described
above. After reaction, the solvent was removed in vacuo, the
residue was dissolved in 40 ml of 0.5N sodium hydroxide, and,
after a small amount of insoluble material was filtered off,
methyl iodide (2.1 g, 0.015 mole) was added portionwise with
a vigorous stirring was continued for 1 hr. At this point,
it started to precipitate. The mixture was kept at room
temperature. The solution was then neutralized with 0.5N
hydrochloric acid. The precipitate that formed was collected
by filtration and recrystallized from water, giving 2.1 g (55%)
of a pure sample.
4-(N'-Benzoylcarbamoyl)amino-5-imidazolecarboxamide (iv).
Method A.
Compound III (500 mg) was heated in 20 ml of
IN hydrochloric acid at refluxing temperature for 30 min. On
cooling, white crystals precipitated. The precipitate was
filtrated and recrystallized from 120 ml of water to yield
150 mg (43%) of a pure sample; mp 250°; UV xfjL1 nm (e):
c
H
Nriw: 11
max
Anal. Calcd
5 c> nm
52.75;
N, 25.65.
1 2 ii°3
236 (15,400),
271for
(12,400);
A?"
(e):H,
3194.03;
(9,200).
Found: C, 52.65; H, 4.06; N, 25.44.
Method B.
5-Amino-4-imidazolecarboxamide (2 g, 20 mmole)
was dissolved in 50 ml of acetonitrile by slight heating.
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To the solution was added benzoyl isocyanate '
(2.56 g,
22 ramole) with a vigorous stirring. Immediately, crystals
formed. The crystals were collected by filtration and
recrystallized from aqueous ethanol. A yield of 1.1 g (50.5%)
of product was obtained, which was identical with that prepared
by method A in all respects.
Xanthosine (VII).
A solution of III (500 mg, 1.3 mmole) in 10 ml of IN
sodium hydroxide was refluxed for 1 hr. An aliquot of the
solution showed a single spot on paper chromatogram. The pH
of the solution was then adjusted to 3 with Amberlite IR-120
(H form). The filtrate, after removal of the resin, was
concentrated under reduced pressure to give a crude product.
Recrystallization from a small amount of water gave 250 mg
(68.3%) of a pure sample, which was proved to be in good
agreement with an authentic sample by comparison of physical
properties.
Anal. Calcd for c l o H i2°6 N 4 : C ' i2-25> H ' 4.26; N, 19.71.
Found: C, 42.56; H, 4.55; N, 19.99.
2-Mercaptoinosine (VIII)^:3
Method A.
A solution of
I (840 mg) in 20 ml of 0.5N
sodium hydroxide was heated to reflux for 30 min. After
cooling, the solution was adjusted to pH 3 by adding Amberlite
IR-120 (H+ form) portionwise. The resin was removed by
filtration and the filtrate was concentrated to dryness in
vacuo. The residue was dissolved in a small amount of water
and the solution was allowed to stand at room temperature,
giving a yellow crystal. Yield was 300 mg (46%). This
compound was shown to be identical in all respects with a
sample
previously reported.
Anal. Calcd for c 1 0 H 1 2 ° 5 N 4 S H 2 0 : C ' 37-74'' H ' 4-4<>;
N, 17.07. Found: C, 37.62; H, 4.39; N, 17.06.
Method B.
Compound III (500 mg) was dissolved in 20 ml
of 0.5N sodium hydroxide saturated with hydrogen sulfide at
room temperature and the solution heated in an autoclave at
100° for 2 hr. An aliquot of the reaction mixture exhibited
a single spot of VIII on paper chromatogram. The solution was
then acidified with Amberlite IR-120 (H form) as described
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above and after removal of the resin, was concentrated to
dryness.
The residue was crystallized from a small amount
of water, which was identified with an authentic sample.
N -Benzoylguanosine (V).
To a solution of ethanolic sodium ethoxide (prepared from
1.2 g (50 mg-atoms) of metallic sodium and 30 ml of ethanol)
was added 0.78 g (2 mraole) of III with stirring.
When the
mixture was refluxed for 3-5 min, the solution jellified.
After cooling, ca. 10 ml of water was added and the clear
solution was neutralized to pH 7 by adding 2N hydrochloric
acid portionwise.
The solution was concentrated to dryness
in vacuo, the residue was dissolved in a small amount of
water by slight heating, and the solution was allowed to
stand at room temperature to give a crystalline product.
Recrystallization from water with charcoal afforded 480 mg
(62%) of white crystals; mp 232° (dec); [ a ] " -17.5° (c=0.5,
DMSO); Rf 0.55
(solvent A ) ; 0.60 (solvent B ) ; UV x p H 1 nm ( e ):
max
nn 7
269 (18,700), 283 (18,800); x£j|x
(14,800); X ^
1 3
nm (e) : 263 (15,500), 292
nm (e) : 265 (12,500).
Anal. Calcd for C 1 7 H 1 7 O g N 5 H 2 0 : C, 50.35; H, 4.73; N, 17.28.
Pound: C, 49.81; H, 5.21; N, 17.62.
Guanosine (IX).
Method A.
To 30 ml of concentrated ammonium hydroxide
was added III (780 mg) and the mixture was heated in an
autoclave on a steam bath for 2 hr.
Solvent was removed in
vacuo, 30 ml of water was added, and the residue was dissolved
by heating.
This was allowed to stand at room temperature.
The resulting crystals were collected by filtration and
recrystallized from water with charcoal, giving 410 mg (72%)
of pure crystals.
The infrared and ultraviolet absorption
spectra of the compound were identical with those of an
authentic sample.
Anal. Calcd for C 1 Q H 1 3 O 5 N 5 : C, 42.40; H, 4.63; N, 24.73.
Found: C, 42.56; H, 4.43; N, 24.71.
Method B.
A solution of V (390 mg) in 20 ml of 0.1N
sodium hydroxide was refluxed for 30 min.
The solution was
then neutralized with IN hydrochloric acid and concentrated
to dryness in vacuo.
The residue was crystallized from water
gave 100 mg (35%) of an analytically pure material, which was
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identified by direct comparison of an authentic sample.
Anal. Calcd for c 1 0 H 1 3 O 5 N 5 : C, 42.40; H, 4.63; N, 24.73.
Found: C, 42.59; H, 4.91; N, 25.10.
N-Methylguanosine (X).
Compound III (1 g) was aminated with 20 ml of 30% methylamine
in an autoclave at 100° for 2 hr. The solvent was removed
in vacuo, the residue was dissolved in 15 ml of water,
and the N-methylbenzamide that formed was extracted with two
10 ml portions of chloroform. Concentration of the water
layer in vacuo gave a residue, which was crystallized from
water. The resulting crystals were collected by filtration
and recrystallized from water to yield 0.3 g (29%) of a pure
sample. This compound was shown to be identical with an
by direct comparison.
authentic sample ' 6 /
Anal. Calcd for Cn^gOgNg H 2 0 : C ' 41.90; H, 5.39;
N, 22.24. Found: C, 41.82; H, 5.43; N, 22.19.
N -Dimethylguanosine (XI).
The same procedure as described for the preparation of.X
was applied to 500 mg of III, using 30% dimethylamine. The
crude product that obtained was recrystallized from water to
afford 210 mg (54.7%) of XI as white crystals. This compound
was confirmed to be identical with an authentic XI ' '
by direct comparison of their physical properties.
Anal. Calcd for Cj^H-.O-N : C, 46.30; H, 5.50; N, 22.50.
Found: C, 46.51; H, 5.31; N, 22.59.
9-g-D-Ribofuranosyl-9H-s- (5-pheny)triazolo[2,3-a]purine (VI).
A solution of III (500 mg) and hydrazine (500 mg) in 50 ml of
ethanol was refluxed for 2 hr. After cooling, 50 ml of water
was added and the solution was concentrated to dryness in vacuo.
After the residue was dissolved in 4 ml of water, the solution
was brought pH 4 with 0.5N hydrochloric acid and allowed to
stand at room temperature overnight. The resulting crystals
were filtered and recrystallized from 300 ml of water,
affording 280 mg (56.5%) of white crystals; mp 248-258° (dec);
Rf 0.73 (solvent A); 0.40 (solvent B) ; UV \&* X nm (e) : 250
(22,300); X P i J nm (e): 255 (47,000), 300 (8,700).
ItlaX
Anal. Calcd for C. _H.,O,N,: C, 53.09; H, 4.40; N, 21.67.
1 / 16
J
o
Found: C, 53.10; H, 4.21; N, 21.87.
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Nucleic Acids Research
The Diacetyl Derivative of III.
Compound III (500 mg) was suspended in 10 ml of pyridine
with stirring,and to this was added 8 ml of acetic anhydride.
The mixture was heated at 65° for 1 hr, giving a clear solution.
The solution was kept at the same temperature for 3 hr. After
the solvent was removed in vacuo, ethanol was added and the
mixture was concentrated in vacuo. This procedure was repeated
several times to decompose acetic anhydride completely.
Finally, the residue was dissolved in a small amount of
ethanol and it allowed to stand at room temperature overnight
to furnish a crystal. Recrystallization from ethanol gave
200 mg (33%) of pure crystals; mp 158°; [a]^ 5 +50.0°(c=0.5,
DMSO); Rf 0.78 (solvent A); 0.84 (solvent B); UV A * ^ nm (e):
235 (19,400), 313 (13,200).
Anal. Calcd for c 2 i H 23°9 N 5 H 2 0 : C ' 50-48'* H ' 4.65;
N, 14.02. Found: C, 50.84; H, 5.06; N, 14.09.
ACKNOWLEDGEMENT
We are grateful to professor M. Ikehara of the Osaka
University and Dr. I. Kumashiro of Ajinomoto Co., Inc. for
their helpful comments and suggestions. Thanks are also
due to Dr. M. Kainosho for the nmr spectra.
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Nucleic Acids Research
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250