1-(BETA-D-RIBOFURANOSYL)-1,2,4-TRIAZOLE ACID ESTERS

ESTERS DE DERIVES ACIDES DE 1-(B-D-RIBOFURANOSYL)-1,2,4-TRIAZOLES

English Abstract

S P E C I F I C A T I O N
1-(.beta.-D-RIBOFURANOSYL)-1,2,4-TRIAZOLE
ACID ESTERS
Abstract of the Disclosure
Phosphate and carboxylic acid esters of
1-(.beta.-D-ribofuranosyl)-1,2,4-triazoles are prepared by a
variety of methods and their antiviral activity reported.

Claims

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as follows:
1. A process for the preparation of a 1-(.beta. -D-ribofurano-
syl)-1,2,4-triazole nucleoside of structure
<IMG> (I)
wherein R1 is selected from the group consisting of (a)
carboxamido, (b) thiocarboxamido, and (c) carboxamidino groups
and physiologically acceptable acid addition salts of (c); and
wherein X is hydrogen or C1-C18 acyl, at least one X being
hydrogen and at least one X being acyl or when X in the 5'-
position is hydrogen or C1-C18 acyl, the X in the 2'- and 3'-
positions together represent the group
<IMG>
or one X in said 2'- or 3'-position is a group of formula
<IMG>
and the other is H or C1-C18 acyl, which comprises:
A. acylating a 1-(2,3-O-isopropylidene-.beta.-D-ribo-
furanosyl)-1,2,4-triazole-3,carboxamide, 3-thio-
carboxamide or -3-carboxamidine followed by deisopropylidenation
or
17

B. selectively deacylating a (2,3,5-tri-O-acyl-.beta.-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide,-3-thiocarboxamide
or -3-carboxamidine, to give a compound of structure (I) wherein
X is hydrogen or C1-C18 acyl, at least one X being hydrogen and
at least one X being acyl,
C. treating a 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-
nucleoside of structure (I) wherein R1 is the same as above and
X in 5'-position is hydrogen or C1-C18 acyl, the remaining X's
are hydrogen with the tri-n-butyl amine salt of pyrophosphoric
acid, to give a 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-R1 2',3'-
cyclic phosphate, or
D. cleavage of a 1-(.beta.-D-ribofuranosyl)-1,2,4-tria-
zole-3-R1, 2', 3'-cyclic phosphate, as defined in C, at the
cyclic phosphate group to produce a triazole-nucleoside of
structure (I) with a monophosphate group
<IMG>
in the 2'- or 3'- position and, when desired C1-C18 acylating
otherwise free glycosyl hydroxyl groups and separating 2'-
and 3'-phosphates; and, when desired converting a compound of
structure (I) in which R1 is carboxamidino into a physio-
logically acceptable acid addition salt thereof.
2. A process according to claim 1A comprising acylating
a 1-(2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide and deisopropylidinating the resultant 1-(5-O-acyl-
2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carbox-
amide to produce 1-(5'-O-acyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-
3-carboxamide.
3. A process according to claim 1A comprising acylating
a 1-(2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
thiocarboxamide followed by deisopropylidenation to produce a
1-(5'-O-acyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-thiocarbox-
amide.
18

4. A process according to claim 1A comprising acylating
a 1-(2,3-O-isopropylidene-.beta.-?-ribofuranosyl)-1,2,4-triazole-3-
carboxamidine followed by deisopropylidenation to produce a
1-(5'-O-acyl-.beta.-?-ribofuranosyl)-1,2,4-triazole-3-carboxamidine
and converting said 3-carboxamidine to a corresponding
hydrochloride salt.
5. A process according to claim 1, wherein X is hydrogen
or C1-C18 acyl, at least one X being hydrogen and at least one
X being acyl.
6. A process according to claim 1C, wherein X in the 5'-
position is hydrogen or C1-C18 acyl and X in the 2'- and 3'-
positions together represent a group
<IMG>
7. A process according to claim 6, wherein X in the 5'-
position is hydrogen.
8. A process according to claim 1, which comprises treat-
ing 1-.beta.-?-ribofuranosyl-1,2,4-triazole-3-carboxamide 2',3'-
cyclic phosphate with a dilute acid to cleave the cyclic
phosphate group.
9. A process according to claim 1, which comprises
treating 1-(2,3-O-isopropylidene-.beta.-?-ribofuranosyl)-1,2,4-
triazole-3-carboxamide with benzoyl halide followed by treat-
ment to remove the isopropylidene group to give 1-(5'-O-benzoyl-
.beta.-?-ribofuranosyl)-1,2,4-triazole-3-carboxamide.
19

10. A process according to claim 1, which comprises
treating 1-(2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamide with an excess of acetic anhydride
containing a catalytic amount of 4-dimethyl-aminopyridine,
followed by treatment to remove the isopropylidene group to
give 1-(5'-O-acetyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide.
11. A process according to claim 1, which comprises
treating 1-(2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamide with an excess of butyric anhydride
containing a catalytic amount of 4-dimethylaminopyridine,
followed by treatment to remove the isopropylidene group to
give 1-(5'-O-butyryl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide.
12. A process according to claim 1, which comprises
reacting 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
with pyrophosphoric acid and tri-n-butyl-amine in dimethyl-
formamide to give 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide 2',3'-cyclic phosphate.
13. A 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole nucleoside of
the structure
<IMG>

wherein R1 is selected from the group consisting of (a)
carboxamido, (b) thiocarboxamido, and (c) carboxamidino groups
and physiologically acceptable acid addition salts of (c);
and wherein X is hydrogen or C1-C18 acyl, at least one X being
hydrogen and at least one X being acyl and when X in the 5'-
position is hydrogen or C1-C18 acyl, the X in the 2'- and 3'-
positions together represent the group
<IMG>
or one X in said 2'- or 3'-position is a group of formula
<IMG>
and the other is H or C1-C18 acyl, whenever produced by the
process of claim 1, or its obvious chemical equivalents.
14. A 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole nucleoside
of structure
<IMG>
wherein R1 is selected from the group consisting of (a)
carboxamido. (b) thiocarboxamido, and (c) carboxamidino groups
and physiologically acceptable acid addition salt of (c); and
wherein X is hydrogen or C1-C18 acyl, at least one X being
hydrogen and at least one X being acyl, whenever produced by
the process of claim 5, or its obvious chemical equivalents.
21

15. A compound 1-(5'-O-acyl-B-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamide, whenever produced by the process of
claim 2, or its obvious chemical equivalents.
16. A compound 1-(5'-O-acyl-.beta.-D-ribofuranosyl)-1,2,4-
triazole-3-thiocarboxamide, whenever produced by the process
of claim 3, or its obvious chemical equivalents.
17. A compound 1-(5'-O-acyl-.beta.-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamidine hydrochloride, whenever produced by
the process of claim 4, or its obvious chemical equivalents.
18. 1-(5'-O-Benzoyl-.beta.-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamide whenever produced by the process of
claim 9, or its obvious chemical equivalents.
19. 1-(5'-O-acetyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-
carboxamide whenever produced by the process of claim 10, or
its obvious chemical equivalents.
20. 1-(5'-O-butyryl-.beta.-D-ribofuranosyl)-1,2,4-triazole-
3-carboxamide whenever produced by the process of claim 11, or
its obvious chemical equivalents.
21. A 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole nucleotide
of structure
<IMG>
wherein R1 is selected from the group consisting of (a)
carboxamido, (b) thiocarboxamido and (c) carboxamidino groups
and physiologically acceptable acid addition salts of (c);
and wherein R is hydrogen or C1-C18 acyl, whenever produced
22

by the process of claim 6, or its obvious chemical equivalents.
22. A 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole nucleoside of
structure I, as defined in claim 1, wherein X in the 5'-
position is hydrogen and X in the 2'- and 3'-positions together
represent a group
<IMG>
whenever prepared by the process of claim 7, or its obvious
chemical equivalents.
23. 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
2',3'-cyclic phosphate whenever produced by the process of
claim 12, or its obvious chemical equivalents.
23

Description

10~0004
In our United States Patent 3,798,209, dated March 19,
1974, are described and claimed compounds of structure
1 ~
HO
'k~'~
HO OH
wherein Rl is carboxamido, thiocarboxamido or carboxamidino and
its physiologically acceptable acid addition salts, as well as
5'-phosphates and 3',S'-cyclic phocphates thereof, and compounds
in which otherwise free glycosyl hydroxyls bear C1-C18 acyl
groups. The compound~ are diAclosed in the above Patent to
possess potent antiviral activity. In one e~bodiment of the
present invention, we provide analogous compounds having the
same aglycon whose glycosidic moiety is of structure -
xo~~l
1~1 :~ - '
XO OX
wberein at least one X i9 Cl-C18 acyl and at least one X is
hydrogen. According to another embodiment of thi~ invention
there-are provided analogous nucleotides, viz., 2',3'-cyclic
;~;. pho~phates and mixed 2'- and 3'-phosphates of nucleosides of
structure (a). Compounds prepared according to this invention
~: exhibit significant antiviral activity in in vivo animal testing, ~ -
and may be adminlstered generally as in our U.S. Patent 3,927,216,
~20 dated Dece~ber 16, 1975.
: There are thus provided in accordance with the invention,
~ -D-ribofuranosyl)-1,2,4-triazole nucleosides of structure . ~ :
.: :
'
.,,.,. . . :, : ~ . . .. .

1060004
R~
N (I)
o
XO ~ \
1/
~ ~X
wherein Rl i~ selected from the group consisting of ta) carbox-
amido, (b) thiocarboxamido, and (c3 carboxamidino groups and
physiologically acceptable acid addition salts of (c); and where-
in X i9 hydrogen or Cl-C18 acyl, at lPast one X being hydrogen
and at least one X being acyl or when X in the 5'-position is
hydrogen or Cl-C18 acyl, the X in the 2'- and 3'-positions
together represent the group
0~ \ ' .
OH
or one X in said 2'- or 3'-position in a group of formula
o = P - OH
OH
and the other is H or Cl-C18 acyl.
The nucleoside~ of the invention can be prepared in ~ .
accordance with another aspect of the invention, in a process
which~comprise
A, acylating a 1-(2,3-O-isopropylidene-~-D-ribo- : -
~ ~ .
furanosyl)-1,2,4-triazole-3-carboxamide, 3-thiocarboxamide or
-3-carboxam~dine followed by deisopropylidenation,or
B. electively deacylating a (2,3,5-tri-O-acyl-~-D-
~; 20 ribofuranosyl)-1,2,4-triazole-3-carboxamide,-3-thiocarboxamide
- or -3-carboxamidine, to give a compound of structure (I) wherein
~ .
X i~ hydrogen or Cl-C18 acyl, at least one X being hydrogen and
at least one X being ac~l,
C. treating a l-(~-~-ribofuranosyl)-1,2,4-triazole-
nucleoside of ~tructure I wherein Rl is the same as above and
.
_. .
~ ~ - 2 -
. ,~...,~

1060004
X in 5'-position is hydrogen or Cl-C18 acyl, the remaining X's
are hydrogen with the tri-n-butyl amune salt of pyrophosphoric
acid, to give a l-(~-D-ribofuranosyl)-1,2,4-triazole-3-R1 2',3'-
cyclic phosphate, or
D. cleavage of a l-(~-D-ribofuranosyl)-1,2,4-triazole-
3-Rl, 2',3'-cyclic phosphate, as defined in C, at the cyclic
phosphate group to produce a triazole-nucleoside of structure(I)
with a monophosphate group
O = P - OH
OH
in the 2'- or 3'-position, and, when desired Cl-C18 acylating
otherwise free glycosyl hydroxyl groups and separating 2'- and
3'-phosphates, and, when desired, converting a compound of
structure (I) in which Rl is carboxamidino into a physiologically
acceptable acid addition salt thereof.
Preferred 5'-O-acyl compounds are prepared by a variety
of methods chosen according to the particular acyl group desired.
In the main (Method~ 1-3, infra), syntheses of 5'-O-acyl compounds
involve isopropylidene blocking of the 2'- and 3'-positions,
acylation at the 5'-position, and deisopropylidenation. Methods
1 and 2 involve acylation reaction in solvent media (e.g., pyridine)
and are employed fox the higher molecular weight acyl groups. In
Method 1 anhydride reactants are employed while Method 2 will
serve where the corresponding acyl haLides are more readily
obtainable. Lower molecular weight acyl group~ whose correspond-
ing anhydrides are liquid at normal te~peratures are attached by ~;
Method 3, employing 4-alkylaminopyridine as catalyst. A fourth
method of`choice, particularly for preparation of 5-O-benzoyl
compounds, involves selective deacylation. ~ -
Following Examples 1-5 illustrate 5'-O-acylation of the
preferred 1-(3-D-ribofuranoqyl)-1,2,4-triazole-3-carboxamide. -~
Esse~tially the same procedures may be employed in forming corres-
ponding 3-thiocarboxamides and 3-carboxamidines, mutatis mutandis.
~~~ '

1060004
In each of following Examples(1-3) of preferred
embodiments of the invention, the starting material 1-(2,3-0-
Isopropylidene-~- D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
was prepared as follows:
1-(2~3-o-Isopropylidene-~-D-ribofuranosyl)-l~2~4-triazole-3
carboxamide
~ D-Ribofuranosyl)-1,2,4-triazole-3-carboxamide
(30.0 g, 123 mole) was suspended in a mixture of acetone (400 ml)
and 2,2-dimethoxy-propane (200 ml). The mixture was cooled in
an ice bath and 7~/~ per-chloric acid (6 ml) was added. The
mixture was kept at room temperature for 3 hr and at 5
overnight. The resulting orange solution was neutralized with
2N potassium hydroxide, filtered, and evaporated to dryness.
The solid residue was treated with methanol and the insoluble
product was removed by filtration. The methanolic solution
was concentrated to a small volume and the crystalline product
wa~ collected. Recrystallization from a mixture of ethyl-
acetate and methanol gave 1-(2,3-0-isopropylidene-~-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide (31.0 g, 90/O)
with mp 153-154.
Anal- Calcd for CllH16N4s C~ 46-47; H~ 5-67; ~ 19.71.
Found: C, 46.38; ~, 5.73~ ~, 19.50.
~ .'.
:: .
.
": . , , . ~:

1060004
Depending upon the particular 5-0-acyl derivative
under preparation, one or more of the following methods was
employed.
Method l. A mixture of 1-(2,3-0-isopropylidene-~-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide, anhydrous
pyridine and the appropriate acyl anhydride is stirred at 60
until the reaction is complete as shown by thin-layer chromato--
graphy (24-72 hr). Water is added to the mixture after the re-
action is complete and the product is collected by filtration or
evaporation and extraction.
The intermediate l-(S-0-acyl-2,3-0-isopropylidene-~-
D-ribofuranosyl)-1,2,4-triazole-3-carboxamide is converted
to the 1-(5-0-acyl-~-D-ribofuranosyl)-1,2,4-triazole-3-carbox-
amide by treatment with aqueous acid. A mixture of the isopro-
pylidene intermediate is heated at 100 with a 4:1 (volume/volum~
mixture of acetic acid and water until the reaction is complete
as shown by thin-layer chromatography (2-5 hr). The solvent ~-
i9 removed by evaporation and the 5-0-acyl product is obtained
by crystallization.
Alternatively, the isopropylidene group may be
removed from the intermediate 5'-0-acyl-2',3'-0-isopropylidene
derivative by treatment with a 9:1 (volume/volume) mixture of
trifluoroacetic acid and water at room temperature for 5 min.
The reaction mixture is evaporated to dryness and the 5'-0-acyl
product is purified by crystallization.
.~
,~,,.~. .. . . , . : . .

1060004
Method 2. A mixture of 1-(2,3-0-isopropylidene-~-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide and anhydrous
pyridine is cooled in an ice bath and the appropriate acyl
halide is slowly added with stirring. The reaction mixture
is kept at room temperature until the reaction is complete as
shown by thin-layer chromatography (2-24 hr). Water is added
to the mixture and the product is obtained by filtration or
evaporation and extraction. The intermediate 1-(5-0-acyl-2,3-0-
isopropylidene-~-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
is converted to the 1-(5-0-acyl-~-D-ribofuranosyl)-1,2,4-triazole-
3-carboxamide as in Method 1.
Method 3. A mixture of 1-(2,3-0-isopropylidene-~-D-
ribofuranosyl)-1,2,4-triazole-3-carboxamide and an excess of
the appropriate acyl anhydride containing a catalytic amount
(ca. 100 mg) of 4-dimethylaminopyridine is stirred at room
temperature until the reaction is complete as shown by thin
layer chromatography (1~-24 hr). The reaction mixture is
evaporated to dryness and the product is isolated and converted
to the 1-(5-0-acyl-~-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
as in Method 1. Alternatively, the crude 5'-0-acyl-2',3'-0-
isopropylidene product obtained by evaporation may be converted
directly without purification to the 1-(5-0-acyl-~-D-ribofurano-
syl)-1,2,4-triazole-3-carboxamide by the deisopropylidenation
procedures given in Method 1.
-- 6 --

10160004
Method 4. A mixture of 1-(2,3,5-tri-0-benzoyl-~-D-
ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester
and methanol saturated at 0 with anhydrous ammonia was stirred
at 0 for 4 hr. The resulting solution was evaporated to dryness
under reduced pressure and the product was crystallized from
aqueous ethanol to give l-(5-0-benzoyl-~-D-ribofuranosyl)-
1,2,4-triazole-3-carboxamide with mp 182-184.
The particular methods employed in preparing the
compounds of Example 1-5 appear from Table I, which reports
certain of their properties.
: '
'~.
:.
'

1060004
TABLE 1
Properties of
1-(5-0-Acyl-~-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
O
H2N-C~,_N
R-o-~l/o 1
HO OH
Exam- _ Syn- Empirical Analyses
R thesis MP(C) _ormula _ Calcd - Found
1 CH3CO 3 1 H 4 93 4 97
(acetyl) N 19.58 19.63
2 CH3(CH2)2CO- 3155.5-157C12H18~4O6 H 45 787 5 80
(butyryl) N 17.83 17.73
3 CH3(CH2)14CO 1159-16324H42N46 H 58 77 58 80
(Palmitoyl) N 11.61 11.63
4 ~ 2,4 182-la4C15Hl6N4O6 C 51 72 51 91
(benzoyl) N 16.09 16.19
- .
morphousCl9H26~46 H 56 44 56 51 ~;
(adamantoyl) N 13.78 13~83
- ~ .
: ~ ~
~, ~ , . ,
~ - 8 - ~
: .
~ ' :
~'~

1060004
Of course, compounds within the scope of this
invention may be otherwise secured. For example, 5'-0-acyl
analogs may be approached via selective acylation, as may be,
e.g., 2t-0-acyl, 3'-0-acyl, and di-0-acyl analogs, with con-
sequent separation via column chromatography. See, e.g.,
C.A. Decker and L. Goodman, The Carbohydrates, vol. 2A, p. 26
(2d.ed. 1970) Academic Press, ~.Y.
~ ~'
Example 6
Various of the compounds prepared in the preceding
examples are tested for activity in vivo against Influenza
A2 (Japan 305) induced deaths in male Swiss mice (19-20 gm) and
the results compared with those obtained with l~ D-ribofurano-
syl)-1,2,4-triazole-3-carboxamide. The mice were intranasally
inoculated with virus and treated with compound undex test
by oral administration tw1ce daily for 8 days commencing 2
hours pre- and 4 hours post-virus inoculation. Virus dosage
was 3.2 LD50. Infected mice were observed for 21 days. The
`~ results of testing are presented in Table I. All compounds
~; tested were orally non-toxic at the dosages tested.
,` ~ '.
~ ,,:
. .. :.
....
, ~
,~ - 9 - ,:
. .

1060004
,1 ~
~ ~q
,~ a)
~> h
h U ~1
~ I ~1 0 Ir)
U~ H P1 0 It) O O O U'l O O Lr) O ~1 0 Il)
, , I . o,
a~ o oo o o o o o o o o o o
V AV V V V V V /~ V V V
~E~
,~
~_
~
h
u~ ~ ~ _I o ~u~ ~ ~ ~ ~ ~ r~ ~ ~D ~ O c~
O U~ . . . . . . . . ~ . . . . . ..
~ , .
1~ . ~ ''.
~ . . ~
:.
-1
N ~ h a~
O Cq In
H :1 ~ ~ 115 ~O 1` 1` ~ ~D 1` ~ ~) ~ 1` 0 d' 1` 1`
_I ~ hl~ d~9~ ~ d' ~ d' d' d' ~ O d' ~ ~9
q~ Ul ~O~ .. .... .. -. ....
E'3 H J~ Cl~ H O O O O O O O O O O O O O O
,:1 ~ a ...
m o ~ ~
f C ~ ~ E
E~ ~ ~ . X ~ ~ :
~i H ~ O -1 . R
O ~ ~ t~ U
C) U-rl~ O 00 0000 0000 0 00 I
~: , O ~ O ~ ~ 1 ~r),1
h E~ ~ ~ ~ ~ ~ I ~1
O H CQ ~1 ~1 0 0--I ~1 0--I ~1 ~1 0 lo--I O O ~
~:: . 1~ 0
q I h p~
W d' U -i
. . ~ .
u~ n ~ ~ :
~h O tl o u) I` O O Lt~ I` O O U~ I` O O It~ r-- ~ h ~ .~, ~
t~l` O t` ~ O~ O 1` ~ bq U o ~ "
~
.
. O rlrl ~
~ ~ ~a ~ -
P~ R ~
~ o -I ~o ~,. .-.
* ~) ~1 ~I ~L h ~ h
~-~0 1~ _1 11 ~¢ 11
P~ ~ :
.~O *
10~
"

~060004
The compound of Example 4 was similarly tested,
save that the mice were 17-18 g. male Swiss mice, virus dose
was 3.2 LD50, and drug was first administered 15 minutes
prior to virus inoculation.
The results appear in Table II.
: . ;
.: :
.
'

1060004
0 U~
~rl a1
h ~-1
U ~ O ~1
~ ~U O N o O ~r)
u~ H P~ .. ...
00 000
~: ~ V V V V A
a) '1
E~
g
gQ
h al bq
.r~ ) N IS~
tl U~rl (11 -. - . .
O ~ ~ `D ~ ~ S) N r-
0 ~
~ : .
I~
~I
0 .1 h al
N ~ ~ O ~1 O ~1 ~ --i
H I ~1 ~ h0 .. ...
H ~1 h O Pl 0 0 0 0 0
~: ~~ H V V V V A ~
H ~) C : .
3~1 g - l o o o o o
oU ,, ~ ,, ,, ,, ,, ,, ~ , 0
~,~ ~ o
q I h E~ ~ ~ l
OH U~ O . o~
~: U h
E~
_ d' fd
. ~ ~ a) o ..
l` O O It) I -. h U
~0 .~ t` ~) O ~ I ~ ~Q 3
~u
. ~ _
,~ U~ ~ ~ - .-: -
, ~ o ~ a ,4 u , , ",,
12
~. :'
: :'

1060004
The l~ D-ribofuranosyl)-1,2,4-triazole-3-Rl
2',3'-cyclic phosphates of the invention are prepared by an
adaptation of the procedure of T. Ueda and I. Kawai,
Chem. Pharm. Bull (Japan) 18, 2303 (1970) wherein the corresponding
nucleoside is refluxed in dimethylformamide with the tri-n-
butyl amine salt of pyrophosphoric acid. Product is purified by
cellulose ion exchange chromatography. 5'-0-acyl 2~,3'-cyclic
phosphates may be similarly prepared, commencing with the 5'-0-
acylated nucleosides.
Mixed (2',3') phosphates are secured by cleavage of
corresponding cyclic phosphates, as with dilute acid, If desired,
otherwise free glycosyl hydroxyls of the resulting mixture may
be acylated and 0-acyl 2'-and 3'-phosphates separated, one from
the other.
..
.
1 ,
:
~,,~ ',
', ' .

1060004
` Example 7
Preparation of l~~-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide
. ............................................................. :
21,3~-cyclic phosphate
A solution of l-~-D-ribofuranosyl-1,2,4-triazole-3-
carboxamide (1.22 g., 5.0 mmole), pyrophosphoric acid (10.0
mmole) and tri-n-butyl-amine (40.0 mmole) in dimethylformamide
(100 ml) was refluxed for 2.5 hr. The solvent was removed
in vacuo and the residue was dissolved in LN ammonium hydroxide
(100 ml). The solution wasextracted with ether and the
aqueous phase was concentrated to a small volume. The solution
was diluted with water to 60 ml and the pH was adjusted to 8.5 ~
with ammonium hydroxide. This solution was applied to a DE 52 -
cellulose (130 g) column in the bicarbonate form. Elution was
with a linear gradient of water (1000 ml) to 0.05 M triethylamine
bicarbonate ~1000 ml) and fractions of 20 ml were collected. ~-
The product was contained in fractions 35-50 which were combined
and evaporated to dryness. The residue was dissolved in water
(20 ml) and passed through a Dowex 50* X 8 (20 ml) column in the
ammonium form. The column was eluted with water and the solution
containing the product was evaporated to dryness. The residue
was dissolved in methanol and a small amount of impurity was -
removed by filtration. The filtrate was evaporated to dryness ~
and the residue was dissolved in water. The solution was ~ ~-
,
lyophilized to afford the ammonium salt of the 2',3'-cyclic phos-
phate.
~; Anal- Calcd for C8H14N507P.H2o.
.
N, 21.13. Found: C, 28.16; H, 4.73 N, 20.53. -
Alkali metal salts or the free 2',3' cyclic phosphate
may be obtained from the ammonium salt by ion exchange.
14 -
* trademark

~060004
After brief treatment with dilute acid the 2',3'-
cyclic phosphate was converted to the 2'(3')-phosphate as shown
by thin layer chromatography on silica gel with 7:3 (v~v)
acetonitrile: 0.1 N aqueous ammonium chloride.
Example 8
The compound of Example 7 was tested for antiviral
activity by the virus rating (VR) method of Sidwell, et al
Appl. Microbiol 22, 797 (1971) against type 1 herpes simplex
virus (HSV/l), type 13 rhinovirus (RV~13), type 3 parainfluenza
1~ virus (PIV/3), influenza virus type A, strain NWS (I Sj, _ ;~
and vaccinia virus ( W ), in the cell lines shown in Table III
below. V.R. > 1.0 is indicative of definite antiviral
activity, V.R. of 0.5 - 0.9 is indicative of moderate antiviral
activity, and V.R. < 0.5 suggests slight or no apparent anti-
viral activity. To demonstrate the antiviral activity of the
compounds resulting when the cyclic phosphate was opened by
acid hydrolysis, we acidified the solution (in cell culture medium)
containing the highest concentration of compound to pE 1.5 with
lN HCl. Formation of a mixture of l-(~-D-ribofuranosyl?-1,2,4-
triazole 2'-and 3l-phosphates was confirmed by thin layer
chromatography. This solution and similar solution which has not
.
been acidified were incubated side-by-side at 37C for 2 hr.
The acidified solution was then neutralized by adding lN NaOH and
the two incubated solutions were diluted and tested in cell
culture along with a similar solution that had not been incubated.
The resulting VR data follow.
,, , ' .
.:

1060004
T A B L E III
Virus Rating of Certain
1,2,4-triazole-3-carboxamide
l-~-D-Ribotides
:
RV/13 PIV/3
Compound KB RK-13 KB XB KB CE
Ammonium salt of
-D-ribofuranosyl)-
1,2,~-triazole-
3-carboxamide
2 ,3 -cyclic
phosphate0.8,1.2 1.1 0.6 0.2,0.3 1.1,1.1 0.4 .
~ : .
:- .
--do--
(incubated) 0.8 -- -- 0.2 1.0 0,4
mixed l-(~-D- ~.
ribofuranosyl)-
1,2,4-triazole-
3-carboxamide
2'- and 3l-phosphates
(incubated) 0.8 -- -- 0.6,0.6 1.0 0.5
-,:
The mixture of 2'- and 3'-monophosphates exhibited
. activity similar to that of the cyclic phosphate, save in the ::
~ . - : ..
case of RV/13, which proved more sensitive to the non-cyclic ~ -~
phosphate,s.
:~ :
-
',~,:: ; , ,. -
..-:
:-::
,, ~ ,
~ - 16-
;' ~ .
' - ':
. .