U.S. patent number RE29,835 [Application Number 05/782,294] was granted by the patent office on 1978-11-14 for 1,2,4-triazole nucleosides.
This patent grant is currently assigned to ICN Pharmaceuticals. Invention is credited to Roland K. Robins, Joseph T. Witkowski.
United States Patent |
RE29,835 |
Witkowski , et al. |
November 14, 1978 |
1,2,4-Triazole nucleosides
Abstract
As antiviral agents and intermediates therefor, 3-substituted
1-(.beta.-D-glycosyl)-1,2,4-triazoles, O-acylated analogs thereof,
and 5'- and 3',5'-cyclic phosphates of the triazole nucleosides,
"glycosyl" being .[.a pentofuranosyl moiety, preferably one whose
2'-oxygen is trans to the triazole aglycon, e.g., xylofuranosyl,.].
ribofuranosyl, .[.2-0-methylribofuranosyl, etc.,.]. the triazole
aglycon being 3-substituted with cyano, methylcarboxylate,
carboxamidoxime, carboxamido-, thiocarboxamido, or carboxamidine.
Preparation of these nucleosides is by silylation of the
substituted triazole followed by glycosylation with the appropriate
blocked glycosyl halide. Alternatively, acid-catalyzed fusion of
the requisite 1,2,4-triazole with an O-acylated pentofuranose
yields the nucleosides.
Inventors: |
Witkowski; Joseph T. (Laguna
Niguel, CA), Robins; Roland K. (Santa Ana, CA) |
Assignee: |
ICN Pharmaceuticals (Irvine,
CA)
|
Family
ID: |
27386778 |
Appl.
No.: |
05/782,294 |
Filed: |
March 28, 1977 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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149017 |
Jun 1, 1971 |
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Reissue of: |
240252 |
Mar 31, 1972 |
03798209 |
Mar 19, 1974 |
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Current U.S.
Class: |
536/26.11;
536/26.9; 536/28.7 |
Current CPC
Class: |
C07H
13/04 (20130101); C07H 19/04 (20130101); C07H
19/056 (20130101) |
Current International
Class: |
C07H
19/056 (20060101); C07H 13/04 (20060101); C07H
13/00 (20060101); C07H 19/00 (20060101); C07H
19/04 (20060101); C07H 019/06 (); C07H
019/10 () |
Field of
Search: |
;536/23,29 |
References Cited
[Referenced By]
U.S. Patent Documents
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3541079 |
November 1970 |
Schramm et al. |
|
Primary Examiner: Brown; Johnnie R.
Attorney, Agent or Firm: Boswell; K. H. Sears; Mary Helen
Irons; Edward
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This is a continuation-in-part of our copending U.S. application
Ser. No. 149,017 of the same title filed Jun. 1, 1971 and now
abandoned.
Claims
We claim:
1. A compound of structure ##STR8## wherein G is .[.a
pentofuranosyl moiety whose 2'-oxygen is trans to the aglycon.].
.Iadd..beta.-D-ribofuranosyl .Iaddend.and R.sub.1 is selected from
the group consisting of ##STR9## (d) physiologically acceptable
acid addition salts of (c), ##STR10##
2. A compound selected from the group consisting of 5'-phosphates
and 3',5'-cyclic phosphates of a compound according to claim 1.
3. A compound selected from the group consisting of (I) compounds
of structure: ##STR11## wherein .[.one of R.sub.2 and.]. R.sub.3
.[.are.]. .Iadd.is .Iaddend.hydroxyl.[., the other being.].
.Iadd.and R.sub.2 is .Iaddend.hydrogen, and wherein R.sub.1 is
selected from the group consisting of ##STR12## (d) physiologicaly
acceptable acid addition salts of (c), ##STR13## (II) a
5'-phosphate of a compound (I); and (III) a 3',5'-cyclic phosphate
of a compound (I). .[.
4. A compound according to claim 3 wherein R.sub.2 is
hydrogen..].
5. A compound according to claim .[.4.]. .Iadd.3 .Iaddend.wherein
otherwise free glycosyl hydroxyls are acyl-blocked.
6. A compound according to claim .[.4.]. .Iadd.3 .Iaddend.wherein
R.sub.1 is (a), (b), (c) or (d).
7. A compound according to claim .[.4.]. .Iadd.3 .Iaddend.wherein
R.sub.1 is (a), (b) or (d).
8. The ammonium or alkali metal salt of a 5'-phosphate (II)
according to claim 7. .[.9. A compound according to claim 3 wherein
R.sub.3 is hydrogen..]. .[.10. A compound according to claim 9
wherein otherwise free glycosyl hydroxyls are acyl-blocked..].
.[.11. A compound according to claim 9 and wherein R.sub.1 is (a),
(b), (c) or (d)..]. .[.12. A compound according to claim 9 wherein
R.sub.1 is (a), (b) or (d)..]. .[.13. The ammonium or alkali metal
salt of a 5'-phosphate (II) according to claim
12..]. 14. A compound (I) according to claim .[.4.]. .Iadd.3.
.Iaddend.
A compound (II) according to claim .[.4.]. .Iadd.3. .Iaddend. 16. A
compound (III) according to claim .[.4.]. .Iadd.3. .Iaddend. .[.17.
A compound (I) according to claim 9..]. .[.18. A compound (II)
according to
claim 9..]. .[.19. A compound (III) according to claim 9..]. 20.
The ammonium salt of the 5'-phosphate of
1-(.beta.-D-ribofuransoyl)-1,2,4-triazole-3-carboxamide.
21. 1(-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide.
22.
1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-thiocarboxamide.
23. 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamidine. 24.
1-(2,3,5-tri-O-acetyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxa
mide. 25. 1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
hydrochloride. .[.26.
1-(.beta.-D-xylofuranosyl)-1,2,4-triazole-3-carboxamide..]. 27.
1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
3',5'-cyclic
phosphate. 28. The ammonium salt of the compound of claim 27.
Description
BACKGROUND OF THE INVENTION
Presently, the only two known nucleosidic antibiotic agents having
5-membered heterocyclic rings are showdomycin and pyrazomycin,
which have respectively been reported to have structures (1) and
(2): ##STR1## Among the presently known synthetic nucleosidic
antiviral agents, the more important are 5-iodo-2'-deoxyuridine
(5-IDU) 9-.beta. -D-arabinofuranosyl adenine (Ara-A) and 1-.beta.
-D-arabinofuranosyl cytosine (Ara-C). Of these agents, only 5-IDU
(in 0.1% wt. solution) is commercially available specifically as an
antiviral agent, and this compound suffers the disadvantage of low
solubility and high toxicity, from whence arises the undesirably
great dilution in which it is now employed.
Alsonso et al. in J. Heterocyclic Chem. 7, 1269-72 (1970) report
the preparation of 1-(.beta.
-D-ribofuranosyl)-4-carboxamido-1,2,3-triazole but make no mention
of biological activity. Witkowski and Robins, in "The Chemical
Synthesis of the 1,2,4-Triazole Nucleosides Related to Uridine,
2'-Deoxyuridine, Thymidine and Cytidine," J. Org. Chem. 35, 2635-41
(1970) suggest various 1,2,4-triazole nucleoside analogs as
candidates for biological testing, but no particular efficacy is
noted and our later testing has shown that, of the analogs there
reported, only 5-bromo-3-nitro-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole exhibits even slight antiviral
activity and even this activity is believed to be attributable
merely to the cytotoxicity of the compound.
BRIEF SUMMARY OF THE INVENTION
According to this invention, there are provided compounds of
formula ##STR2## wherein "G" is .[.a pentofuranosyl moiety,
preferably characterized by 2'-oxygen trans to the triazole
aglycon.]. .Iadd..beta.-D-ribofuranosyl, R.sub.1 being cyano-,
carboxamido-, thiocarboxamido-, -methylcarboxylate, carboxamidine,
or a physiologically acceptable acid addition salt of the latter
group. The invention also embraces the O-acylated analogs of the
foregoing compounds and the 5'-phosphates and 3',5'-cyclic
phosphates thereof as well as the ammonium and alkali metal salts
of the phosphates. Preferred embodiments include compounds of
structure ##STR3## where R.sub.1 is as previously defined and
.[.one of.]. R.sub.2 .[.or R.sub.3 .]. is hydrogen, .[.the other
being.]. .Iadd.and R.sub.3 is .Iaddend.hydroxyl, as well as
5'-phosphates and 3',5'-cyclic phosphates thereof. Compounds
according to this invention exhibit antiviral activity and, in
nonacylated or "deblocked" embodiments, exhibit substantially
greater aqueous solubility than earlier reported nucleosidic
antiviral agents. The preferred 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide nucleoside of the
invention has exhibited a broad spectrum of antiviral activity as
well as, in laboratory animals, antitumor activity.
DETAILED DESCRIPTION OF THE INVENTION
Methyl 1,2,4-triazole-3-carboxylate and 3-cyano-1,2,4-triazole are
precursors in alternative synthetic routes to 1,2,4-triazole
nucleosides of the invention. The former compound can be
conventionally prepared by oxidation of 3-methyl-1,2,4-triazole
followed by esterification of the resulting acid according to the
procedure of Cipens and Grinsteins' Latvijas PSR Zinatnu Akad.
Vestis., Kim. Ser. (1965) (2) 204-08 (see C.A. 63, 13243, 1965).
3-cyano-1,2,4-triazole is prepared by a multi-step synthesis
reported by Cipens et al., supra, or more conveniently, by the
addition reaction of cyanogen and hydrazine to form
1-cyanoformimidic acid hydrazide, followed by acid-catalyzed ring
closure in triethylorthoformate to the 3-cyano compound. By one
route, the trimethylsilyl derivative of the methyl
1,2,4-triazole-3-carboxylate is formed in quantitative yield by
reaction with hexamethyldisilazane under reflux and reacted with an
appropriate O-acylated halo sugar GX to form, for example, a
mixture of O-benzoylated 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyle ester
and 5-carboxylic acid methyl ester, i.e.: ##STR4## wherein B.sub.z
is benzoyl. The 3-carboxylic acid methyl ester is separated by
fractional crystallization or, preferably, by column chromatography
over silica gel. Debenzoylation and aminolysis gives the 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide.
The preferred method of synthesis is by the acid-catalyzed fusion
of an appropriately substituted triazole with the O-acyl blocked
sugar (in this case 1-O-acetyl-2,3,5-tri-O-benzoyl-.beta.
-D-ribofuranose) followed by deblocking and aminolysis, i.e.:
##STR5## Alternatively, fusion can be had with
3-cyano-1,2,4-triazole with later conversion of the
3-cyano-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole to the active 3-carboxamido
nucleoside by reaction with hydrogen peroxide in the presence of
aqueous ammonia, albeit in lesser yields than when fusion occurs
with methyl 1,2,4-triazole-3-carboxylate.
The 3-cyano-1-(2,3,5-tri-O-acetyl-.beta. -D-ribofuranosyl)-1,2,4
triazole, whether obtained by the silylation or fusion procedure,
is a useful intermediate to formation of the active 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-thiocarboxamide nucleosides of
the invention as by reaction of the same with hydrogen sulfide in
the presence of triethylamine followed by deacylation where the
blocked nucleoside is employed as the intermediate. Similarly, the
nucleosidic 1,2,4-triazole-3-carboxamidines can be made from the
3-cyano-1,2,4-triazole nucleoside either with or without
intermediate formation of the 3-carboxamidoxime analog, i.e.:
##STR6## where G is a glycosyl moiety as above-defined. The
carboxamidine is obtained in free form from the synthesis involving
the carboxamidoxime intermediate. Contrarywise, the preferred
preparation directly from the 3-cyano compound in the presence of
ammonium chloride yields the carboxamidine in the form of its
hydrochloride salt, and other physiologically acceptable acid
addition salts (e.g., hydrobromic, hydroiodic, citric, acetic,
sulfuric, phosphoric acid addition salts) may be obtained therefrom
by ion exchange or alternatively by neutralization of the hydrogen
chloride salt with sodium bicarbonate, followed by reaction of the
resulting free carboxamidine with an appropriate acid.
The 3-carboxamido, 3-thiocarboxamido and 3-carboxamidine
nucleosides of the invention may be converted to the corresponding
5'-phosphate or ammonium or alkali metal salt of the phosphate and
administered as such. Similarly, 5'-phosphates of the intermediate
3-methyl carboxylates, 3-cyano- and 3-carboxamidoxime compounds of
the invention may be formed prior to operations at position 3 of
the triazole aglycon leading to formation of the active
nucleotides. Those skilled in the art are fully acquainted with the
method and manner of phosphorylating nucleosides. In a preferred
embodiment of the invention, the 5'-phosphate of 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide is formed by
reaction of 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester with POCl.sub.3 in trimethyl phosphate, followed
by hydrolysis in ice water to yield the free 5'-phosphate.
Formation of the ammonium salt and aminolysis of the methyl ester
occur simultaneously on tratment with aqueous ammonia. Alkali metal
salts or the free 5'-phosphate of the 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide nucleotide can then
be obtained from the ammonium salt of the phosphate by ion
exchange.
For enhanced cellular transport, the 3',5'-cyclic phosphates of the
active carboxamido, thiocarboxamido and carboxamidine compounds of
the invention may be secured by dicyclohexylcarbodiimide (DCC)
cyclization of corresponding 5'-phosphates. Alternatively,
5'-phosphates of the cyano-, methylcarboxylate and carboxamidoxime
intermediates may be cyclized prior to operations on position 3 of
the aglycon leading to said active cyclic nucleotides. In either
case, the cyclic phosphates may be employed either in free form or
in the form of ammonium or alkali metal salts obtained as in the
case of the 5'-phosphates.
While the invention has been described with particular reference to
acetyl or benzoyl blocking of the 2', 3' and 5' hydroxyls of the
glycosyl moiety, it will be understood that any acyl group may be
employed to preserve those hydroxyls against side reactions such as
dehydration during synthesis of the active agents of the invention.
Sutherland et al., in Biochim. et Biophys Acta 148, 106 (1967) have
reported that acylation of cyclic nucleotides enhances cellular
transport. Similarly, otherwise free glycosyl hydroxyls of the
nucleotides and nucleosides of the invention may be acylated for
enhanced lipid solubility or, e.g., provided with alkyl or alkaryl
sulfonyl groups such as, e.g., tosyl, mesyl, brosyl, nisyl, etc. to
the same end.
While the 1'-"leaving group" of the sugar reagents discussed above
has been characterized as acetyl or, in the silyation procedure, as
halo-, it will be understood that any displaceable moiety may be
employed subject to amenability to byproduct separation.
.[.While, for the sake of example, reference above has been to
.beta.-D-ribofluranosyl and xylofuranosyl moieties, it will be
appreciated that the invention includes employment of other
glycosyl moieties as well, i.e., 2'-deoxy-.beta.-D-ribofuranosyl,
2'-deoxy-.alpha.-D-ribofuranosyl and .beta.-D-arabinofuranosyl. In
the case of the 2'-deoxy triazole nucleosides precursor sugars
employed may include 2-deoxy-1,3,5-tri-O-acetyl-D-ribofuranose or
1-O-acetyl-2-deoxy-3,5-di-O-(para-toluoyl)-D-ribofuranose, the
latter arising from treatment of
2-deoxy-3,5-di-O-(para-toluoyl)-D-ribofuranosyl chloride with
mercuric acetate in tetrahydrofuran..].
.[.The arabinosyl triazole nucleosides are made by the silylation
procedure, employing 2,3,5-tri-O-benzyl-D-arabinofuranosyl
chloride..].
.[.Given biological activity of the ribofuranosyl triazoles of the
invention, activity in the correspondingly substituted
arabinofuranosyl triazoles would appear to follow, especially in
view of the known antiviral activity of "Ara-A" and "Ara-C"
discussed above. Similarly, biological activity of the
corresponding 2'-deoxy-.beta.-D-ribofuranosyl triazoles is
suggested by the known antiviral activity of 5-iodo-2'-deoxy
uridine, once given efficacy of the ribofuranosyl triazoles of the
invention, especially as against essentially DNA viruses like
Herpes Types 1 and 2. However, when these compounds were tested
against the viruses of Example 16, infra, essentially no antiviral
activity was seen, apparently owing to the inability of enzymes
present in the tissue culture to phosphorylate free nucleosides
whose glycosyl moieties lack trans 2'-oxygen atoms. Hence, these
nucleosides should be synthetically phosphorylated to the believed
active 5'-phosphate form prior to administration. Alternatively,
the 5'-phosphate may be cyclized before administration, in vivo
cleavage yielding the active 5'-phosphate metabolite. The
phosphorylation procedure is essentially like that described above
for preparation of the ribotides..].
The invention is further illustrated and described in the following
examples, in which all parts and percentages are by weight and all
temperatures in degrees centigrade unless otherwise qualified. All
evaporative procedures were carried out in a rotary evaporator
under diminished pressure at 35.degree. C.
EXAMPLE 1
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazoles-3-carboxylic
acid methyl ester and
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-5-carboxylic
acid methyl ester (silylation procedure)
(A) Preparation of methyl
N-(trimethylsilyl)-1,2,4-triazole-3-carboxylate. A suspension of
methyl 1,2,4-triazole-3-carboxylate (14.0 g., 110 mmole) and
hexamethyldisilazane (100 ml.) was refluxed with stirring until
evolution of ammonia ceased (ca. 2 hrs.). The excess
hexamethyldisilazane was removed under diminished pressure to
provide 20.6 g. (100%) of the N-trimethylsilyl derivative of methyl
1,2,4-triazole-3-carboxylate.
(B) Ribosylation with halo sugar: A solution containing methyl
N-(trimethylsilyl)-1,2,4-triazole-3-carboxylate (20.6 g., 110
mmole) and 2,3,5-tri-O-benzoyl-D-ribofuranosylbromide (52.5 g., 100
mmole) in anhydrous acetonitrile (300 ml.) was kept at 25.degree.
for 3 days. The solvent was removed and the residue was
crystallized from ethanol. Recrystallization of the material from
ethyl acetate-ethanol and column chromatography of the filtrates
over silica gel with chloroform provided pure
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester (25.1 g., 44.0%) with M. P. 137- 139.degree..
Analysis.--Calcd. for C.sub.30 H.sub.25 N.sub.3 O (percent): C,
63.04; H, 4.41; N, 7.35. Found (percent): C, 62.91; H, 4.17; N,
7.10.
The faster migrating component from the silica gel column was
crystallized from ethanol to provide
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-5-carboxylic
acid methyl ester (13.2 g., 23.1%) with M.P. 122-124.degree..
Analysis.--Found (percent): C, 63.20; H, 4.35; N, 7.12.
EXAMPLE 2
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester (fusion procedure)
A mixture of methyl 1,2,4-triazole-3-carboxylate (12.7 g., 100
mmole) and 1-O-acetyl-2,3,5-tri-O-benzoyl-.beta.-D-ribofuranose
(55.4 g., 110 mmole) was heated in an oil bath maintained at
160-165.degree.. After the sugar had melted, bis(p-nitrophenyl)
phosphate (400 mg.) was added with stirring and the mixture was
heated under diminished pressure at 160-165.degree. for 15-20 min.
Crystallization of the residue from ethyl acetate-ethanol provided
42.5 g. (74.5%) of product with M.P. 137-139.degree..
EXAMPLE 3
1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester
A mixture of methyl 1,2,4-triazole-3-carboxylate (12.7 g., 0.10
mol) and 1,2,3,5-tetra-O-acetyl-.beta. -D-ribofuranose (31.8 g.,
0.10 mol) was heated in an oil bath maintained at 160-165.degree.
until the sugar had melted. Bis(p-nitrophenyl) phosphate (250 mg.)
was added and heating at 160-165.degree. was continued with
stirring under diminished pressure for 15-20 min. The residue was
dissolved in hot benzene, the solution was filtered and cyclohexane
was added to the filtrate to give the crystalline product (30.0 g.,
77.8%) with M.P. 107-109.degree..
Analysis--Calcd. for C.sub.15 H.sub.19 N.sub.3 O.sub.9 (percent):
C, 46.75; H, 4.97; N, 10.91. Found (percent): C, 46.88; H, 5.03; N,
10.64.
EXAMPLE 4
1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl
ester
A solution of
1-(2,3,5-tri-O-benzoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester (25.0 g., 43.8 mmol) and sodium methoxide (400
mg.) in methanol (200 ml.) was refluxed for 45 min. The solution
was neutralized with Bio-Rad AG50-X2 (H), filtered and the filtrate
was concentrated to a syrup. Crystallization of the syrup from
methanolethyl acetate provided 8.0 g. (70.5%) of product with M.P.
117-199.degree..
Analysis.--Calcd. for C.sub.9 H.sub.13 N.sub.3 O.sub.6 (percent):
C, 41.70; H, 5.06; N, 16.21. Found (percent): C, 41.57; H, 5.13; N,
16.16.
EXAMPLE 5
(A) 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(Method 1)
A solution of 1-(2,3,5-tri-O-benzoyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester
(16.0 g., 28.0 mmole) in methanol (300 ml., presaturated with
anhydrous ammonia at 0.degree.) was kept in a sealed pressure flask
at 25.degree. for 3 days. The solvent was removed and the product
was crystallized from ethanol to give 6.7 g. (98%) of material with
M.P. 174-176.degree.. Recrystallization of the product from aqueous
ethanol provided a second crystalline form of the nucleoside with
M.P. 166-168.degree..
Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.5 (percent):
C, 39.34; H, 4.95; N, 22.94. Found (percent): C, 39.08, H, 5.10; N,
22.67.
(B) 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(Method 2)
A solution of 1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester
(10.0 g., 26.0 mmole) in methanol (70 ml.) saturated at 0.degree.
with anhydrous ammonia was kept in a sealed pressure flask at
25.degree. for 18 hrs. The product was crystallized from ethanol to
give 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (5.70
g., 90.0%).
(C) 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(Method 3)
A mixture of 3-cyano-1-(2,3,5
-tri-O-acetyl-.beta.-D-ribofuranosyl)-1,2,4-triazole (705 mg., 2.0
mmole), 28% aqueous ammonia (20 ml.) and 30% hydrogen peroxide (3.0
ml.) was stirred at 25.degree. for 6 hrs. An additional 3.0 ml.
portion of 30% hydrogen peroxide was then added and stirring at
25.degree. was continued for 12 hrs. The excess hydrogen peroxide
was destroyed by addition of platinum black, the solution was
filtered, the filtrate was evaporated to dryness. The residue was
dissolved in methanol and silic gel (5.0 g.) was added to the
solution. The solvent was removed and the silica gel mixture was
applied to a silica gel column. Elution with ethyl acetate-methanol
(1:1) provided 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (250 mg.,
51.2%).
EXAMPLE 6
1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
3',5'-cyclic phosphate ammonium salt
To 1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
5'-phosphate ammonium salt (5.7 g., 15.9 mmole) in pyridine was
added 4-morpholine-N,N'-dicyclohexylcarboxamidine (4.65 g., 15.9
mmole) and the resulting solution was evaporated in vacuo several
times with pyridine to an anhydrous syrup. The syrup was dissolved
in 1 liter of pyridine and added dropwise (over a one hour period),
through a reflux condenser, into a refluxing anhydrous solution of
dicyclohexylcarbodiimide (16.4 g., 79.6 mmole) in 3 liters of
pyridine. The solution was refluxed for a further two hours and 200
ml. water was added slowly. After ca. 12 hours the solution was
evaporated in vacuo and to the residue was added 200 ml. water and
50 ml. ether. The suspension was stirred vigorously and then
filtered. The aqueous layer was separated and extracted with 2X
100ml. ether. The aqueous layer was passed through Dowex 50
(NH.sub.4.sup.+ form, 100-200 mesh) and the eluant evaporated to a
syrup. Ethanol ca. 100 ml. was added to the syrup and the resulting
mixture set at room temperature ca. 12 hours. The crude precipitate
was filtered and the filtrate set at room temperature for two
weeks. The resulting precipitate (1.2 g.) was dissolved in 5 ml.
warm water and then 40 ml. ethanol was added. The resulting
crystals were filtered and dried 12 hours at 78.degree. C. over
P.sub.2 O.sub.5, in vacuo to give 1.05 g. of 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide 3',5'-cyclic
phosphate ammonium salt: .alpha..sub.D.sup.25.degree. =67.degree.
(c.=1,H.sub.2 O); IR (KBr) 1688 cm..sup.-1 ##STR7## M.P. 245 d.
Analysis.--Calcd. for C.sub.8 H.sub.11 N.sub.4 O.sub.7
P.multidot.NH.sub.3 (percent): C, 29.73; H, 4.36; N, 21.66. Found
(percent): C, 29.86; H, 4.67; N, 21.47.
EXAMPLE 7
1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
5'-phosphate ammonium salt
A solution of 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester
(259 mg., 1.00 mmole), trimethyl phosphate (3.0 ml.) and phosphoryl
chloride (0.20 ml.) were stirred at 0.degree. for 1.5 hrs. Ice
water as added and the solution was neutralized with aqueous sodium
hydrogen carbonate. The solution was extracted with chloroform and
the aqueous phase was cooled to 0.degree. and saturated with
ammonia. The solution was kept at 25.degree. for 16 hrs. then it
was filtered and the filtrate was concentrated to a small volume.
Addition of ethanol gave a precipitate which was dissolved in water
and passed through a Bio-Rad AG50W-X2 (NH.sub.4) column (20 ml.).
Concentration of the fractions containing the nucleotide provided
the ammonium salt of 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide 5'-phosphate (190
mg., 53.0%).
Analysis.--Calcd. for C.sub.8 H.sub.16 N.sub.5 O.sub.8 P.H.sub.2 O
(percent): C, 26.74; H, 5.05; N, 19.50. Found (percent): C, 26.78;
H, 5.23; N, 19.68.
EXAMPLE 8
1-(.beta. -D-ribofuranosyl)- 1,2,4-triazole-3-thiocarboxamide
A mixture of 3-cyano-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole (8.0 g., 22.7 mmole),
triethylamine (14.0 ml.) and ethanol (200 ml.) was stirred at
25.degree. while hydrogen sulfide gas was passed into the solution
for 2 hrs. The solvent was removed and the residue was treated with
a solution of sodium methoxide (600 mg.) in methanol (150 ml.) for
three hours at 25.degree.. After the solution was neutralized with
Bio-Rad AG 50W-X2(H), it as filtered, and the solvent was removed.
Crystallization of the product from aqueous ethanol provided 4.5 g.
(76.0%) of the thiocarboxamide with M.P. 173-175.degree..
Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.4 S (percent):
C, 36.92; H, 4.65; N, 21.53; S, 12.32. Found (percent): C, 37.12;
H, 4.90; N, 21.26; S, 12.02.
EXAMPLE 9
3-cyano-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole
A mixture of 3-cyano-1,2,4-triazole (9.41 g., 0.10 mol) and
1,2,3,5-tetra-O-acetyl-.beta. -D-ribofuranose (31.8 g., 0.10 mol)
was heated in an oil bath maintained at 150.degree..
Bis(p-nitrophenyl) phosphate (100 mg.) was added with stirring and
heating at 150.degree. under diminished pressure was continued for
15 min. The residue was dissolved in chloroform, the solution was
filtered and the solvent was removed. Crystallization of the
residue from ether provided 28.2 g. (80%) of product with M.P.
96-97.degree..
Analysis.--Calcd. for C.sub.14 H.sub.16 N.sub.4 O.sub.7 (percent):
C, 47.73; H, 4.58; N, 15.90. Found (percent): C, 47.79; H, 4.63; N,
15.95.
EXAMPLE 10
1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamidine
hydrochloride
A mixture of 3-cyano-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole (7.04 g., 20.0 mmole) ammonium
chloride (1.07 g., 20.0 mmol) and anhydrous ammonia (150 ml.) was
heated in a bomb at 85.degree. for 18 hours. After removal of
excess ammonia, the residue was crystallized from
acetonitrile-ethanol to provide 5.30 g. (95%) of product with M.P.
177.degree.-179.degree. dec.
Analysis.--Calcd. for C.sub.8 H.sub.14 ClN.sub.5 O.sub.4 (percent):
C, 34.35; H, 5.05; Cl, 12.68; N, 25.04. Found (percent): C, 34.39;
H, 5.06; Cl, 12.73; N, 24.99.
EXAMPLE 11
1-(.beta. -D-ribofuranosyl)-1,2,4-triazole-3-carboxamidoxine
A solution of 3-cyano-1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole (3.0 g., 8.52 mmol) and excess
hydroxylamine in ethanol (100 ml.) was refluxed with stirring for 2
hrs. The solvent was removed and the product was crystallized from
aqueous ethanol to provide 2.0 g. (90.7%) of the carboxamidoxine
with M.P. 212-214.degree. dec.
Analysis.--Calcd. for C.sub.8 H.sub.13 N.sub.5 O.sub.5 (percent):
C, 37.07; N, 5.06; N, 27.02. Found (percent): C, 36.80; H, 4.92; N,
27.23.
.[.EXAMPLE 12.].
.Badd..[. 1-(.beta.
D-xylofuranosyl)-1,2,4-triazole-3-carboxamide.]..Baddend.
.[.A mixture of tetra-O-acetyl-D-xylofuranose (12.7 g., 40.0 mmol)
methyl 1,2,4-triazole-3-carboxylate (5.08 g., 40.00 mmol) and
bis-(p-nitrophenyl) phosphate (50 mg.) was heated under reduced
pressure for 20 min. in an oil bath maintained at 160-165.degree..
The residue was cooled and dissolved in chloroform. The crude
product was purified by chromatography on silica gel with
chloroform-acetone (20:1). A portion (2.0 g.) of the purified
1,2,3,5-tri-O-acetyl-.beta.
-D-xylofuranosyl)-1,2,4-triazole-3-carboxylic acid methyl ester was
treated for 16 hrs. at 25.degree. with methanol saturated with
methanol saturated with ammonia. The solvent was removed and the
residue was crystallized from aqueous ethanol to provide 0.70 g. of
product with M.P. 194.degree.-196.5.degree...].
.[.Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.5
(percent): C, 39.34; H, 4.95; N, 22.94. Found (percent); C, 39.18;
H, 4.82; N, 22.97..].
EXAMPLE .Badd..[.13.]..Baddend. .Iadd.12.Iaddend.
1-(2,3,5-tri-O-acetyl-.beta.
-ribofuranosyl)-1,2,4-triazole-3-carboxamide
A solution of 1-(.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (488 mg., 2.00 mmol)
in pyridine (10.0 ml.) containing acetic anhydride (1.0 ml.) was
added and the solution was stirred at room temperature for 2 hrs.
The solvent was removed and water was added to the residue. The
mixture was extracted with methylene chloride (three 20-ml.
portions) and the organic layer was extracted with aqueous sodium
hydrogen carbonate and water. The organic phase was dried over
magnesium sulfate, filtered, and evaporated to a syrup which was
applied to a silica gel column. Elution with chloroform-methanol
(19:1) provided the pure product as an amorphous solid (560 mg.,
75.6%).
Analysis.--Calcd. for C.sub.14 H.sub.18 N.sub.4 O.sub.8 (percent);
C, 45.40; H, 4.90; N, 15.13. (percent): (percent); C, 45.36; H,
466; N, 14.90.
1-(2,3,5-tri-O-acetyl-.beta.
-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide, when given orally
twice daily for 9 days at dosages of 125 and 250 mg./kg./day to
mice infected with influenza A.sub.2 (Jap 305) significantly
increased the number of survivors as well as mean survival time of
the treated animals relative to controls.
.[.EXAMPLE 14.].
.Badd..[. 1-(.beta.
-D-arabinofuranosyl)-1,2,4-triazole-3-carboxamide.]..Baddend.
.[.A solution containing methyl
N-(trimethylsilyl)-1,2,4-triazole-3-carboxylate (4.12 g., 22.0
mmol) and 2,3,5-tri-O-benzyl-D-arabinofuranosyl chloride (100 ml.)
was kept at 25.degree. for 48 hrs. The solution was evaporated to
dryness, the residue was dissolved in methylene chloride and the
solution was washed with aqueous sodium hydrogen carbonate and
water. The methylene chloride solution was dried over magnesium
sulfate, filtered, and concentrated to a syrup which was applied to
a silica gel column packed in benzene. Elution of the column with
benzene-ether (7:3) provided 1.9 g. of syrupy product which was
treated with methanol (50 ml.) saturated at 0.degree. with
anhydrous ammonia in a pressure flask at 25.degree. for 48 hrs. The
solvent was removed and the product was crystallized from methylene
chloride-cyclohexane to provide 1.3 g. (12.6%) of
1-(2,3,5-tri-O-benzyl-.beta.
-D-arabinofuranosyl)-1,2,4-triazole-3-carboxamide with M.P.
100-102.degree...].
.[.Analysis.--Calcd. for C.sub.29 H.sub.30 N.sub.4 O.sub.5
(percent): C, 67.69; H, 5.88; N, 10.89. Found (percent): C, 67.82;
H, 5.70; N, 10.94..].
.[.A suspension of palladium black and methanol (20 ml.) was
prepared by reduction of palladium chloride (300 mg.) with hydrogen
and to this was added a solution of 1-(2,3,5-tri-O-benzyl-.beta.
-D-arabinofuranosyl)-1,2,4-triazole-3-carboxamide (600 mg.) in
methanol (25 ml.) The mixture was shaken on a hydrogenation
apparatus at 25 p.s.i. for 2 hrs. at 20.degree.. The catalyst was
removed by filtration and the solution was passed through a column
of Amberlite IR 45 (OH) (15 ml.). The solvent was removed and the
residue was crystallized from ethanol to give 250 mg. (88.5%) of
product with M.P. 189-191.degree...].
.[.Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.5
(percent): C, 39.34; H, 4.95; N, 22.94. Found (percent): C, 39.25;
H, 4.86; N, 23.09..].
.[.EXAMPLE 15.].
.[.(A) 1-0-acetyl-2-deoxy-3,5 -d-O-p-toluoyl-D-ribofuranose.].
.[.A mixture of 2-deoxy-3,5 -di-O-p-toluoyl-D-ribofuranosyl
chloride (15.6 g., 40.0 mmol), mercuric acetate (12.7 g., 40.0
mmol) and tetrahydrofuran (200 ml.) was stirred at 20.degree. for
10 hours. The solvent was removed and chloroform was added to the
residue. The mixture was extracted with 30% aqueous potassium
iodide (four 60-ml portions) and water. The organic phase was dried
over magnesium sulfate, filtered and concentrated to a syrup (16.0
g., 97.0%). Crystallization of a portion of the product from
cyclohexane-benzene provided pure material with M.P. 88-91.degree.
..].
.[.Analysis.--Calcd. for C.sub.23 H.sub.24 O.sub.7 (percent): C,
66.98; H, 5.87. Found (percent): C, 67.23; H, 5.97..].
.[.(B) 1-(2-deoxy-3,5
-di-O-p-toluoyl-.alpha.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester and 1-(2-deoxy-3,5
-di-O-p-toluoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxylic
acid methyl ester.].
.[.A mixture of 1 -O-acetyl-2-deoxy-3,5
-di-O-p-toluoyl-D-ribofuranose (4.54 g., 11.0 mmol), methyl
1,2,4-triazole-3-carboxylate (1.27 g., 10.0 mmol) and
bis(p-nitrophenyl) phosphate (10 mg.) was heated with stirring
under diminished pressure for 10-15 min. in an oil bath maintained
at 150.degree.. The residue was dissolved in methylene chloride and
the solution was filtered, then washed with aqueous sodium hydrogen
carbonate and water. The organic phase was dried over magnesium
sulfate, filtered, and evaporated to a syrup. Column chromatography
of this anomeric mixture over silica gel with chloroform provided
the .alpha.-anomer which was crystallized from
cyclo-hexane-methylene chloride to give 780 mg. of pure product
with M.P. 94-95.degree...].
.[.Analysis.--Calcd. for C.sub.25 H.sub.25 N.sub.3 O.sub.7
(percent): C, 62.62; H, 5.26; N, 8.76. Found (percent): C, 62.44;
H, 5.16; N, 8.64..].
.[.Subsequent fractions from the silica gel column contained a
mixture of the .alpha.- and .beta.-anomers. These fractions were
combined and the mixture was further purified by column
chromatography over silica gel with chloroform to provide the
.beta.-anomer as a syrup..].
.[.(C)
1-(2-deoxy-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide.].
.[.A solution of
1-(2-deoxy-3,5-di-O-p-toluoyl-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-car
boxylic acid methyl ester (650 mg., 1.35 mmol) in methanol (20 ml.)
saturated at 0.degree. with anhydrous ammonia was kept at
25.degree. for 3 days in a sealed pressure bottle. The solvent was
removed and the residue was applied to a silica gel column. Elution
with ethyl acetate-methanol (9:1) provided
1-(2-deoxy-.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(250 mg., 80.8%) as an amorphous solid..].
.[.Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.4
(percent): C, 42.10; H, 5.30; N, 24.55. Found (percent): C, 41.86;
H, 5.12; N, 24.49..].
.[.(D)
1-(2-deoxy-.alpha.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide.].
.[.A solution of
1-(2-deoxy-3,5-di-O-p-toluoyl-.alpha.-D-ribofuranosyl)-1,2,4-triazole-3-ca
rboxylic acid methyl ester (780 mg., 1.63 mmol) in methanol (25
ml.) saturated at 0.degree. with anhydrous ammonia was kept in a
sealed pressure flask at 25.degree. for 3 days. The solvent was
removed and the crude product was applied to a silica gel column.
Elution with ethyl acetate-methanol (9:1) provided
1(2-deoxy-.alpha.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(350 mg., 94.4%) as an amorphous solid..].
.[.Analysis.--Calcd. for C.sub.8 H.sub.12 N.sub.4 O.sub.4
(percent): C, 42.10; H, 5.30; N, 24.55. Found (percent): C, 41.88;
H, 5.48; N, 24.29..].
.[.By the same procedure employed to convert the separated methyl
ester anomers to their respective carboxamides, the anomeric
mixture of the methyl esters formed in part (B) above may be
reacted with ammonia to yield a mixture of the .alpha.- and
.beta.-anomers of the carboxamide..].
EXAMPLE .Badd..[.16.]. .Baddend..Iadd.13.Iaddend.
Antiviral agents of the invention are tested for activity against
both small and large viruses of both DNA and RNA types by the virus
rating (VR) method of Sidwell et al., Appl. Micbrobiol. 22, 797
(1971). 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 antiviral activity. The
results reported below, which for comparison sake include data
taken with various known antiviral agents, were obtained by testing
on MIcrotest II (Falcon Plastics) plastic panels with a monolayer
of KB or RK13 cells.
TABLE I
__________________________________________________________________________
COMPARATIVE ANTIVIRAL ACTIVITY Type 1 Type 2 Pseudo- Vac- Para-
Type 13 herpes herpes Myxoma rabies cinia Adeno influenza Rhino No.
Compound V.R. V.R. V.R. V.R. V.R. V.R. V.R. V.R.
__________________________________________________________________________
1 1-(.beta.-D-Ribofuranosyl)- 1.5, 1.0, 1.2 1.3 1.7 0.0, 0.0 1.0,
0.9 0.5, 0.5 1.0, 1.0, 0.5 1,2,4-triazole-3-carbox- amide. 2
Ammonium salt of the 0.7, 0.8 0.9 -- -- 0.5 0.0 0.5 0.3
5'-phosphate of 1-(.beta.-D- ribofuranosyl)-1,2,4-
triazole-3-carbox- amide. 3 1-(.beta.-D-Ribofuranosyl)- 1.0, 0.5 --
0.2 0.1 0.3 0.0 0.0, 0.0 0.1, 0.2 1,2,4-triazole-3-thiocar-
boxamide. [4] [1-(.beta.-D-Xylofuranosyl)- [0.7, 1.2] -- [0.95]
[0.8] [0.7] [0.1] [0.7, 0.8] [0.4, 0.5] 1,2,4-triazole-3-carbox-
amide.] 5 1-(.beta.-D-Ribofuranosyl)- 0.8, 1.0 -- 1.1 0.6 -- 0.1
0.7, 0.8 0.6 1,2,4-triazole-3-carbox- amidine hydrochloride. 6
1-(.beta.-D-Ribofuranosyl)- 0.65, 0.8 -- 0.7 0.5 -- 0 0 0.6, 0.5
1,2,4-triazole-3-carbox- amide 3',5'-cyclic phos- phate ammonium
salt. 7 5-IDU 1.4, 1.0, 1.6 1.8 0.8 0.6 1.3 0.2 0.0 0.0 8 Ara-C
1.2, 1.1 0.8 0.6 0.4 0.8 0.2 0.1 0.1 9 Ara-A 0.7, 1.1 0.6 0.8, 0.8
0.6, 1.1 0.7, 0.9 0.0, 0.0 0.1 0.1
__________________________________________________________________________
Compound 1 above has been tested in rabbits and hamsters and at
nontoxic doses displays significant anti-HSV keratitis activity.
The compound significantly inhibits the development of HSV-induced
lesions in mousetails when administered locally to the infection.
It has also been tested in mice against influenza A2, influenza B
and parainfluenza 1, with highly significant antiviral activity
seen in these experiments. This compound, it should be noted from
Table 1 above, displays a spectrum of antiviral activity
substantially broader than that exhibited by IDU, Ara-A or Ara-C.
In addition to the antiviral activity documented above,
1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide has been
found to inhibit growth of the bacteria Pseudomonas aeruginosa and
the fungi Candida albicans and Cryptococcus diffluens.
All of the compounds .[.(1-6).]. .Iadd.(1-3,5,6) .Iaddend.above
exhibited low cytotoxicity and were soluble in aqueous media.
1-(.beta.-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide also
possesses interesting antitumor activity. Three groups of C 57
Blk./6 mice (6 animals per ground) were given subcutaneous implants
of adenocarcinoma-755. Two groups received the compound (100
mg./kg. .times. 7 days and 200 mg./kg. .times. 7 days)
intraperitoneally, whereas the third control group received only
saline. On the 16th day the treated animals showed 18% and 63%
inhibition of the tumor respectively as compared to the controls.
In a similar experiment DBA/2 mice inoculated with L-1210 leukemia
(1 .times. 10.sup.5 cells per animal) showed a 31% increase in
median survival time over controls when treated with the compound
(250 mg./kg. .times. 7 days). When given at a dosage of 250 mg./kg.
.times. 14 days, the compound produced 80% survivors from Swiss
mice bearing intraperitoneal implants of Ehrlich ascites carcinoma.
Marginal in vivo activity against Novikoff hepatoma was also
seen.
We have also discovered that 1,2,4-triazole-3-carboxamide itself
exhibits significant antiviral activity. When tested by the
foregoing procedure, the following VR data was obtained: Herpes
Type 1-0.6, 1.0; Vaccinia--0.8; and Parainfluenza 9.6, 0.6. VR
results when this compound was tested against Adeno and Rhino Type
13 viruses were, respectively, 0.0 and 0.3.
* * * * *