CA2159632A1 - Novel 5'-substituted nucleosides and oligomers produced therefrom - Google Patents

Abstract

Disclosed are novel 5'-substituted nucleosides and oligonucleotide analog compounds prepared therefrom having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2- internucleoside linkages instead of the naturally occurring backbone of phosphodiester internucleoside linkages. The present invention also discloses a method of synthesizing oligonucleotide compounds having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2 internucleoside linkages instead of the naturally occurring backbone of phosphodiester internucleoside linkages, this process comprising preparation of 5'-substituted nucleoside compounds, for example, as illustrated in Figure 1, and utilizing them as synthons in automated DNA synthesizers. Oligonucleotide analogs of the present invention are useful as nuclease resistant, sequence specific antisense compounds.

Description

215~63~

NOVEL 5' SUBSTITUTED NUCLEOSIDES AND
OLIGOMERS PRODUCED THEREFROM

Field of the Invention The present invention relates to novel 5'-substituted nucleosides and to oligonucleotide analogs prepared therefrom having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1 R2 internucleoside linkages instead of the 10 naturally occurring backbone of phosphodiester internucleoside linkages.
The present invention also relates to a method of synthesizing oligonucleotide compounds having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1 R2 internucleoside linkages instead of the naturally occurring backbone of 15 phosphodiester internucleoside linkages, this process comprising preparation of 5'-substituted nucleoside compounds, for example, as illustrated in Scheme 1, and utilizing them as synthons in automated DNA
synthesizers. Oligonucleotide analogs of the present invention are useful as nuclease resistant, sequence specific antisense compounds.
R~ck~round of the Invention An antisense compound binds to or hybridizes with a nucleotide sequence in a nucleic acid (RNA or DNA) to inhibit the function (or synthesis) 25 of the nucleic acid. Because they can hybridize with both RNA and DNA, antisense compounds can interfere with gene expression at the level of transcription, RNA processing or translation.
As discussed, e.g., in Klausner, A., Biotechnology. 8:303-304 (1990), the development of practical applications of antisense technology is 30 hampered by a number of technical problems. Thus, natural, phosphodiester-linked antisense oligomer compounds are susceptible to rapid degradation by nucleases that exist in target cells and elsewhere in the body; both exonucleases, which act on either the 3' or the 5' terminus of the nucleic acid,and endonucleases, which cleave the nucleic acid at internal phosphodiester 35 linkages between individual nucleosides. As a result of such'nuclease action,the effective half life of many administered antisense compounds is very short, necessitating the use of large, frequently administered, doses.

WO 94122890 _ 9 ~ 3 ~ -2- PCTIUS94/02993 The high cost of producing antisense DNA or RNA on currently available DNA synthesizers is another problem. Armstrong, L., Rusiness Week, March 5, 1990, page 89, estimated the cost of producing one gram of antisense DNA to be about $100,000.
There is also a problem regarding delivery of antisense agents to targets within the body (and cell). Thus, antisense agents targeted to genomic DNA must permeate the plasma and the nuclear membrane to gain ~ccess to the nucleus. The consequent need for increased hydrophobicity to enhance membrane permeability must be balanced against the need for increased hydrophilicity (water solubility) in body fluids such as the plasma and cell cytosol.
Also, oligonucleotide compounds such as antisense DNA are susceptible to steric reconfiguration around chiral phosphorous centers. This results in stability problems, too, whether the compounds are free within the body or hybridized to target nucleic acids.
To overcome the stability and drug delivery limitations, various oligonucleotide analogs have been investigated. In order to be of practical utility, such analogs should have good cell penetration properties, be resistant to nuclease degradation, have good sequence specific hybridization to target nucleic acids, and be synthesized by chemical methods that are not too difficult or costly.
Recent efforts to overcome the foregoing problems and prepare antisense compounds that are stable, nuclease resistant, relatively inexpensive to manufacture and which can be delivered to and hybridized with nucleic acid targets throughout the body have involved synthesizing oligonucleotide analogs that consist of oligonucleotide analog sequences with internucleoside linkages that differ from the 'normal' internucleoside phosphodiester linkage, either by introducing modifications in the phosphodiester structure or by using non-phosphate internucleoside linkages that approximate the length and orientation of the normal phosphodiester internucleoside linkage. Uhlman, E. and Peyman, A., Chemical Reviews, 9(4):544-584 (1990).
Among the modified phosphodiester linkages that have been reported are phosphorothioates, alkylphosphotriesters, methylphosphonates and alkylphosphoramidates. Also, a variety of non-ionic oligonucleotide analogs sequences containing non-phosphate internucleoside linkages, such as carbonate, acetate, carbamate, sulfone, sulfoxide, sulfonamide and dialkyl-or diaryl- silyl derivatives have been synthesized and reported. More WO 94/22890 215 9 6 3 ~ PCT/US94/02993 recently, chimeric oligonucleotide analogs comprising nucleoside linkages containing two carbon atoms and one nitrogen atom or one oxygen atom, as well as those containing three carbon atoms, have been reported. See, e.g., International Patent Publication WO 9202534.
The prior art describes 1-mononucleosides and - mononucleotides substituted at the 5'- position by a variety of substituents, 2-dinucleosides phosphates substituted at the 5'-position of the 3'-terminal nucleoside by methyl, ethyl, propyl and allyl; it also discloses the corresponding pairs of stereoisomers (Padyukov, A, et al. (1980), Collection of C7echoslov~k Chemic~l Communic~tions. vol 45, 2550-2557). This reference reports that no significant differences were observed between two stereoisomers in pancreatic ribonuclease degradation. The prior art shows that rates of cleavage of 5'-substituted dinucleotide phosphates by nuclease vary unpredictably with the steric configuration at the 5'-position, the nucleoside base and the nuclease used. The prior art is, however, devoid of disclosure of the novel 5'-substituted oligonucleotides of this invention; nor does it contain any suggestion of their excellent nuclease stability or hybridization properties.
The present invention provides novel 5'-substituted nucleosides, as well as oligonucleotide analogs of two bases and longer derived therefrom which are resistant to nucleases and will bind in a sequence specific manner to complementary nucleic acid sequences. Also provided is a method of synthesizing such oligonucleotide analogs using the nucleoside derivatives described in this specification. Another advantageous feature of the present invention is the relative ease with which optically pure isomers of the 5'-substituted nucleoside analogs of the invention as compared to phosphorothioate, methylphosphonate and phosphoroamidate nucleosides used in the prior art to synthesize oligonucleotide analogs.

Summ~ry of the Invention The present invention provides novel 5'-substituted nucleoside analogs and oligonucleotide analogs of 2 to about 60 bases containing 3~-O-PO2H-O-5'-CR1 R2 substituted internucleoside linkages instead of the naturally occurring phosphodiester internucleoside linkages.
More particularly, in one aspect, the present invention provides novel nucleoside analogs having the structure of Formula I below:

9 6 3 ~ -4-Q-(cHR)n~ o E3 RR2 ~

L(CHR)n R4 E

Fonnula wherein:
Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower 10 alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of 15 H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower 20 alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4; and each B is independently select from the group consisting of adenine, cytosine, guanine, thymine, uracil or a modification thereof that does not substantially interfere with the affinity of an oligonucleoside or chimeric oligonucleotide analog for its antisense counterpart wherein the bases are 30 selected from the group consisting of adenine, cytosine, guanine, thymine and uracil;
or an optical isomer thereof or a pharmaceutically acceptable salt thereof.

2 1 ~

In another embodiment, the invention provides oligonucleotide analogs having the structure of Formula ll below:

Q-(CHR)n~B

R4_ ~oYB
R3 ~
/ R4 E q O B
R3,~
L(CHR)n R4 E
Formub 11 wherein:
Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and 1 0 phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4 W094/22890 '~5~6~ -6- PCT/US94/02993 each B is independently selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or a modification thereof that does not substantially interfere with the affinity of an oligonucleoside or chimeric oligonucleotide analog for its antisense counterpart wherein the bases are 5 selected from the group consisting of adenine, cytosine, guanine, thymine and uracil;
each W is selected from 3'-(OPO2HO)C(R1 R2)-5' and a natural phosphodiester internucleoside linkage with the proviso that at least one W is 3'-(OPO2HO)C(R1 R2)-5'; and q is 0 or an integer from 1 to 60.
As used herein, the term 'oligonucleotide' means nucleic acid compounds which contain only 'natural' phosphodiester internucleoside linkages. On the other hand, the term 'chimeric oligonucleotide analogs' means compounds that comprise sequences containing both 'synthetic' 15 oligonucleoside linkages and oligonucleotide linkages. By the term 'oligonucleotide analogs,' we mean both oligonucleotide analogs that contain only synthetic (as opposed to the naturally occurring phosphodiester) internucleoside linkages and chimeric oligonucleotide analogs.
The present invention also provides a method of synthesizing 20 oligonucleotide compounds having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2 internucleoside linkages instead of the naturally occurring backbone of phosphodiester internucleoside linkages, this process comprising joining a 5'-end nucleoside, a middle unit, and 3'-end nucleoside, by conventional 25 synthetic organic procedures known in the art.

WO 94/22890 21~ 9 ~ ~ ~ PCT/US94/02993 net~iled nescription of the Invention The nucleoside analogs of the present invention have the structure of Formula I below:

Q-(CHR)n~B

L(cHR)n R4 E

Fomlula wherein:
Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower 10 alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower 15 alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of 20 H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and F4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower 25 alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4; and each B is independently select from the group consisting of adenine, cytosine, guanine, thymine, uracil or a modification thereof that does not WO 94/22890 f~ J S 9 6 3 ~ PCT/US94102993 substantially interfere with the affinity of an oligonucleoside or chimeric oligonucleotide analog for its antisense counterpart wherein the bases are selected from the group consisting of adenine, cytosine, guanine, thymine and uracil or a naturally occurring modification thereof;
or an optical isomer thereof or a pharmaceutically acceptable salt thereof.
The oligonucleotide analogs of the invention have the structure of Formula ll below:

Q-(CHR)n~B

W ,~ ~ Y
R3 ,~
/ R4 E q o B
R3,~

L(CHR)n R4 E

Fonnu~
wherein:
Q is selected from the group consisting of H, OH, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, 15 amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, 20 phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of H, OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower 25 alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:

WO 94/22890 ~ l $ ~ 6 3 ~ PCTIUS94/02993 each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4 each B is independently selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or a modification thereof that does not substantially interfere with the affinity of an oligonucleoside or chimeric oligonucleotide analog for its antisense counterpart wherein the bases are selected from the group consisting of adenine, cytosine, guanine, thymine and uracil or a naturally occurring modification thereof;
each W is selected from 3'-(OPO2HO)C(R1 R2)-5' and a natural phosphodiester internucleoside linkage with the proviso that at least one W is 3'-(OPO2HO)C(R1 R2)-5'; and q is 0 or an integer from 1 to 60.
As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
"Alkyl" means a saturated aliphatic hydrocarbon which may be either straight- or branched-chain. Preferred groups have no more than about 12 carbon atoms and may be methyl, ethyl and structural isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
"Lower alkyl" means an alkyl group as above, having 1 to 7 carbon atoms. Suitable lower alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and n-heptyl.
"Aryl" means phenyl, naphthyl, substituted phenyl and substituted naphthyl.
"Substituted phenyl (or naphthyl)" means a phenyl (or naphthyl) group in which one or more of the hydrogens has been replaced by the the same or different substituents selected from halo, lower alkyl, nitro, amino, - acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, and trifluoromethyl.
"Heteroaryl group" means, pyridyl, furyl, thienyl, or imidazolyl.
"Substituted heteroaryl" means a heteroaryl group in which one or more of the hydrogens has been replaced by the the same or different wo 94n2890 ~ ~ S ~ 6 ~ ~ PCT/US94/02993 substituents selected from halo, lower alkyl, nitro, amino, acylamino, hydroxyl,lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, and trifluoromethyl.
"Lower alkenyl" means an unsaturated aliphatic hydrocarbon having 2 to 8 carbon atoms, such as ethylene, propylene, butylene, isobutylene, 5 etc., including all structural and geometrical isomers thereof.
"Halo" means bromo, chloro or fluoro.
An ~O-, S-, or N-protecting group" is a radical attached to an oxygen, sulfur, or nitrogen atom, respectively, which radical serves to protect the oxygen, sulfur, or nitrogen functionality against undesired reaction. Such protecting groups are well known in the art; many are described in "The Peptides." E. Gross and J. Meienhofer, Eds. Vol 3 Academic Press, NY
(1981). The N-protecting groups can be N-acyl, N-alkoxycarbonyl, N-arylmethoxy-carbonyl, trifluoromethylacyl and N-arylsulfonyl protecting groups. Suitable O-protecting groups include benzyl, tert-butyl, methyl, tosyl, dimethoxytrityl, tert-butyl-dimethylsilyl, and carbobenzoxy groups. S-Protecting groups include methyl, tert-butyl, benzyl, and carbobenzoxy groups.
The abbreviation "iPr2" as used herein refers to diisopropyl.
The present invention provides a method of synthesizing oligonucleotide compounds having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2 internucleoside linkages instead of the naturally occurring backbone of phosphodiester internucleoside linkages, this process comprising preparation of 5'-substituted nucleoside compounds, for example, as illustrated in Figure 1, and utilizing them as synthons in automated DNA synthesizers.
Scheme 1 illustrates the preparation of various 5-substituted nucleoside analogs of the invention which, in turn, are useful for the preparation of 3'-O-PO2H-O-5'-CR1 R2 linked dinucleosides and oligonucleotide analogs of the invention. As shown in Scheme 1, (when B is thymidine) 3'-t-butyldimethylsilyloxy-2'-deoxy-5'-formyl-5'-deoxy-thymidine 2 is a key intermediate for the preparation of various 5'-substituted nucleoside analogs of the invention, including ones containing the 5'-methyl group 5, the 5'-nitromethyl group 8, the 5'-aminomethyl group 9, the 5'-epoxy group 7, and the 5'-azidomethyl group 13.
Thus, as shown in Scheme 1A, reaction of methylmagnesium bromide with the aldehyde 2 yields the 5'-methyl thymidine 3 which, upon treatment with dimethyltrityl chloride followed by desilylation and reaction with chloro-2-cyanoethyl-N,N-diisopropyl-phosphoramidite, affords the 5'-WO 94/22890 ~ 63~ PCT/US94/02993 substituted nucleoside synthon 5. The synthon 5, may be utilized for the synthesis of 5'-modified DNA employing a DNA synthesizer such as ABI 380B
Oligomer synthesizer. As shown in Scheme 1B, the 5'-epoxy compound 7 may be prepared by the reaction of the aldehyde 2 with diazomethane, 5 whereas the 5'-nitromethyl compound 8 may be prepared by the reaction of the aldehyde 2 with nitromethane. The chemical reduction of the nitro group with lithium aluminum hydride affords the desired 5'-aminomethyl compound 9, which may be acylated to yield the trifluoroacetyl amide 10. As illustrated in scheme 1C, azide ring opening of the epoxy compound 7 provides the 10 corresponding azidomethyl analog 12. The amide 11 and azido analog 13 may be prepared, respectively, from compounds 10 and 12 via deprotection of t-butyldimethylsilyloxy group followed by reaction with chloro-2-cyanoethyl-N ,N-diisopropyl-phosphoramidite.

WO 94/22890 2 ~ 5 ~ 6 3 2 PCT/US94102993 Scheme 1 General Sy.lU-esls of Tar~et ~cn~ er (B = T) A. 5'-Alkyl '~Q 1 ''sation TBSO~ Et~N~ 7a2c ~ CH~M~Br ; ~ Dmt CI, 4;DMAP

DmtrO--~O B DmtrO~O B
\ Y CIP(OCH2CH2CN)~
HO 4-DAMP, EtNiPr2 OP(OCH 2CH2CN)(N-iPr2) e. 5'-Amlnoalkyl '1c ~ ncallon K2C02 ~ CH2N2 TBSO~

1. DmtCI

2 LiAlH4 H2N ODmtr CF3CONH ODmtr CF3CONH ODmtr ~o B \~oyB 1. nBu4N F \~0 B
)_/Y (CF3C0)20 y 2. Cl(P(OCH2CH2CN)(N-iPr2) )~
TBSO TBSO OP(OCH2C H2CN)(N-ipr2) C. 5'-~ o-'~yl Mo~iilitallon O N 3 ODmtr ODmtr 1. LiN3 \~Y ,N3~B
2. DmtrCI 2. Cl(P(OCH 2C H2CN)(N -i Pr2) TBSO DMAP/pyr TBSO Op(ocH2cH2cN)(N-ipr2) WO 94/22890 2 I ~ ~ ~ 3 ~ PCT/US94/02993 -~3-This invention also contemplates pharmaceutically acceptable salts of the compounds of Formula 1. It is well known in the pharmacological arts that nontoxic addition salts of pharmacologically active amine compounds do not differ in activities from their free base. All stereoisomers as5 well as optical isomers related to the novel antisense agents described in this disclQsure are also considered to be within the scope of this invention.
Pharmaceutically acceptable salts include both acid and base addition salts. ~Pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which 10 are not biologically or otherwise undesirable. Suitable pharmaceutically acceptable acid addition salts can be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 15 cinnamic acid, mandelic acid, methanesulfonic acid, and p-toluenesulfonic acid, and the like.
Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the 20 like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, 25 tripropylamine, ethanolamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, dicyclohexylamine, Iysine, arginine, histidine, caffeine, procain, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, peperizines, piperidine, polyamine resins and the like. Particularly preferred organic non-30 toxic bases are isopropylamine, diethylamine, ethanol-amine and dicyclohexylamine.
In one embodiment, the compounds of the present invention comprise oligomeric antisense agents, as shown in Formula ll, of about 6 to about 60 bases, preferably from about 9 to about 50 bases, more preferably 35 from about 12 to about 25 bases, most preferably from 15 to 18 bases. An important feature of this invention is that the methyl group present at the 5'-position of the sugar interferes with the hydrolysis of the phosophodiester bond by nucleases. The methyl group present at the 5'-position unexpectedly WO 94/22890 S 9 6~ PCT/US94/02993 enhanced the nuclease stability. Another important feature of this invention is the discovery that the novel 5'-substituted nucleosides of the invention may be incorporated at multiple sites within an oligonucleotide analog, and have no appreciable effect on the hybridization stability of the resulting (antisense) oligonucleotide analog to its natural 'sense' target oligonucleotide when compared to the hybridization stability to the same 'sense' target of the corresponding unmodified antisense oligonucleotide.
These antisense agents can be formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, forparenteral injection or oral administration, in solid or liquid form, for rectal or topical administration, or the like.
The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as WO 94/22890 ~ PCT/US94/02993 for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b)binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium 5 carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as, for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as, for example, kaolin and bentonite, and (i) lubricants, as, 10 for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose 15 or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and 20 can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
Examples of embedding compositions which can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, 25 if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other 30 solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, particularly cottonseed oil, ground-nut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and 35 fatty acid esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

$63~ -16-Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of 5 these substances, and the like.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary 10 temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any 15 preservatives, buffers or propellants as may be required. Opthalamic formulations, eye ointments, powders and solutions are also contemplated.
Actual dosage levels of the active ingredient in the compositions may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and 20 method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the active ingredients administered to a host in single or divided doses may be in amounts, for example, of from about 25 0.5 mg to about 10 mg per kilogram of body weight. Dosage unit compositions may contain such amounts or such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of 30 administration, rates of absorption and excretion, combination with other dnugs and the severity of the particular disease being treated.
The present invention is further directed to a method of inhibiting the expression of a gene that comprises administering to a host mammal in need of such inhibition an inhibition-effective amount of a compound of 35 Formula ll, in which that compound hybridizes to a nucleotide sequence of the gene.whose expression is to be inhibited. In a preferred embodiment, the compound of Formula ll is dissolved or dispersed in a physiologically tolerable carrier.

WO 94/22890 2 1 5 ~ ~ 3 ~ PCT/US94/02993 As discussed elsewhere herein, inhibition of the expression of a gene can be effected by interfering with transcription, translation, or RNA
processing. Hence, the activity of an antisense molecule can be at the level of messenger RNA or genomic DNA. So, for example, when an antisense molecule hybridizes to messenger RNA, translation is inhibited. When an antisense molecule hybridizes to genomic DNA, transcription is inhibited. An antisense molecule may also bind to other nucleic acid species in a cell, including heterogeneous nuclear RNA (hnRNA) and pre-messenger RNA.
A host mammal in need of the inhibition of the expression of a gene suffers from a disease state in which the expression of the gene is implicated. Such disease states include a variety of cancers, in which the expression of an oncogene or oncogenes is implicated, cystic fibrosis, Huntington's chorea, and other such disease states in which the aberrant expression of a normal gene or the expression of an abnormal gene is responsible, in whole or in part, for the disease condition.
As used herein, an "inhibition-effective amount" is the amount of a compound of the present invention which is sufficient to inhibit the expression of the gene whose expression is to be inhibited. Means for determining an inhibition-effective amount will depend, as is well known in the art, on the nature of the gene to be inhibited, the type of inhibition desired (i.e., inhibition of translation or transcription or both), the mass of the subject being treated, and the like.
It is to be understood that the compound of the Formula ll used in the inhibition of the expression of a gene must hybridize to a sequence of that gene in such a way as the expression of that gene is inhibited. That is, the nucleotide bases used to make a compound of the Formula ll (B in Formula ll as defined above) must hybridize to the nucleotide sequence of the gene whose expression is to be inhibited. Such sequence can readily be ascertained from the known sequence of that gene, and the appropriate antisense molecule of Formula ll can therefore be prepared. Hybridization of greater than about 90 percent homology (identity), and more preferably about 99 percent homology, is contemplated in the present invention.

The following examples further illustrate the invention and are - 35 not to be construed as limiting of the specification and claims in any way.

WO 94/2~890 ~ 1~N594/02993 FxAMpl FS:

Example 1 N~-Ren7oyl-3'-t-butyldimethylsi~yloxy-~'-deoxy-5'(RS)-methyl-~denosine A solution of DMSO (2 mM, 142 ~I) in methylene chloride (0.5 ml) was added to a stirred solution of oxalyl chloride (2.~ ml) at -78C under nitrogen. After 5 minutes, a solution of N6-benzoyl-3'-t-butyldimethylsilyloxy-2'-deoxy-adenosine (1 mM, 469 mg) in DMSO/CH2CI2 (0.4 ml/1.16 ml) was 1 0 added over a ten minute period. Stirring was continued for 20 minutes and then triethylamine (5 mM, 700 ~I) was added. The reaction mixture was stirred for an additional 5 minutes. Methyl- magnesium bromide ( 3 M in ether, 2 mM, 666 1ll) was added via syringe, and the reaction mixture was stirred for 1 hour at -78C, followed by warming to room temperature 1 5 overnight. Water was added and the mixture was extracted into chloroform (2x40 ml), washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by flash chromatography (silica gel; 90%
EtOAc/Hexane). Yield 390 mg (81%). Mol. Wt.:483.3 for C24H33NsoSi. FAB-MS: (M+H)+ = 484.2.
Example 2 N6-Ben7Oyl-3'-t-butyldimethylsilyloxy-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-~denosine 4,4-Dimethoxytrityl chloride (0.992 mM, 336 mg) was added to a 25 solution of N6-benzoyl-3'-t-butyldimethylsilyloxy-2'-deoxy-5'(RS)-methyl-adenosine (0.824 mM, 400 mg), triethylamine (1.12 mM, 156 ~11) and 4-dimethylaminopyridine (0.04 mM, 4.88 mg) in pyridine/ethylene chloride (1 :1;
32 ml) and stirred under nitrogen at 60C for 1 hour. Then the reaction mixture was heated to 100C and progress of the reaction was monitored by 30 thin layer chromatography (TLC). As required, more dimethoxytrityl chloride (2x200 mg) was added while maintaining the temperature at 100C. The reaction was stopped after 18 hours by addition of water, extraction into ethyl acetate and drying the pooled organic layers over anhydrous sodium sulfate.
The crude product was purified by flash chromatography (silica gel; 20%
35 EtOAc/Hexane). Yield: 307 mg (54%). Mol Wt: 785.3 for C4sHs1 NsO6Si.
FAB-MS: (M+H)+ = 786.3.

_19_ Example 3 N6-Ren7Oyl-~'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-~denosine Tetra-n-butyl ammonium fluoride (1 M in THF; 0.764 mM) was 5 added dropwise via syringe to a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-adenosine (0.382 mM, 300 mg) in anhydrous THF (4 ml). The reaction was stirred under nitrogen for 6 hours at room temperature. After a standard aqueous work-up, the crude product was purified by flash chromatography (silica gel; 100% EtOAc). Yield: 260 mg (97%) Mol. Wt. 671.4 for C3gH37N~O6. FAB-MS: (M + H)+ = 672.4.

Example 4 ~16-Ren7Oyl-?'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-~lenosine-3~-o-(Rs)-~-cyanoethyl-N .N-diisopropyl-phosphor~midite) 1 5 A solution of chloro-2-cyanoethyl-N ,N-diisopropyl-phosphoramidite (0.345 mM, 66.2 ~I) in anhydrous THF (2 ml) was added to a solution of N6-benzoyl-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-adenosine (0.235 mM, 160 mg), diisopropylethylamine ( 0.92 mM, 200 ~I) and 4-dimethylaminopyridine (8 mg) in anhydrous THF (2 ml), and stirred under 20 nitrogen for 3 hours. After an aqueous work-up, the crude product was purified by flash chromatography (silica gel; 50% EtOAc/Hexane. Yield: 140 mg (68.4%) Mol. Wt.: 871.0 for C4gHs4N7O7P. FAB-MS: (M + H )+ = 872.3.

Example 5 3'-t-Butyldimethylsilyloxy-~'-deoxy-5'(RS)-methyl-thymidine To a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-thymidine (14 mM, 5 9) in 140 ml of methylene chloride was added at room temperature (8.98 9, 21 mmol) of Dess-Martin periodinane reagent. After 30 minutes of reaction time, the mixture was diluted with 150 ml of ether, and a mixture of 30 Na2S2O3 (19.9 9, 126 mmol) in 150 ml of saturated sodium bicarbonate was added, and stirring continued for 15 minutes. The reaction mixture was poured over 1~0 ml of ethyl acetate and the organic layer was separated, washed with saturated sodium bicarbonate solution (2x150 ml), dried over anhydrous sodium sulfate and concentrated in vacuo. The crude aldehyde in 35 600 ml of benzene was azeotroped (Dean Stark) and the solvent was removed in vacuo to afford 4 9 of the crude aldehyde.
To a solution of the above aldehyde (4 9) in 150 ml of THF at -78C was added dropwise methylmagnesium bromide in ether (3.0 M, 90 ml, WO 94/22890 3 % -20- PCT/US94/02993 20 eq.); after stirring for 1 hour, the reaction mixture was poured over a mixture of 1000 ml of ice/saturated ammonium chloride and 10 ml of acetic acid. The resulting mixture was extracted with ethyl acetate (5x200 ml) and the organic layer was washed with saturated sodium bicarbonate (2x450 ml) and brine (1x450 ml), dried over anhydrous sodium sulfate and concentrated in vacuo. The product was purified by flash chromatography (100 9 silica gel;
50% EtOAc/Hexane). Yield: 1.93 9.

Example 6 3'-t-Rutyldimethylsilyloxy-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine To a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-5'(RS)-methyl-thymidine (300 mg, 0.8 mmol) in 2.7 ml of pyridine at room temperature was added 98 mg (1 eq.) of dimethylamino-pyridine, 0.6 ml (5 eq.) of triethylamine and 1.37 9 (5 eq.) of dimethoxytrityl chloride. The reaction mixture was allowed to react at 75C overnight. The mixture was diluted with ethyl acetate, washed with water, followed by saturated ammonium chloride solution, and the organic layer was dried with anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel; 30% EtOAc/Hexane). Yield: 225 mg.

Example 7 ~'-Deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine Tetra-n-butyl ammonium fluoride (1.1 M in THF; 0.68 mM, 0.9 ml) was added dropwise via syringe to a solution of 3'-t-butyldimethylsilyloxy-2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine (0.68 mM, 460 mg) in anhydrous THF (3.44 ml). The reaction was stirred under nitrogen for 4 hours at room temperature. After a standard aqueous work-up the crude product was purified by flash chromatography (silica gel; 60 - 75% EtOAc/Hexane).
Yield: 340 mg (92%).

WO 94/22890 2 1 ~ ~ ~ 3 2 PCT/US94/02993 Example 8 ~'-Deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine-3'-O-(RS)-(2-cy~noethyl-N .N-diisopropyl-phosphoramidite) To a solution of 2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-5 thymidine (0.58 mM, 320 mg) in 6 ml of methylene chloride at -40C was - added diisopropylethylamine (1.5 eq., 155 mg), followed by chloro-2-cyanoethyl-N,N-diisopropyl-phosphoramidite (170 mg, 1.3 eq.). The reaction mixture was stirred at -30C for 2 hours, and at 0C for two additional hours.
Methanol (0.1 ml) was added to the mixture and the resulting reaction mixture 10 was stirred at 0C for 1/2 hour, diluted with ethyl acetate, and washed with saturated ammonium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel; 40% EtOAc/Hexane). Yield: 376 mg (65%).
Example 9 3'-t-Rutyldimethylsilyloxy-~'-deoxy-5'(RS)-(1 -c~rbmethoxy- 1 -phenylsulfonyl)methyl-thymidine To the crude aldehyde from Example 5 (3 mmol) was added 20 dropwise at -78C a solution of sodium methyl phenylsulfonylacetate in 6 ml of THF. After allowing the mixture to react at -78C overnight, the reaction mixture was worked-up according to the procedure described in Example 5.
The product was purified by flash chromatography (silica gel; 50%
EtOAc/Hexane). FAB-MS: (M + H )+ = 569.
Example 10 3'-t-Butyldimethylsilyloxy-2'-deoxy-5'-epoxyethyl-5'-deoxy-thymidine The crude aldehyde from Example 5 (0.5 mmol) was added 30 dropwise via syringe at -78C to a solution of diazomethane (approx. 1.5 mmol) in ether. After 2 hours of reaction time, the mixture was allowed to stand at -78C for overnight. The reaction mixture was worked-up according to the procedure described in Example 5. The product was purified by flash chromatography (silica gel; 50% EtOAc/Hexane). Yield: 100 mg. FAB-MS
35 shows the desired mol ion for the title epoxide.

wo 94,22890 2 ~ 6 ~ ~ -22- PCT/US94/02993 Example 11 Oli~omer Synthesis Usin~ 2'-Deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine-3'-O-(RS)-(2-cyanoethyl-N .N-diisopropyl-phosphoramidite) The synthesis of the oligomers listed in Table 1 was conducted 5 using an ABI 380B synthesizer in accordance with the manufacturer's protocols. Synthesis of strands at one micromole scale using monomer (e.g 2'-deoxy-5'-dimethoxytrityl-5'(RS)-methyl-thymidine-3'-O-(RS)-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite) was carried out. Monomer was dried just before DNA synthesis. The purification of DNA product was conducted with 10 DMT + reverse phase HPLC with slicing through major peak.

Oligomer Sequence number 1 5'-l l ~ T~T-3' (SEQ ID NO:1) 2 5'- 1 1 I m I I I TT-3' (SEQ ID NO:2) 3 5'-m 1 1 I TT'T~T-3' (SEQ ID NO:3) 4 5'- ~ TT-3' (SEQ ID NO:4) 5'-TT~T TT~T~ TT~T-3' (SEQ ID NO:5) 6 5'-GGG TGT GTG rTA GCG GG-3' (SEQ ID NO:6) 7 5'-GGG TGT GTG TT~A GCG GG-3' (SEQ ID NO:7) 8 5'-GGG TGT GTG~ T~T~A GCG GG-3' (SEQ ID
NO:8) 9 5'-CCC GC~T ~A~A CAC ACA CCC-3' (SEQ ID
25 NO:9) In the above Table 1, the ~ in the sequence signifies the location of a 5'-methyl-phosphodiester bond instead of a natural phosphodiester bond.
Example 11 Analysis of Nuclease Stability For the nuclease stability studies, oligonucleotides were labelled at the 5' terminus with 32p by using T4 polynucleotide kinase and 35 standard end-labelling procedures. Unincorporated 32P-ATP was removed by passing over a NucTrap column followed by purification over Sephadex G-25. A trace (1-1011M) of 32P-labelled oligonucleotide was combined with unlabelled oligomer at a concentration of 1 IlM. These conditions minimize any variation incurred due to differences in oligomer labelling efficiencies andsimulate concentrations used in typical antisense experiments. The oligomer was added to Dulbecco's minimal essential medium (DMEM) cell culture media containing 20% fetal calf serum (FCS) which serves as a source of 3' 5 exonuclease activity. After incubation at 37C for 2.5 hours, the mixture was denatured for 2 minutes at 90C and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide/8 M urea; 19:1 acrylamide:bis-acrylamide; 89 mM Tris/89 mM boric acid/2 mM EDTA). As a 0 hour control, the oligonucleotide was added to serum-free DMEM media prior 10 to PAGE analysis. Gels were dried between sheets of cellulose drying film, imaged, and quantitative data obtained using a Phosphorimager. The gel lanes demonstrate the effect observed for the various modified oligonucleotides as compared to the control phosphodiester oligomer.
Multiple reactions were run for each oligonucleotide, and the results proved to 15 be reproducible. Furthermore, more dramatic results were obtained in experiments in which only a trace of 32P-labelled oligonucleotide (i.e., 1-10 ~M total oligomer) was added to media containing 10% or 20% FCS.
Oligodeoxynucleotide #1 (5'-TT I I I I I I TT~T-3'; SEQ ID NO:1;
containing a 3'-end cap modification with a methyl group at the 5' carbon of 20 the sugar at the penultimate thymidylate residue) was added to a final concentration of 3 nM to tubes on ice containing RPMI 1640 media with L-glutamine (GIBCO). Also added were: HEPES (GIBCO) to a final concentration of 20 mM, and fetal bovine serum to 10%. Total reaction volume was 400 ~11. Tubes which were incubated for 0 time (controls) did not 25 receive serum. Tubes were incubated at 37C for 5, 30, 60, and 120 minutes.
Time 0 tubes were kept on ice. An extra 120 min reaction lacking serum was also incubated at 37C as a control to check for chemical degradation of the oligomer. As a positive control, d(T)11 oligodeoxynucleotide (Oligomer #2 (SEQ ID NO:2), prepared in the same manner as the other, experimental 30 oligomers) was assayed at time 0 and at 120 minutes without serum.
Reactions were stopped by extraction with equal volumes of a 24:1 mixture of chloroform and isoamyl alcohol, five times. The final aqueous layer was filtered through a Spin-X, 0.22 ~M cellulose acetate filter unit. Fifty microliters of final sample, kept at 40C, was loaded onto a Gen Pak Fax 35 anionic exchange column by automatic injection, and eluted in 15 mM sodium phosphate, 1 mM EDTA mobile phase, pH 8.0, with a 0-0.5 M NaCI gradient utilizing a LKB HPLC system. The gradient went from 0 to 0.4 M NaCI in 55 WO 94n2890 2 ~ ~ 9 6 3 ?~ PCT/US94/02993 minutes, was held to 60 minutes, increase to 0.5 M NaCI at 65 minutes, and held to 70 minutes. Elution was monitored at 269 nm.
Parent oligomer and reaction product peaks were integrated for peak areas. Peak retention times were compared to controls and d(T) 5 oligomer standards that had been previously analyzed on this HPLC system.
The results of the assay show that the parent oligomer (Oligomer #1, an 11-mer (retention time of 41.76 min)), was rapidly digested, by 3'-~5' exonucleolytic activity present in the 10% fetal bovine serum. The product was an extremely stable, N-1 reaction product, a 10-mer (retention time of 10 38.64 min), the result of cleavage of the 3'-terminal thymidylate residue by the enzyme. The 10-mer remained undigested for up to 120 minutes in the presence of serum, based on the resulting peak areas.
Thus, the 3'-modification provides protection to the remaining oligomer against further digestion by inhibiting the activity of the 3'->5' 15 exonuclease present in the serum. It is concluded that the methyl group present at the 5' position of the sugar interferes with the hydrolysis of the phosophodiester bond by the nuclease enzyme.

Example 13 Hybridiz~tion (Tm) Testin~
Two nanomoles of each of the 1 7-mer antisense oligonucleotides of Table 1 (oligomer numbers 6, 7, 8 and 9) were combined with two nanomoles of their respective complementary sense strands in a buffer containing 50 mM Na2HPO4/100 mM NaCI/1 mM EDTA, denatured at 25 85C, and annealed by slow cooling to room temperature. The annealed DNA was heated from 35C to 85C and absorbance data at 260 nM
collected at 0.2 C intervals. From this data, melting curves were generated and values for melting temperature (Tm) derived.
The Tm for the sense and antisense control (normal, unmodified 30 phosphodiester [PDE]) strands annealed together is 68 +/- 0.5 degrees. The Tm for each of Oligomers 6 (SEQ ID NO:6) and 7 (SEQ ID NO:7) was 68C.
The Tm observed for Oligomer # 8 (SEQ ID NO:8) annealed with sense PDE
strand was 67.8 degrees. The Tm for Oligomers 8 (SEQ ID NO:8) and 9 (SEQ
ID NO:9) annealed together is 66.8 degrees. The results show that there is 35 virtually no loss of duplex stability when the normal PDE bond is replaced with this substituted PDE bond. The results further show that the methyl-PDE
bond does not at all interfere in Watson Crick base paring even when both members of the pair that anneal to each other are methyl-PDE modified (i.e., 215~6:~

Oligomers 8 and 9). This result indicates that the racemic 5'-methyl analogs of nucleosides can be incorporated at multiple sites within an oligonucleotide, and have essentially no effect on hybridization stability.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

10 (i) APPLICANT: Saha, Ashis (ii) TITLE OF INVENTION: NOVEL 5' SUBSTITUTED NUCLEOSIDES AND
OLIGOMERS PRODUCED THEREFROM

15 (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterling Winthrop, Inc.
(B) STREET: 9 Great Valley Parkway (C) CITY: Malvern (D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19355 25 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

WO 94/22890 2 ~- ~ 9 5 3 PCT/US94/02993 (viii) ATTORNEY/AGENT INFORMATiON:
(A) NAME: Newman, Irving (B) REGISTRATION NUMBER: 22,638 (C) REFERENCE/DOCKET NUMBER: PRF 161 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 889-8824 10 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 20 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (10A11) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

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(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 94/22890 ~ 1 5 9 ~ ~ 2 PCT/US94/02993 (ii) MOLECULE TYPE: DNA (genomic) 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
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(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 20 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (8^9) (D) OTHER INFORMATION: /note= "The bases are linked by a 5~-methyl-phosphodiester bond."
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (9^10) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

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W O 94/22890 æ 1 ~ 9 6 ~ ~ PCTrJS94/02993 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (6^7) (D) OTHER INFORMATION: /note= nThe bases are linked by a 5'-methyl-phosphodiester bond."

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l l l l l l l l l I T 11 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (2^3) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

WO 94/22890 2 I ~ ~ B ~ 2 PCTIUS94/02993 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (5^6) (D) OTHER INFORMATION: /notee "The bases are linked by a 5'-methyl-phosphodiester bond."

(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (6^7) (D) OTHER INFORMATION: /note= nThe bases are linked by a 5'-methyl-phosphodiester bond."

(ix) FEATURE:
(A) NAME/KEY: misc_feature 1 5 (B) LOCATION: (9^10) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (10A11) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester link."

(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (11^12) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

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2~ ~963~ -30-(i) SEQUENCE CHARACTERISTICS:
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(A) NAME/KEY: misc_feature (B) LOCATION: (10^11) (D) OTHER INFORMATION: /note= ~The bases are linked by a 5'-methyl-phosphodiester bond."

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(ix) FEATURE:
(A) NAMEtKEY: misc_feature (B) LOCATION: (10^11) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (1 1 ^12) (D) OTHER INFORMATION: Inote= "The bases are linked by a 5~-methyl-phosphodiester bond."

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(A) NAME/KEY: misc_feature (B) LOCATION: (5~6) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: (6^7) (D) OTHER INFORMATION: /note= "The bases are linked by a 5'-methyl-phosphodiester bond."

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Claims (12)

1. A compound having the structural formula:

Formula I
wherein:
Q is selected from the group consisting of H, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphates, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy:
each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;
each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4; and each B is independently selected from the group consisting of adenine, cytosine, guanine, thymine or uracil, or an optical isomer thereof or a pharmaceutically acceptable salt thereof.

2. A compound having the structural formula II below:

Formula II

wherein:
Q is selected from the group consisting of H, NHR, CHO, phosphate, lower-alkyl, lower alkenyl, protected O-, protected N-, lower alkoxy, lower alkenyloxy, benzyloxy, dimethoxytrityloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, halogen, phosphonium salt and phosphonate;
L is selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), H, OH, NHR, phosphate, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, halogen, epoxyethyl, phosphonium salt, phosphonate and t-butyldimethylsilyloxy;
each R is independently selected from the group consisting of OZ, SZ and NHZ;
each R1 and R2 is independently selected from the group consisting of H, OH, lower alkyl, lower alkenyl, lower cycloalkyl, epoxyethyl, amino lower alkyl, amino lower alkoxy, lower alkoxy and lower alkenyloxy;
each R3 and R4 is independently selected from the group consisting of H, lower alkyl, lower alkenyl, lower alkoxy and lower alkenyloxy;
each Z is independently selected from the group consisting of H, lower alkyl, lower alkenyl, aryl, acetyl and protecting groups for O-, S-, and N-;

each E is independently selected from the group consisting of -OP(OCH2CH2CN)(N-iPr2), NHR, lower alkyl, lower alkenyl, lower alkoxy, lower alkenyloxy, amino-lower alkyl, amino-lower alkoxy, N3, epoxyethyl, phosphonium salt and phosphonate;
each n is independently 0 or an integer from 1 to 4 each B is independently selected from the group consisting of adenine, cytosine, guanine, thymine or uracil, each W is selected from 3'-(OPO2HO)C(R1R2)-5' and a natural phosphodiester internucleoside linkage with the proviso that at least one W is

3'-(OPO2HO)C(R1R2)-5'; and in at least one W, R1 or R2 is other than hydrogen; and q is an integer from 1 to 60.
3. The compound of claim 2, wherein W is 3'-(O-PO2H-O)-5'-CH(CH3)-.

4. The compound of claim 2, wherein at least one W is a natural phosphodiester internucleoside linkage.

5. The compound of claim 2, wherein q is 2, 3 or 4.

6. The compound of claim 2, wherein q is from 9 to 50.

7. The compound of claim 2, wherein q is from 12 to 25.

8. The compound of claim 2, wherein q is from 15 to 18.

9. A method of synthesizing oligonucleotide compounds having from 2 to about 60 bases and having an internucleoside backbone containing one or more 3'-O-PO2H-O-5'-CR1R2 internucleoside linkages instead of the naturally occurring backbone of phosphodiester internucleoside linkages, this process comprising using a compound of claim 1 as a synthon in a conventional synthetic organic process for synthesizing oligonucleotides.

10. The method of claim 9, wherein the synthesis is carried out in an automated DNA synthesizer.

11. A method of inhibiting the expression of a gene that comprises administering to a host mammal in need of such inhibition an inhibition-effective amount of a compound of claim 2, said compound hybridizing to a nucleotide sequence of said gene.

12. A pharmaceutical composition comprising a compound of claim 2 dissolved or dispersed in a physiologically tolerable carrier.