CA2077314A1

CA2077314A1 - Antisense oligonucleotides with phosphoramidate internucleotide linkage throughout

Info

Publication number: CA2077314A1
Application number: CA002077314A
Authority: CA
Inventors: Antonius Lobberding; Burkhard Mielke; Wolfgang Springer; Udo Stropp; Jorg Baumgarten; Helga Rubsamen-Waigmann; Lothar Biesert; Haryadi Suhartono
Original assignee: Individual
Current assignee: Bayer AG
Priority date: 1991-09-03
Filing date: 1992-09-01
Publication date: 1993-03-04
Also published as: EP0530614A1; DE4129318A1; JPH05202090A

Abstract

Novel antisense oligonucleotides with phosphoramidate internucleotide linkage throughout Abstract The invention relates to oligonucleotides of the general structure I

I

and to the use thereof for the treatment of genetically related disorders and for defence against exogenous nucleic acids.

Le A 28 522

Description

~ ~ J 7-~L~

The specific regulation o unwanted gene expression by complementary nucleic acids, so-called an~isense oligonuceotides, would represent a great advance in chemotherapy. It could be of Lmportance for the treatment of genetically related disorders and for defence against exogenous nucleic acids, fox example after infection by bacteria, fungi or viruses.

The antisense principle is based on the ability of the so-called antisense oligonucleotides to hybridise with the mRNA to be inhibited, and in this way to prevent protein biosynthesis.

There is evidence in the literature for the ac~ion of antisense oiigonucleotides. Thus, inter alia, their antiviral action, for example ~gain~t retroviruses such lS as HIV, is discussed ~P. C. Hoyle, E. C. Cooper, Adv.
Appl. Biotechnol. Ser. 2, 1989, 35).

The use of nature-identical phosphodiester oligonucleotides is prevented by their great sensitivity to enzymatic degradation by endogenous endo- and exonucleases.

The present invention relates to antisense oligonucleotide analogues with achiral phosphoramidate internucleotide linkage which are derived from 5'-amino-2',5'-dideoxyfur~noses and may be modified at the 5~ end Le A 28 522 - 1 -by lipid residues. These oligonucleotide analogues have an increased resistance to endo- and exonucleases and display anti-viral actions.
Oligonucleotide analogues which contain one or more phosphoramidate internucleotide linkages have been described previously (W. sannwarth~ Helv. Chim. Acta 71 (1988), 1517; M. Mag, J.W. Engels, Nucleic Acids Res. 17 (1989), 5973; S. M. Gryaznov, N. I. Sokolova, Tetrahedron Lett. 31 (1990), 3205).
These compounds do not have improved properties compared with unmodified phosphodiester oligonueleotides. Antiviral activities have not been disclosed for these compounds.
Syntheses of oligonucleotide sequences which eontain phosphoramidate internueleotide linkages throughout have not been described.
However, introduetion of phosphoramidate internucleo-tide linkages throughout, or almost throughout, results in adequately nuelease-stable molecules with antiviral aetion. It is also possible to use these structures as gene probes (Review:
J. A. Matthews, L. J. Xrieka, Analytical Bioehemistry 169 ~0 (1988), 1).
The invention relates to oligonueleotides of the general strueture I:

B

D E
O=P~X
A ~ B

D E
_ I ~ n where A can be NX or NR2 throughout, ~ can be, independently of one another, a na~urally occurring or modified pyrimidine or purine nucleobase or else OH, oR2 or H, D can be O, S, NH or NR2, E can be, independently of one another, H, OH, oR2, azide or halogen, 10 X can be -O~, S~, -oR2, -SR2 or alkyl, R2 can be alkyl, aryl, aralkyl or cycloalkyl.

By alkyl iB meant here a branched or unbranched alkyl radical with 1 to 20 C atom~. Alkyl radicals with 1 to 10 C atoms are preferred, particularly preferably methyl, ethyl, propyl, allyl, 2,2-dimethylallyl, benzyl.

Le A 28 522 - 3 _ a~L~

By aryl i~ meant here phenyl, naphthyl, optionally ~ub~titutad, for example by halog0n or nitro.
By aralkyl i5 meant here an aralkyl radical with 1 to 18 carbon atoms in the straight-chain or branched alkyl moiety and 6 to 12 C atom in the aryl moiety, where aryl represents phenyl or n~phthyl.

By cycloalkyl is meant here a cyclic alkyl radical with 3 to 8 carbon atoms, preferably with 3 to 6 carbon atoms, par~icularly preferably cyclopentyl and cyclohexyl.

Y can be -NH2, -NHR2, -NR2 or N ? --OR
H o~

R~ can be alkyl, aryl, hetaryl, aralkyl, cycloalkyl, lipid.

By alkyl is meant here a branched or unbranched alkyl radical with 1 to 30 C atoms. Alkyl radicals with 1 to 18 C atoms are preferred, and alkyl radicals with 6 to 18 C atoms are very particularly preferred.
2~
By aryl is meant here, for example, phenyl or naphthyl, optionally substituted, for example by halogen or nitro.

By hetaryl is meant, for example, pyridyl, acridinyl or carbazolyl.

Le A 28 522 - 4 -By aralkyl is meant an aralkyl radical with l to 18 C atoms in the traight-chain or branched alkyl moiety and 6 to 12 C atoms in the aryl moiety, where aryl represen~s phenyl or naphthyl.

By cycloalkyl is meant cyclic alkyl radical with 3 to 8 carbon atoms, preferably with 3 to 6 carbon atoms, particularly preferably cyclopentyl and cyclohexyl.
n can be 3 to 50.

n i~ preferably 10 to 30 and particularly preferably 18 to 28.

Le A 28 522 - 5 -~s~
2318g-7387 The activated monomers are synthesised by known processes (~. Mag., J. W. Engels, Nucleic Acids Res. 17 (1989), 5979).
HO ~ T N3 ~ T ~N ~ T

OH OH OH
1a 3a 4a M~rrHN ~ T Mn~THN ~ T

OH ~
N~P~o ~ CN

~a 6a where T indicates thymine Le A 28 522 - 6 -2~i_t~

M~rr= - C ~ O~H3 _~ _ Thymidine is reacted with lithium azide, tetra-bromomethane and triphenylphosphine in dimethylformamide. The azido group is reduced with triphenylphosphine in pyridine to the amino groups, and the amino group is subsequently protected with the monomethoxytrityl group. The activated derivative 6a can be obtained from 5a using cyanoethyl N,N,N',N'-tetraisopropyl-phosphoro-diamidite.

HO ~ B Ts ~ B N3 ~ B

OH OH OH
1~-e) ~(~e) 3(~e~

Le A 28 522 - 7 -~7~

H N~B MM~ ~B MM HN~B

OH OH ~
_,~N~P~o~

4(b-e) 5(b-e) 6(b~) b: B = N~lo o N

NH- C ~
c: B = N 3~N

Ts -- ~SO2 ~ CH3 Le A 28 522 - 8 -o d: B~ ~ ~ NH o - N N~N~-C

e. B= ~ ~ NH O
N N ~H-C - CH2 ~

N~-benzoyl-2'-deoxycytidine lb, N4-benzoyl-2'-deoxy-adenosine lc, N2-isobutyryl-2'-deoxyguanosin~ ld and N2-phenylacetyl-2'-deoxyguanosine le are prepared by the transient protection method (G.S.Ti., B. L. Gaffney, R. A. Jones, J. Am. Chem. Soc. 104 (1982), 1316;
F. Benseler, L. W. McLaughlin, Synthesis 1986, 45).

The monotosylates 2 (b-e) are obtained from the building blocks 1 (b-e) and are converted with lithium a~ide into the 5'-azido compounds 3 (b e).

The synthetic route described for the thymidine derivative is utilised for sub equent reaction step~.

The lipid residues, for example undecanol, are activated analogously by reacting with cyanoethyl N,N,N',N'-tetra-Le A 28 522 - g -~ 23189-7387 isop~opyl-phosphorodiamidite to yield, for example to 7 ,OCH2CH2CN
CH3-(CH2)l0 ~ `N

7 ) \

Linkage of the 5~-deoxyaminonucleotide building blocks to give oligonucleotides The linkage of the monomer building blocks to give oligonucleotides is preferably carried out under ~he conditions of solid-phase synthesis based on a described process (M. Mag, J. W. Engels, Nucl. Acids Res, 17, 5973 (1989); W. Bannwar~h, Helv. Chim. Acta 71, 1517 (1988)).

The oligonucleotide analogues according to the invention are linked in the final reaction step, for example, with the phosphoramidite rea~ent 7. Af~er ths protec~ive groups have been removed and the sequenco ha3 been cleaved off the ~olid phass by base treatment, the reaction product is preferably isolated ~y preparative HPLC, the chromatographic behaviour being determined in each case by the lipid residue which permits easy removal from by-products.
Oligonucleotide sequences such as the following have been synthesized in this way.

RNHGNHCNHTNHCNHCNHGNHANHGNHGNHCNHTNHINHANIiaNHANHTG

Le A 28 522 - 10 -R = CH3-(cH2)lo - O -P - 8 OH

The corresponding pharmaceutical formulations contain, besides the pho~phoramidate oligonucleotide~, the auxiliaries customary for parenteral formulations such S as, for example, buffers and/or stabilisers or liposome formulations. Topical application is also conceivable.
Examples of formulations which can be employed for this purpose are ointments, creams, solutions or plasters which, besides the acti~e compound, contain the pharmaceutical auxiliaries which are suitable for this application.

Svnthesis examples Example 1:

5'-O-(4-Toluenesulphonyl)-N~-phenylacetyl-2'-deoxyguanosine N2-Phenylacetyl-2'-deoxyguanosine (13.9 g; 36 mmol) and 4-toluenesulphonyl chloride (21.0 g; 108 mmol) are stirred in anhydrous pyridine (140 ml) at room temperature for 70 min. After this, water (5 ml) is added at 0C, the mixture is stirred at room temperature for 20 min, concentrated under high vacuum and codistilled several times with toluene, the residue i8 taken up in ethyl acetate (150 ml) and washed once each with 5%

Le A 28 522 NaHC0~ solution and NaCl solution (50 ml each)~ and the organic phase is dried (MgS04) and concen~rated. The crude product is purified by chromatography (dichloro-methane/methanol 15:1).
Yield: 11.3 g (58~).

Example 2:
5'-Azido-N2-phenylacetyl-2',5'-dideoxyguanosine Lithium azide (5.0 g, 100 mmol) is added to a solu~ion of 5'-0-(4-toluenesulphonyl)-N2-phenylacetyl-2'-deoxy-guanosine (11.0 g; 20 mmol) in N,N-dimethylformamide (120 ml) and the solution is stirred at 50C for 7.5 h.
It is cooled, concentrated under high vacuum and codistilled several times with toluene, and the residue is taken up in ethyl acetat2 (500 ml) and extracted by shaking twice each with NaHC03 solution (60 ml each time) and NaCl solution (100 ml each time). The combined aqueous phase is back-extracted several times with ethyl acetate. The combined organic phases are dried (MgS0~) and concentrated. The crude product is purified by chromatography (dichloromethane/methanol 13:1 ~ 10:1).
Yield: 8.0 g (95~).

Example 3:
5'-Amino-N2-phenylacetyl-2',5'-dideoxyguanosine 5'-Azido-N2-phenylacetyl-2',5'~dideoxyguanosine (8.2 g;
29 mmol) and triphenylphosphine (7.9 g; 30 mmol) are dissolved in pyridine (150 ml) and left at room Le A 28 522 - 12 -temperature for 16 h. Water (25 ml) is then added and the mixture is stirred for 2 h. The reaction solution is poured into water (200 ml), filtered and the aqueous phase is extracted twica with ethyl acetate (100 ml each time). ~he combined organic phase is then extracted five times with water (100 ml each time). The combined aqueous phase is freeze-dried and provid0s the crude product as a yellowish powder which is immediately reacted further.
Yield: 7.1 g (92%) Example 4:
5~-(4-Methoxytriphenylmethyl)amino-N2-phenylacetyl-2~,5'-dideoxyguanosine 5'-Amino-NZ-phenylacetyl-2~,5~-dideoxyguanosine (7.1 g;
18.5 mmol) is dissolved in pyridine (200 ml), chloro(4-methoxy)triphenylmethane (17.0 g; 55.5 mmol), 4-(N,N-dimethylamino)pyridine (490 mg; 3.8 mmol) and triethylamine (2.7 ml; 19 mmol) are added, and the mixture is stirred at room temperature for 2.5 h. It is then concentrated under high vacuum and codistilled several times with toluene, the residue is taken up in dichloromethane (500 ml) and extracted by shaking twice each with NaHCO3 solution and NaCl solution, and the organic phase is dried (MgSO4) and concentrated.
Purification by chromatography follows (dichloromethane/methanol 20:1 10:1).
Yield: 2.4 g (20%).

Le A 28 522 - 13 Example 5:
5~-(4-methoxytriphenylmethyl)amino-N2-phenylacetyl-2',5'-dideoxyguanosine 3'-0-(2-cyanoethyl-N,N-diisopropyl-amino)phosphite 5'-(4-Methoxy~riphenylmethyl)amino-N2-phenylacetyl-2',5'-dideoxyguanosine (2.2 g; 3.3 mmol) and tetrazole (176 mg;
2.5 mmol) are dissolved, s~ringently excluding air and moisture, in absolute dichloromethane/acetonitrile (1:1;
70 ml), 2-cyanoethyl N,N,N~,N'-tetraisopropyl-phosphoro-diamidite (1.65 ml; 5.3 mmol) is added, and the mixture is stirred at room temperature for 1.5 h. After the reaction is complete, butanol ~3 ml) is added, and the mixture is then stirred for 10 min, diluted with dichloromethane (500 ml), rapidly extracted once each with 5~ NaHCO3 solution (150 ml) and NaCl solution (250 ml), dried (MgSO4) and concentrated. The crude product is dissolved in dichloromethane/ether (1:1, 14 ml) and precipita~ed in n-pentane (450 ml) cooled in dry ice.
Yield: 1.67 g (59%).

Exam~le 6:
Undecane l-O-(2-cyanoethyl-N,N-diisopropylamino)phosphite Bis-(diisopropylamino)-(2-cyanoethoxy)-pho~phine (3.3 g, 11 mmol) is added to a stirred solution of undecanol (1.72 g, 10 mmol), diisopropylamine (0.5 g, 5 mmol) and tetrazole (350 mg, 5 mmol) in 30 ml of dichloromethane under argon over the course of 20 min.

Le A 28 522 - 14 -2318g-7387 After the reaction is complete, the mixture is ex~racted by shaking with cold 10% strenqth sodium carbonate solution. The organic phase is evaporated, and ~he product is chromatographed on silica gel.
Yield: 3.3 g (73%).

Example 7:
Synthesis of the exemplary sequence detailed below:

R~GN}~CN~T~ HCNHC~C,"~ANXGNHG~CNHTNHCNHA~`.~GNHANHTC

R = CH3-(CH2)l0-- P
OH

The synthesis wa~ carried out in an Applied Biosystems 380B automatic DNA synthesiser. Used as solid phase was a commercially available (Applied Biosystems) 10 ~mol (or 1 ~mol) controlled pore glass support to which 2-deoxy-cytidine that is protected at the 5'-position by a 4,4'-dimethoxytrityl group is bonded via the 3'-hydroxyl group.
The 4,4'-dimethoxytrityl protective group is eliminated by treatment with 2.5% strength dichloroacetic acid in methylene chloride, and the excess acid and the protective group are subsequently washed out with acetonitrile. The chain extension is carried out by adding a 0.1 M solution of 5'-N~-mono-methoxytrityl-protected nucleotide building block 6a in acetonitrile in the presence of tetrazole.
Subsequent Le A 28 522 - 15 -231~9-7387 oxidation to pentavalent phosphorus is carried out by treatment with a 0.2 M iodine solution in pyridine/H2O/THF/1:1:8O To avoid wrong sequences, subsequently unreacted 5'-hydroxyl or 5'-amino groups (in the subsequent cycles) are blocked by treatment with acetic anhydride/dimethylaminopyridine in pyridine/
acetonitrile.
The next reaction cycle starts with elimination of the monomethoxytrityl protective group by treatment with 2.5% strength dichloroacetic acid. The reaction with subsequent 5'-amino-nucleotide building blocks 6 is carried out in analogy to the description given above, using the compound of formula 6a to introduce thymine and the corresponding adenine-, cytosine- or guanine-containing compound to introduce the respective other bases (A), (C) or (G).
In the last reaction step, a 0.1 M solution of unde-canol ~-cyanoethyl phosphoramidite reagent 7 in acetonitrile is coupled on in the presence of tetrazole after acid elimination of the last monomethoxytrityl protective group. Iodine oxidation is followed by cleavage off the polymeric support and removal of the protective groups by treatment with concentrated ammonia at 55C
~or 16 h.
The reaction product is isolated by preparative HPLC on an RP 18 column with a linear rising gradient of acetonitrile in 0.1 M triethylammonium acetate; yield: 2.8 mg (4.8%).
Nuclease stability of olignonucleotide amidates Besides the chain length, sequence and cell perme-ability, ~r~t7~
Lmportant for the biological action of antisense oligonucleotides is the nuclease resistance. The synthesised oligonucleotides with 5'-amidate inter-nucleotide linkage were tnerefore compared with natural S oligonucleotide diesters in respect of their nuclease stability.

The antisense oligonucleotide diesters and amidates were for this purpose radioactively labelled at the S' end with 32P-ATP and their degradation with various defined nucleases and cell extracts was examined by polyacryl-amide gel electrophoresis and autoradiography and quantitatively determined in a scintillation counter by isola~ion of the degradation products from the gel.

Natural oligonucleotide diesters have only low nuclease stability. They are completely degraded within 30 minutes to 1 hour. Oligonucleotide amidates, by contrast, are 10 to 100 times more resistant to nucleases and are therefore particularly well suited for use as antiviral antisense oligonucleotide inhibitors.

Antiviral actions:

In vitro translation assay The antiviral, translation-inhibiting activity of the amidates was shown in a cell-free in vitro translation assay. For this, in vitro synthesised TAR-tat mRNA was placed in a rabbit reticulocyte translation system and Le A 28 522 - 17 -the ta~ protein synthesis was measurad quantitatively with and without oligonucleotide.

In detail: 1 ~g of in vitro synthesised TAR-tat mRNA is mixed with a required amount of oligonucleotide (0.2-2 ~9) in a volume of 2,5 .~1. For the translation, 10 ~1 of preincubated translation mix (from Promegs) with radioactive 35S-cysteine are pipetted into the 2.5 ~l and incubated at 30C for 90 min. An aliquot of the mixture is removed and heated with SDS loading bUf fer and fractionated on a 6-20~ discontinuous SDS PAGE. After fixing and drying, the gel is autoradiographed and the resulting tat protein bands are evaluated by densitometry.

Result: the inhibition of the translation of the tat protein by the assayed amidates is 3 times greater than that of diester oligonucleotides of identical sequence in various concentrations.

Antiviral action in a cell culture assay The phosphoramidate oligonucleotides were tested on the virus isolates detailed below:
HI~-lD3~ Isolation by Dr. von Briesen and Prof.
Dr. H. Rubsamen-Wai~mann, Georg-Speyer-Haus, Frankfurt am Main, 1985.

Le A 28 522 - 18 -HIV-2RoD Isolation by Pasteur Institute, Paris, 1986.

Used as assay system were peripheral blood lymphocytes ( PBL ) from HIV-seronegative donors which had been preactivated with phytohaemagglutinin. The substances were added to the cell culture immediately after the infection. The cell suspensions were cultivated with various concentrations o-f the compounds in an incubator for 4-6 days. Virus replication was detected by evaluation under the light microscope of the HIV-induced syncytia formation.

Table 1:
Inhibition of virus-induced syncytia formation in the presence of phosphoramidate oligonucleotide 8 Test HIV-l HIV-2 ~g/ml ~mol/l ~g/ml ~mol/l 1 5-10 0.9-1.9 _ 2 2-5 0.4-0.9 15-25 2.8-4.7 3 2-5 0.4-0.9 15-25 2.8-4.7 4 5-lO 0.9-1.9 ~ ~ l The test substance distinctly inhibits HIV-1-induced syncytia formation in concentrations above 2-5 ~g/ml, that is to say 0.4-O.g ~mol/l.

Le A 28 522 - 19 -

Claims

1. Oligonucleotides of the general structure I:

where A can be NH or NR2 throughout, B can be, independently of one another, a naturally occurring or modified pyrimidine or purine nucleobase or else OH, OR2 or H, D can be O, S, NH or NR2, E can be, independently of one another, H, OH, OR2, azide or halogen, X can be -O?, S?, -OR2, SR2 or alkyl, Y can be -NH2, -NHR2, -NR22 or R1 can be alkyl, aryl, hetaryl, aralkyl, cycloalkyl, lipid.
R2 can be alkyl, aryl, aralkyl or cycloalkyl n can be 3 to 50.

2. Oligonucleotides according to claim 1 wherein n is 10 to 30.

3. Oligonucleotides according to claim 1 wherein n is 18 to 28.

4. Pharmaceuticals comprising one or more of the oligonucleotides of claim 1.

Le A 28 522 - 20 -

5. An olignonucleotide of the general structure where each A can be, independently of other A's NH or NR2 throughout, each B can be, independently of other B's, a naturally occurring pyrimidine or purine nucleobase or a pyrimidine or purine nucleobase that is protected on an amino group by an acyl protecting group, or B can be OH, OR2 or H, each D can be, independently of other D's, O, S, NH
or NR2, each E can be, independently of other E's, H, OH, OR2, azide or halogen each X can be, independently of other X's -O?, -S?, OR2 , -SR2 or alkyl, Y can be -NH2, -NHR2 or R1 can be branched or unbranched alkyl, alkenyl, alkynyl, aryl that is optionally substituted by halogen or nitro, a 5- or 6-membered nitrogen-containing aromatic ring that is optionally fused to one or more benzene, naphthalene or hetero-cyclic rings, aralkyl, cycloalkyl or a lipid, R2 can be branched or unbranched alkyl, alkenyl, alkynyl, aryl that is optionally substituted by halogen or nitro, aralkyl or cycloalkyl, R3 can be H or can be a group of formula wherein B, D, E and X are as defined above, and n can be 2 to 48.

6. Use of an oligonucleotide as claimed in any one of claims l to 3 and 5 as an antiviral agent.

7. Use of an oligonucleotide as claimed in any one of claims 1 to 3 and 5 as a gene probe.

8. A commercial package containing, as active pharmaceu-tical ingredient, an oligonucleotide as claimed in any one of claims 1 to 3 and 5, together with instructions for its use as an antiviral agent.