Note: Descriptions are shown in the official language in which they were submitted.
~~~~~~2
BOEHRINGER MANNHEIM GMBH 3489/OA/WO
Oligo-2'-deoxynucleotides and their use as
pharmaceutical agents with antiviral activity
Oligonucleotides whose sequences are complementary to
the RNA or DNA of a viral sequence or to an oncogene are
of potential interest for the therapy of viral
infections since they can inhibit the expression of
viral genes. The underlying method, denoted antisense
principle, is described for example by Zamecnik, P.C.
and Stephenson, M.L. (1978) in Proc. Natl. Acad. Sci.
USA 75, 280.
However, it has turned out that when such antisense
oligonucleotides are introduced into cells,
preferentially intrinsic cell enzymes rapidly degrade
these oligonucleotides by cleavage of phosphodiester
bridges, and they thus become ineffective.
Therefore many attempts have been made to synthesize
antisense oligonucleotides which are resistant to
enzymatic degradation (Uhlmann, E. and Peyman, A. (1990)
in "Antisense oligonucleotides: A new therap.
Principle", Chem. Rev. 90, 543 - 584). Up to now such
modifications have been primarily carried out at the
internucleotide bridges i.e, on the phosphorus atom.
Thus for example oligonucleoside-phosphorothioates and -
oligonucleoside-phosphorodithioates, as well as non-
ionic oligonucleoside-methylphosphonates, oligo-
nucleoside-methylphosphorothioates, oligonucleoside-
alkylphosphotriesters and oligonucleoside-alkyl-
phosphoramidates have been described which are resistant
to enzymatic degradation. A disadvantage of these
210062
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compounds is for example their chirality with regard to
the phosphorus atom. This means that in each case there
are two pairs of diastereoisomers. This non-uniformity,
however, limits their pharmacological effectiveness or
requires a complicated separation of the isomers before
use as therapeutic agents.
A further known class of compounds which has been
proposed for antisense therapy are oligonucleotides
which contain intercalating or reactive ligands. Thus
for example acridine-modified oligomers or
oligonucleotides which can be cross-linked (psoralen-,
azidoproflavin-substituted) have been described (Helene,
C. and Thuong, N.T. in "Antisense RNA and DNA", Curr.
Commun. Mol. Biol.; Cold Spring Harbor Laboratory, Cold
Spring Harbor NY, 1987). The production of such
oligonucleotides is, however, very complicated.
In addition it is known that those oligodeoxynucleotides
which have a configuratively changed glyconic part
(alpha DNA) can be used as antisense oligonucleotides.
Such an alpha DNA which is comprised exclusively of
purine-2'-deoxynucleosides and pyrimidine-2'-deoxy-
nucleosides in the alpha-D configuration is not or only
very slowly degraded by intrinsic cell enzymes and would
therefore be suitable for antisense therapy. However, a
disadvantage of these compounds is the very complicated
and tedious synthesis (Cohen, J.S. in Topics in
Molecular and Structural Biology, "Oligonucleotides:
Antisense Inhibitors of Gene Expression", MacMillan
Press, Lt. 1989).
2103062
- 3 -
The present invention seeks to provide oligonucleo-
tides which are not enzymatically degraded in
eukaryotic cells, which can be easily produced and are
suitably as pharmaceutical antiviral agents based on
the antisense principle.
In accordance with the invention there is provided a
compound having the general formula(I):
B
R20
(I)
in which: B represents a base selected from the group
consisting of adenine, thymine, cytosine, guanine, 1-
deazaadenine, 3-deazaadenine, 7-deazaadenine, 1-deaza-
guanine, 3-deazaguanine, 7-deazaguanine, 1-deazah.ypo-
xanthine, 3-deazahypoxanthine, 7-deazahypoxanthin, 7-
deazapurines substituted at C-7, purines substituted
at C-8, and pyrimidines substituted at C-5; R1 repre-
sents a hydrogen atom or a reporter group or an inter-
calator group or a phosphoramidite group or a H-phos-
phonate group or a P-methyl-phosphoramidi.te group, and
RZ represents a hydrogen atom, a mono, di or triphos-
phate or a protecting group, the 5'-O group and the
amino groups of the base being independently unpro-
tected or protected by protecting groups.
B
H H
2103062
- 3a -
The invention is particularly concerned with oligode-
oxyribonucleotides in which at least two 2'-deoxy-(3-D-
erythro-pentofuranosyl groups are replaced by 2'-
deoxy-~i-D-threo-pentofuranosyl groups at both the 5'
and 3' end and which are composed of 6 to 100 nucleo-
tide building blocks.
The invention in addition concerns oligodeoxyribo-
nucleotides in which at least 20% of the 2'-deoxy-(3-D-
erythro-pentofuranosyl groups are replaced by 2'-
deoxy-(3-D-threo-pentofuranosyl groups in consecutive
nucleotide building blocks and which are composed of
6-100 nucleotide building blocks.
Suprisingly such oligonucleotides ( also denoted
oligonucleotides in the following) are resistant or
substantially resistant to nucleolytic degradation by
cellular enzymes such as for example phosphodi-
esterases, exonucleases and endonucleases. In
addition they form stable double-stranded hybrid
structures with natural 2'-deoxyribonucleotides under
,the natural conditions in eukaryotic cells although
they presumably at least in part have left-helical DNA
structures as demonstrated by
.B
21030~~
- 4 -
their CD spectra. The oligonucleotides according to the
present invention are suitable for inhibiting the
expression of viral genes and oncogenes in eukaryotic
cells and can therefore be used therapeutically as
antisense oligonucleotides.
30 ~ and particularly preferably all of the 2'-deoxy-B-
D-erythro-pentofuranosyl groups are replaced by
2'-deoxy-B-D-threo-pentofuranosyl groups.
If all the 2'-deoxy-B-D-erythro-pentofuranosyl groups
are replaced by 2'-deoxy-B-D-threo-pentofuranosyl groups
in an oligonucleotide, it is expedient that this oligo-
nucleotide be designated an oligodeoxyxylonucleotide.
The structure of such an oligonucleotide according to
the present invention is shown schematically in Fig. 1
(section from an oligodeoxyxylonucleotide).
In the following the nucleotides which contain 2'-deoxy-
B-D-erythro-pentofuranosyl groups are designated
2'-deoxyribonucleotides and the nucleotides which
contain 2'-deoxy-B-D-threo-pentofuranosyl groups are
designated 2'-deoxyxylonucleotides or building blocks.
In addition 2'-deoxyribonucleotide building blocks are
referred to as dB (e.g. dA, dT, dC, dG) and 2'-
deoxyxylonucleotide building blocks are referred to as
dxB (e. g. dxA, dxT, dxC, dxG).
It is preferred that the dxBs and dBs occur
consecutively in blocks in the oligonucleotides
according to the present invention.
210062
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Accordingly preferred oligonucleotides according to the
present invention are:
d ~xscB(B)nxsxB}
d { (B) m (xB) n (B) o}
d { (xB) m(B) n (xB) o}
in which n, m and o is at least 4, provided that the
total length of the oligonucleotides according to the
present invention does not exceed 100 nucleotide
building blocks.
In an equally preferred embodiment, one or several
nucleotide building blocks dB can be replaced by dxB at
specific positions on the oligonucleotide (e. g.
recognition sequences of endonucleases) in order to
prevent cleavage by endonucleases.
All natural or modified nucleobases are suitble as
bases. Particularly preferred modified bases are
5-methylcytosine or deazapurine such as 1-deazaadenine,
3-deazaadenine, 7-deazaadenine, 1-deazaguanine,
3-deazaguanine, 7-deazaguanine, 1-deazahypoxanthine,
3-deazahypoxanthine, 7-deazahypoxanthine and those bases
which are substituted at the C-5 of pyrimidines, at the
C-7 in the case of 7-deazapurines or at the C-8 of
purines.
In addition the oligonucleotides according to the
present invention can contain modifications at the
internucleotide bridges in which case the modifications
are preferably present at all internucleotide bridges of
the oligonucleotide according to the present invention.
21fl~0~~
- 6 -
In this connection phosphorothioates, methylphosphonates
and phosphoroamidates are preferred.
The 3' and/or 5' ends the oligonucleotides according to
the present invention can contain all suitable terminal
groups known to a person skilled in the art. Hydrogen,
mono-, di- or triphosphate, a reporter group or an
intercalator group is preferred for the 3' end and for
the 5' end. The other nucleotide building blocks can
also be modified by reporter groups or intercalator
groups.
A reporter group within the meaning of the invention is
understood as a hapten such as e.g. biotin or
digoxigenin or a fluorescent dye residue. Suitable
intercalator groups are described by Helene, C., loc.
cit. and are preferably phenanthroline, acridine,
actinomycin or its chromophore or heavy metal complexing
agents such as EDTA. Those groups which lead to cross-
linking of nucleic acids such as e.g. psoralen are also
advantageous.
Oligonucleotides according to the present invention are
preferably composed of 15 to 30 nucleotide building
blocks. The sequence of bases in the oligonucleotides
according to the present invention depends on the
sequence of the virus or oncogene towards which the
oligonucleotide is intended to be directed. Thus for
example oligonucleotides according to the present
invention are particularly suitable for the therapy of
HIV I infections in which all bases represent A or T and
all 2'-deoxy-f3-D-erythro-pentofuranosyl groups are
replaced by 2'-deoxy-f3-D-threo-pentofuranosyl groups
since the viral sequence of HIV I contains several
clusters with poly A or poly T sequences. Further
2~0~00~
particularly preferred oligonucleotides are those which
are complimentary to certain genome regions which are
important for the replication of viral genes. In the
case of the HIV genome these are for example the
regulatory region of the rev gene (Matsukura, M. et al.
(1989) Proc. Natl. Acad. Sci. USA, 86, 4244).
The oligonucleotides according to the present invention
are produced in a well-known manner for example by the
phosphate triester, phosphite triester or H-phosphonate
method in a homogeneous phase or on a support. The two
latter methods are preferably used in which the
synthesis is usually carried out using automated
synthesizers.
The invention therefore in addition concerns a process
for the production of oligodeoxyribonucleotides in which
at least two 2'-deoxy-!3-D-erythro-pentofuranosyl groups
are replaced by 2'-deoxy-I3-D-threo-pentofuranosyl groups
at both the 5' and 3' end and which are composed of 6 to
100 nucleotide building blocks by means of the process
of oligonucleotide synthesis in which a start nucleoside
is bound to a solid support and subsequently the desired
oligonucleotide is synthesized by stepwise coupling
using appropriate activated monomeric nucleotide
building blocks, if desired trivalent phosphorus is
oxidized to pentavalent phosphorus during or after the
synthesis, the oligonucleotide is cleaved from the
support using a first base, heterocyclic protecting
groups are cleaved with a second base, the 5' protecting
group is cleaved with an acid and the oligonucleotide is
purified if desired.
~~o~osz
_8_
The invention in addition concerns a process for the
production of oligodeoxyribonucleotides in which at
least 20 % of the 2'-deoxy-!3-D-erythro-pentofuranosyl
groups are replaced by 2'-deoxy-f3-D-threo-pentofuranosyl
groups in consecutive nucleotide building blocks and
which are composed of 6 to 100 nucleotide building
blocks by a process of oligonucleotide synthesis in
which a start nucleoside is bound to a solid support and
subsequently the desired oligonucleotide is synthesized
by stepwise coupling using appropriate activated
monomeric nucleotide building blocks, if desired
trivalent phosphorus is oxidized to pentavalent
phosphorus either during or after the synthesis, the
oligonucleotide is cleaved from the support using a
base, heterocyclic protecting groups are cleaved with a
second base, the 5' protecting group is cleaved with an
acid and the oligonucleotide is purified if desired.
Oligonucleotides are preferably produced which are
composed of 15 to 30 nucleotide building blocks.
It is expedient to oxidize trivalent phosphorus to
pentavalent phosphorus after each coupling of a
nucleotide building block when using phosphoramidites as
nucleotide building blocks or after synthesis of the
total oligonucleotide when using phosphonates as
nucleotide building blocks. Iodine (e. g.
iodine/H20/lutidine) or, in the case of the production
of 2'-deoxyxylonucleoside-3'-phosphorothioates and -3'-
phosphorodithioates, sulphurization reagents (e. g.
sulphur in pyridine/carbon disulphide) are preferably
used for the oxidation.
210302
g _
Ammonia at 60°C is preferably used for the alkaline
cleavage; in order to remove the 5' protecting group,
80 ~ aqueous acetic acid or tetrabutylammonium fluoride,
in the case of a silyl protecting group is preferably
used at room temperature.
After cleavage by base or acid it is expedient to
neutralize and purify. Reverse phase HPLC or anion
exchange HPLC is preferably used for the purification in
which case it is subsequently desalted.
The procedure for such oligonucleotide syntheses is
generally known to a person skilled in the art and is
described for example by Gait, M.J. in "Oligonucleotide
synthesis, a practical approach", IRL Press, LTD. 1984,
Narang, S.A., "Synthesis and application of DNA and
RNA", Academic Press 1987.
The supporting material is composed of inorganic
(Controlled Pore Glas, Fractosil~) or organic polymeric
material (e.g. polystyrene) known to a person skilled in
the art.
In order to produce the oligonucleotides according to
the present invention, a monomeric 2'-deoxyxylo-
nucleoside or 2'-deoxyribonucleoside, which serves as
the start nucleoside for the oligonucleotide synthesis,
is preferably coupled to the supporting material by
means of a coupling reagent (cf. e.g. Gait, M.J.
loc.cit.).
A further process for the production of the
oligonucleotides according to the present invention is
the phosphate triester method (cf. Gait, M.J. loc.cit.).
2103062
-~o-
The invention in addition concerns 2'-deoxyxylo-
nucleoside-3'-phosphoroamidites, -3'-H-phosphonates and
-P-methyl-phosphoroamidites protected by bases and
sugars. These compounds are suitable as nucleotide
building blocks for the production of the
oligonucleotides according to the present invention.
The nucleotide building blocks according to the present
invention particularly preferably contain the bases
adenine, guanine, cytosine, thymine, uracil, 5-methyl-
cytidine or deazapurines such as e.g. 1-deazaadenine,
3-deazaadenine, 7-deazaadenine, 1-deazaguanine,
3-deazaguanine and 7-deazaguanine. Further preferred
bases are those which are substituted at the C-5 of
pyrimidines, at the C-7 of 7-deazapurines or at the C-8
of purines.
The nucleotide building blocks according to the present
invention preferably contain protecting groups on the
heterocyclic bases, 5'-OH or 3'-OH protecting groups.
Amino protecting groups such as e.g. benzyl,
formamidine, isobutyryl or phenoxyacetyl group are
preferably used as protecting groups on the heterocyclic
bases.
A triphenylmethyl, monomethoxytrityl, dimethoxytrityl,
t-butyl-dimethylsilyl, t-butyldiphenylsilyl, t-butyl-
methoxyphenylsilyl or pixyl group is preferably used as
the 5'-OH protecting group of the sugar part.
B
~~0~062
- 11 -
3'-O-(2-cyanoethyl)-N,N-diisopropyl-aminophosphanes and
3'-O-methyl-N,N-diisopropylamino-phosphanes are
preferred as phosphoramidites. The H-phosphonates are
preferably used as salts.
In a particularly preferred embodiment the nucleotide
building blocks according to the present invention are
labelled with 32P or 35S.
The nucleotide building blocks according to the present
invention which are capable of coupling are produced
from the corresponding 2'-deoxyxylonucleosides (produced
for example analogously to Fox and Miller J. Org. Chem.
28 (1963) 936, Hansske and Robins J. Am. Chem. Soc. 105
(1983) 6736-6737 or F. Seela and H.P. Muth, Helvetica
Chimica Acta 74 (1991) 1081 - 1090).
The production of monomeric nucleotide building blocks
is carried out according to methods familiar to a person
skilled in the art, for example as described in Gait,
M.J. loc.cit..
The oligonucleotides according to the present invention
can be elongated at the 3' and/or 5' end by ligation
with further oligonucleotides according to the present
invention or known oligonucleotides. Such ligation
reactions using DNA or RNA ligase are familiar to a
person skilled in the art and described for example in
Maniatis et al., Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory, New York (1982).
The oligonucleotides according to the present invention
can also be produced enzymatically using a DNA
- 12 -
polymerase and/or RNA polymerase. In this case
nucleotides having the general formula
Ri0 0 B
OR,
H
H
are used in which B represents A, T, C, G or a base from
the group comprising 1-deazaadenine, 3-deazaadenine,
7-deazaadenine, 1-deazaguanine, 3-deazaguanine,
7-deazaguanine, 1-deazahypoxanthine, 3-deaza-
hypoxanthine, 7-deazahypoxanthine, 7-deazapurines
substituted at C-7, purines substitued at C-8,
pyrimidines substituted at C-5, R1 represents a hydrogen
atom or a reporter group or an intercalator group and R2
represents a mono-, di- or triphosphate.
The invention therefore in addition concerns nucleotides
having the general formula
a10 0 B
OR, H
H H
in which B represents A, T, C, G or a base from the
group comprising 1-deazaadenine, 3-deazaadenine,
7-deazaadenine, 1-deazaguanine, 3-deazaguanine,
7-deazaguanine, 1-deazahypoxanthine, 3-deaza-
hypoxanthine, 7-deazahypoxanthine, 7-deazapurines
~~0300~
- 13 -
substituted at C-7, purines substituted at C-8,
pyrimidines substituted at C-5, R1 represents a hydrogen
atom or a reporter group or an intercalator group and R2
represents a mono-, di or triphosphate.
The invention in addition concerns the use of
oligodeoxyribonucleotides in which at least two 2'-
deoxy-13-D-erythro-pentofuranosyl groups are replaced by
2'-deoxy-f3-D-threo-pentofuranosyl groups at both the 5'
and 3' end and which are composed of 6 to 100 nucleotide
building blocks for the production of a pharmaceutical
agent with antiviral activity.
In addition the invention concerns oligodeoxyribo-
nucleotides in which at least 20 % of the 2'-deoxy-13-D-
erythro-pentofuranosyl groups are replaced by 2'-deoxy-
B-D-threo-pentofuranosyl groups in consecutive
nucleotide building blocks and which are composed of 6 -
100 nucleotide building blocks for the production of a
pharmaceutical agent with antiviral activity.
The oligonucleotides according to the present invention
and their salts can be used as medicines, e.g. in the
form of pharmaceutical preparations which can be
administered orally e.g. in the form of tablets, coated
tablets, hard or soft gelatin capsules, solutions,
emulsions or suspensions. They can also be administered
rectally e.g. in the form of suppositories or
parenterally e.g. in the form of solutions for
injections. In order to produce pharmaceutical
preparations, these compounds can be processed in
therapeutically inert organic and inorganic vehicles.
Examples of such vehicles for tablets, coated tablets
and hard gelatin capsules are lactose, maize starch or
derivatives thereof, tallow, stearic acid or salts
z~o3os~
- 14 -
thereof. Suitable vehicles for the production of
solutions are water, polyols, sucrose, inverted sugar
and glucose. Suitable vehicles for injection solutions
are water, alcohols, polyols, glycerol and vegetable
oils. Suitable vehicles for suppositories are vegetable
oils and hardened oils, waxes, fats and semi-liquid
polyols.
The pharmaceutical preparations can also contain
preservatives, solvents, stabilizing agents, wetting
agents, emulsifiers, sweetners, dyes, flavouring
materials, salts to alter the osmotic pressure, buffers,
coating agents or antioxidants as well as if desired,
other therapeutically active substances.
The invention is elucidated further by the following
examples and figures:
Fig. 1 shows a section from an oligodeoxyxylonucleotide.
Fig. 2 shows the hydrolysis of the oligonucleotide
d(GTAGAAxTxTCTAC) by calf spleen
phosphodiesterase (H = hypochromicity of the
final product obtained, the inserted figure shows
the elution profile of the HPLC of the final
product, a = (Et3NH)OAc pH 7/acetonitrile (95:5),
b = 0 - 20 ~ acetonitrile in mobile solvent a.
Fig. 3 shows the melting curve of the d(A12)/d (xTl2)
hybrid (b) in comparison to hybrid d(A12)/d(T12)
(a) and of the single-stranded oligonucleotides
d(A12) (c) and d(T12) (d).
210300
- 15 -
Fig. 4 shows the CD spectrum of d (T12) (a), T (b), xT
(c) and d (xTl2) (d) .
Fig. 5 shows the CD spectrum of the hybrids d(A12) /
d(T12) (a) and d(A12) / d(xTl2) (c) as well as
of the single-stranded oligonucleotide
d (A12 ) (b) .
Example 1
1-(2'-deoxy-13-D-threo-pentofuranosyl)thymine
The synthesis was carried out according to Fox and
Miller [J. Org. Chem. (1963), 28, 936].
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for 6 in ppm): 11.25 (s, br. NH); 7.80 (s, H-C(6)); 6.06
(dd, J(H-C(1'),HH-C(2') - 2.7 Hz, J(H-C(1'),Ha-C(2') -
5.5 hz, H-C(1')); 5.25 (3'-OH); 4.69 (5'-OH); 4.23 (m,
H-C(3')); 3.76 (m, H-C(4')); 3.70 (m, H2-C(5')); ca.2.5
(Ha-C(2')); 1.84 (d, J(H~-C(2'),Ha-C(2') - - 16.0 Hz,
H~-C(2')); 1.66 (s, CH3).
Example 2
1-f2'-deoxy-5'-(4,4-dimethoxytriyhenylmethyl)-I3-D-threo-
pentofuranosy ~ -thymine
500 mg 1-(2'-deoxy-f3-D-threo-pentofuranosyl)thymine
(2.06 mmol) were dried by co-evaporating several times
with anhydrous pyridine (5 ml in each case). The oily
residue was dissolved in 15 ml anhydrous pyridine and
210306
- 16 -
admixed successively with 1.0 g 4,4'-dimethoxytri-
phenylmethyl chloride (3 mmol) and 0.5 ml diisopropyl-
ethylamine (3 mmol). It is stirred for 3 hours at 40°C
under nitrogen, afterwards the solution is poured into
50 ml 5 % aqueous NaCH03 and extracted twice with 100 ml
dichloromethane each time. The combined organic phases
are dried over sodium sulfate and the solvent is removed
by evaporation. After co-evaporating several times with
toluene, the residue is purified on silica gel 60H by
means of flash chromatography (column: 6 x 15 cm,
CH2C12-MeOH, 98:2). The fractions which contained the
main product are concentrated to a colourless, foamy
residue (780 mg, 69 % of theoretical yield).
TLC (silica gel, CH2C12-MeOH, 95:5) Rf 0.6.
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for s in ppm): 11.30 (s, NH); 7.62 (s, H-C(6)); 7.45-
6.86 (m, 13H, aromat. H); 6.12 (dd, J = 6.2, 2.7 Hz, H-
C(1')); 5.22 (d, J = 3.4 Hz, 3'-OH); 4.20 (m, H-C(3'));
4.10 (m, H-C(4')); 3.73 (s, 6H, 2x OCH3); 3.40 and 3.19
(2m, H2-C(5')); ca. 2.51 (m, Ha-C(2')); 1.87 (d, J =
-15.4 Hz, HH-2'); 1.66 (s, CH3).
Elemental analysis for C31H32N207 (544.6): calculated:
C 68.37, H 5.92, N 5.14; found: C 68.44, H 6.00, N 5.15.
~1 ~3~62
- 17 -
Example 3
1-f(2'-deoxy-13-D-threo-pentofuranosyl-5'-O-(4,4'-
dimethoxytri~henylmethyl)]-thymine-(3'-H-phosphonate).
triethylammonium salt
1.06 g 1,2,4-triazole (15.3 mmol) was added to a
solution of 0.4 ml phosphorus oxytrichloride (4.6 mmol)
and 5.1 ml N-methylmorpholine (46 mmol) in 36 ml
dichloromethane. After stirring for 30 minutes, the
solution is cooled to 0°C and a solution of 500 mg
1-[2'-deoxy-5'-(4,4'-dimethoxytriphenylmethyl)-f3-D-
threo-pentofuranosyl]-thymine in 12 ml dichloromethane
is slowly added to this. After stirring for a further 10
minutes at room temperature, the reaction mixture is
poured into 50 ml 1 M aqueous triethylammonium
bicarbonate solution, pH 8.0, shaken and the phases are
separated. The aqueous phase is extracted with 30 ml
CH2C12 and the combined organic extracts are dried over
Na2S04 and evaporated to a colourless foam. Flash
chromatography on silica gel 60H (column: 6 x 15 cm,
firstly with CH2C12-Et3N, 92:8, then with CH2C12-MeOH-
Et3N, 88:10:2) yielded a main fraction which was pooled
and concentrated by evaporation. The residue was
dissolved in 15 ml CH2C12 and extracted twice with 1 M
aqueous triethylammonium bicarbonate solution, pH 8.0,
the organic phases were dried over sodium sulfate and
concentrated by evaporation. 480 mg (74 ~ of theoretical
yield) of the desired product is obtained in the form of
a colourless foam.
TLC (silica gel, CH2C12-MeOH-Et3N, 88:10:2): Rf 0.3.
~io~os~
- 18 -
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for d in ppm): 11.30 (s, NH); 7.66 (s, H-C(6)); 7.63-
6.86 (m, 13H, aromat. H), 6.13 (dd, J = 6.8, 2.8 Hz, H-
C(1')); 5.76 and 5.28 (d, J = 119 Hz, PH); 4.62 (m, H-
C(3')); 4.12 (m, H-C(4')); 3.73 (s, 6H, 2x OCH3); 3.34
and 3.16 (m, H2-C(5')); 2.73 (q, CH3CH2NH); ca. 2.5 (m,
Ha-C(2')); 2.14 (d, J = -15.2 Hz, H~-C(2')); 1.66 (s,
CH3); 1.03 (t, CH3CH2NH).
31P-~ ((D6)DMSO): 0.88 ppm (1J(P-H) - 587 Hz; 3J(P-H-
4') - 8.8 Hz).
Elemental analysis for C37H48N309P (709.8): calculated:
C 62.61, H 6.82, N 5.92; found: C 62.81, H 7.00, N 5.99.
Example 4
i-f2'-deoxv-5'-O-(4,4'-dimethoxytriphenylmethyl)-t3-D-
threo-Dentofuranosyl]-thymine-3'- ~(2-cyanoethyl)-N,N-
diisopropyl-phosphoramiditel
45 ~,1 chloro-!3-cyanoethoxy-(N,N-diisopropylamino)-
phosphane (0.2 mmol) is added dropwise over a period of
2 minutes under nitrogen at room temperature to a
solution of 100 mg 1-[2'-deoxy-5'-(4,4'-dimethoxy-
triphenylmethyl)-f3-D-threo-pentofuranosyl]-thymine (0.18
mmol) and 0.1 ml N-ethyldiisopropylamine (0.57 mmol) in
dry tetrahydrofuran. It is stirred for 30 minutes and
afterwards the reaction is stopped by addition of 4 ml
5 ~ aqueous NaHC03. The reaction mixture is extracted
twice with 5 ml CH2C12 each time. The combined organic
phases are dried over sodium sulfate and concentrated by
evaporation. Flash chromatography (silica gel 60H,
~~ o~os~
- 19 -
column: 3 x 6 cm, ethyl acetate-CH2C12-Et3N, 45:45:10)
yields two partially overlapping main fractions of the
diastereomers (95 mg, 72 ~ of theoretical yield).
TLC (silica gel, CH2C12-EtOAc-Et3N, 45:45:10) Rf 0.7
and 0.6.
31p-~R (CDC13): 148.5 ppm (more mobile compound in
TLC); 151.8 ppm (less mobile compound in TLC).
Example 5
9-(2'-deoxy-f3-D-xylofuranosyl)adenine (2'-deoxy-
xvloadenosine)
The compound was prepared according to the instructions
of Hansske and Robins [J. Am. Chem. Soc. (1983), 105,
6736-6737].
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for 8 in ppm): 8.35 (s, H-C(8)); 8.15 (H-C(2)); 7.34 (s,
br., NH2) 6.25 (dd, J(H-C(1'),Ha-C(2')) - 2.2 Hz; J(H-
C(1'),HH-C(2')) - 8.7 Hz; H-C(1')); 5.97 (s, br. 3'-OH);
4.69 (s, br., 5'-OH); 4.33 (m, H-C(3')); 3.89 (m, H-
C(4')); 3.67 (m, H2-C(5')); 2.78 (m, Ha-C(2')); 2.25
(dd, J(Ha-C(2'),H~-C(2')) - -14.5 Hz; H~-C(2')).
2.~~3a~2
- 20 -
Example 5a
2'-deoxy-xylo-inosine
2'-deoxy-xylo-adenosine (78 mg, 0.31 mmol) is suspended
in 3 ml water and admixed with adenosine deaminase (calf
spleen, 50 ~,g). It is stirred for 2 h at room
temperature and evaporated to dryness; yield 75 mg
(97 %) colourless crystals with a melting point of 208-
210°C.
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for d in ppm): 12.4 (br. NH); 8.31 (s, H-C(8); 8.07 (s,
H-C(2); 6.24 (dd, J = 8.8, 1.6 Hz, H-C(1'); 5.45 (d, J =
4.0 Hz, 3'-OH); 4.70 (m, 5'-OH); 4.36 (m, J = 3.5 Hz, H-
C(3'); 3.92 (m, H-C(4'); 3.71 and 3.60 (2 m, H2-C(5');
2.76 (m, Ha-C(2'); 2.25 (d, J = -15 Hz, HH-C(2').
13C-~R ((D6)DMSO): 156.7 (C-6); 147.8 (C-4); 145.9 (C-
2); 139.1 (C-8); 124.0 (C-5); 85.6 (C-4'); 82.6 (C-1');
69.1 (C-3'); 59.9 (C-5'); 41.1 (C-2').
The W spectrum (in MeOH) exhibits an absorbance maximum
at 249 nm (Emax = 10500).
Elemental analysis:
calculated: C 47.62; H 4.80; N 22.21
found: C 47.76; H 4.98; N 22.15.
~~o~os~
- 21 -
Example 6
9-f2'-deoxy-5~-O-(4,4'-dimethoxytriphenylmethyl)-fi-D-
xyloDentofuranosyl]-6- Lf(dimethylamino)-
methylidene]amino)adenine
A solution of 2'-deoxy-xyloadenosine (502 mg, 2 mmol) in
dimethylformamide (10 ml) was stirred for 18 h with
dimethylformamide-diethylacetal (1.7 ml, 10 mmol) at
room temperature and subsequently concentrated by
evaporation [TLC (silica gel, CH2C12-acetone-Et3N (20 .
10: 1)) Rf 0.1]. The oily residue (0.8 g) was dissolved
without further purification in pyridine (50 ml) and
concentrated to half its volume. After addition of 4,4'-
dimethoxytriphenylmethyl chloride (760 mg, 2.24 mmol)
and ethyldiisopropylamine (0.37 ml, 2.1 mmol), it was
stirred for 3.5 h at room temperature. After removing
pyridine by evaporation and re-steaming with toluene
(2 x 30 ml), the residue was dissolved in CH2C12-
acetone-Et3N (20:10:1, v/v/v, 5 ml) and chromatographed
on silica gel 60 (6 x 10 cm, 0.5 bar; mobile solvent:
CH2C12-acetone-Et3N, 20:10:1). Yield: 520 mg (43 ~)
colourless foam.
TLC (silica gel, CH2C12-acetone, Et3N, 20:10:1) Rf 0.48.
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for 6 in ppm): 8.93 (s, =CH-); 8.43 (s, H-C(8)); 8.34
(s, H-C(2)); 7.17-7.40 (m, DMT); 6.75-6.84 (m, DMT);
6.41 (m, H-C(1')); 5.75 (d, J = 5.0 Hz, 3'-OH); 4.33 (m,
H-C(3')); 4.20 (m, H-C(4')); 3.7 (m, 2 CH3 and H2-C(5));
3.20 and 3.13 (2 s, 2 OCH3); 2.78 (m, Ha-C(2')); 2.3 (d,
H~-C ( 2 ' ) ) .
210~~~~
- 22 -
Elemental analysis:
calculated: C 67.09; H 5.96; N 13.81
found: C 66.89; H 6.03; N 13.64
Example 6a
9-(2'-deoxy-f3-D-threo-pentofuranosyl)N6-benzoyladenine
2'-deoxy-xyloadenosine (100 mg, 0.40 mmol) is suspended
in dry pyridine (10 ml) and evaporated to a third of its
volume. Trimethylchlorosilane (0.25 ml, 2 mmol) is added
and it is allowed to stir for 15 minutes at room
temperature. Subsequently benzoyl chloride (0.23 ml,
2 mmol) is added and it is stirred for a further 2 hours
at room temperature. After cooling to 0°C and adding
0.5 ml H20, 25 % aqueous ammonia is added after a
further 5 minutes and it is stirred for a further 30
minutes at room temperature. After evaporating the
pyridine, the residue is dissolved in water and
extracted with ethyl acetate (10 ml). The organic phase
is concentrated by evaporation and admixed with pyridine
(3 ml) and 25 % aqueous ammonia (1 ml). After one hour,
it is concentrated by evaporation and chromatographed on
silica gel 60 (70 ml) using the mobile solvent ethyl
acetate/acetone/ethanol H20 (18:3:2:2) during which a
main product is eluted. The yield is 25 mg (60 %), the
melting point is 175 - 177°C.
TLC (silica gel, ethyl acetate/acetone/ethanol/H20,
18:3:2:2): Rf 0.4.
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
~~o~os~
- 23 -
for d in ppm): 11.2 (br, NH); 8.77 (s, H-C(8)); 8.70 (s,
H-C(2)); 8.05 and 7.60 (2 m, benzoyl-H); 6.47 (d, H-
C(1')); 5.55 (d, 3'-OH); 4.73 (t, 5'-OH); 4.42 (m, H-
C(3')); 4.00 (m, H-C(4')); 3.74 (m, H2-C(5')); 2.83 (m,
Ha-C(2')); 2.38 (m, HH-C(2')).
Elemental analysis:
calculated: C 57.46; H 4.82; N 19.71;
found: C 57.36; H 4.95; N 19.64.
Example 6b
9-[2'-deoxy-5'-O-(4,4'-dimethox~triphenyl)-f3-D-threo-
pentofuranosyll-N6-benzoyl-adenine
NBzdxA (280 mg, 0.79 mmol) is dried by evaporating with
pyridine (30 ml) and the residue is dissolved in
pyridine (15 ml). 4,4'-dimethoxytriphenylmethyl chloride
(315 mg, 0.93 mmol) and ethyl diisopropylamine (0.14 ml,
0.85 mmol) are added to this and stirred for 3 hours at
room temperature. Subsequently it is concentrated by
evaporation and re-vapourized several times with toluene
(50 ml) .
The residue is eluted on silica gel 60 (60 ml) with
CH2C12/acetone (12:5) to obtain a main zone from which
428 mg (82 %) of the title compound is obtained as a
colourless foam after removing the solvent by
evaporation.
TLC (CH2C12/acetone, 12:5): Rf 0.47.
2103062
- 24 -
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the 1H-NMR spectrum (results
for s in ppm): 11.2 (br. NH); 8.77 (s, H-c(2)); 8.07 -
6.77 (m, benzoyl- and DMT-H); 6.53 (d, H-C(1')); 5.51
(d, 3'-OH); 4.37 (m, H-C(3')); 4.28 (m, H-C(4')); 3.72
(m, 2 OCH and H2-C(5')); 2.79 (m, Ha-C(2')); ca. 2.5 (m,
H~-C ( 2 ' ) )
Elemental analysis:
calculated: C 69.39; H 5.36; N 10.65
found: C 69.48; H 5.84; N 10.51
Example 7
9-f2'-deoxy-5'-O-(4,4'-dimethoxytriphenylmethyl)-13-D-
xylopentofuranosyl]-6-j[(dimethylamino)methylidenel_
aminoladenine-3'(H-phosphonate) triethylammonium salt
1,2,4-triazole (0.94 g, 13.6 mmol) is added to a
solution of PC13 (360 ~cl, 4.1 mmol) and N-methyl-
morpholine (4.5 ml, 41 mmol) in CH2C12 (35 ml) and the
reaction mixture is stirred for 30 min at room
temperature. After cooling to 0°C, a solution of the
protected nucleoside from example 6 (480 mg, 0.79 mmol)
in CH2C12 (10 ml) is added dropwise and the solution is
stirred for 10 min at room temperature. Subsequently the
reaction mixture is poured into triethylammonium
bicarbonate buffer (1 M, pH 8, 50 ml), the phases are
separated and the aqueous phase is extracted twice with
CH2C12 (30 ml). The combined organic phases are dried
over Na2S04 and concentrated by evaporation. The residue
is chromatographed on silica gel 60 (6 x 10 cm, 0.5 bar,
mobile solvent: 600 ml CH2C12-Et3N, 92:8; CH2C12-MeOH-
Et3N, 88:10:2). After concentrating the main zone by
21030~~
- 25 -
evaporation, the residue is dissolved in CH2C12 (15 ml)
and extracted twice with Et3NH+HC03 (1 M, pH 8, 20 ml).
The organic phase is dried over Na2S04 and concentrated
by evaporation; yield 390 mg (64~) colourless foam.
TLC (silica gel, CH2C12-MeOH-Et3N, 88:10:2): Rf 0.65.
The product (dissolved in (D6)DMSO) is characterized by
the following signals in the iH-NMR spectrum (results
for 6 in ppm): 10.5 (s, br, NH); 8.95 (s, =CH-); 8.45
(s, H-C(8)); 8.41 (s, H-C(2)); 7.39-6.75 (m, DMT); 6.79
and 5.29 (P-H); 6.45 (m, H-C(1')); 4.79 (H-C(3')); 4.28
(m, H-C(4')); 3.71 (m, 2 CH3 and H2-C(5)); 3.20 and 3.13
(2 s, 2 OCH3); 2.96 (m, CH2); 2.5 (m, H2-C(2'));
1.10 (t, CH3).
Elemental analysis:
calculated: C 62.08; H 6.77; N 12.67
found: C 62.12; H 6.90; N 12.46
Example 7a
9- f 2' -deoxv-5' -O- ( 4 . 4' -dimethoxytriphenylmethvl ) -f3-D-
threo-pentofuranosyl]-N6-benzoyladenine-3'-(H-
phosphonate) triethylammonium salt
1,2,4-triazole (359 mg, 5.2 mmol) is added to a solution
of PC13 (136 ~,1, 1.56 mmol) and N-methylmorpholine
(1.72 ml, 15.6 mmol) in CH2C12 (13 ml). The solution is
stirred for 30 min at room temperature, subsequently
cooled to 0°C, and the completely protected nucleoside
(DMT-NBzdxA, 200 mg, 0.3 mmol, dissolved in CH2C12 (8
ml)) is added. After stirring for 30 min at room
temperature, the solution is poured into 1 mol/1 TBK
2~o~os~
- 26 -
buffer (pH 8, 20 ml) and the organic phase is separated.
After extracting the aqueous phase again with
dichloromethane (30 ml), the combined organic phases are
dried over Na2S04 and concentrated by evaporation. The
residue is chromatographed on silica gel 60 (50 ml).
31 mg (16~) of the starting material is eluted with
CH2C12-triethylamine (92:8); the desired H-phosphonate
is eluted with CH2C12-methanol-triethylamine (88:5:2).
After withdrawing the solvent, the residue is dissolved
in CH2C12 (30 ml) and extracted with 1 mol/1 TBK buffer
(pH 8, 30 ml). After drying the organic phase over
Na2S04, it is concentrated by evaporation. 158 mg (64 %)
of a colourless foam is obtained.
TLC (silica gel, CH2C12-MeOH-Et3N, 88:5:2): Rf 0.3.
31P-NMR ((D6)DMSO): 1.37 ppm (iJ(P-H) - 594 Hz; 3J(P-H-
3') - 8.7 Hz).
Elemental analysis:
calculated: C 64.22; H 6.25; N 10.21
found: C 64.47; H 6.48; N 10.51
Example 8
9-f2'-deoxy-5'-O-(4.4'-dimethoxytriphenylmethyl)-13-D-
xvlonentofuranosvl]-6-[[(dimethylamino)met~lidenel
amino]adenine-3'-[2-(cyanoethyl)-N,N-diisopropyl-
phosphoramiditel
Ethyl diisopropylamine (110 ~,1, 0.63 mmol) is added to a
solution of the protected nucleoside from example 6
(122 mg, 0.2 mmol) in dry THF (1.5 ml). Subsequently
chloro-f3-cyanoethyloxy-(N,N-diisopropylamino)phosphane
~~n~ss~
- 27 -
(50 ~1, 0.22 mmol) is added dropwise within 2 minutes
under N2. After the reaction mixture has been stirred
for 30 minutes at room temperature, aqueous NaHC03 (5 %,
4 ml) is added and extracted twice with CH2C12 (5 ml).
The combined organic phases are dried over Na2S04,
concentrated by evaporation and the residue is
chromatographed on silica gel 60H (3 x 6 cm, 0.5 bar,
mobile solvent: CH2C12-EtOAc-Et3N, 45:45:10); yield:
156 mg (96 %) colourless oil.
TLC (silica gel, CH2C12-EtOAc-Et3N, 45:45:10) Rf 0.50
and 0.53 (2 diastereoisomers).
31p_NMR ((D6)DMSO): 151.5, 146.7 ppm.
Example 9
i-f2'-deoxy-5'-O-(4,4'-dimethoxytriphenylmethyl)-3'-O-
succinyl-a-D-threo-pentofuranos~l]-thymine
70 mg 4-dimethylaminopyridine (0.54 mmol) and 230 mg
succinic anhydride (2.3 mmol) are added to a solution of
250 mg of the nucleoside from example 2 (0.46 mmol) in
10 ml dry pyridine and the mixture is stirred for 70
hours at 40°C. Afterwards 3 ml water is added to the
reaction mixture and it is evaporated to a dry residue.
Traces of pyridine are removed by co-evaporation with
toluene. The remaining oil is dissolved in CH2C12 and
extracted by shaking successively with 10 % aqueous
citric acid and water. The organic phase is dried over
sodium sulfate and concentrated by evaporation. The
residue is taken up in 2 ml of a mixture of CH2C12 and
pyridine (95:5) and the solution is slowly poured into
50 ml of a mixture of n-pentane/ether (1:1) while
21(13~fi~
- 28 -
stirring. The precipitate which comes down is filtered
and purified by means of flash chromatography on silica
gel 60 (column: 10 x 6 cm, acetonitrile-water (9:1)).
The desired compound (180 mg, 61 % of theoretical yield)
is obtained as the main fraction in the form of a
colourless powder.
TLC (silica gel, acetonitrile-water, 9:1) Rf 0.8.
The product is characterized by the following signals in
13C-~R (solution in (D6)DMSO) (results in ppm): 173.6,
171.2 (2 C=O); 163.8 (C-6); 158.3 (DMT); 150.5 (C-2);
144.7 (DMT); 135.5 (C-4); 135.4-126.9 (10 signals, DMT);
113.3 (quart.-C, DMT), 109.0 (C-5); 85.8 (DMT); 83.6 (C-
4'); 80.6 (C-1'); 72.3 (C-3'); 61.2 (C-5'); 55.1 (OCH3,
DMT); 40.6 (C-2'); 29.1, 29.0 (2 CH2); 12.3 (CH3-
thymine).
Example 10
Fractosil-bound 1-f2'-c~eoxy-5'-O-(4.4'-
8imethoxytriphenylmethyl)-3'-O-succinyl-f3-D-threo-
pentofuranosyl~ -thymine
40 mg 4-nitrophenol (0.29 mmol) and 60 mg N,N-
dicyclohexylcarbodiimide (0.3 mmol) are added to a
solution of 100 mg nucleoside from example 9 (0.16 mmol)
in 1 ml of a mixture of dioxan-5 % pyridine while
stirring at room temperature. After 2 hours, the
precipitated dicyclohexylurea is removed by filtration
and 200 mg Fractosil~ 200 (Merck, 450 ~Eq. NH2/g) and
1 ml dimethyldormamide are added to the filtrate. After
addition of 0.2 ml triethylamine, the suspension is
shaken for 4 hours at room temperature. Then 60 ~,1
2103Q6~
- 29 -
acetic anhydride is added and the shaking is continued
for a further 30 minutes. Afterwards the nucleoside
bound to the silica gel is filtered off, washed with
dimethylformamide, ethanol and ether and dried in a
vacuum.
The amount of nucleoside bound to silica gel was
determined as follows: 5 mg of the Fractosil support was
treated with 10 ml 0.1 M 4-toluenesulfonic acid in
acetonitrile. The supernatant was measured
spectrophotometrically at 498 nm during which 50.4 wmol
nucleoside per g Fractosil was found (EpMT - 70000).
Example 10a
Fractosil-bound 9-[2'-deoxy-5'-O-(4,4'-
dimethoxytriphenylmethyl)-b-D-xylopentofuranosyl]-6-
ffdimethylamino)methylidenelamino]adenine
N,N-dimethylaminopyridine (60 mg, 0.49 mmol) and
succinic anhydride (200 mg, 2 mmol) are added to a
solution of the completely protected nucleoside from
example 6 (250 mg, 0.41 mmol) in pyridine (10 ml) and
the solution is stirred for 72 hours at 40°C. After
addition of water (3 ml) it is evaporated to dryness and
the residue is re-vapourized with toluene (50 ml). It is
dissolved in CH2C12 and extracted with 10 % aqueous
citric acid solution (30 ml) and water (30 ml). The
organic phase is dried over Na2S04 and concentrated by
evaporation. 278 mg (96 %) of a colourless material is
obtained which is reacted without further purification.
The succinate (142 mg, 0.20 mmol) is dissolved in a 5
solution of pyridine in dioxane (1.25 ml) and admixed
with p-nitrophenol (50 mg, 0.36 mmol) and dicyclohexyl-
2103afi2
- 30 -
carbodiimide (80 mg, 0.40 mmol) at room temperature. It
is allowed to stir for 3 hours at this temperature and
subsequently the precipitated dicyclohexylurea is
filtered off. Subsequently dimethylformamide (1.25 ml)
and Fractosil 200 (450 ~eq NH2/g) are added. After
addition of triethylamine (250 ~,1), the suspension is
shaken for 4 hours at room temperature and subsequently
acetic anhydride (75 ~1) is added. The shaking is
continued for 30 minutes, the nucleoside bound to the
silica gel is filtered off, washed with
dimethylformamide, ethanol and ether and dried in a
vacuum. The method described in example 10 was used to
determined the nucleoside bound to the silica gel; the
concentration of the nucleoside bound to the silica gel
is 27 ~mol/g Fractosil.
Example 11
Solid chase synthesis of the oliqonuoleotide d(xTl2~
The synthesis of the oligomer was carried out on a
1 ~mol scale using the 3'-hydrogenphosphonate from
example 3. The synthesis followed the standard protocol
of the DNA synthesizer for the 3'-H-phosphonate method
(Applied Biosystems Users Manual for the DNA Synthesizer
380 B) .
The 4,4'-dimethoxytrityl protecting group of the
oligomer was removed by treatment with 80 ~ acetic acid
for 5 minutes at room temperature. It was purified by
HPLC on a RP-18 column and a gradient system of 0.1 M
Et3NHOHAc pH 7/acetonitrile (95:5) [A] and acetonitrile
[B]. The pure oligomer was desalted on a 4 x 25 mm HPLC
21~~Q62
- 31 -
cartridge (RP-18 silica gel) and subsequently
lyophilized.
Example iia
Solid phase synthesis of the oliQOnucleotide
d(GTAGxAxAxCTAC)
The synthesis of the oligonucleotide was carried out on
a 1 ~,M scale using the nucleoside-3'-O-(2-cyanoethyl)-
N,N-diisopropylamino-phosphoramidites of the bases A, G,
C and T as well as the corresponding phosphoramidites of
the 3'-xylo-nucleosides xT and xA from examples 4 and 8.
The synthesis followed the standard protocol of the DNA
synthesizer for the phosphoramidite method (Applied
Biosystems Users Manual for the DNA synthesizer 380 8).
The NH2 protecting groups were cleaved with 25 ~ aqueous
ammonia solution at 60°C for 48 hours. The removal of
the 5'-O-dimethoxytrityl protecting groups, the
purification by HPLC and the desalting were carried out
as described in example 11.
Example 12
Solid phase synthesis of further olig~onucleotides
The solid phase synthesis of the oligomers d(xTCG xTCG
CxTG xTCxT CCG CxTxT CxTxT CCxT GCC xA) and
d(GxTxAGAATTCxTxAC) was carried out in an anlogous
manner to that described in example 11.
2~o~osz
- 32 -
Example 12a
Phosphodiesterase cleavage of the oliqomer
d ( GTAGAAxTxTCTAC ) ( I~' ig' . 2 )
0.2 A260 units of the oligonucleotide was dissolved in
200 ~,1 0.1 M Tris-HC1 buffer (pH 8.3) and treated for 45
minutes at 37°C with 12 ~g calf spleen phosphodiesterase
(Boehringer Mannheim, Catalogue No. 108 251) and
subsequently for 30 minutes at 37°C with 2 ~g alkaline
phosphatase (Boehringer Mannheim, Catalogue No. 108
154 ) .
Afterwards the reaction mixture was analyzed by HPLC
(RP-18, eluting agent 0.1 M (Et3NH)OAc (pH
7)/acetonitrile (95:5) [A], followed by 0-20 °s
acetonitrile [B] in [A], flow rate ca. 1 ml/min.).
It turns out that the oligomer is only partially
hydrolyzed; afterwards the phosphodiesterase only
cleaves off the non-modified 5'-terminal nucleotides
with a t/2 value of 5.5 min. (hypochromicity of the
final product 9 ~, TM=36°C). As the HPLC profile
(inserted figure) shows, the major portion of the
oligonucleotide is present in an unchanged form.
Example 12b
Phosphodiesterase cleavage of the oligomer
d(GxTxAGAATTCxTxAC)
The cleavage of this oligonucleotide by calf spleen
phosphodiesterase was examined in an analogous manner to
2103062
- 33 -
that set forth in example 12 a. In this case only a
very slow and negligible hydrolysis is observed (hypo-
chromicity of the final product 3$, TM = 27°C).
Example 13
Hybridization complex d(xTl2) with d(A12Z
a) Melting curves
2 ~mol of each of the oligonucleotides was used for
this. The measurement was carried out in a thermo-
statically controlled cell using a Shimadzu 210-A UV
spectrophotometer in conjunction with a "Kipp and
Zonen BD 90" (trademark) recorder. The increase in UV
absorbance at either 260 or 284 nm was recorded as a
function of time while the temperature of the solution
was increased linearly (10-80°C) by 20°/hour (Lauda
PM-350 (trademark) programmer combined with a Lauda
RCS 6 (trademark) bath with R 22 unit). The
respective temperature was measured in the reference
cell using a Pt resistor. The hypochrocnicity values
were calculated from the initial and final absorbance.
The melting curves obtained in this way are shown in
Fig. 3. Those for the hybrid d(A12) / d(xTl2) (b)
have a shape which is characteristic for a cooperative
melting of double strands (cf. curve (a) for d(al2) /
d(T12)) which is not exhibited by the single -stranded
homopolymers (dCAl2) (c) and d(T12) (d).
b) CD spectroscopy (Fig. 3)
2 ~.mol of each of the respective single strands (dAl2)
and d(T12) or d(xTl2) were hybridized (60 mM cacody-
late buffer pH 7.0, 1 M NaCl, 100 mM MgCl2, 8°C). A CD
2103062
- 3~1 -
spectrum o1 the llybr ids obtained a.; well a:: «l: the
single strands c~.('1'l~ ) , d ( x'1'l~ ) arld a (Al J ) mu.l oI: tlic
monomers '1' and x'f was recorded on a Jasc:o ~~W .~ ( trademark )
spectropolari.meter w.itli a thermally control lc~d (Lauda
RCS G ) 1 cm cuvette . 'flie spectra obta fined X11."c'_ S11UW11 111
f figures 4 alld 5 .
It is appparent that the CD spectra of the hybrids
differ clearly from the sums and differences of the
single strands. 'This shows that a new type of double-
stranded hybrid structure is formed.
Example 14
Determination of viral gene expression of IIIV-I infected
H 9 cells
Chronically infected li 9 (A'fCC CRL 8543) cells were
cultured in the presence of various concentrations of
the oligomer d(x'fCG x'fCG Cx'fG x'fCx'f CCG Cx'fx'f Cx'fx'f CCx'f
GCC xA) from example 12 in 200 ~,1 culture medium (RPMI
1640 containing 15 o foetal calf serum, 4 nlnaol
L-glutamine, 50 nmol 2-mercaptoetlianol, 50 units
penicillin and 50 ~g streptomycin/ml). After G days
culture, 100 ~,1 of the culture supernatant was removed
and the amount of p24 gag protein was determined by
means of radioimmunoassay.
Whereas in the control experiment (without oligomer) the
amount of p24 gag protein increased within 5 days to
values around 200 ng/ml, protein synthesis when using
the oligomer was 50 ng/ml to ca. 5 Ilg/Inl dependlIlg on
the dose.
A
21~3~fi~
- 35 -
Example 15
2.6 Diamino-9-(f3-D-ribofuranosyl)purine (2) jij
5.66 g (20 mmol) guanosine (1) (dried at 80°C/P205) is
silylated by heating for 10 hours to 140°C with 200 ml
hexamethyldisilazane (HMDS) and 0.5 ml
trimethylchlorosilane (TCS); excess HMDS is subsequently
removed at normal pressure by distillation and the
remaining viscous syrup is transferred with a mixture of
35 ml absolute toluene and 2 ml HMDS into a 100 ml
autoclave. After addition of 4 ml of a 0.5 M solution
(2 mmol) of trimethylsilyl triflate ((CH3)SiS03CF3) in
benzene, a pressure of 5 atm NH3 is applied at +5°C for
30 min; the autoclave is then heated for 48 h to 150°C.
After carefully discharging the ammonia, the content of
the autoclave is washed out with 150 ml methanol and,
after addition of 150 ml water, heated for 4 h on a
steam bath. After removing the methanol, it is diluted
with water to 250 ml, the boiling solution is
decolourized with a small amount of active charcoal and
filtered during which it is rewashed with 100 ml hot
water. After concentration to 250 ml, the yellow
filtrate crystallizes slowly on cooling and standing at
24°C. After filtration and further evaporation of the
mother liquor to 100 ml, 4.23 g (75.5%) colourless
crystals of 2 [1] is obtained.
TLC (silica gel, mobile solvent EtOAc-MeOH; 60:40,
Rf = 0.55)
~io~o6z
- 36 -
Example 15
2.6-Diamino-9-(2-O-p-toluenesulfonyl-!3-D-
ribofuranosyl ) purine ( 3 y f 2 ~,
2.40 g (2) and 2.2 g dibutyl-tin oxide are suspended in
200 ml MeOH and boiled for 2 h under reflux during which
a clear solution is slowly formed. After cooling to room
temperature and addition of 18 ml triethylamine, 24 g
p-toluenesulfonic acid chloride is slowly added and
subsequently it is stirred for a further 15 min. Excess
solvent is withdrawn at 40°C and the residue is taken up
with 200 ml water. After extraction with ether (2 x
100 ml), the aqueous phase is evaporated to 100 ml and
stored overnight at 0°C. The crude product which
precipitates in this process is collected and purified
by column chromatography (silica gel, mobile solvent B).
The mother liquor is evaporated to dryness, adsorbed
onto silica gel and also chromatographed. 2.85 g (7
of compound (3) [2] is obtained. F.p. - 134°C; TLC
(silica gel, mobile solvent EtOAc-MeOH, 85:15) Rf =
0.45; 1H-NMR ([D6]DMSO): 7.82 (s, H-C(8)); 6.83 (s,
NH2); 5.91 (d, J(H-C(1'),H-C(2') - 7.5 Hz; H-C(1'));
5.67 (s, NH2); 5.41 (m, Ha-C(2')); 4.29 (m, H-C(3'));
4.01 (H-C(4')); 3.55 (m, CH2); 3.17 (d, HB-C(C2')); 2.29
(s, -CH3).
Example 16
2'-amino-(2'-deoxy-f3-D-threo-pentofuranosvl)adenine (4)
A 1 M solution of LiEt3BH in THF (34 ml) is added to a
solution of (3) (1.4 g; 2.75 mmol) in 30 ml dry DMSO
210362
- 37 -
under an argon atmosphere. The resulting colourless
solution is stirred overnight at room temperature and
7 ml water is added dropwise over a period of 30 min.
Subsequently the solvent is withdrawn and the residue
is desalted on a Dowex (trademark) 1x2 (OH-) column (4
x 30 cm) with 500 ml water. The reaction product can
now be eluted from the column with a MeOH/water
mixture (1:1) and after evaporation with appropriate
fractions can be obtained. Recrystallization from
water (100 ml) yields 760 mg (98~) pure (4) [3]. F.p.
- 175°C; 1H-NMR ([D6]DMSO): 7.95 (s, H-C(8)); 6.78 (s,
NH2); 6.07 (d, H-C(1')); 5.81 (s, NH2); 4.65 (m, OH-
C(5')); 4.30 (br, H-C(3')); 3.84 (m, H-C(4')); 3.72
(m, CH2-C(5')); 2.73 (m, Ha-C(2')); 2.23 (d, J(Ha-
C(2' ), H~j-C(2' ) )_ -14.5 Hz; H~j-C(2'
Example 17
(2'-deoxv-(3-D-threo-pentofuranosyl)quanine (5)
Compound (4) is dissolved in 100 ml water while heat-
ing and, after cooling to ca. 30°C, adenosine deamin-
ase (200 ~,1) is added. After the reaction mixture has
been stirred overnight at 30°C, the reaction course is
monitored by means of TLC and UV spectroscopy. After
withdrawing the solvent, the reaction product is
evaporated several times with acetone and finally 650
mg (920) (5) is obtained. F.p. > 250°C; TLC (silica
gel, mobile solvent iPrOH-25~NH3aq-H20; 3:1:1): Rf =
0.61. 1H-NMR ([D6]DMSO): 7.95 (s, H-C(8)); 6.49 (s,
NH2); 6.05 (d,H-C(1')); 5.43 (s, OH-C(3')); 4.67 (s,
OH-C(5')); 4.33 (d, H-C(3')); 3.87 (s, H-C(4')); 3.65
(m, CH2-C(5')); 2.70 (m, Ha-C(2')); 2.20 (d, H~j-
C(2')).
A
210~06~
- 38 -
Example 18
N2,3',5'-triisobutyryl-(2'-deoxy-f3-D-threo-
pentofuranosyl)qwanine (6)
500 mg (5) is dried by evaporating several times with
absolute pyridine and dissolved in 20 ml pyridine while
heating gently. After cooling to 0°C in an ice bath,
1.95 ml (18.5 mmol) isobutyryl chloride is added slowly
under nitrogen while stirring vigorously. The turbid,
gelatinous reaction mixture is stirred for a further
1 hour at room temperature and subsequently hydrolyzed
with a solution of 2.5 g NaHC03 in 40 ml H20. After
concentrating the solution to ca. 20 ml and allowing it
to stand at 0°C overnight, the reaction product is
aspirated and re-washed with 20 ml ether. After column
chromatography using silica gel (5 cm x 30 cm), mobile
solvent CHC13-MeOH; 8:2, 820 mg amorphous (6) [4] is
obtained. F.p. - 91°C, TLC (mobile solvent CHC13-MeOH;
8:2) Rf = 0.40: 1H-NMR ([D6]DMSO): 12.05 (br, NH); 11.70
(br, NH); 8.14 (s, H-C(8)); 6.14 (d, H-C(1')); 5.30 (s,
OH-C(3')); 4.68 (s, OH-C(5')); 4.36 (s, H-C(3')); 3.94
(m, H-C(4')); 3.72 (m, CH2-C(5')); 2.77 (m, Ha-C(2'));
2.28 (d, H~-C(2')); 1.13 (m, 7H-ibut.); C22H31N507
(477.51): calc.: C 55.34, H 6.54, N 14.67;
found: C 55.18, H 6.57, N 14.73.
Example 19
N2-isobutyryl-(2'-deoxy-I3-D-threo-pentofuranosyl)guanine
1.2 g (2.5 mmol) (6) is dissolved in 40 ml MeOH and
cooled to 0°C in an ice bath. Subsequently 1 N aqueous
2103x62
- 39 -
NaOH solution is added in portions until the pH value
increases to 12. After 50 min, the reaction is stopped
by addition of ion exchanger (Dowex W x 8 ; pyridinium
form) and the neutral solution is filtered. After
washing the resin with MeOH, the solvent is withdrawn
and the residue is re-crystallized from a very small
amount of water. After completing the crystallization by
allowing it to stand overnight in a refrigerator, 560 mg
(72 ~) white needles of compound (7) is obtained.
F.p. > 260°C; TLC (silica gel, mobile solvent CHC13-
MeOH, 9:1) Rf = 0.42; 1H-NMR ([D6]DMSO): 12.05 (br, NH);
11.70 (br, NH); 8.20 (S, H-C(8)); 6.14 (d, H-C(1'));
5.30 (s, OH-C(3')); 4.68 (s, OH-C(5')); 4.36 (s, H-
C(3')); 3.94 (m, H-C(4')); 3.72 (m, CH2-C(5')); 2.77 (m,
Ha-C(2')); 2.28 (d, HH-C(2')); 1.13 (m, 7H-i-but.);
C14H19N505 (337.3): C 49.85, H 5.68; N 20.67;
found.: C 49.98, H 5.81, N 20.75.
Example 20
N2-isobutyrvl-5'-O-dimethoxytrityl-(2'-deoxy-13-D-threo-
pentofuranosyl)g~uanine (8)
300 mg (7) (0.89 mmol) is dried by evaporating several
times with absolute pyridine and subsequently dissolved
in 5 ml absolute pyridine. After addition of
dimethylaminopyridine (16 mg, 0.13 mmol) 150 mg
(0.4 mmol) 4',4'-dimethoxytrityl chloride is added under
argon. After stirring for 4 hours at room temperature
and subsequent addition of 45 ml 5 ~ NaHC03 solution, it
is extracted with CH2C12 (3 x 50 ml). The combined
organic phases are dried over Na2S04, filtered and the
solvent is withdrawn. After chromatography on silica gel
(20 x 5 cm, mobile solvent CHC13-MeOH, 8:2) and
evaporation of the main zone, 423 mg (74 ~) amorphous
z~o3o6z
- 40 -
(8) is obtained. TLC (silica gel, mobile solvent CHC13-
MeOH, 8:2) Rf = 0.38
iH-NMR ([D6]DMSO): 12.09 (br, NH); 11.77 (br, NH); 8.07
(s, H-C(8)); 6.22 (d, H-C(1')); 5.30 (s, OH-C(3')); 4.34
(s, H-C(3')); 4.21 (s, H-C(4')); 3.71 (s, 3H-OCH3); 3.20
(m, H-C(5')); 2.74 (m, Ha-C(2')); 2.30 (m, HH-C(2'));
1.12 (m, 7H-i-but.);
Example 21
N2-isobutyryl-5'-dimethoxytrityl-(2~-deoxy-f3-D-threo-
pentofuranos~l)gwanine 3~-phosohonate (9a)
The compound was prepared and processed as described for
compound 16.
Example 22
N2-isobutyryl-5'-dimethoxytrityl-(2'-deoxy-f3-D-threo-
pentofuranosyl)guanine 3'x(2-cyanoethvl)-N,N-
diisopropylphosphoramidite [9b1 ,
Compound 9b was prepared and processed as described in
[6].
Example 23
9-(2~-deoxv-13-D-threo-pentofuranosyl)g~uanine 3~-j3-
~N-'Fractosil'carbamoyl)propanoate] j9c]
The compound 9c was prepared and processed as described
in [6].
213~62
- 41 -
Example 24
4-benzovlamino-1-(2'-deoxy-A-D-ervthro-pentofuranosyl)-
2(iH)-pyrimidinone (10)
Compound 10 was prepared as described by Ti et al.
Example 25
02,3'-anhvdro-1-~2'-deoxy-5'-O-(4-methoxybenzo~l)-13-D-
threo-centofuranosyl)-4-benzoylcytosine (ii) (3~
A solution of diisopropylazodicarboxylate (DIAD, 3 ml,
15 mmol) and p-methoxybenzoic acid (2.3 g, 15 mmol) in
dry DMF (11 ml) is added dropwise to a solution of (10)
(3.3 g, 10 mmol) and PPh3 (4 g, 15 mmol) in dry DMF
(20 ml) within 5 minutes. The reaction mixture is
allowed to stir for 15 min at room temperature and
subsequently admixed with the same amount
triphenylphosphine and DIAD. It is allowed to stir for a
further 30 minutes at room temperature. Subsequently the
solution is poured into ice-cooled Et20 (250 ml) and the
suspension which is obtained is cooled for 2 h. The
white residue is filtered off and washed with Et20.
Compound (11) is obtained by re-crystallization from
ethanol as colourless platelets (3.42 g, 76 %) melting
point 188°C, Rf (CH2C12/CH30H): 0.38. W ((MeOH): ~ max
(E) - 318, 254 (30440, 17150). 1H-NMR [(D6)-DMSO]: 2.59-
2.71 (m, Jgem = 12.5 Hz, 2H, H-2'J3, H-2'a); 4.41 (m, 2H,
H-5'); 4.58 (m, 1H, H-4'); 5.47 (m, 1H, H-3'); 6.00 (m,
1H, H-1'); 6.52 (d, J5,6 = 7.25 Hz, 1H, H-5); 7.01, 7.84
(AB, q, 4H, C6H4); 7.68 (d, 1H, H-6); 7.45, 7.95 (m, 5H,
arom. H). C24H21N307 (447.4): calc: C 64.44, H 4.73, N
9.39; found: C 64.49, H 4.71, N 9.31.
2~O~Q~~
- 42 -
Example 26
4-aminoi-i- (2' -deoxy-li-D-threo pentofuranosyl) -2 ( iH)-
pyrimidinone tit)
A solution of (11) (2.0 g, 4.5 mmol) dissolved in
ethanol/water (1:1, 250 ml) is admixed with Dowex ion
exchanger (OH-form; 250 ml, suspended in water). It is
allowed to stir for 16 h at 50°C. The ion exchanger is
filtered off and washed intensively with water. The
filtrate and slurry are concentrated by evaporation, the
oily residue is taken up in a small amount of
methanol/ethyl acetate (1:1, 50 ml) and concentrated in
an oil pump vacuum. Compound (12) is obtained as a
colourless foam (965 mg, 95 %). The W data for compound
(12) were identical to the data for (11). 1H-NMR [(D6)-
DMSO]: 1.82 (m, J2'f3,2'a = 14.5 Hz, H-2'l3); 2.57 (m, 1H,
H-2'a); 3.68 (m, 2H, H-5'); 3.81 (m, 1H, H-4'); 4.23 (m,
1H, H-3'); 4.73 (br s, 1H, 5'-OH); 5.19 (br s, 1H, 3'-
OH); 5.73 (d, J5,6 = 7.25 Hz, 1H, H-5); 6.02 (m, 1H, H-
1'); 7.11 (br s, 2H, NH2); 7.84 (d, iH, H-6).
Example 27
1-(4-benzoylamino-2'-deoxy-l3-D-threo-pentofuranosyll-
cytosine (13) [41
Nucleoside 12 (454 mg, 2 mmol) which has previously been
dried three times by evaporation with dry pyridine, is
suspended in absolute pyridine (10 ml) and admixed with
(CH3)3SiC1 (1.28 ml, 10 mmol) under argon. The solution
is stirred for 15 minutes, afterwards benzoyl chloride
(0.87 ml, 10 mmol) is added and the solution is stirred
for 2 h at room temperature. Subsequently the mixture is
~~o~c~~~
- 43 -
cooled in an ice bath and water (2 ml) is added. After
addition of 25 % ammonia solution (2 ml) it is stirred
for a further 15 minutes at room temperature. The
solution is evaporated almost to dryness, the residue is
dissolved in water (28 ml) and washed with ethyl acetate
(10 ml). After concentrating the aqueous phase by
evaporation, product (13) begins to crystallize out on
cooling. White needles are obtained by re-
crystallization from methanol. (450 mg, 68 %) melting
point: 177°C (MeOH). TLC (CH2C12/CH30H 9:1): Rf 0.36. UV
(MeOH):~.max (E) - 302, 258 (10500, 22230). C16H17N305
(331.70): calc.. C 58.01, H 5.17, N 12.68; found: C
58.14, H 5.29, N 12.71.
1H-NMR [(d6)-DMSO]: 2.01 (d, JH-2'f3, H-2'a = 14.5 Hz,
iH, H-2'!3,); 2.57 (m, iH, H-2'a); 3.78 (m, 2H, H-5', H-
5 "); 3.98 (m, iH, H-4'); 4.35 (m, iH, H-3'); 4.76 (t,
J5'-OH, H-5', H-5 " - 5.5 Hz, 1H, 5'-OH); 5.09 (d, J3'-
OH, H-3'= 2.75, 1H, 3'-OH); 6.00 (dd, J = 7.25 Hz, 1H,
H-1'); 7.31 - 8.02 (m, 5H, arom. H); 7.63 (d, JH-5,H-6 =
7.5 Hz, 1H, H-6); 8.28 (d, JH-6; H-5= 7.5 Hz, 1H, H-6);
11.19 (brs, 1H, NH).
Example 28
1-f5'-0,4-N-Bis(4,4'-dimethoxytrityl)-2'-deoxy-13-D-
threo-pentofuranosyl]cytosine (14a)
Dry (12) (520 mg, 2.2 mmol) which can be obtained by
evaporation with dry pyridine, is dissolved in absolute
pyridine (10 ml). 4,4'-dimethoxytrityl chloride (1.5 g,
4 mmol) and 4-dimethylaminopyridine (150 mg, 1.26 mmol)
is added to this solution.
2103p~2
- 44 -
After five hours the TLC shows almost no more starting
material. The reaction mixture is poured into cold
saturated sodium hydrogencarbonate solution (50 ml) and
extracted with three portions of ethyl acetate (30 ml).
The collected organic phases are concentrated by
evaporation and the residue is purified by column
chromatography (column 6 x 30 cm, 0.1 bar,
CH2C12/MeOH/Et3N at first 93:5:2, then 88:10:2).
The suitable fractions of the non-polar substance (14a)
are collected, the solvent is removed by evaporation,
the residue is dissolved in a small amount of
dichloromethane and added dropwise while stirring to
petroleum ether as a result of which (14a) precipitates
as a white solid. (680 mg, 40%).
TLC (CH2C12/CH30H 9:1): Rf 0.46. W (MeOH): ~ max (E) -
280, 230 (18100, 43000). 1H-NMR [CDC13]: 2.17 (d, JH-
2'~, H-2'a = 14.3 Hz, 1H, H-2'~,); 2.52 (m, 1H, H-2'a);
3.46 (m, 2H, H-5', H-5 "); 3.74 (s, 12H, OCH3); 3.96 (m,
1H, H-4'); 4.35 (m, 1H, H-3'); 4.99 (d, JH-5, H-6 = 7.75
Hz, 1H, H-5'); 6.01 (dd,J = 6.25 Hz, 1H, H-1'); 6.75 -
7.38 (m, 26H, arom.H); 7.51 (d, JH-5,H-6 = 7.75 Hz, 1H,
H-6). C51H49N308 (831.96): calc.. C 73.63, H 5.94, N
5.05; found: C 73.62, H 6.08, N 5.06.
Example 29
1-f5'-O-(4.4'-dimethoxytrit_yl)-2'-deoxy-~-D-threo-
pentofuranosyl] cytosine (14b) [21
The fractions of the polar substance (14b) are also
collected, the solvent is removed by evaporation, the
residue is taken up in a small amount of dichloromethane
~~03062
- 45 -
and added dropwise while stirring to petroleum ether as
a result of which (14b) precipitates as a white solid.
(580 mg, 55 %). TLC (CH2C12/CH30H 9:1) Rf 0.36. W
(MeOH):/~,max (e) - 234, 274 (22200, 8970). 1H-NMR [(d6)-
DMSO]: 1.80 (d, JH-2'!3, H-2'a = 14.5 Hz, 1H, H-2'f3,);
2.50*) (m, 1H, H-2'a); 3.18 (m, 2H, H-5', H-5 "); 3.74
(s, 6H, OCH3); 4.06 (m, 1H, H-4'); 4.15 (m, 1H, H-3');
5.12 (d, J3'-OH,H-3' - 3.70 Hz, iH, 3'-OH); 5.64 (d, JH-
5,H-6 = 7.43 Hz, 1H, H-1'); 6.04 (dd,J = 6.5 Hz, 1H, H-
1'); 6.87 - 7.44 (m, 15H, arom. H); 7.66 (d, JH-5, H-6
- 7.43 Hz, 1H, H-6). C30H31N306 (529.59): C 68.04, H
5.90, N 7.93; found: C 68.03, H 5.93, N 7.89.
Example 30
1-f4-benzoylamino-2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-
f3-D-threo-pentofuranosyl~ aytosine (15)
Method A [8]: compound 13 (270 mg, 0.8 mmol) is dried by
evaporation with dry pyridine. The residue is dissolved
in absolute pyridine (10 ml). DMT-C1 (0.6 g; 1.8 mmol)
and 4-dimethylaminopyridine (200 mg; 1.8 mmol) is added
under argon and the solution is stirred for 4 hours at
room temperature. The solution is shaken in 5 % NaHC03
solution (27 ml), extracted twice with dichloromethane
(50 ml), the collected organic phases are dried over
Na2S04 and the solvent is removed by evaporation. After
repeated evaporation with toluene, the residue is
purified by flash chromatography (column 6 x 20 cm,
0.1 bar, CH2C12/MeOH/Et3N 93:5:2). The product (15) is
precipitated from petroleum ether as a white powder
(330 mg, 64 %).
z~o3~s~
- 46 -
Method B: After the processing (without column
chromatography), the reaction mixture of (14a) and (14b)
is dissolved in absolute pyridine (10 ml). (CH3)3SiC1
(1.28 ml, 10 mmol) is added to the solution under argon.
After 15 minutes benzoyl chloride (1.16 ml, 10 mmol) is
added and the reaction mixture is stirred for three
hours at room temperature. After cooling to 0°C water,
(2 ml) is added and after a further 5 minutes 25 %
ammonia solution (4 ml) is added. After stirring for 30
minutes at room temperature, the solution is
concentrated by evaporation and the rubber-like residue
is extracted in dichloromethane (20 ml) and 5 % NaHC03
solution (40 ml). The aqueous phase is extracted with 2
portions dichloromethane, the organic phases are
combined and the solvent is removed by evaporation. The
isolation is carried out in the same manner as described
above. (530 mg, 36.5%)
TLC (CH2C12/MeOH 9:1):Rf 0.54. W (MeOH):~,max (e) - 301,
256, 236 (14540, 28880, 37040). 1H-NMR [(d6)-DMSO]: 2.00
(d, JH-2'B, H-2'a = 14.5 Hz, 1H, H-2'!3,); 2.50*) (m, 1H,
H-2'a); 3.39 (m, 2H, H-5', H-5 "); 3.75 (s, 6H, OCH3);
4.23 (m, 2H, H-4', H-3'); 5.07 (d, J3'-OH,H-3' - 2.50
Hz, 1H, 3'-OH); 6.05 (dd,J = 7 Hz, 1H, H-1'); 6.89 -
7.62 (m, 18H, arom.H); 7.53 (d, JH-5,H-6 = 7.43 Hz, 1H,
H-1'); 8.00 (d, JH-5,H-6= 7.43 Hz, 1H, H-6); 11.20 (brs,
1H, NH); C37H35N307 (633.70): C 70.13, H 5.57, N 6.63;
found: C 70.15, H 5.64, N 6.46.
~1030~2
- 47 -
Example 31
1-f4-benzovl-2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-f3-D-
threo-Dentofuranosyl]cytosine-(3'-hydrogen phosphonate)
16
PC13 (500 ~,1; 5.75 mmol) and 1,2,4-triazole (1.35 g;
19.4 mmol) are added to a solution of N-methylmorpholine
(6.5 ml; 57.5 mmol) in 23 ml absolute dichloromethane
under argon. The solution is stirred for 30 minutes,
after which it is cooled to 0°C and admixed with (15)
(400 mg; 0.63 mmol), which had previously been dried by
evaporation with absolute acetonitrile, dissolved in
7 ml absolute dichloromethane and added slowly dropwise
at room temperature. After 20 minutes stirring at room
temperature, the solution is poured into 32 ml TBK
buffer (pH 7.5 - 8.0) and the phases are separated. The
aqueous phase is extracted 4 times with dichloromethane
(15 ml), the pooled organic phases are dried over
Na2S04, filtered and the solvent is removed by
evaporation. The light yellow foam is chromatographed
(silica gel column 6 x 20 cm, 0.5 bar, 1 litre
CH2C12/Et3N 98:2, CH2C12/MeOH/Et3N 88:10:2). The residue
of the main zone is dissolved in CH2C12, extracted by
shaking 10 times with 0.1 M TBK buffer (10 ml), the
aqueous phase is reshaken several times with CH2C12, the
collected organic phases are dried over Na2S04 and the
solvent is removed by evaporation. The H-phosphonate is
present as a colourless foam (260 mg; 51%).
TLC (CH2C12/MeOH/Et3N 88:10:2): Rf 0.35. iH-NMR [CDC13]:
1.18 (t, J = 7.25 Hz, 9H, 3xCH3CH2NH); 2.47 (d, J H-2'a,
H-2'!3 = -l5Hz, iH, H-2'!3); 2.62 (m, 1H, H-2'a); 2.90 (q,
J = 7.25 Hz, 6H, 3xCH3CH2NH); 3.55 (m, 2H, 5'-CH2); 3.77
(s, 6H, OCH3); 4.27 (m, 1H, H-4'); 4.78 (m, 1H, H-3');
5.31, 7.81 (d, JH-P = 625 Hz, 1H, H-P); 6.16 ("dd", J =
z~o3o6z
- 48 -
6.75 Hz, 1H, H-1'); 6.80 - 7.90 (m, 18H, arom H); 7.21
(d, JH-5,H-6 = 7.25 Hz, 1H, H-5); 8.08 (JH-6,H-5 =
7.25 Hz, 1H, H-6). 31P-NMR [d6-DMSO]: 0.60 1J(P,H =
590 Hz; 3J(P,H-C4' - 8.4 Hz). 31P-NMR [CDC13]: 3.30
1J(P,H = 625 Hz; 3J(P,H-C4' - 8.9 Hz).
Example 32
1-f4-benaovl-2'-deoxy-5'-O-(4,4'-dimethoxvtrityl)-fi-D-
threo-oentofuranosyl~ cytosine-3'-j(2-cyanoethyl)-N,N-
diisoDropylphosphoramidite,lisbl
Compound 16b was prepared and processed as described in
[6].
Example 33
i-f2'-deoxv-b-D-threo-pentofuranosyl)c~sosine 3'-[3-(N-
'Fractosil'carbamoyl)propanoate,> [16c~
Compound 16c was prepared and processed as described in
[6].
Literature references for examples 15 - 33
[1] H. Vorbriiggen, Liebigs Ann. Chem.
K. Krolikiewicz,
1976, 745.
[2] M. J. Robins, .G. Wood, N. Dalley,
S K.
P. Herdewijn, Balzarini, deClercq, J. Med.
J. E.
Chem. 1989, 32, 1763.
[3] S. Czernecki, M. Valery,
J. Synthesis
1991, 239
[4] G. S. Ti, B. L. Gaffney, R. Jones, J. Amer.
A.
Chem. Soc. 1982, 104, 1315.
2~0306~
- 49 -
[5] B. C. Froehler, P. G. Ng, M. D. Mateucci Nucleic
Acids Res. 1986, 14, 5399.
[6] H. Rosemeyer, F. Seela, Helv. Chim. Acta 1991, 74,
748.
Example 34
Inhibition of HIV virus expression by
oliconucleotides according' to the present invention
5'- xTxTT CCC AGG CTC AGA TCT GGT CxTxT T*- 3' (tar region)
5'- xTxTT CGT CGC TGT CTC CGC TTC TTC CTG CCA xTxTT*- 3'
(rev region)
5'- xTxTC TGC TAG AGA TTT TCC ACA CxTxT T*- 3'
(primer binding
site)
* due to the commercial availability of the CPG carrier
material, it was started with unmodified 2'-deoxy-ribo-
thymidine.
Virus HIV III B H 9
Stock solutions of oligomers: 1 mg x ml-1 in PBS buffer
Dilution series of the oligomers: 0.05 - 50 ~g x ml-1
Test system I: MT-2 (T-lymphoblastoid cells)
Test system II: PBMC (peripheral blood monocytic cells)
2143Q6~
- 50 -
Test procedure:
2 x 104 or 2 x 105 infected MT-2 or PBMC cells
respectively were added to 100 ~cl in each case of the
respective oligonucleotide solution.
After a 7 day incubation at 37°C, it was monitored for
syncytia formation and the reduction of the cytoplastic
effect was measured by means of a colour test with MTT
or a p24 ELISA. The results are shown in Table I.
Table I
Oligonucleotide Concentration CPE reduction
region ~,g/ml
tar 0.05 59.1
rev 0.05 40.7
pbs 0.05 42.2
Any oligonucleotides according to the present invention
(e. g. analogous to example 11 and 22) can be synthesized
using the monomers described in the examples.
Example 35
1-(2-deoxy-8-D-threo-pentofuranosyl)thymine cyclic
3',5'-monophosphates, triethylammonium (2)
General information concerning the production of
deoxyxylonucleoside-5'-mono, di and triphosphates
~1 o~os~
- 51 -
In order to produce the 5' mono, di or triphosphates,
the 3'-OH group is firstly benzoylated and then it is
phosphorylated with POC13 in trialkylphosphate,
preferably trimethylphosphate or triethylphosphate to
form 5'-phosphate. Subsequently the 3' protecting group
is removed with a base such as ammonia, during which a
corresponding protecting group which has been introduced
into the heterocyclic base in some nucleotides is also
cleaved off if necessary. A corresponding protecting
group strategy can also be applied to the production of
thiophosphates.
A solution of (1-(2-deoxy-13-D-threo-pentofuranosyl)-
thymine (1) (59 mg, 0.24 mmol) in trimethylphosphate
(1 ml) is incubated for five hours at 4°C with POC13
(40 ~1, 0.42 mmol).
Triethylammonium bicarbonate (1 mol, pH 7.6, 10 ml) is
added and the reaction mixture is concentrated by
evaporation after stirring for one hour at room
temperature.
The residue is chromatographed on DEAF Sephadex
(HC03- form, column 30 x 2 cm). The chromatography is
carried out with 400 ml water and subsequently with a
linear gradient of triethylammonium bicarbonate (0 -
0.3 mol/1, 1200 ml). The cyclophosphate elutes at
0.2 mol/1. The fractions containing the product are
concentrated by evaporation, taken up three times in 50
ml water and again concentrated by evaporation.
The product is again chromatographed on DEAF Sephadex
under the same conditions. The vapourizable salts are
~~o3o6z
- 52 -
removed by repeated co-evaporation with water, ethanol
and acetone.
1394 A267 units (65 %) of a colourless solid. TLC (2-
propanol/25 % aq. ammonia/water, 7:1:1): Rf 0.45;
(EtOAc/acetone/EtOH/water, 15:3:4:3): Rf 0.09. HPLC (5
MeCN in 0.1 M triethylammonium acetate, pH 7.0;
0.6 ml/min): tR 14.9 min.
Electrophoresis: EUP 0.64. W (MeOH):~.max267 nm. 31P-
NMR (D20/0.1 M Tris-HC1 buffer, 1:1) - 5.03 (d, J =
17.3). iH-NMR ((D6)DMSO): 11.28 (s, NH); 10.79 (br, NH);
7.87 (s, H-C(6)); 6.12 (d, J = 7.9, H-C(1')); 4.69 (br,
H-C(3')); 4.40 - 4.14 (m, CH2(5')); 3.64 (br, H-C(4'));
3.04 (q, CH2(Et)); 2.66 (m, Ha-C(2')); 1.96 (d, J =
-16.2, HB-C(2')); 1.76 (s, Me-C(5)); 1.20 (t, Me-(Et)).
Example 36
9-(2-deoxv-13-D-threo-pentofuranosyl)adenine cyclic
3',5~-monophosphates, triethylammonium (4a)
Method A:
The reaction of compound 3 (9-(2-deoxy-li-D-threo-
pentofuranosyl)adenine (25 mg, 0.1 mmol) with POC13 is
carried out in trimethyl phosphate as described for
compound 2 in example 35 but in the presence of 2-t-
butylimino-2-diethyl-amino-1,3-dimethyl-perhydrodiaza-
phosphorine (BEMP) (41 mg, 0.15 mmol). The reaction is
completed after 12 hours. The processing is carried out
as described in example 35. The final purification after
chromatography on Sephadex is carried out by thin layer
chromatography using EtOAc/acetone/EtOH/water, 15:3:4:3
as the solvent system (Rf 0.13).
zio~QOz
- 53 -
Method B:
The reaction is carried out with compound 3 (20 mg,
0.08 mmol) as described for method A but without
addition of BEMP. After chromatography on DEAE Sephadex
adenine (5 mg, 46 %) is firstly eluted with water and
then compound 4a) is eluted with butter. A colourless
substance is obtained (567 A260 antis, 39 %).
Method C:
POC13 (20 ~,1, 0.21 mmol) is added to a solution of
imidazole (86 mg, 1.26 mmol) in MeCN (0.5 ml). It is
stirred for 30 minutes at room temperature, cooled to
0°C and the solution of 9b) (35 mg, 0.1 mmol) in
MeCN/dimethylformamide (1:1, 1 ml) is added. The
reaction mixture is incubated for 2 hours at 4°C and
subsequently for 14 hours at room temperature. After
neutralization with 1 mol/1 aqueous triethylammonium
bicarbonate (12 ml), the solution is stirred for 1 hour
and concentrated by evaporation. The residue is treated
with 25 % aqueous ammonia solution (20 ml) for 16 hours
and concentrated by evaporation. The purification of
compound 4a) is carried out on DEAF Sephadex as
described. The unprotected nucleoside 3 is obtained
after elution with water (24 %), compound 4a) is
obtained as a colourless compound (843 A260 units, 57 %)
using a solution of 0.2 mol/1 triethylammonium
bicarbonate.
21030~~
- 54 -
Example 37
i-f3-O-benzvol-2-deoxy-5-o-(4,4'-dimethoxytriphenyl-
methvl)-f3-D-threo-pentofuranosyllthymine (6a).
Benzoyl cyanide (75 mg, 0.57 mmol) and triethylamine
(80 ~,1, 0.57 mmol) are added to a solution of compound
5a) (265 mg, 0.49 mmol) in MeCN (4 ml). It is stirred
for 1 hour at room temperature, concentrated by
evaporation and the residue is chromatographed on a
silica gel column (2 x 17 cm) in CH2C12/acetone, 6:1.
Compound 6a) is obtained as a white foam (255 mg, 81 %).
TLC (CH2C12/acetone, 6:1): Rf 0.56. iH-NMR ((D6)DMSO):
11.32 (s, NH); 7.72 - 7.17 (m, 14 arom. H and H-C(6));
6.80 - 6.61 (m, 4 arom. H); 6.18 (d, J = 5.7, H-C(1'));
5.68 (m, H-C(3')); 4.48 (m, H-C(4')); 3.69 (m, CH2(5')
and 2s, 2 Me0); 2.88 (m, Ha-C(2')); 2.26 (d, J = -16.2,
HB-C(2')); 1.55 (s, Me). Anal. calcd. for C38H36N208
(648.71): C 70.36, H 5.59 N 4.32; found: C 70.31,
H 5.70, N 4.36.
Example 38
1-(3-O-benzoyl-2-deoxy-f3-D-threo-pentofuranos 1)th
ymine
7a .
The solution of 6a) (200 mg, 0.31 mmol) in 80 ~ acetic
acid (3 ml) is stirred for 15 minutes at room
temperature, diluted with 100 ml and concentrated by
evaporation. The residue is taken up once each time with
50 ml water, 2-propanol (50 ml) and 50 ml methanol,
concentrated by evaporation and chromatographed on
~~o3os~
- 55 -
silica gel (2 x 12 cm) in CH2C12/MeOH 95:5 in which
compound 7a) is obtained as a white foam (98 mg, 92 %).
TLC (CH2C12/MeOH, 95:5): Rf 0.30. 1H-NMR ((D6)DMSO):
11.29 (s, NH); 7.98 - 7.51 (m, 5 arom. H and H-C(6));
6.14 (dd, J = 7.8, 2.3, H-C(1')); 5.54 (m, H-C(3'));
4.98 (t, OH-C(3')); 4.19 (m, H-C(4')); 3.82 (m, CH2
(5')); 2.87 (m, Ha-C(2')); 2.20 (dd, J = -15.4, 2.0 H~-
C(2')); 1.69 (s, Me). Anal. calcd. for C17H18N206
(346.34): C 58.96, H 5.24, N 8.09; found: C 59.11,
H 5.35, N 7.99.
Example 39
1-(2-deoxy-f3-D-threo-pentofuranosvl)thymine 5'-
monoohosphate, triethylammonium (8a).
Method A:
The solution of compound 7a) (80 mg, 0.23 mmol) in
PO (OMe)3 (0.5 ml) is incubated for 8 hours at 4°C with
POC13 (46 ~1, 0.48 mmol). The reaction mixture is added
to cold 1 molar aqueous triethylammonium bicarbonate
(15 ml), incubated for two hours at room temperature,
concentrated by evaporation and again taken up twice in
water (50 ml) and concentrated by evaporation. The
residue is incubated for three hours with 25 % aqueous
ammonia colution (50 ml), concentrated by evaporation
and chromatographed on DEAE Sephadex (HC03 form, 2 x
30 cm). It is eluted with water (600 ml) and then with a
linear gradient of triethylammonium bicarbonate (0 -
0.3 mol/1, 1200 ml). The fractions that contain
phosphate elute at 0.2 - 0.25 mol/1. They are
concentrated by evaporation, again taken up in water and
210300
- 56 -
concentrated and again chromatographed on DEAE Sephadex
under the same conditions. Triethylammonium bicarbonate
is removed by repeated evaporation with water and
ethanol. A colourless amorphous product is obtained.
(1443 A267 units, 71 %). TLC (EtOAc/acetone/EtOH/water,
15:3:4:3): Rf 0.06, (2-propanol/25 % aq. ammonia/water,
7:1:1): Rf 0.10. HPLC (0.1 M triethylammonium
acetate/5 % MeCN; 0.6 ml/min): tR 8 min.
Electrophoresis: EUp 0.87. W (H20):~l.max267 nm. 31P-NMR
(D20/0.1 M Tris-HC1 buffer): 1.36
Method B:
9-f2-deoxy-5-0-(4,4~-dimethoxvtriphenylmethvl)-f3-D-
threo-pentofuranos~l]adenine (5b)
The compound is obtained from compound 7a) (80 mg,
0.23 mmol) as in method A. Only the removal of the
benzoyl group is carried out with 0.1 mol/1 NaOH in 50
aqueous methanol (15 ml) at 50°C for two hours. The
solution is neutralized with Dowex 50 (H+ form), the ion
exchanger is removed by filtration and the neutral
solution is concentrated by evaporation. The further
purification is carried out as described in method A.
1214 A276 units (60 %) are obtained.
Example 40
Compound 3 (300 mg, 1.19 mmol) in pyridine (15 ml) is
incubated with 4,4'-dimethoxytrityl chloride (508 mg,
1.5 mmol) and ethyldiisopropylamine (255 ~,1, 1.5 mmol)
for 12 hours at room temperature. The solution is
concentrated by evaporation at 30°C. The residue is
210306
- 57 -
again taken up in toluene, concentrated by evaporation
and chromatographed on silica gel (4 x 11 cm).
CH2CL2/acetone/Et3N (40:40:3), White foam (476 mg,
72 ~). TLC (CH2C12/acetone/Et3N, 40:40:3): Rf 0.64. 1H-
NMR ((D6)DMSO): 8.26 (s, H-C6)); 8.16 (s, H-C(2)); 7.41
- 7.18 (m, 9 arom. H, NH2); 6.84 - 6.77 (m, 4 arom. H);
6.35 (d, J = 8.2, H-C(1')); 5.94 (d, J = 5.2, OH-C(3'));
4.32 (m, H-C(3')); 4.19 (m, H-C(4')); 3.71 (s, 2 OMe);
3.23- 3.02 (m, CH2-(5'); 2.78 (m, Ha-C(2')); 2.29 (d, J
- -14.8, H~C(2')).
Example 41
9-f3-0-benzoyl-2-deoxy-5-0-(4,4~-dimethoxytriphenyl-
methyl)-Q-D-threo-pentofuranosyl]adenine (6b).
The solution of compound 5b) (400 mg, 0.72 mmol) in MeCN
(4 ml) is incubated with benzoyl cyanide (105 mg,
0.8 mmol) and triethylamine (130 ~,1, 0.39 mmol) at room
temperature for three hours. 25 mg (0.19 mmol) benzyl
cyanide and 25 ~,1 triethylamine are in addition added
and it is stirred for a further 15 hours. The solution
is concentrated by evaporation and the residue is
chromatographed on silica gel (2 x 15 cm) using
CH2C12/acetone (1:2) to give a white foam (305 mg,
64 ~). TLC (CH2C12/acetone, 1:2): Rf 0.60. 1H-NMR
((D6)DMSO): 8.09 (s, H-C(8)); 8.07 (s, H-C(2)); 7.68-
7.13 (m, 14 arom. H, NH2); 6.75 - 6.69 (m, 4 arom. H);
6.41 (d, J = 5.2, H-C(1')); 5.82 (m, H-C(3')); 4.57 (m,
H-C(4')); 3.69 (s, OMe); 3.67 (s, OMe); ca 3.3 - 3.2 (m,
CH2(5')); 3.05 (m, Ha-C(2')); 2.91 (d, J = -14.3, H~-
C(2')).
- 58 -
Example 42
9-(3-0-benzoyl-2-deoxy-fi-D-threo-pentofuranosyl)adenine
7b
Compound 6b) (260 mg, 0.40 mmol) is taken up in 80
acetic acid (10 ml) and stirred for 80 minutes at room
temperature. The solution is diluted with 50 ml water
and concentrated by evaporation. The residue is taken up
twice with 20 ml water each time and once in 20 ml 2-
propanol and concentrated by evaporation. The product is
purified on silica gel (2 x 12 cm) and chromatographed
in CH2C12/MeOH 93:7, Colourless solid (120 mg, 85 ~).
M.p. 166-168°C (2-propanol).
TLC (CH2C12/MeOH, 93:7): Rf 0.30. 1H-NMR ((D6)DMSO):
8.28 (s, H-C(8)); 8.07 (s, H-C(2)); 7.85 - 7.47 (m, 5
arom. H); 7.27 (s, NH2); 6.38 (d, J = 5.2, H-C(1'));
5.68 (m, H-C(3')); 5.01 (br, OH-C(5')); 4.33 (m, H-
C(4')); 3.76 (m, H-C(5')); 3.02 (m, Ha-C(2')); 2.83 (d,
J = -14.7, H~-C(2')); Anal. calcd. for C17H17N504
(355.35): C 57.46, H 4.82, N 19.71; found: C 57.15,
H 5.02, N 19.49.
Example 43
9-(2-deoxy-f3-D-threo-pentofuranosyl)adenine 5'-
phosphate, triethylammonium (8b).
Compound 8b) is prepared from compound 7b) (53 mg,
0.15 mmol) as in method A for the production of compound
8a). The duration of the POCl3 treatment is 4 hours. An
amorphous product (1222 A258 units, 53 ~) is obtained.
The product is dissolved in ca. 200 ~,1 methanol and
~~Q306~
- 59 -
precipitated with 20 ml ether. The precipitate is
separated by centrifugation and dried over phosphorus
pentoxide. Thin layer chromatography is carried out with
2-propanol/25 % ammonia/water, 7:1:2): Rf 0.24,
(EtOA/acetone/EtOH/water, 15:3:4:3): Rf 0.06. HPLC
(0.1 M triethylammonium acetate/5 % MeCN; 0.6 ml/min)
tR 13 min. Electrophoresis: EUp 0.68. W (MeOH):~.
max259 nm. 31P-NMR (D20/MeOD, 1:1): 2.94. 1H-NMR
(D20/MeOD, 1:1): 9.89 (s, H-C(8)); 9.71 (s, H-C(2));
7.84 (d, J = 8.0, H-C(1')); 6.08 (m, H-C(3')); 5.77 -
5.61 (m, CH2(5')); 5.01 (m, H-C(4')); 4.68 (q, CH2);
4.48 (m, Ha-C(2')); 3.92 (d, H~-C(2')); 2.74 (m, CH3).
Example 44
Compound 5a) is prepared analogous to
Rosemeyer, H.; Seela, F. Helv. Chim. Acta 1991, 74, 748.
Rosemeyer, H.; Krecmerova M.; Seela F. Helv. Chim. Acta,
1991, 74, 2054.
Fox, J.J.; Miller, N.C. J. Org. Chem. 1963, 28, 936.
Data for compound 4a:
824 A260 units (53.8 %) of colourless solid TLC (2-
propanol/25 % aqueous ammonia/water, 7:1:1): Rf 0.54.
HPLC (5 % MeCN in 0.1 M triethylammonium acetate, pH 7,
0.6 ml/min): tR 15 min.
Electrophoresis: EUP 0.35. W (MeOH):iL max260 nm. 31P-
z~o~osz
- 60 -
NMR (D20/0.1 M Tris-HC1 buffer, 1:1): -5.11 (d, J =
19.4). iH-NMR ((D6)DMSO): 9.97 (br, NH); 8.33 (s, H-
C(8)); 8.15 (s, H-C(2)); 7.34 (s, NH2); 6.40 (d, J =
7.8, H-C(1')); 4.63 (br, H-C(3')); 4.42 - 4.10 (m,
CH2(5')); 3.94 (br, H-C(4')); 3.05 (q, CH2); 2.85 (m,
Ha-C(2')); 2.28 (d, J = -14.6, H~-C(2')); 1.18 (t, Me).
