Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini

The invention relates to novel oligonucleotides, the process for their preparation and their biological uses as mediators of the action of interferon. The oligonucleotides according to the invention have the formula: ##STR1## in which Y and T are identical or different and represent particularly O, S, Z and W are identical or different and represent particularly O, S, one at least of the elements Y and Z being different from oxygen, X represents particularly --CHOHCH.sub.2 OH, .SIGMA. is a whole number equal to or greater than 2, A represents adenine or one of its derivatives. These oligonucleotides have antiviral use.

BACKGROUND OF THE INVENTION 
The invention relates to novel oligonucleotides, the process for their 
preparation and their biological use as mediators in the development of 
the action of interferons, particularly in the development of a part at 
least of the antiviral action of interferons. 
It is known that interferons constitute a family of proteins characterised 
particularly by their antiviral properties. 
It has been observed that the antiviral effect of interferons is mediated 
by the synthesis of particular proteins. Specific tests have enabled the 
function of two of them to be identified, which are both enzymes 
(BAGLIONI. C., 1979, Interferon induced enzymatic activities and their 
role in the antiviral state. Cell 17, 255-264). 
One of them is a polymerase oligonucleotide (2-5A synthetase). This 
polymerase oligonucleotide catalyses, after activation by bicatenary RNAs 
and from ATP, the synthesis of a family of oligonucleotides. 
These oligonucleotides are short chains of adenosines connected by 
phosphodiester linkages 2'.fwdarw.5' (KERR I. M. et BROWN R. E., 1978, 
pppA2'-p5'A2'p5'A: An inhibitor of protein synthesis synthesized with an 
enzyme fraction from interferon treated cells. PNAS 75, 256-260) of which 
the general formula may be represented by pppA (2' p5'A).sub.n. These 
oligonucleotides may be denoted by "oligonucleotides 2'-5'" particularly 
"oligoadenylates 2'-5'" or by (2'-5') (A).sub.n. One of these 
oligoadenylates may be represented by the following formula: 
##STR2## 
It is composed of short chains containing several adenosine groups 
(adenine+ribose) joined to one another through phosphodiester linkages, as 
shown, and in which the position at 5' of the adenine nucleus of the 
terminal adenosine is linked to a variable number of phosphate groups (up 
to 3 on the 2'.fwdarw.5' oligoadenylate shown). 
When the oligoadenylate 2'.fwdarw.5' is totally desphosphorylated, that is 
to say when the position at 5' of the adenine nucleus of the terminal 
adenosine is free from the abovesaid variable number of phosphate groups, 
the resulting compound is denoted by "nucleus(2'.fwdarw.5')A3'," which is 
an abbreviation for "riboadenylyl (2'.fwdarw.5') riboadenylyl 
(2'.fwdarw.5') riboadenosine". 
The 2'-5' nuclei corresponding to the dephosphorylated (2'-5') 
oligoadenylates are also called "cores". 
In the rest of the description, the 2'-5' oligoadenylates induced in the 
treated cells by interferon will also be denoted by "unmodified 2'-5' 
oligoadenylate". 
It is accepted that the expression "oligoadenylates 2'.fwdarw.5'" mentioned 
above and used below will denote also, for convenience of language, the 
nucleus (2'-5') (A).sub.n partly or entirely dephosphorylated. 
The discovery of these 2'.fwdarw.5' oligoadenylates has revealed a novel 
class of biologically active oligonucleotides, which are assumed to show 
an important role as mediators of the action of interferon, particularly 
in the activation of L. endoribionuclease, which is present both in the 
cells treated by interferon and in those untreated, and in the inhibition 
of the synthesis of proteins. However the phosphodiester 2'.fwdarw.5' 
linkages of these adenylates are rapidly cleaved by an enzyme denoted by 
2'-phosphodiesterase (cf. the BAGLIONI reference mentioned above). 
L endoribonuclease as well as 2-phosphodiesterase are present at levels 
substantially equal in the treated cells as well as in the cells untreated 
with interferon. 
When the cell is treated with interferon, the concentration of 
oligonucleotide 2'-5' polymerase increases. Infection by certain viruses 
of cells so treated results in the production at the viral replication 
site of NRA bicatenaries activating oligonucleotide 2'-5' polymerase. 
There results an increase, transitory and possibly localised, at the 
replication site of the virus, of the concentration of oligoadenylate 
2'.fwdarw.5' (Nilsen T. W. et Baglioni C., 1984. Interferon 5, J. Gresser 
Ed., Academic Press, New York). These oligonucleotides activate themselves 
by specifically binding therein endoribonuclease L which degrades the 
viral RNA messengers. 
When the interferon is removed from the culture medium, the activity of the 
oligonucleotide 2'-5' polymerase decreases and the cell loses its 
antiviral state. 
The synthesis of the proteins induced by interferon is transient and, 
consequently, the cells kept in the tissue cultures do not maintain a high 
level of these proteins. 
In addition, the 2'.fwdarw.5' oligoadenylates induced in the cells treated 
with interferon exhibit the drawback of having a low metabolic stability. 
In fact, the unmodified 2'.fwdarw.5' oligoadenylates are, on the one hand, 
rapidly hydrolysed by a specific phosphodiesterase degrading the molecule 
progressively from its ribose 2' terminal, on the other hand, are degraded 
under the action of a phosphatase on the side of the first ribose 
connected with the variable number of phosphate groups. (Lebleu B. et 
Content J., 1982, Interferon 3, J. Gresser Ed. Academic Press, New York). 
Researches have been undertaken to find similar compounds to the unmodified 
2'.fwdarw.5' oligoadenylates and having increased activity, in comparison 
with the 2'.fwdarw.5' oligoadenylates induced in cells treated with 
interferon (BAGLIONI C. et coll., 1981, Analogs of (2'-5')oligo(A). 
Endonuclease activation and inhibition of protein synthesis in intact 
cells. The Journal of Biological Chemistry, vol. 256, n.degree. 7, p. 3, 
253-3 257). 
Various researches have been carried out to synthesise (enzymatically 
and/or chemically) modified analogs of 2'.fwdarw.5' oligoadenylates 
induced in cells treated with interferon, which would be resistant to the 
degradation actions, without losing their biological activity. 
Among these researches, it is possible to cite enzymatic synthesis by means 
of 2'.fwdarw.5A synthetase of "cordycepine 2.fwdarw.5A" from 2' 
deoxyadenosine triphosphate (DOETSCH et coll., 1981). 
Cordycepine has been considered as inhibiting the synthesis of the proteins 
in an acellular system and the corresponding dephosphorylated compound 
("core" or "nucleus") has been considered as blocking the blastic 
transformation of human lymphocytes. 
The results have however been disputed (CHAPEKAR M. S. et coll., 1983, 
Biochem. Res. Comm., 115, 137-143) and it seems that the effects observed 
have been caused by the accumulation of toxic degradation products of 
cordycepine. 
It is also possible to cite, among the researches carried out, the chemical 
synthesis of an analog of the nucleus of 2'.fwdarw.5' oligoadenylates, in 
a xylose series (IMBACH J. L. et coll., 1981, Tetrahedron Letters, vol. 
22. n.degree. 47, p. 4 699-4 702), named "xylo 2'-5'A". This analog is 
shown to present greater stability with respect to phosphodiesterases than 
the nucleus of unmodified 2'-5' oligoadenylates, an interesting activity 
with respect to a DNA virus, such as Herpes, but not with respect to RNA 
virus (EPPSTEIN D., et coll., 1983, Nature, 302, 723-724). 
There can also be mentioned the chemical synthesis and the modification at 
its 2' terminal end of a 2'-5' oligoadenylate into a compound called 
"tailed 2'-5' A" in which a hexylamine chain has been associated with a 
morpholine nucleus, itself condensed by a phosphate group onto the OH 
group at the 2' position of the terminal ribose. This derivative is very 
stable with respect to phosphodiesterases and activates L endoribonuclease 
in an acellular system (IMAI J. et coll., 1982. J. Biochem. Chem., 257, 12 
739-12 741), but its antiviral activity has not been established. 
Among these researches, it is possible also to mention the chemical 
synthesis of modified derivatives of 2'-5' oligoadenylates such as the 
derivatives of 2'-5'A triphosphates (represented by the formula 
pppA2'p5'A2'p5A) in which the phosphorus atoms at the beta and gamma 
positions of the triphosphate group at the 5' position are separated by a 
methylene group. 
Another modification to obtain modified 2'-5'A oligoadenylates relates to 
the replacement of a hydroxyl group at the 3' position by an OCH.sub.3 
group either in the terminal adenosine, or in all the adenosines (J. A. J. 
DEN HARTOG et coll., 1981, J. Org. Chem., 46, 2 242-2 251). 
However it is shown that these two latter groups of compounds were weakly 
active, even inactive and did not show satisfactory metabolic stability 
(cf. the reference mentioned above and BAGLIONI et coll., 1981, J. Biol. 
Chem., 256, 2 353-2 357). 
Other analogs, such as 5'S-methylthiophosphorothioates have been 
synthesised. Certain of these analogs are revealed to be stable. But a 
priori, the apparent differences of properties of these analogs does not 
seem to permit their use on human cells for therapeutic purposes to be 
envisaged (HAUGH M. C., CAYLEY P. J. et coll., 1983, Europ. J. Biochem., 
132, 77-84). 
Investigations have also borne on the incidence of the modification of the 
one or more phosphate groups carried by the carbon at the 5' position of 
2'-5' oligoadenylates with respect to antimitogenic activity (cf. TORRENCE 
et coll., 1983, J. Medicinal Chemistry 26, n.degree. 12, 1674-1678). The 
compounds prepared within the scope of these researches are shown to 
present antimitogenic activity, but it has been found that certain of them 
do not activate endoribonuclease L in an in vitro cellular system, which 
prevents the establishment of a correlation between antimitogenic activity 
and antiviral action. 
Other similar oligonucleotides of (2'-5') (A).sub.m have been synthesised 
enzymatically by replacing the adenosine, particularly by 8-azaadenosine, 
toyocamycine, sangivamycine, formycine, 8-bromoadenosine, tubercidine and 
guanosine. It was shown that the majority of these compounds were degraded 
in cellular extracts. Only inhibition tests of the synthesis of proteins 
and of cellular proliferation have been carried out in intact cells, but 
the antiviral activity has not been established (B. G. HUGHES and R. K. 
ROBINS, 1983, Biochemistry, 22, n.degree.9, 2 127-2 135). None of the 
analogs of (2'-5')(A) synthesized hitherto have shown stability properties 
-both with respect to phosphodiesterases and phosphases-, and sufficient 
biological activity to be able to envisage using them in the therapeutic 
treatment of viral infections. 
GENERAL DESCRIPTION OF THE INVENTION 
Applicants have discovered new oligonucleotides having a structure 
different from that of unmodified 2'-5'A oligoadenylates and its known 
analogs, having an antiviral activity similar to that of interferon, which 
are resistant with respect to degradation by 2'-phosphodiesterase and by 
phosphatases permitting their use in the treatment of viral infections to 
be contemplated, in so far as they are associated with appropriate 
carriers enabling them to cross the cell membrans. 
One of the aspects of the invention is to propose new oligonucleotides 
which can be recognized by L endoribonuclease, that is to say which can 
form with L endoribonuclease an active complex. 
Another aspect of the invention is to provide new oligonucleotides which 
have an increased resistance with respect to degradation by 
2'-phosphodiesterase. 
Another aspect of the invention is to provide new oligonucleotides which 
have an increased resistance relative to phosphatases. 
Another aspect of the invention is to provide new biologically active 
oligonucleotides, which show particularly an effective antiviral activity, 
in so far as they are associated with appropriate carriers enabling them 
to cross the cell membrans. 
Another aspect of the invention is to provide new oligonucleotides liable 
to be used in the preparation of biologically active compounds, which 
present particularly an efficient antiviral activity. 
These various aspects are achieved by novel oligonucleotides comprising a 
chain containing n nucleoside units, identical or different, n being equal 
to or higher than 2, these nucleoside units being joined by 2'-5' 
linkages, which comprise a group of linkages containing at least one 
phosphorus atom and in which: 
The nucleosidic "first unit" of the above-said chain is linked through its 
carbon at the 5' position to a variable number of phosphate groups, and 
one of the oxygen atoms of at least one of the phosphate groups, which 
oxygen atom joined only to the phosphorus of the phosphate groups and not 
taking part in the linkage between two phosphate groups, is replaced by an 
atom of sulfur, of selenium or an NH group, and/or 
one at least of the linkages between two adjacent phosphate groups 
comprises an NH group or a sulfur atom; and/or 
the nucleosidic "last unit" of the above-said chain is linked, through its 
carbon atom at the 2' position: 
either to a phosphoglyceryl group, 
or to a phosphate group, which is joined to the carbon at the 5' position 
of a "modified nucleoside group", in which the direct bond between the 
carbon at the 2' and 3' positions has been eliminated and the carbons at 
the 2' and 3' positions are respectively bearers of aldehyde groups or of 
alcohol groups, possibly esterified. 
These various aspects of the invention are preferably achieved through 
novel oligonucleotides comprising a chain containing n nucleoside units, 
identical or different, n being greater than or equal to 2, these 
nucleoside units being connected by 2'.fwdarw.5' linkages, which comprise 
a group of linkages containing at least one phosphorus atom, and in which: 
the first nucleoside unit of the above-said chain is linked, through its 
carbon at the 5' position, to phosphate groups and one of the oxygen atoms 
of at least one of the phosphate groups, which oxygen atom connected only 
to the phosporus of the phosphate groups and not taking part in the 
linkage between two phosphate groups, is replaced by a sulfur atom, an 
atom of selenium or an NH group, and/or one at least of the linkages 
between two adjacent phosphate groups comprises an NH group or a sulfur 
atom; 
and possibly the "last nucleoside unit" of the abovesaid chain is joined, 
through its carbon atom at the 2' position: 
either to a phosphoglyceryl group; 
or to a phosphate group, which phosphate group is connected through the 
carbon at the 5' position of a "modified nucleoside group", in which the 
direct linkage between the carbon at the 2' and 3' positions has been 
eliminated, and the carbon atoms at the 2' and 3' positions are bearers of 
aldehyde groups, or of alcohol groups, possibly esterified. 
A nucleoside unit denotes a compound constituted by a pentose linked to a 
purine or pyrimidine base, in which the pentose can be in the pyran or 
furan form. 
In the rest of the description, the formulae will represent the pentoses 
generally in furan form. 
The nucleoside units are according to the invention advantageously 
constituted by adenosines, adenosine denotes the compound constituted by 
ribose linked to adenine and may be represented by the formula: 
##STR3## 
in which the ribose is in furan form, but may also be in pyran form and in 
which A represents adenine. 
Within the scope of the invention, adenine denotes the molecule represented 
by the following formula: 
##STR4## 
The nucleoside units according to the invention may also be constituted by 
adenosine derivatives, adenosine derivatives denoting the compound 
constituted by ribose, joined to an adenine derivative. Among these 
derivatives of adenine, may be mentioned those of the following formula: 
##STR5## 
The corresponding adenosine derivatives will be respectively denoted by 
8-azaadenosine, sagivamycin toyocamycin, formycin, tubercidine, 
8-bromo-adenosine. 
The number of nucleoside units constituting the oligonucleotides of the 
invention is not limited within the above values, provided that the 
oligonucleotides obtained can be associated with a physiologically 
acceptable vehicle. 
This number can rapidly be limited to the extent that the increase in this 
number and the corresponding more difficult synthesis would not be 
supported by a sufficient increase in activity. 
The number of nucleoside units should be selected so that the molecular 
weight is preferably comprised between 1 500 to 5 000 daltons. 
In a preferred class of oligonucleotides according to the invention, the 
value of n is not higher than 10, and is preferably 7 or 8. 
The oligonucleotides in which the value of n is 3 or 4 are particularly 
preferred. 
In a preferred class of oligonucleotides according to the invention, the 
first nucleoside unit is linked to one or several phosphate groups. 
Preferably, the number of these phosphate groups is 1 to 3. 
In a preferred class of compounds of the invention, the first nucleoside 
unit is linked to the following phosphate groups: 
##STR6## 
in which R', R", R"' represent, independantly of one another: 
a hydrogen atom, 
an alkyl radical having from 1 to 4 carbon atoms, in particular methyl, 
an ethyl radical substituted at the beta position by a cyano, aryl or 
arylsulfonyl group, 
a trihalogenoethyl radical. 
In a preferred class of compounds according to the invention, the linkage 
2'.fwdarw.5' joining two nucleoside units and comprising at least one 
phosphorus atom is a phosphodiester linkage, a phosphotriester linkage, or 
an alkylphosphonate linkage. 
The linkage 2'.fwdarw.5' phosphodiester which joins two adjacent nucleoside 
units in the oligonucleotides according to the invention may be 
represented as follows: 
##STR7## 
The phosphotriester 2'.fwdarw.5' linkage which joins two adjacent 
nucleoside units in the oligonucleotides of the invention may be 
represented as follows: 
##STR8## 
in which R.sub.1 represents an alkyl radical having from 1 to 4 carbon 
atoms; 
an alkyl radical having from 1 to 4 carbon atoms, in particular methyl, 
an ethyl radical substituted at the beta position by a cyano, aryl or 
arylsulfonyl group, 
a trihalogenoethyl radical. 
The phosphonate 2'.fwdarw.5' linkage which joins two adjacent nucleoside 
units in the oligonucleotides according to the invention may be 
represented as follows: 
##STR9## 
in which R.sub.2 can represent an alkyl having from 1 to 4 carbons, in 
particular methyl. 
By convention, in an oligonucleotide according to the invention containing 
n nucleoside units, below, the nucleoside unit of rank n, when the last 
element of the chain is a phosphoglyceryl group, will be denoted by the 
expression "last nucleoside unit". 
In this case, the oligonucleotide according to the invention will be 
denoted by (2'-5') (A).sub.n PGro. 
When the last element of the oligonucleotide according to the invention is: 
either a nucleoside group of the formula: 
##STR10## 
A having the above-indicated meanings; 
or a "modified nucleoside group" as defined below; the definitions 
nucleoside unit of rank n-1 will be denoted by the expression "last 
nucleoside unit" by convention. 
By "modified nucleoside group" is defined a nucleoside group in which: 
the direct linkage between the carbon 2' and the carbon 3' joining the 
linkage directly has been eliminated and may be represented by the 
following formula: 
##STR11## 
in which the pentose is in furan form, but may also be in pyran form and 
in which A represents adenineor a derivative of adenire as defined above; 
the carbons at the 2' and 3' positions are bearers of aldehyde functions or 
of alcohol functions, optionally esterified; in the case where the carbons 
at the 2' and 3' positions bear alcohol functions, the oligonucleotides 
according to the invention will be denotable by (2'-5') (A).sub.n Ox Red. 
The oligonucleotides according to the invention may be represented by the 
following formula (I): 
##STR12## 
in which: 
Y and T, identical or different, represent independantly of one another, O, 
S, Se or NH; 
Z and W, identical or different, represent independantly of one another, O, 
S or NH; 
X represents: 
##STR13## 
A represents adenine or one of its derivatives as defined above; 
.SIGMA. is a whole number equal to: 
n when X represents -CHOHCH.sub.2 OH; 
n-1 when X is different from -CHOHCH.sub.2 OH 
n being a whole number greater than or equal to 2; 
m is a whole number equal to 0 and preferably greater than or equal to 1; 
provided that: 
either one at least of the two elements Y or Z is different from oxygen; 
or X represents: 
##STR14## 
or one at least of the elements Y or Z is different from oxygen and X 
represents: 
##STR15## 
A preferred class of oligonucleotides according to the invention is 
constituted by those corresponding to the following formula (I): 
##STR16## 
in which: 
Y and T are identical or different and represent O, S, Se, NH; 
Z and W are identical or different and represent O, S, NH; 
one at least of the elements Y and Z being different from oxygen; 
X is selected from the group constituted by: 
##STR17## 
the alcohol functions of these radicals being possibly esterified by 
R.sub.3 COOH carboxylic acids, R.sub.3 representing an alkyl radical of 1 
to 5 carbon atoms, or a phenyl radical: 
.SIGMA. is a whole number equal to: 
n when X represents --CHOHCH.sub.2 OH, 
n-1 when X represents: 
##STR18## 
n being a whole number greater than or equal to 2; 
m is a whole number greater than or equal to 1; 
A is a base selected from among adenine and its derivatives, particularly 
those of the formula: 
##STR19## 
In a preferred clss of oligonucleotides according to the invention, the 
number m varies preferably from 1 to 3. 
Among the groups connected to the first nucleoside unit and of formula: 
##STR20## 
one at least is such that Y represents Se, S or NH and/or Z represents S 
or NH, and the other groups: 
##STR21## 
representing phosphate groups. 
In a preferred class of oligonucleotides according to the invention, X 
represents S, and Z represents O. 
A preferred class of oligonucleotides according to the invention is that in 
which X is either a modified nucleoside group which can be represented by: 
##STR22## 
or the group --CHOHCH.sub.2 OH. 
The alcohol functions of the radicals: 
##STR23## 
are possibly esterified by an R.sub.3 COOH carboxylic acid, in which 
R.sub.3 represents an alkyl radical of 1 to 5 carbon atoms or a phenyl 
radical. 
A preferred class of oligonucleotides according to the invention is 
constituted by those of the following formula (II): 
##STR24## 
in which: 
Y.sub.1, Y.sub.2, Y.sub.3, T are identical or different and represent O, S, 
Se, NH; 
Z.sub.1 and Z.sub.2 are identical or different and represent O, S, NH; one 
at least of the elements Y.sub.1, Y.sub.2, Y.sub.3, Z.sub.1, Z.sub.2 being 
different from oxygen; 
X is selected from the group constituted by: 
##STR25## 
the alcohol functions of these radicals being possibly esterified by 
R.sub.3 COOH, carboxylic acids, R.sub.3 representing in alkyl radical of 1 
to 5 carbon atoms or a phenyl radical; 
represents a whole number equal to n, when X represents --CHOHCH.sub.2 OH, 
and .SIGMA. represents a whole number equal to n-1 when X is a different 
from --CHOHCH.sub.2 OH, n being a whole number greater than or equal to 2; 
--A is a base selected from among adenine and its derivatives, particularly 
those of the formula: 
##STR26## 
The phosphorus of the group: 
##STR27## 
will be called alpha phosphorus. 
The phosphorus of the group: 
##STR28## 
will be called beta phosphorus. 
The phosphorus of the group: 
##STR29## 
will be called gamma phosphorus. 
Within the class of oligonucleotides of formula (II), a preferred class of 
oligonucleotides according to the invention is constituted by the 
oligonucleotides of formula (III): 
##STR30## 
in which: 
Y.sub.1 represents S, Se, or NH; 
Z.sub.1 represents O, NH or S; 
.SIGMA., X and A having the above-indicated meanings. 
Within this class of oligonucleotides, a preferred class of 
oligonucleotides is constituted by those in which: 
X represents: either: 
##STR31## 
A representing adenine; or: 
--CHOHCH.sub.2 OH. 
Another preferred class of oligonucleotides according to the invention is 
constituted by those of the following formula (IV): 
##STR32## 
in which: 
Y.sub.3 represents S, Se or NH, 
.SIGMA., X and A having the above-indicated meanings. 
Within this oligonucleotide class, a preferred class of oligonucleotides 
according to the invention is constituted by those in which: 
X represents: either: 
##STR33## 
A representing adenine; or: 
--CHOHCH.sub.2 OH. 
Another class of preferred oligonucleotides according to the invention is 
constituted by that of the following formula (V): 
##STR34## 
in which: 
Y.sub.1 represents Se, S or NH; 
.SIGMA., X and A having the above-indicated meanings. 
Within this class, a preferred class of oligonucleotides according to the 
invention is constituted by those in which: 
X represents: either: 
##STR35## 
A representing adenine; or: 
--CHOHCH.sub.2 OH. 
Another preferred class of oligonucleotides according to the invention is 
constituted by those of the following formula (VI): 
##STR36## 
in which: 
Z.sub.1 represents S or NH; 
.SIGMA., X and A having the above-indicated meanings. 
Within this class, a preferred class of oligonucleotides is constituted by 
those in which: 
X represents: either: 
##STR37## 
A representing adenine; or: 
--CHOHCH.sub.2 OH. 
Another preferred class of oligonucleotides according to the invention is 
constituted by those of the following formula (VII): 
##STR38## 
in which: 
Y.sub.2, Y.sub.3, T are identical or different and represent O, S, Se, NH; 
Z.sub.2 represents O, S, NH; one at least of the elements Y.sub.2, Y.sub.3, 
Z.sub.2 being different from oxygen; 
X is selected from a group constituted by: 
##STR39## 
.SIGMA., is a whole number equal to n, when X represents --CHOHCH.sub.2 OH 
and is a whole number equal to n-1, when X is different from 
--CHOHCH.sub.2 OH, n being a whole number varying from 2 to 10; 
A is a base selected from among adenine and its derivatives, particularly 
those of the formula: 
##STR40## 
Within this class of nucleotides a preferred class is constituted by those 
in which T represents oxygen. 
Another preferred class of the oligonucleotides according to the invention 
is constituted by those of the following formula (VIII): 
##STR41## 
in which: 
Y.sub.3 represents S, Se, NH; 
T represents O, S, Se, NH; 
X is selected from the group constituted by: 
##STR42## 
.SIGMA. represents a whole number equal to n, when X represents 
--CHOHCH.sub.2 OH and a whole number equal to n-1 when X is different from 
--CHOHCH.sub.2 OH, n being a whole number varying from 2 to 10; 
A is a base selected from among adenine and its derivatives, particularly 
those of the formula: 
##STR43## 
Within this class of nucleotides a preferred class is constituted by those 
in which T represents oxygen. 
Particularly preferred oligonucleotides according to the invention have the 
formula: 
##STR44## 
The invention also relates to the salts which can be obtained by reaction 
of the abovesaid oligonucleotides with suitable bases, in particular the 
quaternary ammonium salts, such as the triethylammonium salt, inorganic 
salts, such as the sodium salt. 
The invention also relates to a process for the preparation of the 
oligonucleotides. 
To prepare the oligonucleotides according to the invention, either a full 
chemical synthesis, or an enzymatic synthesis followed by chemical 
modifications may be resorted to. 
As regards the chemical synthesis, reference may be made to the procedure 
described in Methods of Enzymology, 79, 1981, 233-234. 
As regards the enzymatic synthesis of the oligonucleotides of formula (I) 
according to the invention, it comprises: 
the polymerisation of compounds of following formula (XIX): 
##STR45## 
in which: 
Y represents O, S, Se or NH; 
Z represents O, S, or NH; 
one at least of the elements Y and Z being preferably different from 
oxygen; 
m is greater than or equal to 3; 
A has the above-indicated meanings; 
to obtain a compound of the following formula (Ibis): 
##STR46## 
in which: 
Y and T, identical or different, represent O, S, Se, NH; 
Z and W, identical or different, represent O, S, NH; 
one at least of the elements Y and Z being preferably different from 
oxygen; 
.SIGMA. is a whole number equal to n-1, in being greater than or equal to 
2; 
m is a whole number greater than or equal to 1; 
A has the above-indicated meanings; 
and if necessary the following chemical steps namely: 
possible oxidation of the glycol group to introduce aldehyde functions on 
the carbons at the 2' and 3' positions of the last nucleoside unit and to 
obtain the compound of formula (Iter): 
##STR47## 
The possible reduction of the two aldehyde functions into alcohol functions 
to obtain the compound of formula (Iquater): 
##STR48## 
The possible hydrolysis, under conditions avoiding beta-elimination, to 
obtain the compound of the following formula (Iquinquies): 
##STR49## 
A preferred method of producing oligonucleotides according to the invention 
of the following formula (XX): 
##STR50## 
in which: 
Y and T, identical or different, represent O, S, Se, NH; 
Z and W, identical or different, represent O, S, NH; 
Y and Z do not simultaneously represent oxygen; .SIGMA. is a whole number 
varying from 1 to 9; 
A is a base selected from among adenine or its derivatives, particularly 
those of formula: 
##STR51## 
comprises: 
1. the polymerisation of a compound of the following formula (XIXbis): 
##STR52## 
in which: Y represents O, S, or NH; 
Z represents O, S, or NH; 
Y and Z do not simultaneously represent oxygen; 
A has the above-indicated meanings; 
2. the possible oxidation of the terminal glycol group, particularly by the 
periodate ion to convert the glycol into two aldehyde functions and to 
obtain a compound of the following formula (XXI): 
##STR53## 
in which Y, Z, T, W, .pi. and A have the above-indicated meanings; 
3. the possible reduction of the aldehyde functions, particularly by sodium 
borohydride to convert the two abovesaid aldehyde functions into alcohol 
functions and to obtain the compound of the following formula (XXII): 
##STR54## 
4 the possible hydrolysis, particularly controlled acid hydrolysis, to 
remove the ribose nucleus and to obtain the compound of the following 
formula (XXIII): 
##STR55## 
Among the compounds of the formula (XIX) are available in commerce, those 
of the following formula (XIXbis): 
##STR56## 
in which Y represents sulfur and Z represents oxygen, as well as those in 
which Y represents oxygen and Z represents the NH group, A representing 
adenine or one of its derivatives as define above. 
The oxidation of the glycol group of the last nucleoside unit to eliminate 
the direct linkage between the carbon at the 2' position and the carbon at 
the 3' position and to introduce two aldehyde functions can be carried out 
by periodic acid under rigorously controlled pH conditions to avoid beta 
elimination. 
The expression "rigorously controlled pH conditions" means the maintenance 
of the reaction medium at pH 4.0, at 0.degree.-4.degree. C. and in 
darkness. 
The reduction of the aldehyde functions into alcohol functions can be 
effected by sodium borohydride. The hydrolysis to convert: 
##STR57## 
into a --CHOHCH.sub.2 OH group is preferably a controlled acid hydrolysis, 
carried out according to conventional methods. 
A process for the obtaining according to the invention of the 
oligonucleotides of the following formula (V): 
##STR58## 
in which: 
Y.sub.1 represents NH, Se, S; 
X represents: 
##STR59## 
.SIGMA. is a whole number equal to n, when X represents --CHOHCH.sub.2 OH, 
n-1 when X is different from --CHOHCH.sub.2 OH, n varying from 2 to 10; 
is characterised in that the compounds of the following formula (XXIV): 
##STR60## 
is polymerised and the steps 2.degree., 3.degree. and 4.degree. are 
carried out as indicated above. 
Within the class of process which have just been defined, a process 
according to the invention for producing oligonucleotides of the following 
formula (Vbis): 
##STR61## 
in which: 
.SIGMA., X have the previously indicated meanings; 
A has the previously indicated meaning, and preferably represents adenine; 
is characaterized in that a compound of the following formula (XXIVbis): 
##STR62## 
is polymerised and the steps 2, 3 and 4 are carried out as indicated 
above. 
A process according to the invention for producing oligonucleotides of the 
following formula (VI): 
##STR63## 
in which: 
Z.sub.1 represents NH or S; 
X is selected from the group constituted by: 
##STR64## 
.SIGMA. is a whole number equal to 1, when X represents --CHOHCH.sub.2 OH 
and equal to n-1 when X is different from CHOHCH.sub.2 OH, n varying from 
2 to 10: is characterised in that the compound of the following formula 
(XXV): 
##STR65## 
in which Z.sub.1 represents S or NH and A has the above-indicated meaning, 
is polymerised: and the steps 2, 3 and 4 are carried out as indicated 
above. 
A process for obtaining oligonucleotides according to the invention of the 
following formula (IV): 
##STR66## 
in which: 
Y.sub.3 represents NH, S or Se; 
.SIGMA., X and A have the above-indicated meanings; 
is characterised in that the compound of the following formula (XXVI): 
##STR67## 
in which: 
Y.sub.3 represents S, Se, NH; 
A has the above-indicated meanings, is polymerised; and the steps 2, 3 and 
4 are carried out as indicated above.

EXAMPLE 1 
This example relates to the preparation of oligonucleotides 
(2'-5')(A).sub.n synthesised enzymatically and modified chemically. These 
compounds may be represented by the following formulae: 
##STR68## 
and their corresponding dephoshporylated derivatives. 
MATERIALS AND METHODS 
Materials 
The media come from Eurobio (Paris) and the serums from Flow Laboratories. 
For example interferon of human leucocytes (Hu IFN .alpha.) purified to a 
specific activity of 2.times.10.sup.6 IU/mg of proteins, is used. 
The polyacid ribocytidylic riboinosinic-polyacid [denoted below by the 
abbreviation poly (rI). poly(rC)] is obtained, for example, from PL 
Biochemicals. The type III-R bacterial alkaline phosphatase comes from 
sigma and is preserved at 4.degree. C. 
The [.gamma..sup.32 P] ATP (specific activity 2 000 Ci/mM) and the 
(2'-5')(A)-pCp[.sup.32 p] (specific activity 3 000 Ci/mM) come from 
Amersham. 
The sodium boro [.sup.3 H] hydride (specific activity 30 Ci/mol) is 
supplied by the Commissariat a l'Energie Atomique. 
The diethylaminoethyl-trisacryl (denoted below by DEAE trisacryl) was 
obtained from l'Industrie Biologique Francaise. 
Cells and virusas 
HeLa cells are kept in monolayers in a medium marketed under the name RPMI 
1640, particularly by Laboratoires Eurobio, Paris, completed by 10% (v/v) 
foetal calf serum, 50 IU/ml of penicillin and 50 .mu.g/ml of streptomycin. 
L929 cells were grown in a minimum essential medium supplemented with 5% 
(v/v) of donor horse serum, 3 g/l of bactotryptose phosphate broth, 3.4 
g/l of glucose and antibiotics as mentioned above. The Indiana strain of 
the vesicular stomatitis virus (VSV) was used and allowed to grow in L929 
cells. 
Ensymatic synthesis of (2'-5')(A).sub.n of oligoadenylates 
(2'-5')(A).sub.n oligoadenylates were synthesised enzymatically by the 
method described by MINKS et coll. (J. Biol. Chem., 1979, 254, 5 058-5 
064). 
The preparation of the (2'-5')(A).sub.n compounds may be summarised as 
follows. 
Cytoplasmic extracts were prepared from HeLa cells treated with 200 units 
per ml of interferon of human leucocytes for 48 hours. The extract was 
incubated with 5 mM of ATP and 20 ug/ml of poly(rI).poly(rC) for 2 hours, 
brought to boiling for 3 min at 100.degree. C. and centrifuged at 10 000 g 
for 10 min. [.gamma..sup.32 p] (2'-5')(A).sub.n oligomers were synthesised 
by incubating cellular extracts with [.gamma.-.sup.32 p] ATP under the 
same conditions. 
Fractionation of the (2'-5')(A).sub.n oligoadenylates 
Approximately 4 000 units of optical density at 260 nm of (2'-5')(A).sub.n 
were synthesised, that is to say 100 .mu.moles, n ranging from 2 to 15 
(for subsequent fractionation) in extracts of HeLa cells treated with 
interferon at 37.degree. C. for 2 hours as described above. The proteins 
were precipitated by incubation of the mixture at 100.degree. C. for 5 min 
and centrifugation at 15 000 x g for 10 min. The supernatant liquor was 
diluted 3 times with water and adjusted to pH 805 with 0.1 M KOH before 
being charged onto a column (2.5.times.64 cm) of trisacryl M DEAE, 
equilibrated with a buffer at pH 8.5 of 0.25 M triethylammonium 
bicarbonate. The column was washed with 1 500 ml of this buffer and the 
(2'-5')(A).sub.n loigoadenylates were eluted with a linear gradient (1 500 
ml/1 500 ml) at pH 8.5 of 0.125-0.45 M triethylammonium bicarbonate. The 
oligomers at the individual peaks were identified by high performance 
liquid chromatography (HPLC). The fractions were concentration under 
vacumm under reduced pressure and co-evaporated with water several times, 
in order to remove the triethylammonium bicarbonate buffer. Amounts in mg 
of each of the oligomers can be obtained in purified form and checked by 
HPLC. 
Synthesis and purification of the nuclei or "cores" of (2'-5')(A).sub.n 
The dephosphorylated oligoadenylates which have also been denoted by 
"cores" or nuclei (400 units of A.sub.260) obtained by enzymatic digestion 
of (2'-5')(A).sub.n of alkaline phosphatase were fractionated by ion 
exchange chromatography on a column (1.5.times.25 cm) of DEAD-trisacryl M. 
Each dephosphorylated oligoadenylate was obtained in pure form by elution 
of the column with a linear gradient (300 ml/300 ml) of buffer at pH 8.5 
of triethylammonium bicarbonate (0-100 mM). 
Chemical modification of oligoadenylates (2'-5') (A).sub.n 
The oxidation was carried out, by means of periodate, of the 
(2'-5')(A).sub.n for 15 hours under controlled conditions, in order to 
avoid beta-elimination. Conventionally, 100 .mu.l of sodium metaperidate 
16 mM in an 0.2 M sodium acetate buffer at pH 4.0 was added to 100 .mu.l 
of (2'-5')(A).sub.n 1 mM in distilled water at 4.degree. C. The mixture 
was shaken at 4.degree. C. in darkness for 15 hours. The excess periodate 
was destroyed immediately after the oxidation phase with 10 .mu.l of 
ethylene glycol and the dialdehyde derivative at the 2', 3' position, 
according to the invention, corresponding to the (2'-5')(A).sub.n compound 
was reduced at 4.degree. C. for 5 hours by 100 .mu.l of 0.1 M sodium 
borohydride in an 0.1 M borate buffer at pH 9.0. In certain cases, the 
dialdehyde at the 2', 3' position is reduced with sodium boro[.sup.3 H] 
hydride. The mixture was then acidified with 0.1 M acetic acid and it was 
desalted on a Sephadex G-15 column. The O-phosphoglycerylated derivative 
according to the invention of the (2'-5')(A).sub.n compound was obtained 
by controlled acid hydrolysis of the riboseoxygen linkage with 0.005 M 
sulfuric acid at 80.degree. C. for 30 minutes. The 2'-5' oligoadenylates 
according to the invention which include a terminal O-phosphoglycerylated 
group will be denoted below by (2'-5')(A).sub.n PGro. 
The diagram below summarsies, by way of example, the principal chemical 
modifications effected from the unmodified (2'-5')(A).sub.4 
oligoadenylates to obtain the (2'-5')(A).sub.4 oligoadenylates according 
to the invention. 
##STR69## 
The oxidation, for example by periodate ion, of the alpha-glycol group of 
the (2'-5')(A).sub.n molecule introduces two aldehyde functions at the 2' 
and 3' positions, which results in compound (IX). 
##STR70## 
The two aldehyde functions were reduced, for example, with sodium 
borohydride into two alcohol functions, which leads to the compound (X). 
##STR71## 
The controlled acid hydrolysis with, for example, dilute sulfuric acid 
gives a (2'-5') oligoadenylate including a 2'-phosphoglycerated terminal 
group. The dephosphorylated nuclei corresponding to the compounds of 
formulae (IX), (X), (XI) are obtained by treatment of the latter, with 
bacterial alkaline phosphatase. 
The compounds of the following formulae were thus respectively obtained: 
##STR72## 
Analysis by high performance liquid chromatography of phosphorylated and 
unphosphorylated (2'-5')(A).sub.n s and of their O-phosphoglycerylated 
derivatives 
The (2'-5')(A).sub.n oligoadenylates, the (2'-5')(A).sub.n -PGro 
(2'-O-phosphoglycerylated derivative of (2'-5')(A).sub.n) and the 
corresponding dephosphorylated oligoadenylates (nuclei) were isolated and 
characterised on a column marketed under the name .mu.Bondapak C in an 
aluminium phosphate buffer (Brown R. E. et coll., 1981, Methods Enzymol. 
78B, 208-216 et Knight M. et coll., 1980, Nature, 288, 189-192). The 
column was equilibrated with a 50 mM ammonium phosphate buffer at pH 7.0 
for the separation of the phosphorylated (2'-5')(A).sub.n or with a 4mM 
ammonium phosphate buffer at pH 6.5 for the separation of the 
dephosphorylated nuclei and it was eluted for 25 minutes with 25 ml of an 
0-50% linear gradient of methanol/water (1:1 v/v). All the separations 
were carried out with an HPLC chromatograph marketed under the name Varian 
5 000. 
Analysis by high voltage electrophoresis of (2' -5')(A).sub.n 
oligoadenylates and their degradation products 
The individual (2'-5')(A).sub.n oligoadenylates and their degradation 
products such as inorganic phosphates (Pi), ATP and AMP were separated by 
electrophoresis on paper marketed under the name Whatman DE81 in 8.7% 
(v/v) formic acid at pH 1.8 for 0.5 to 6 hours at 60 V/cm in a high 
voltage electrophoresis apparatus marketed under the name Gilson. The 
locations of the radioactive components were marked and they were 
quantified by autoradiography with a film marketed under the name Kodak 
X-Omat AR. 
Activity of the phosphodiesterase in the HeLa cell extracts 
The stability of the (2'-5')(A).sub.n s and of the analogous nuclei 
according to the invention was determined in a HeLa cell extracts by 
measuring the disappearance of the oligonucleotides. The (2'-5')(A).sub.5 
(5 .mu.l) was incubated to the final concentration of 0.02 mM with 5 .mu.l 
of HeLa cellular extracts (22 mg of proteins per ml) in the presence of 
4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (Hepes) 20 mM at pH 
7.6 of magnesium acetate 2.5 mM, of 33 mM ammonium chloride, of 
dithiothreitol and 1 mM phenylmethylsulfonyl fluoride (buffer 1). The 
reaction was stopped by heating to 100.degree. C. for 2 minutes and it was 
centrifuged at 10 000 Xg for 10 minutes. The (2'-5')(A).sub.3 (1 nM) was 
added to with the supernantant liquor as internal reference and the 
residual products were quantified by HPLC as described above. 
Activity of phosphates in HeLa cell extracts 
The (2'-5')(A).sub.n oligoadenylates and their analogs according to the 
invention were incubated in HeLA (5 .mu.l) cellular extracts for different 
periods of time in 20 .mu.l tests, in the above-defined buffer 1 (cf. test 
with phosphodiesterase) or in the same buffer completed with ATP 1 mM, GTP 
(guanosine triphosphate) 0.1 mM, CTP (cytosine triphosphate) 0.6 mM, 
creatine phosphate 100 mM, creatine phosphokinase in the proportion of 160 
.mu.g/ml and all 20 aminoacids in the proportion of 500 uM each (buffer 
2). The reaction was stopped by heating at 100.degree. C. for 20 minutes, 
the proteins precipitated were removed by centrifugation at 10 000 Xg for 
10 minutes. The activity of the phosphatase was determined by measuring 
the disappearance of[.gamma.-.sup.32 p](2'-5')(A).sub.n and the 
concomitant appearance of .sup.32 Pi, released after high voltage 
electrophoresis or by measuring the accumulation of oligoadenylate nuclei, 
by high performance liquid chromatography. 
Cellular Micro-injection 
HeLa cells were cultivated on small fragments of glass (2 mm2) at densities 
which permitted about 200 cells to be attached to each of the glass 
fragments as described in Huez G. et coll., 1981, Proc. Natl Acad. Sci., 
78, 908-911. Micro-injections were carried out according to the method 
originally described by Graessmann (1983, Methods Enzymol., 101, 482-492). 
An average volume of 0.5 nl (approximately 1/10th of the cellular volume) 
was injected into the cytoplasm of each of the cells with glass 
micro-pipettes of 0.5-1 .mu.m diameter. The injections were checked under 
a phase contrast microscope marketed by Leitz-Diavert with a magnification 
of 320. 
Test of Antiviral Activity 
The cells were infected at times indicated, generally one hour after the 
micro-injection, with a vesicular stomatitus virus (VSV) at a multiplicity 
of 10 for one hour at 37.degree. C. in an RPMI 1640 medium supplemented 
with foetal calf serum 5% (v/v). The unadsorbed viruses were carefully 
removed by three washings with RPMI containing 10% foetal calf serum 
(v/v). 
The titer of virus produced 18 hours later was determined by known methods 
(Stewart W. E. ., 1970, J. Virol., 6, 795-799). To summarise 10.sup.6 L929 
cells were placed in Petri dishes for tissue culture (2 cm diameter). 24 
hours after incubation, 0.05 ml of the dilute virus suspensions (dilution 
factor 50) were carefully spread on the monolayer of cells. One hour 
later, the virus suspension was removed by suction and 2 ml of molten 
agarose (1.6% v/v) was spread in a minimum essential medium completed with 
2% (v/v) foetal calf serum on the monolayer of cells. The plates were 
incubated for 18 hours in an incubator with CO.sub.2. The plates were then 
developed by a 1% (v/v) solution of neutral red in an isotonic buffered 
phosphate saline solution. 
RESULTS 
Synthesis and Chemical Modification of (2'-5')(A).sub.n 
mg amounts of (2'-5')(A).sub.n were synthesised enzymatically in HeLa cell 
extracts treated with interferon and fractionated in one step by ion 
exchange chromatography on trisacryl DEAE as previously described. 
Chemical modifications were then made as indicated above, to obtain the 
modified (2'-5')(A).sub.n s according to the invention. 
Stability of (2'-5')(A).sub.n and of its Analogs 
In order to test the stability of the compounds according to the invention 
with respect to 2-phosphodiesterase the (2'-5')(A).sub.n nuclei and their 
O-phosphoglyceryl derivatives were incubated for 8 hours in extracts 
prepared either from untreated HeLa cells, or from cells treated with 
interferon, and their disappearance was followed by high performance 
liquid chromatography. For the HeLa cellular extracts treated with 
interferon and according to the results published (Minks M. A., 1979, J. 
Biol. Chem., 254, 5 058-064; Williams B. R. G. et coll., 1978, Eur. J. 
Biochem., 92, 455-462; Schmidt A et coll., 1979, Proc. Natl Acad. Sci. 
U.S.A., 76, 4 788-4 792; Verhaegen-Lewalle M. 1982, Eur, J. Biochem., 126, 
639-643), the unmodified nuclei were rapidly degraded. On the contrary, 
the (2'-5')(A).sub.n -PGro nucleus or the nucleus of the (2'-5')(A).sub.n 
derivative bearing two alcohol functions according to the invention were 
stable under the same conditions. Similar results were obtained in 
extracts prepared from cells untreated with interferon. 
FIG. 1 relates to the stability of the unmodified (2'-5')(A)5 nucleus 
compared with that of the (2'-5')(A).sub.4 PGro. nucleus. 
In abscissae, is shown time, expressed in hours, and in ordinates the 
percentage of nuclei degraded. 
The dephosphorylated nuclei of (2'-5')(A).sub.n (represented by triangles 
in FIG. 1) and their corresponding 2'-O-phosphoglyceryl derivatives 
(2'-5')(A) PGro nuclei) (represented by circles in FIG. 1) were incubated 
with HeLa cellular extracts completed (solid line) or not (dashed line) by 
1 mM ATP and an ATP regenerating system. 
The nuclei were introduced at an initial concentration of 0.02 mM. The 
incubations were stopped at the times indicated by boiling and the 
denatured proteins were removed by centrifugation. The residual nuclei of 
the supernatant liquor were analysed by HPLC as indicated above. 
Antiviral Activity of (2'-5')(A).sub.n Oligoadenylates and of Their 
Phosphoglycerylated Derivatives 
To test the biological activity of charged compounds such as 
(2'-5')(A).sub.n and their analogs according to the invention in intact 
cells, recourse was had to micro-injection with micropipettes, considering 
that it permits the introduction of predetermined amounts of compounds 
into the cytoplasm, and without disturbing, significantly, cellular 
metabolism (Graessmann M. 1983, Methods Enzymol., 101, 482-492). 
Table 1 below relates to the antiviral activity of unmodified 
(2'-5')(A).sub.5 and of its 2'-O-phosphoglycerylated derivatives according 
to the invention. 
TABLE 1 
______________________________________ 
ANTIVIRAL ACTIVITY OF 2'-O-PHOSPHOGLYCERYL 
DERIVATIVES OF THE (2'-5')(A).sub.n COMPOUNDS OF THE 
INVENTION [(2'-5')(A).sub.n -PGro] COMED WITH THE 
ACTIVITY OF UNMODIFIED (2'-5')(A).sub.n 
OLIGOADENYLATES 
Concen- Titer of 
Compound tration virus (pfu/ 
% of 
Test n.degree. 
Tested (.mu.m) 200 cells) 
control 
______________________________________ 
1 -- -- 1.6 .times. 10.sup.3 
100.0 
(2'-5')(A).sub.5 
10 1.5 .times. 10.sup.3 
93.7 
(2'-5')(A).sub.4 PGro 
10 2.5 .times. 10.sup.1 
1.5 
2 -- -- 1.6 .times. 10.sup.4 
100.0 
(2'-5')(A).sub.4 PGro 
10 1.7 .times. 10.sup.2 
1.1 
(2'-5')(A).sub.4 PGro 
1.0 2.5 .times. 10.sup.3 
15.6 
(2'-5')(A).sub.4 PGro 
0.1 2.1 .times. 10.sup.4 
131 
______________________________________ 
The tests whose results have been collected in Table 1 were carried out as 
follows. 
HeLa cells which grew on glass fragments were micro-injected with 0.5 nl 
with each of the (2'-5')(A).sub.5 or products relating to the 
concentrations indicated. 
One hour later, the cells were infested with vesicular stomative virus 
(infection multiplicity=10) and the yield of the virus was determined 18 
hours after, by testing of plates in L929 cells. 
Considering that 0.5 nl represents a approximately 1/10th of the cell 
volume, it could be estimated that the final intracytoplasmic 
concentrations of the oligomers was about 1/10th of the values in Table 1. 
As shown by Table 1, unmodified (2'-5')(A).sub.5 does not effect the 
production of vesicular stomatitus virus when it is micro-injected into 
the HeLa cells at an intercytoplasmic concentration of about 1 .mu.m. 
On the contrary, the (2'-5')(A).sub.4 PGro reduced the growth of the virus 
about 100 fold at the same concentration and was still active at the final 
concentration of 100 nM. Conclusion 
The chemical modifications introduced into the 2'-5' oligoadenylates, 
according to the invention, resulted in derivatives which were always 
active with respect to endoribonuclease and which were stable with respect 
to the degradation of the phosphodiesterase in acellular extracts, and had 
an increased antiviral biological activity in the cells. 
EXAMPLE 2 
Synthesis of .gamma.S-(2'-5')(A).sub.n Ox Red 
It is recalled that by .gamma.S-(2'-5')(A).sub.n Ox Red are denoted the 
compounds which can be represented by the following formula: 
##STR73## 
in which .SIGMA. is a whole number equal to or greater than 1. 
The starting material is adenosine 5'-O-(3-thiotriphosphate) (called below 
.gamma.S ATP) of formula (XXXI): 
##STR74## 
which is polymerised, for example, enzymatically by means of a partly 
purified preparation of 2-5A, to obtain the compound of formula (XXXII), 
called below .gamma.S-(2'-5')(A).sub.n : 
##STR75## 
in which .SIGMA. is a whole number equal to or greater than 1. 
It is then purified, for example, by ion exchange chromatography on 
DEAE-triacryl. An oxidation is carried out, for example by the periodate 
ion, of the terminal glycol, to obtain the compound of formula (XXXIII): 
##STR76## 
in which .SIGMA. is a whole number equal to or greater than 1. 
Then the aldehyde groups are reduced, for example by sodium borohydride to 
obtain .gamma.S-(2'-5')(A).sub.n Ox Red. 
Then, for example, by molecular filtration it is purified and analysed by 
HPLC chromatography. 
Metabolic Stability 
The oligoadenylates .gamma.S(2'-5')(A).sub.n according to the invention and 
comprising a sulfur atom on the phosphorus group at the gamma position of 
the triphosphate group linked to the carbon at the 2' position at the 
first oligonucleoside unit and which can be obtained as indicated above 
and in particular the S(2'-5')(A) Ox Red have a metabolic stability in an 
acellular system higher than that of derivatives protected only at their 
3'OH ends. 
FIGS. 2 and 3 below show the percentages of degradation in an extract of 
HeLa cells, respectively of unmodified (2'-5')(A).sub.4, of 
S-(2'-5')(A).sub.4 Ox Red according to the invention and of 
(2'-5')(A).sub.4 Ox Red according to the invention. 
FIG. 2 relates to a medium containing ATP (1 mM) (very close to the in vivo 
conditions). 
FIG. 3 relates to a medium without ATP. 
In each of FIGS. 2 and 3 are shown as abscissae the time (in hours) and in 
ordinates the percentage of undegraded products. 
In FIGS. 2 and 3, the curve marked by triangles relates to 
.gamma.S(2'-5')(A).sub.4 Ox Red, the curve marked by dots relates to the 
compound (2'-5')(A).sub.4 Ox Red and the curve marked by squares relates 
to the unmodified compound (2'-5')(A).sub.4. 
Biological Activity 
(a) Binding to endoribonuclease 
The various analogs according to the invention bind to endoribonuclease 
with an affinity almost identical to that conventionally used in this 
field of "radiobinding" described initially by Knight et coll., 1980. 
(b) Antiviral activity 
The different compounds were micro-injected by means of micropipettes into 
the cytoplasm of HeLa cells. As shown by the results indicated in Table 2 
below, .gamma.S-(2'-5')(A).sub.n Ox Red exhibits an antiviral activity 
distinctly greater than that of the unmodified compound (2'-5')(A).sub.n. 
TABLE 2 
______________________________________ 
ANTIVIRAL ACTIVITY OF ANALOGS OF (2'-5')(A).sub.n 
CONCEN- 
DERIVATIVE TRATION TITER OF VIRUS 
______________________________________ 
1 -- -- 4.0 .times. 10.sup.5 
(2'-5')(A).sub.n 
10 .mu.M 1.2 .times. 10.sup.5 (N.S.) 
2 -- -- 3.3 .times. 10.sup.5 
(2'-5')(A).sub.n Ox.Red 
100 nM 2.6 .times. 10.sup.2 
(2'-5')(A).sub.n Ox.Red 
10 nm 5.2 .times. 10.sup.3 
3 -- -- 3.8 .times. 10.sup.5 
.gamma.S(2'-5')(A).sub.n 
10 .mu.M 3.3 .times. 10.sup.5 (N.S.) 
.gamma.S(2'-5')(A).sub.n 
1 .mu.M 2.4 .times. 10.sup.5 (N.S.) 
4 -- -- 2.3 .times. 10.sup.4 
.gamma.S ATP 10 .mu.M 1.3 .times. 10.sup.4 (N.S.) 
5 -- -- 3.8 .times. 10.sup.5 
.gamma.S(2'-5')(A).sub.n Ox.Red 
1 .mu.M &lt;10 
.gamma.S(2'-5')(A).sub.n Ox.Red 
10 nM &lt;10 
.gamma.S(2'-5')(A).sub. n Ox.Red 
1 nm &lt;10 
.gamma.S(2'-5')(A).sub.n Ox.Red 
10 pM 1.1 .times. 10.sup.2 
.gamma.S(2'-5')(A).sub.n Ox.Red 
1 pm 2.5 .times. 10.sup.3 
core 
.gamma.S(2'-5')(A).sub.n Ox.Red(1) 
10 .mu.M 6.2 .times. 10.sup.4 
______________________________________ 
N.S.: Difference with respect to the control not significant 
&lt;10: corresponds to totally protected cells 
(1): the same product dephosphorylated by alkaline phosphatase 
The tests, to determine the antiviral activity, were carried out as 
follows. 
HeLa cells (about 200 per experimental spot) attached to a glass support 
were each micro-injected with 5.times.10.sup.-10 ml of (2'-5')(A).sub.n or 
an analog of (2'-5')(A).sub.n at the concentrations indicated. 
The cells were infected one hour later with the virus of vesicular 
stomatitis (infection multiplicity=10) and the viral multiplication was 
determined 18 hours later by a lysis areas test on L929 mouse fibroblasts. 
Since 5.times.10.sup.-10 ml represents about 1/10th of the cellular 
volume, the final intracellular concentrations in 2-5 A were about 1/10th 
of the values indicated in this table. 
The invention also relates to the salts that the above oligonucleotides can 
form with bases in particular inorganic or organic bases. Among the 
inorganic salts, are preferred the salts of sodium or potassium. Among the 
organic salts, the amine, alkylamine and arylamine salts are prefered, in 
particular those of secondary amines, such as diethylamine, piperazine, or 
other tertiary amines, such as methylamine, pyridine, methylpiperazine 
etc. Among all the latter, the physiologically acceptable salts are 
prefered. The salts may be freeze dried. The compounds according to the 
invention have biologically interesting properties, in particular 
properties of the interferon type, and more particularly an antiviral 
activity. 
The compounds according to the invention are capable of inhibiting the 
synthesis of DNA, in particular the replication in the cells and/or 
degradation of viral RNA, thus preventing the synthesis of proteins, more 
particularly viral proteins in cells infected with the virus at nanomolar 
concentrations. 
The compounds according to the invention are stable and resist degradation 
by phosphodiesterases and the time of resistance with respect to 
phosphatases is increased. 
A prefered class of compounds according to the invention resist degradation 
by phosphatases. 
The oligonucleotides according to the invention are hence suitable 
substitutes for interferon and its known applications. They may be 
prepared reproducibly in highly purified form, as biological reagents, in 
particular as a comparison reference in qualitative and in quantitative 
tests, in cell cultures, of compounds of interferon or other substances 
similar to interferon. 
The invention relates also to the pharmaceutically acceptable salts of the 
oligonucleotides defined above in particular those suitable for in vivo 
administration. 
The invention relates also to pharmaceutical compositions associating the 
above-said oligonucleotides, preferably in the form of pharmaceutically 
acceptable salts, with a pharmaceutical vehicle. 
The invention thus provides pharmaceutical compositions having an activity 
similar to that of interferon by using a predetermined chemical compound 
in the form of high purity, not having toxicity, being stable and easy to 
manipulate. 
The composition according to the invention may be in the form of 
preparations administrable orally or rectally, by using suitable solids or 
liquids for such a type of administration or in the form of sterile 
injectable preparations containing any one at least of the nucleotides in 
association with suitable sterile liquid vehicles, preferably isotonic. 
Other suitable forms of preparations consisting of pommades in which the 
oligonucleotides of the invention are associated with vehicles in a 
pommade. 
Any one of the conventionally used techniques of preparation for 
associating interferon with pharmaceutical supports may be used to prepare 
the pharmaceutical compositions according to the invention. 
The oligonucleotides according to the invention may be associated with 
other suitable vectors, such as liposomes. 
The compositions of the invention have antiviral properties and are in 
particular capable of inhibiting viral diseases which can be followed by 
tumoral disorders, for example diseases induced by hepatitus B virus or 
the various forms of virus of herpes. 
More generally, the compositions of the invention are useful for the 
treatment and the prevention of viral diseases, and for antitumoral 
treatments with respect to tumors capable of being also controlled by 
treatments with interferon. 
It will be noted that the doses at which the compositions are used are 
determined according to the nature of the disease which afflicts the 
patient and the particular conditions of health. 
Suitable dosages are determined by the physician, as practice may require 
in these fields of use.