U.S. patent number 4,981,957 [Application Number 06/756,369] was granted by the patent office on 1991-01-01 for oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini.
This patent grant is currently assigned to Centre National de la Recherche Scientifique. Invention is credited to Bernard Bayard, Bernard Lebleu.
United States Patent |
4,981,957 |
Lebleu , et al. |
January 1, 1991 |
Oligonucleotides with modified phosphate and modified carbohydrate
moieties at the respective chain termini
Abstract
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.
Inventors: |
Lebleu; Bernard (Montpellier,
FR), Bayard; Bernard (Castelnau Le Lez,
FR) |
Assignee: |
Centre National de la Recherche
Scientifique (Paris, FR)
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Family
ID: |
9306290 |
Appl.
No.: |
06/756,369 |
Filed: |
July 18, 1985 |
Foreign Application Priority Data
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Jul 19, 1984 [FR] |
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84 11469 |
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Current U.S.
Class: |
536/25.2;
536/26.21; 536/26.23; 536/25.5; 536/26.26 |
Current CPC
Class: |
C07H
21/00 (20130101); A61P 31/12 (20180101) |
Current International
Class: |
C07H
21/00 (20060101); C07H 021/02 () |
Field of
Search: |
;514/46,47
;536/27,28,29 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0142296 |
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Nov 1981 |
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JP |
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0049399 |
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Mar 1983 |
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JP |
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0950733 |
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Aug 1982 |
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SU |
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Other References
Baglioni et al, The Journal of Biological Chemistry, vol. 256, No.
7, pp. 3253-3257 (Apr. 10, 1981). .
Gosselin et al., Tetrahedron Letters, vol. 22, No. 47, pp.
4699-4702 (1981). .
Haugh et al, Eur. J. Biochem., vol. 132, pp. 77-84 (1983). .
Torrence et al, J. Med. Chem., vol. 26, No. 12, pp. 1674-1678 (Dec.
1983). .
Hughes et al, Biochemistry, vol. 22, No. 9, pp. 2127-2135 (1983).
.
Kariko et al, Chem. Abstr., 103, 16829h, 1985. .
Knight et al., Methods in Enzymology, vol. 79, pt. A., pp. 216-227,
1981. .
Eppstein et al., Nature, vol. 302, pp. 723-724, 1983. .
Justesen et al., Proc. Nat. Acad Sci (Biochem)., vol. 77, pp.
4618-4622, 1980. .
March, Advanced Organic Chemistry, McGraw-Hill Book Co., 1968, New
York, pp. 306 (Acetal Hydrolysis) & 678 (BH.sub.4.sup..crclbar.
+Aldehydes)..
|
Primary Examiner: Griffin; Ronald W.
Assistant Examiner: Crane; L. Eric
Attorney, Agent or Firm: Fleit, Jacobson, Cohn, Price,
Holman & Stern
Claims
We claim:
1. An oligonucleotide of the formula ##STR77## in which Y and T are
identical or different and are O, S, Se or NH, Z and W are
identical or different and are O, S or NH, at least one of the
substituents Y and Z in at least one of the phosphate moieties
being different from oxygen, X is selected from the group
consisting of ##STR78## R is H or ##STR79## R.sub.1 is alkyl of 1
to 5 carbon atoms or phenyl, .SIGMA. is a whole number equal to
n when X is --CHOHCH.sub.2 OR, or
n-1 when X is ##STR80## wherein n is an integer of 2 to 10, m is an
integer of 1 to 3, and A is adenine or a derivative selected from
the group consisting of ##STR81## and the physiologically
acceptable salts thereof.
2. The oligonucleotide of claim 1, having the formula ##STR82## in
which Y.sub.1, Y.sub.2, Y.sub.3 and T are identical or different
and are O, S, Se or NH and Z.sub.1 and Z.sub.2 are identical or
different and are, O, S or NH, at least one of the substituents
Y.sub.1, Y.sub.2, Y.sub.3, Z.sub.1 and Z.sub.2 being different from
oxygen.
3. The oligonucleotides of claim 1, having the formula ##STR83## in
which Y.sub.2, Y.sub.3 and T are identical or different and are O,
S, Se or NH, Z.sub.2 is O, S or NH, at least one of the
substituents Y.sub.2, Y.sub.3 and Z.sub.2 being different from
oxygen, R is hydrogen, and n is an integer between 2 to 10,
inclusive.
4. The oligonucleotide of claim 1, having the formula ##STR84## in
which Y.sub.3 is S, Se or NH, T is O, S, Se or NH, R is hydrogen,
and n is an integer between 2 and 10, inclusive.
5. The oligonucleotide of claim 1 in which T is oxygen.
6. The oligonucleotide of claim 2 in which T is oxygen.
7. The oligonucleotide of claim 3 in which T is oxygen.
8. The oligonucleotide of claim 4 in which T is oxygen.
9. The oligonucleotide of claim 1, wherein X is selected from the
group consisting of ##STR85##
10. The oligonucleotide of claim 9, wherein Z and W are oxygen.
11. The oligonucleotide of claim 10, selected from the group
consisting of oligonucleotides of the formulas ##STR86##
12. A pharmaceutical composition comprising the oligonucleotide of
claim 9 and a pharmaceutically acceptable carrier.
13. A pharmaceutical composition comprising an oligonucleotide of
claim 11 and a pharmaceutically acceptable carrier.
Description
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] COMPARED 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 <10
.gamma.S(2'-5')(A).sub.n Ox.Red 10 nM <10
.gamma.S(2'-5')(A).sub. n Ox.Red 1 nm <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 <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.
* * * * *