CA2088673A1 - Compounds and methods for inhibiting gene expression - Google Patents

Abstract

The present invention relates to compounds, compositions and methods for inhibiting gene expression. The compounds of this invention comprise: 1) oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage or 2) oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.

Description

WO92/n2534PCTtUS91/05531 COMPO~NDS AND ~T~ODS
FOR IN~IBITING G~NE ~P~ESSION
De~criPtion Cro~s ~eference to Relate~ A~Plications 5This application is a continuation-in-part of application U.S. Serial No. 07/562,180, filed August 3, 1990; application U.S. Serial No. 07/582,287, filed September 13, 1990; application V.S. Serial No.
07/582,456, filed September 13, 1990; and application U.S.
Serial No. 07/582,457, filed September 13, 1990.
Technical F~eld The present invention relates to compounds, compositions and methods for inhibiting gene expression.
~ ~~ ~ The compounds of this invention comprise 1) oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage or 2) oligonucleotide sequences of from about 9 to about 200 bases havi~3 a diol at either or both termini.

sacx~round of t~e Inventio~
An antisense compound .Ls a compound that binds to or hybridizes with a nucleotide sequence in a nucleic acid, RNA or DNA, to inhibit the function or synthesis of said nucleic acid. Because of their ability to hybridize with both ~NA and DNA, antisense compounds can interfere with gene expression at the level of transcription, RNA processing or translation.
Antisense molecules can be designed and synthesized to prevent the transcription of specific genes to mRNA by hybridizing with genomic DNA and directly or indirectly in~ibiting the action of RNA
polymerase. An advantage of targeting DNA ic that only small amounts of antisense compounds are needed to achieve a therapeutic effect. Alternatively, antisense compounds can be designed and synthesized to ~ybridize with RNA to inhibit post-transcriptional modification tRNA processing) or protein synthesis (transla~ion) - , ,. . ~ , i : , W092/025~ PCT/US91/0~31 ,~ v ~,J~ 3 - 2 - .

mechanisms. Exemplary target RNAs are messenger RNA
(mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and the like. Examples o~ processing and translation mechanisms include splicing of pre-mRNA to remove introns, capping of the 5' terminus of mRNA, hybridization arrest and nuclease mediated mRNA
hydrolysis.
At the pressnt time, however, the development of practical scientific and therapeutic applications of antisense technologies is hampered by a number of technical problems. Klausner, A., Biotechnoloqv, 8:303-304 (l990). Synthetic antisense molecules are susceptible to rapid degradation by nucleases that exist in target cells. The oligonucleoside sequences of antisense DNA or RNA, for example, are destroyed by exonucleases acting at either the 5' or 3' terminus of the nucleic acid. In addition, endonucleases can cleave the DNA or RNA at internal phosphodiester linkages between individual nucleosides. As a result of such cleavago, the effective half-life of administered antisense compounds is very short, necessitating the use of large, frequently administered, dosages.
Another problem is the extremely high cost of producing antisense D~A or RNA using availabl~
semiautomatic DNA synthesizers. It has recently been estimated that the cost of producing one gram of antisense DNA is abdut $100,000. Armstrong, L., Business Week, March 5, l990, page 89.
A further problem relates to the deliYery of antisense agents to desired targets within the body and cell. Antisense agents targeted to genomic DNA must gain acce~s to the nucleus ti.e. the agents mu~t permeate the plasma and nuclear me~brane). The need for increased membrane permeability (increased hydrcphobicity) must be balanced, however, against the . : . . ,. : ::
. . : : , . . . - :
. : . . .- : : :

W092/025~ PCT/US91/0S~l ,~ ) need for aqueous solubility (increased hydrophilicity) in body fluid compartments such as the plasma and cell cytosol.
` A still further problem relates to the stability of antisense agents whether free within the body or hy~ridized to target nucleic acids.
Oligonucleotide sequences such as antisense DNA are susceptible to steric reconfiguration around chiral phosphorous centers.
~ene targeting via antisense agents is the inevitable next step in human therapeutics. Armstrong, supra at 83. The success~ul application of antisense technology to the treatment of disease however, reguires finding solutions to the problems set forth above.
One approach to preparing antisense compounds that are stable, nuclease resistant, inexpensive to produce and which can be delivered to and hybridize with nucleic acid targets throughout the body is to synthesize oligonucleoside saquences with modi~ications in the normal phosphate-sugar bacXbone ~tructure.
In general, two types o$ oligonucleoside se~uences, with modified backbones have been reported.
The first type includes modi~icat:ions to the normal int~rnucleoside phosphodiester linXage. The second type includes replacement of the phosphodiester linkage with non phosphate internucleoside linkagas. Uhlmann, E. and Peyman, A., ~hemical ~eviews, 9(4):544-584 (1990).
Mod~fied phosphodiester linkages that have been report~d to date are phosphorothioates, alkylphosphotriesters, methylphosphonates and alkylphosphoramidat2s.
Phosphorothioate modified phosphodiester linkagas refer to phosphodiester ~onds in which one or more of the bridging oxygen atoms is replaced by sulfur.
Such linka~es, however, are not suitable for use in ' . ,; ~; , . ' .. . :., .

: ,, ~ ; :

W092/02S3~ PCT/US91/05531 -- 4 -- -- _ V ~

antisense compounds. The retention of the chiral phosphorus center results in steric variation of monothioates. Purther, both mono- and dit~ioates lack sequence specific hybridization and both are rapidly cleared from the plasma. The high affinity of phosphorothioates for glass and plastic also makes synthesis of these compounds difficult and inefficient.
Methyl- and ethylphosphotriesters have been prepared by reacting phosphodiester linked oligonucleosides with anhydrous ~ethanol or ethanol.
~iller, P.S. et al., J. Am. Chem. Soc., 93:66S7-6665(1971).
The triester linkage in oligodeoxyribonucleotide ethylphosphotriesters is stable under no~nal physioloqical pH conditions, although it can be hydrolyzed by strong acid or base.
Methylphosphotriesters are less stable than ethyl- and other alXylphosphotriesters at n~sutral pH, owing to the possibility o~ nucleophilic displacement of the triester methyl group by solvent. Oligod~eoxyribonucleotide ,ethylphosphotriesters appear to ]~e complately re~istant to hydrolysis by exonucleases and are not hydrolyzed by nucleases or esterases found in fetal bovine serum or human blood serum. Uhlmann, supra.
The methylphosphonates have several significant shortcomings in terms o~ therapeutic potential which include poor water solu~ility, chiral phosphorous centers, inability to control high yield stereoselective synthesis, rapid plasma clearance and urinary excretion.
Oligodeoxyribonucleoside phosphoramidates have internucleoside bonds containing nitrogen-phosphorus bonds. These nucleic acid analogs can be prepared from phosphoramadite intermediates or ~y oxidation of H-phosphonate intermediates in the presence of a primary W092/~2534 PCT/US91/05531 ~ 5 -c; , or secondary ~mine. Preparation of the H-phosphonate analogs and the oxidation reaction can be readily carried out in a co~mercial DNA synthesizer.
A variety of non-ionic oligonucleoside segyences containing non-phosphate internucleoside linkages such as carbonate, acetate, carbamate and dialkyl- or diarylsilyl- derivatives have been synthesized and reported.
Althouqh the carbonate linkage is resistant to hydrolysis ~y acid, it is rather easily cleav~d with base, and thus special precautions are required for removaL of the~protecting groups at the end of the synthesis. While stable dupl~xes have b~en observed between po:Ly(dA) analogs containing carboxymethyl ~5 internucleotlde linkages and poly(U) analogs, other bases have not been studied. Thu~s, it is not known whether the ~idelity of duplex formation with other bases will ~e perturbed by the carbonate linkage.
Internucleoside carbamates are reported to be more water soluble than other internucleoside bridges.
T~elut~lity o~ carbamate linX~ges is limited, however, since thymine carbamates do not ~orm hybrids with complementary DNA, while cytosine carbamates do not hybridize to guanine oligomers.
The carbamate linkage, like the carbonate linkage, is stable under physiological conditions.
Unlika the carbonates, however, the carba~ate linkage is stable to hydrolysis by bases, a property i.~ich simplifies tha synthesis of oligomers ~ontaining this linkage. The carbamate linkage is resistant to nuclease hydrolysis.
Like the carbonate and acetate linkages, the car~amate linXage does not re~emble the shape of t~e phosphodiester internucleotide bond. However, molecular models suggest that the linkage should allow the W0~)2/n253~ PCT~US9l/05~31 " ~ ~

oligomer to assume conformations which would allow it to form hydrogen-bonded complexes with complementary nucleic acids. There are conflictin~ reports on the stability of duplexes for~ed by carbamate oligomers and complementary nucleic acids. A carbamate-linked oligomer containing six thymidine units does not form complexes with either A(pA)s or dA(pA)5. On the other hand, a carbamate-linked oligomer containing six deoxycytosine units forms stable complexes with d-(pG) 6 and poly(dG).
The internucleoside linkage of dialkyl- or diphenylsilyl oligomer analogs closely resembles the tetrahedral geometry of the normal phosphodiester internucleotide bond. The oligomers are prepared in solution by reacting a suitably protected nucleoside-3~-O-dialkyl- or diphenylsilyl chloride or trifluoromethanesulp~onyl derivative with a 3'-protected nucleoside in anhydrous pyridine. The former can be prepared by reaction of 5~-O-trityl nucleoside with dialkyl- or diphenyldichlorosilane or with the bis(trifluoromethan~sulphonyl)diisopropylsilane.
Because the dialkyl- and diphenylsilyl linkages are sensitive to hydrolysis by acid, care must be taken in choosing protecting groups ~or the synthesis. Nucleoside dimers and hexamers having siloxane internucleoside linkages and a method of synthesizing such polymers have been reported by Ogilvie and Cormier. See, e.n., Ogilvie, K.X. and Cormiar, J.F., Tetrahedron Letters, 26(35):4159-4162 (1985); Cormier J.F. and Ogilvie, X.K., Nucleic Acids Research, 16(10):4583-4594 ~1988).
Although the carbonate, carbamate and silyl linked oligonucleoside sequences have the requisite nuclease resistance to make them attrActive candidates as antisense reagents, their ability to function in t~is :, :,- - :

WO ~2/0253q PCI`/US91/OSS31 r. ,, ~
r . ~

-- 7 ~

capacity has not yet been reported. Further, the abllity of these oligomers to be taken up by cells in culture has not been reported. A potential drawback with these oligomers is their reported low solubility in aqueous solution. It ls not clear whether sufficient concentrations can be obtained or their effective use in biological experiments, although so;ubility could presumably be increased by introduction of hydrophilic groups into the molecules.
The present invention provides oligonucleotide analog compounds, compositions comprising such compounds, intermediatas for preparing such compounds and methods for synthesizing such novel stable, nuclease resistant, target specific, lipid soluble 15 oligonucleotide analogs.

8UmmarY o~ the ~nvention The present invention provides nucleotide analog compounds comprising oligonucleoside seguences of 20 from about 6 to about 200 ~ases having a three atom internucleoside linkage. ~he three atom internualeoside linkage of such oligonu~leoside sequences has the formula:
-D-D-D-25 where each D is independently ~HR, oxygen or NR6, wherein ~ is independently hydrogen, OH, SH or NH2, R6 is hydrogen or Cl-C2 alkyl, with the proviso that only one D is oxygen or NR6, In a pref~rred e~bodiment, the oligonucleoside 30 sequences comprise bases selected from the group consisting of adenine, cytosine, guanine, uracil, thymine and modifications thereof.

..
- .:

' ' , ,:'; `'', ` :
: .. .. :

W092/02~34 PCT/US91105~31 n~; ~7 3 More particularly, compounds of the present invention comprise oligonucleoside sequences of Formula I:

Lw~- ~
, ,_, ,~

~Rl where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH or NH2, R6 is hydrogen or C1-C2 alkyl with the proviso that only one D is oxygen or NR's;
each W' is independently W or o Oe each R1 is independently OH, SH, NR~R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is Cl-Cl2 acyl;
each y is independently H `or OH;
each B is independently adenine, cytosine, gNianine, t~.ymine, uracil or a modification thereof;
j is an integer from l to about 200:
k is o or an integer from l to about 197; and :`
`

,: ~ ~ , . , .:

WO~2/02534 PCTlUS91/0553i 9 _ q is O or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.
The compounds of the present invention comprise oligonucleotide or oligonucleoside sequences optionally having a diol at either or both termini.
Preferred diols are 1,2-diols (glycols).
Representative glycols are polyalkyleneglycols, preferably polyethyleneglycols or polypropyleneglycols.
Preferred glycols are tetraethyleneglycol and hexaethyleneglycol. Suitable diols may also include polyols that have all but two hydroxyls blocked. J
Where the compounds of the present invention are oligonucleoside sequences having a diol at either or both termini, the compounds of the present invention have Formula II:

2 ~

~ ~ II

W ~B

,~: . , , :.
:.

W092/02534 PCT~US91/05~31 f., _ J

where each z is independently R' or O O
Il 11 Rl- ([(CIH)pO]m ~PI~ O)e([(CIH)~ ]n IP 0)~;

where each R1 is independently OH, SH, NHR~ wherein R2 and R3 are independently hydrogen, or C~-C6 alkyl, or NHR4 wherein R4 is Cl~Cl2 acyl:
each Rs is independently hydrogen or Cl-C1. alkyl;
each of W, W', Y, B, j, k, and ~ is as defined above;
each e and f is independently O to 50 with the proviso .
that at least one of e and f be at least l;
each m and n is independently l to 200; and each p is lndependently 2 to 4.
Xn a pre~erred embodiment, the sum of ~ + k q is from about 9 to about 50 baso~s, more preferably from about 12 to about 25 and most preferably from about 15 to about 18. In this embodiment, compounds of this invention comprise oligonucleotid~es of the formula:
O O o Il 11 11 l l R- (t~1H)p]m ~I~ O)C([(lH)pO]n -1- O)~oligo (N)](O -P-[ (CH)pO]n)e(O -P- ~(CH)p]n~
Rl Oe 3S where R is OH, SH, NR2R3 wherein R2 and ~ are independently hydrogen or C1-C6 alkyl, or NHR4 wherein R4 is Cl-Cl2 acyl;
R~ is hydrogen or C1-C12 alkyl;
oligo (N) is a native or modi~ied oligonucleotide sequence of from about 9 to about 2ao bases;
each e and f is independently O to 50, with the proviso that at least one of e and f be at least l;

~ r; ) ~
~,.J

each m and n is independently l to 200; and each p is independently 2 to 4.
In a preferred embodiment, the oligonucleotide contains, in a homopolymer or heteropolymer sequence, S any combination of dA, dC, dG, T.
Where the glycol is polyethyleneglycol, the compounds of this em~odiment comprise oligonucleotides of the formula:
O o R- ~(C~2CHzO)~ -P- o]e[oligo(N)][o -P- (oCH2CH2) n]f -R~
- Oe Oe . ._ .
where R is OH, SH, NR2R~ wherein R2 and R3 are independently hydrogen or C1-C6 alXyl, or NHR~ wherein R~
is C1-C1z aoyl;
oligo N is an oligonucleotide sequence of from about 9 to about 50 bases;
e and f are independently O to 50, with the proviso that at least one of e and f be at least l;
m and n are independently O to 200 with the proviso that at least one of m and n be l to ~00.
T~e oligonucleotides o~' the present invention can include known internucleosid~l lin~ing groups such as phosphodiester, silyl and other well known linking groups providing they contain an effective amount of the -D-D-D- linking groups of the present invention and/or diol terminating groups of the present invention.

:. . : -. ~ . . ~ . .: , :
., - - .
~ .. ., ::- :

W~'~2/0253~ PCT/US91/0~31 i~ J _ ;J '~

The present invention is also directed to nucleoside dimers of the formula:

~Y
B
W--<`l ~Y
R

where W is -D-D-D- wherein each ~ is independently CHR, oxygen or NR6 wherein R is independently hydrogen, OH, SH or NH2 ~ R6 is hydrogen or Cl-C2 alkyl, with the proviso that only one D is oxygen or NR6;
each ~ is independently adenine, cytosine, guanine, thymine, uracil or a modi~ication thereof;
R7 is OH, t-butyldimethylsilyloxy or a phosphoramidite and R8 is OH, a protecting group or t-butyldimethylsilyloxy.
The present invention further provides a method of inhibiting nuclease degradation of compounds comprising oligonucleoside sequences. This method comprises attaching a diol to either the 5', the 3' terminus or both termini of said compound. The diols are , : - ~, ~ : ; , . .

W092/0~34 c~ PCT/US91/05~31 ~ G , j '~ _~
~,J`J

attached to the 5' and/or the 3' terminus by reacting the oligonucleotide compounds with an alXoxytrityldiolcyanophosphine, preferably a dimethoxytritylglycolcyanophosphine or a monomethoxytritylglycolcyanophosphine.
The present invention further provides a method of inhibiting nuclease degradation oP native or modified nucleotide compounds comprising preparing oligonucleoside sequences of from about 6 to about 200 lO bases having a three atom internucleoside linkage having the formula -D-D-D- as defined herein.
.
The present invention also provides compositions useful in inhibiting gene expression comprising compounds comprising oligonucleoside 15 sequences of from about 6 to about 200 bases having a three atom internucleoside linkage as defined herein and a physiologically acceptable carrier. The compound may have a diol at either or both termini. Preferred diols are polyethyleneglycols.
The present invention further provides a method of inhibiting gene expression comprising administering to a mammal in need o~ such ~reatment an effective amount of a compound comprising an oligonucleoside sequence from about 6 to about 200 bases 25 having a three atom internucleoside linkage as defined herein. The compounds may h~ve a diol at either or both termini. Preferred diols are polyethalyeneglycols.

Brief Description of the Drawinqs ~igure la depicts a synthetic pathway for preparing a nucleoside aldehyde (Compound I).
Figure lb depicts a synthetic pathway for preparing a phosphonium iodide nucleoside ~Compound II).
Figure 2 depicts a syntpetic pathway ~or 35 preparing nuclaoside dimers connected by a 3 carbon ~ : . . .:,: . :
. : . .

::. : : .: .:: . :
:: .: . ~: . : : , - :- : : ~, ;. ~ :

W0~l2/02534 PCTtUS91/05~31 14 ~

internucleoside lin~age utilizing the aldehyde nucleoside and the phosphonium iodide nucleoside of Figures la and lb respectively.
Figure 3 depicts a synthetic pathway for preparing a thymidine dimer utilizing a thymidine aldehyde and a phosphonium iodidP thymidine (compounds I
and II respectively).
Figure 4 depicts a synthetic pathway for preparing a nucleoside dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3'-C-C-N-5'. Dimers are synthesized by reacting nucleosid~ss that contain aldehydes (CHO) with nucleosides that contain amine functionalities (NH2) undex reductive conditions.
Figure 5 depicts a synthetic pathway ~or preparing a nucleoside dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3l-N-C-C-5'. Dimers are synthesized by reacting nucleosides having aldehyde and amine functionalities under reductive conditions.
Figure 6 depicts a synthetic pathway ~or preparing a thymidine dimer connected by a two carbon-one nitrogen atom internucleoside lin~age of the form 3'-C-C-N-5~. ~imers are synthesized by reacting thymidines that contain aldehydes (CHO) with thymidines that contain amine functionalities (NH2) under reductive conditions.
Figure 7 depicts a synthetic pathway for preparing a thymidine dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3'-N-C-C-5'. Dimers are synthesized by reacting thymidines having aldehyde and amine functionalities under reductive conditions.

: . :

,~

~J J

D~tailed De~criP~on of t~e Invention The compounds of the present invention are generally oligonucleotide or oligonucleoside sequences that are resistant to nuclease degradation.
As used herein, "nucleoside" refers to a combina~ion of a purine or pyrimidin~ base with a five-carbon sugar (pentose).
As used herein, "nucleotide" refers to a phosphoric acid ester of a nucleoside.
As used herein, "oligonucleotide" refers to polynucleotides having only phosphodiester internucleoside linkages, e.g. "native" DNA or RNA.
Exemplary nucleosides are adenosine(A), guanosine(G), cytidine(C), uridine(U), deoxyadenosine (dA), deoxyguanosine(dG), deoxycyt:idine(dC) and thymidine(1').
The compounds of the present invention comprise oligonucleoside sequences of from about 6 to about 200 bases having a phosphodiester or a three atom lnternucleoside linkage. The thr~se atom internucleoside linkag~ ~-D-D-D ) contains l) thr~ae carbon atoms, 2) two carbon atoms and one oxygen atom or 3) two carbon atoms and one nitrogen atom.
$he oligonucleoside sequences are saquences of natiYe or modified nucleosides. As used herein, the phrase "internucleoside linkage" refers to atoms and molecules forming a bridge between the sugar moiety caxbon atom at position 3 of one native or modified nucleoside and the sugar moiety carbon atom at position 5 of an adjacent such nucleoside. The sugar ~oiety may be either a ribose or a deoxyribose moiety or an analog thereof. Thus~ the nucleosides include A, C, G, U, dA, dC, dG, T or modifications thereof as for example 5-bromo or 5-iodouracil, 5-methyl cytosine, isocytosine ~S t2-amino-4-oxopyrimidina~, isoguanine (2-oxo-6-,' ,....

- :
, W092/02534 PCT/US91/~5531 aminopurine), inosine (6-oxopurine), 5-vinyluracil and 5-vinylcytosine.
The three atom internucleoside lin~age has the ~ormula:
-D-D-D-where each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH or NH2, oxygen, R5 is hydrogen or C~-C2 al~yl, with the proviso that only one D is oxygen or NR6.
The compounds of the present invention comprise oligonucleoside se~uences of Formula I:

~ ' ~w . W' ~ ~
~Y

where ~ is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH or NH2, ~6 is hydrogen or Cl-C2 alkyl, with the proviso that only one D is oxygen or NR6;

. .. ; .. ~ . :, ~: , . ,. ~ : .

:: - :~ ::
:: . , : .
: : -:: . . . :~

.
:.

WO 9~/0'~534 ~ ~ PCr/US91/05531 ~ r ";J ~1 ' ~,, J _, each W' is independently W o- o le each Rl is independently OH, SH, NR~R3 wherein R- and R3 are independently hydrogen or C~-C6 alkyl or N~R4 wherein RJ is C~-CI2 acyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
j is an integer from 1 to about 200;
k is O or an integer from 1 to about 197; and q is o or an integer from 1 to about 197, wi~h the proviso that the sum of ; + k I q is from about 4 to about 200.
In a preferred embodiment, the sum of j + k +
q is from about 9 to about 50. In a more pre~erred embodiment, the sum of ; + k + q is from about 12 to about 25 and, more preferably from about 15 to about 18.
The compounds of the present invention may have a diol at either or both termini. Pre~erred diols are ~lycols, also known as 1,2-diols, which contain two hydroxyl groups on adjacent carbons. Preferred glycols are polyalkyleneglycols. The term "alkylene" as used herein refers to linear and branched chain radicals having 2 to 4 carbon atoms which may be optionally substituted as herein defined. Representative of such radicals are ethylene, propyle~e, isobutylene, and the like. Preferred polyalkyleneglycols are polyethyleneglycols such as hexaethyleneglycol and tetraethyleneglycol. Suitable diols may also include polyols that have all but two hydroxyls blocked.
The diols are attached to either the 5', the 3' or both termini of the oligonucleosides via phosphodiester linkages. In one embodiment, the diols , .: . . .... . . .

. . . : .:

W092/025~ PCT/US91/05531 - l8 ~ V J ~

are attached to only one terminus of an oligonucleoside sequence.
The terminal diol is linXed to a moiety selected from the group consisting of hydroxyl (OH), sulfhydryl (SH), amino (N~2), alXylamino (NH-alkyl), dialkylamino (N[alkyl].) and amido (NH[acyl]).
Where glycols are present at either or both termini, the compounds of the present invention comprise oligonucleoside seqiuences of Formula II:

~ ~ 1 1 ~ ~ 8 Y

where each Z is independently R' or O O
Il 11 . .
R~- ([(CIH)pO]m -Pl- )~([(ClH)p~O]n-p-o)f;

.. . : , ` : .:
::
; ', . ~ : ' ,: . ;. , , .:

: .......... , : . , -: i ~ . :. ::: .. :: . ::: : :: :: i -where each R~ is independently OH, SH, NHR~R3 wherein R2 and R3 are independently hydrogen or C~-C6 alkyl, or NHR4 wherein R4 is C~-C~2 acyl;
each R5 is independently hydrogen or Cl-C~2 alkyl;
each of W, W', Y, B, j, k, and q is as defined above;
each e and f is independently 0 to 50, with the proviso that at least one of e and f be at least l;
each m and n is independently l to 200; and each p is independently 2 to 4.
In a preferred embodiment, m and n are independently l to 6 and the sum of j + k + q is from ~~~~ ~~ about 9 to about 50. In a more preferred embodiment, ~
the sum of j + k + q is from about 12 to 25, more preferably from about 15 to about 18.
lS In another preferred embodiment, the compounds of the present invention compris~e oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini. In yet another pre~erred embodiment, the compounds of the present invention comprise oligonucleotide sequences of from about 9 to about 200 bases h~ving a (~D-D-D-) linkage of the present invention.
Preferred diols are glycols, also known as l,2-diols, which contain two hydroxyl groups on adjacent carbons. Preferred glycols are polyalkyleneglycols.
The term "alkylene" as used herein refers to linear and branched chain radicals having 2 to 4 carbon atoms which may be optionally substituted as herein defined.
Representative of such radicals are ethylene, propylene, butylene and the like. Pre~erred polyalkyleneglycols are polyethyleneglycols. More preferred are tetraethyleneglycol ar.d hexa2thyleneglyccl.
The diols are attached to either the 5', the 3' or both termini ~f the oligonucleotides via p~osphodiester linkages. In one embodiment, the diols ~ .,: ; .

. . - .
. ~

' ` ;.~, ' :.` , .. ~ : .

WO~2t~2534 PCT/US91/05531 ri r ~
v v rj -" . j are attached to only one terminus of an oligonucleotide sequence.
The terminal diol is linked to a moiety selected from the group consisting of hydroxyl (OH), sulfhydryl ~SH), amino (NH2), alkylamino (NH-alkyl), dialkylamino (N[alXyl]2) and amido (NX[acyl]). As used herein, "alkyl" refers to linear or branched chain radicals having l to 12 carbon atoms which may be optionally substituted as herein defined.
l0 Representative alkyl- and dialkylamino radicals include methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, dimethyl-, diethyl-, dipropyl-, di~utyl-,-dipentyl- and dihexylamines and the like. As used herein, "NH(acyl)"
or "amido" refers to linear or ~ranched chain radicals 15 having 1 to 12 carbon atoms with a termi~al 0-CNH2 group. Representative amido radi.cals ~nclude methanamide, ethanamide, propana~lide, butanamide, pentanamide, hexanamide, heptanan~ide, octanamide, nonanamide, decanamide, undecanamide and dodecanamide.
In one embodiment, the compound~ o~ the pre~ent invention compri~e oligonucleotides o~ the formula:
O o o Il 11 . `11 R ([(CIR)pO]m ~1~ )e(t(cl~)po~n -Pl- o)f[olig~ ~N)~(O -P1-t ~fH)pO~n) ,~ - I-- t(CH~p]m) f R;
R1 Oe 3 S where R is OH, SH, NR2R3 wherein RZ and R3 are independently hydrogen or C1-C6 alkyl, or NHR~ wherein R~
is C~-C12 acyl;
R~ is hydrogen or C1-C12 alkyl;

:.; :

:: :: , . . , - .
- . , .

. ,~ ~ ' ' . . . ` ` ', ':
: . ., . '' . " "' :' ' ` ' ' ~ . ''~ - " .

J ~

oligo (N) is a native or modified oligonucleotide sequence of from about g to about 200 bases;
each e and f is independently O to 50;
each m and n is independently 1 to 200; and each p is independently 2 to 4.
The oligonucleotide sequence is preferably a homopolymer or heteropolymer sequence containing any combination of dA, dC, dG, T or analogs thereof.
In a preferred embodiment, m and n are independently 1 to 8 and, more preferably, both m and n are 4. Preferred oligonucleotide sequences contain from about g to about 50 bases, more preferably about 12 to about 25 hases, and most preferably about 15 to about 18 bases.
In a preferred embodiment, the antisense compounds have polyethyalkyleneglycol at both the 5' and 3' termini and have the formula:
Il I O
R ~(CIH)pO]m -Pl- O)~t~(CIH)po]n -1- O)f~oligo (N)](O -X-25 . Il [ (CH) p]n) e~ ~ I ~ ~0 (CH) p]~) t -R
Rl Oe where R is OH, SH, NR2~3 wherein R2 and R3 are independently hydrogen or Cl-C6 alXyl, or N~R~ wherein R4 is Cl-Cl2 acyl:
R1 is hydrogen or Cl~C12 alkyl;
oligo (N) is~a native or modified oligonucleotide sequence of from about 9 to about 200 bases;
each ~ and f is independently 1 to 50;
each m and n is independently 1 to 200; and each p is independently 2 to 4.

:
~ .

~ .-- : .
. ;: : .
:: :
.

W092/025~ PCT/US91/05~31 r l ~

Where the glycol is polyethyleneglycol, the compounds o~ this embodiment comprise oligonucleotides of the formula:
O O
R- t(CH2CH20)~ -P- O~[oligo(N)~0 -P- tOC~2CH2)n], -R:
Oe Oe where R is 0~, SH, NR2Ri wherein R2 and R3 are independently hydrogen or C~-C~ alkyl, or NHR~ wherein R~
iS C~-C~2 acyl;
oligo N is an oligonucleotide sequence of from about g to about 50 bases; and e, f, m and n are each independently 1 to 50.
In a preferred embodiment, the oligonucleotide contains, in a homopolymer or heteropolymer sequence, any combination of dA, dC, dG, T.
In other preferred embodiments, the polyethyleneglycol is tetraethyleneglycol (TEG) and both m and n are 4 or hexaethyleneglyc:ol and both m and n are 6.
The compounds o~ the p~esent invention are u~e~ul as anti3ensQ a~Qnts. AntlqensQ agents hybridize ~5 with a complementary nucleotide ~;equence in a target nucleic acid to inhibit tha translational or transcriptional function of said target nucleic acid.
The target nucleic acid may be either RNA or DNA.
Antisense compounds of the present invention comprise oligonucleoside sequences of from about 6 to about 200 bases having homopolymer or heteropolymer sequences comprising bases selected from the group consisting of adenine (A), cytosine ~C), guanine (G) ` uracil (U), thymine ~T) 2nd modific~tions of these bases. Particular sequences are selected on the basis o~ their desired target. The sequence selected hybridi~es with the targe~ nucleic acid. Exemplary :: ,-: : ;. , . :: :
:: :
. : . ~ ;.; .. , :,;

WO g2/02534 PCr/US91/0553 t~rgets include the MYC oncosena, the RAS oncogene, and viral nucleic acids.
The compounds of the present invention can be prepared by the following procedures:
~. Com~ounds havina a three_carbon internucleoside linXaqe Oligonuclessides connected by a three-carbon internucleoside linkage are synthesi2ed by reacting nucleosides having aldehyde and ylide functionalities at 3' and 6' positions respectively under Wittiq conditions.
The syntheses of a nucleoside aldehyde and a phosphonium iodide nucleoside from commercially available compounds are illustrated in Figure la and lb, respectiva:Ly. The aldehyde ~Compound I from Fiqure la) i5 synthesized ~rom the known 3'-i~llyl-3'-deoxy-5'-0-tert-butyldimethylsilyl-3'-thymid,ine ~Compound A, Figure l). The allyl compound is regioselectively o~idized with a catalytic amount of osmium tetroxide and N-methylmorpholine oxide as a cooxidant. The resultant diol (Compound B, Figure la) iq cle~ved with sodium ~eriodate to give the aldehyde in almost quantitative yield.
~he synthesis of the phosphoniu~ iodide nucleoside (Compound II, Figure lb) follows from a commercially available 5'-tritylated nucleoside (Compound C, Fiqure lb). The tritylatad nucleoside is silylated at the 3' position with tert-butyldimethylsilyl chloride and the trityl group removed under acidic conditions with high effioiency. The resultant primary hydroxyl (Compound E, Figure lb) is oxidi~ed under Swern Gonditions to give the aldehyde (Compound F, Figure lb). The crude aldehyde is "~
immediately reactad with the ylide derived fro~
methyltriphenylphosphonium bromide to give a 4'-vinyl-. , : -. . i : , , "~

4 P~T/US91/05531 ~ ~ n ~
24 _ ~ ~ v ~

4'-deoxy-3'-tert-butyldimethylsilyl nucleoside in good yield. The vinyl compound i-~ regioselectively hydroborated to give a primary alcohol (Compound G, Figure lb) in good yield. The primary alcohol in turn is converted to the corresponding iodide (Compound ~, Figure lb) using triphenylphosphine-iodine in the presence of imidazole in excellent yield. Finally, the iodide is transformed to the desired phosphonium iodide nucleoside using triphenylphosphine in acetonitrile.
A ylide is prepared from the phosphonium iodide nucleoside using potascium tert-butoxide as a base and immediately reacted with the aldehyde to give-a~
Wittig product (Compound 1, Figure 2) in good yield.
The Wittig product is regioselectively hydrogenated with 10% palladium on carbon ~10~ Pd-C) with hydrogen at atmospheric pressure ln quantitative yield to saturate the double bond of the linkage. The saturated compound (Compound 2, Figure 2) ls desilylated with tetrabutylammonium fluoride to gi.ve the diol tCompound 3, Figure 2~. Tha 5'-primary hydlroxyl of the diol is then rQgioselectively protectQd with dimethoxytrityl chloride and the resultant 3'-hydroxyl (Compound 4, Figure 2) is converted to a phosphoramidite (Compound 5, Figure 2) with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
The nucleoside dimers or hiyher oligomers with trialkylsilyloxy protecting groups are conjugat~d to form oligonucleotides of any desired length. Upon completion of chain elongation, the oligomers are deprotected by standard methods. For furt~er chain length extension in a solid phase synthesizer in which the oligomers are connected by phosphate linkages, the terminal 5'- and 3'-hydroxyl groups of the oligomers are appropriately functionalized, raspectively with .

.: . :: . ~.. . . . -; .

W092/025~ PCT/US91/05~31 ~ J~
- 25 ~

tritylating reagents such as dimethoxytritylchloride and phosphoramidite.
B. ~ompounds havinq a two carbon-one ox~gen atom inter~ucleoside linkaqe Oligonucleoside sequences having ~ two carbon-one oxygen atom internucleoside linkage are synthesized by reacting 3'-silylated, 5'-toluenssulfonyl nucleoside with a 5'-protected nucleoside.
A 3'-acetyl-5'-aldehyde nucleoside is prepar~d from a commercially available 3'-acetyl-nucleoside using standard methods well known to those of skill in the -art. The 3'-acetyl-~'-aldehyde nucleoside is then ~~~~ ~ ~~
converted to a 3'-acetyl-5'-carbomethoxymethylene nucleoside using a modified Wittig reaction.
The 5'-methylene side chain is reduced with sodium borohydride in alcohol, preferably isopropanol, followed by deprotection of the l'-acetyl group with sodium methoxide in an alcohol, preferably methanol.
The 3'-hydroxy ~s then protected with a silyl group. In A preferred embodiment the silyl group i5 a t~bu~yldim~thylsilyl group.
The 3'-silyl-5'-carbomethoxymethyl nucleoside is then further reduced to a 3'-0-silyl-5'-deoxy-3'-(2 "-ethanol) derivative of the nucleoside with diisobutyl aluminum hydride (DIB~L) in tetrahydrofuran (THF). The 5'-ethanol group is converted to ~ p-toluenesulfonyl group with p-toluene sulfonyl chloride in pyridine. The exocyclic amino group of the base moiety of the 5'-p-toluenesulfonyl nucleoside is optionally protected by methods well Xnown and readily apparent to those of skill in the art. A preferred protecting group for '.hr exocyclic amino groups of adenine and cytosine is the benzoyl moiety. A preferred protecting group ~or the exocyclic amino group of :
.

.~ ., .. . ~.

W0~.~/02534 PCT/US91/05531 ~ ~ v ~

guanine is the isobutyl moiety. Guanine may also be protacted at the o6 posi.tion.
The 3'-0-silyl-S'-0-p-toluenesulfonyl nucleoside is then reacted with a 5'-protected S nucl~oside to ~orm a ~-0-silyl-S'-protected nucleoside dimer with a two carbon-one oxygen atom internucleoside linkage. The 5'-0-prot~ecting group is preferably a trityl and, more preferably a dimethoxytrityl. The 3'-0-silyl-5'-0-protected nucleoside is optionally protected at the exocyclic amino groups of the nucleoside base moiety.
- The nucleosid,e dimers are`~deprotected and rederivatized at the 3'-carbon atom position with a cyanophosphine reagent, preferably 2-cyanoethoxydiisopropylaminophosphine for use in a phosphoramidite solid p'hase synthesis method of chain elongation. Gait, su~ra.
The nucleosid,e dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleosides oE any desired length. Upon completion of chain elol~gation, the oligomers are deprotected by standard ~ethods. For further chain length extension in a solid phase synthesizer in which the oligomers are conne,:ted by phosphate linkages, the ~ terminal 5' and 3' hydroxyl groups of the oligomers are appropriately functionaLized, respectively with tritylating reagents su~:h as di~ethoxytritylchloride and j ~hosphoramidite.
~ C. Compounds havinq a two carbQn-one 30 ~ n~qen atom internucleoside_linkage Oligonucleoside sequences connected by a two c~rbon-cne nitrogen atQ~ internucleoside l~nkage o~" the form C-C-N are synthesi;~ed by reacting nucleosides that j contain aldehydes with nucleosides that con~ain a~ine : ~ - ~ , ", '~ ' ' . ~ ;' ' : . : ' ... , : .
. , ~ .

W092/025~ PC~/US~1/05531 ~. , _ functionalities under reductive conditions as illustrated in Figure 4.
Both the aldehyde and the amine compounds are prepared from commercially available compounds. The 5 aldehyde is prepared from 3'-allyl-3'-deoxy-5'-O tert-butyldimethylsilyl thymidine. The allyl compound is regioselectively oxidized with a catalytic amount of osmium tetroxide in the presence of N-methyl morpholine,N-oxide as a cooxidant to give the diol. The diol is, in turn, oxidized with sodium periodate to give the aldehyde in almost quantitative yield.
The amine compound is synthesi2ed from `'~'~
commercially available nucleosides. In a typical procedure, the primary hydroxyl group of a nucleoside is regioselectively transformed into a tosylate group with p-toluene sulfonyl chloride and then converted into an iodide. The 3'-hydroxy of the iodide intermed'iate is protected with tert-butyldimethylsilyl chloride and the azido group introduced by reactirlg with sodium azide.
The azido functionality is efficiently converted to the required amine by reduction usinsl 10% palladium on carbon under a hydrogen atmosphere or Raney Nickel reduction conditions.
The amine and the aldehyde are coupled (reductive amination) in the presence of sodium cyanoborohydride under buffered conditions. The oligonucleoside dimer with a C-C-N internucleoside linkage is formed in good yield. The oligonucleoside is reacted with trifluoroacetic anhydride-triethylamine, to protect the secondary aliphatic nitrogen. The protected oligonucleoside is desilyla~ed with tetrabutylammonium fluoride and the primary hydroxyl group of the resultant diol is selectively protected with dimethoxytrityl chloride. The remaining secondary hydroxyl is .: , . :
. . .
.. ~
.
.

W O 92/02S34 PC~r/US91/05531 ~ ~ ~! r.

transformed to the required phosphoramidite by reacting with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
Oligonucleosides connected by two car~on-one nitrogen atom internucleoside linkage of the form N-C-C
are synthesized by reacting nucleosides having aldehyde and amine functionalities at 3'- and 51_ positions, respectively, under reductive conditions as illustrated in Figure 5, The amine and the aldehyde components are synthesized from commercially available compnunds. The amine is synthesized from 3-azido-3-deoxy thymidine (AZT). The primary hydroxyl group of AZT is protected with dimethoxytritylchloride and the resultant azide regioselectively transformed to the required amine with 10~ palladium on carbon in the presence of a hydrogen atmospherQ or using Raney Nickel.
The aldehyde is synthelsized from commercially available 5'-O-dimethoxytritylth~ymidine. The tritylated thymidine i5 silylated with tert-butyldimethylsilyl chloride and the trityl group is removed under acidic conditions. The resultant primary hydroxyl group is oxidized under Swern conditlons to give tho aldehyde.
The aldehyde is not isolated but immediately reacted with (carbethoxymethylene)triphenylphosphorane to give the unsaturated ester. The unsaturated ester is regioselectively hydrogenated with 10% palladium on carbon to give a saturated ester in quantitative yield.
The saturated ester in turn is converted to the required aldehyde with diisobutyl aluminum hydride ~DI~AL-~) in a highly selective manner.
The amine and the aldehyde are coupled in the presence o~ sodium cyanoborohydrid2 under buffered reductive amination conditions. The N-C-C
internucleoside linkage is obtained in good yield. The secondary aliphatic nitrogen of the oligonucleoside is ., .. .. ,, - . -. .. ,., : : ~ : - , - ~ - . .- : . . .

-, . . -. . : .. .: , . .

WO')2/02S3'1 PCT/US91/0~531 protected with trifluoroacetic anhydride and triethylamine. The protected dimer or higher oligonucleoside sequence is desilylated and the resultant hydroxyl converted to phosphora~idite with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite.
The nurleoside dimers or higher oligomers with trialkylsiloxyl protecting groups are conjugated to form oligonucleosides of any desired length. Upon completion of chain elongation, the oligomers are desilylated by standard methods. For further chain length extension in a solid phase synthesizer in which the oligom~rs are conne~ted by phosphate linkages, the terminal 5' and 3' hydroxyl groups of the oligomers are appropriately ~unctionalized, respectively with tritylating reagents such as dimethoxytritylchloride and phosphoramidite.
D. ~ompounds havinq a diol at çither or ~oth WherQ desired, diols are attached to either or both termini by a modi~ication of the solid phase phosphor~midi~e method. Q~ cl~o~idq Svn~h~si~: A
Practical ~roach, ed. by M.J. Gait, pages 35-81, IRL
Press, Washington, D.C. tl384).
~n accordance with our modification of the solid phase method, a diol is introduced at one, or both, terminalts) of the oligon~cleotide by a procedure in which the diol is reacted with an alkoxytrityl compound to ~orm a tritylated diol. The diol is preferably a glycol, more preferably, a polyalkyleneglycol. The alkoxytrttyl reagent is preferably monomethoxytrityl chloride or dimethoxytrityl chloride and, most preferably dimethoxytrityl chloride.
The tritylated diols are ~hen reacted with a cyanophosphine reagent to form a trityldiolcyanophosphine compound, which compound is used as a phosphoramidite reagent (hereinafter referred W092/02534 PCT/U~91/05531 J,~

to as a "diol phosphoramidite reagent") in the solid phase synthesis of the compounds of the present invention.
The initial step in solid phase synthesis is attachment of a nucleoside to a solid support, preferably a controlled pore glass (CPG) support. The nucleoside is preferably attached to the CPG via a succinate linkage at the 3'-hydroxyl position of the nucleoside. Other means of attaching nucleosides to solid supports are known and readily apparent to those of skill in the oligonucleotide synthesis art.
Alternatively, in order to introduce a diol ~t the 3' terminal, a diol phosphoramidite reagent can be attached to the solid support prior to addition of the first nucleoside. The diol phosphoramidite reagent i~
attached to the solid support using succinatls or other linkages in a manner analogous to methods used for nucleoside attachment. Means of modifying such methods for use with diol phosphoramidite reagents will be readily apparent to those o~ skill in the art. Any num~er of diols can be placed on the solid support prior to add~tion of the fir~ nucleo~,ide. Preferably ~rom 1 to about 50 diols are used. Whe!re diols are attached only to the 5' terminus, no diols are placed on the solid support.
Following attachment of the first nu~leoside ~r the diol~s) to the solid support, chain elongation occurs via the sequential steps of removing the 5'-hydroxyl protecting group (a functionalized trityl group), activating the 5'-hydroxyl group in the presence of a phosphoramidite reagent, i.e., a 5'-trityl nucleoside, 3'-phosphoramidite, capping the unraactad nucleosides and oxidizing the phosphorous linkage.

::-: : ., ;. . .:

W0~2/02534 P~T/US9t/05531 ,~r;~ 31 -J ~ .
The protecting group at the 5'-hydroxyl position of the attaohed nucleosides is removed with acid, preferably trichloroacetic acid.
Activating reagents that can be used in accordance with this method are well known to those of skill in the art. Preferred activating reagents are tetrazole and activator gold (BecXman Instr. Inc., Palo Alto, CA).
The activation step occurs in the presence of the added nucleoside phosphoramidite reagent or diol phosphoramidite reagent, which latter reagent replaces the nucleoside phosphoramidite reagent of conventional synthetic methods when diol is added to the terminal(s) of the polynucleotide. Unreacted chains are terminated --`
or capped with capping reagents such as acetic anhydride and N-methyl imidazole.
The labile trivalent phosphorus linkage is oxidized, preferably with iodine, to the stable, pentavalent phosphodiester lin~age of the oligonucleotide.
After the desired oligonucleotide chain assembly is complete, the phosphalte protecting groups are removed, the chains are separated ~rom the solid support and the base protecting groups are removed by conventional methods. Gaits, su~ra at 67-70.
T~ose skilled in the art will appre~iate that other means of synthesizing oligonucleotides can be modified in an analogous manner to produce diol-terminated antisense oligonucleotides.
The compounds of the present invention are use~ul in treating mammals with heredit~ry disorders or diseases associated with altered genetic expression mechanisms. At present, attempts are underway to develop antisense therapies for use in treating viral infections such as ~IY, cytomegalovirus, herpes simplex, ` ' , . . ~ ,:
'' ', ~ :

:
' `

W092/025~ PCT/US91/OS~31 ~ ~ ~ ' ;' `3 hepatitis ~, papilloma virus and picorna virus; cancers of the lung, colon, cervix, breast and ovary;
inflammatory diseases; and diseases of the i~mune system such as acquired immunodeficiency syndrome (AIDS), hematological neoplasma and hyperproliferative disorders. Armstrong, supra at 89; Klausner, supra at 303, 304.
Compositions of the present invention useful in inhibiting gene expression comprise physiologically acceptable carriers and 1) compounds comprising oligonucleoside sequences of from about 6 to about 200 bases having an internucleoside linkage of the ~ormula -D-D-D- as defined herein, optionally having a diol at either or both termini or 2) compounds comprisinq oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.
Compositions of the pr~sent invention useful in inhibiting genQ expression include one or more of the compounds of this invention formulated into compositions together with one or more non-toxic physiologically acceptabl~ carriers, ad~uvants or veh~cles which are collectively ra~erred to herein ~Ig carriers, ~or parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, and the like.
The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally ~powders, ointments or drops), or as a buccal or nasal spray.
Compositions suitable ~or parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconsti~ution into ,. ~ , , ~ , , , :
~ .. . .. . ... .

W~2/0253~ PCTiUS9t/05~31 sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, qlycerol, 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 r . _ . _ _ . . .
agents. Prevention of the action o~ microorganisms can be ensurecl by various antibacterial and antifungal lS 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 in~ectable form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate ~nd gelatin.
~ If desired, and for more effective distribution, the compounds can be incorporated into slow release or targeted delivery systems such as 2~ polymer matrices, liposomes, and microspheres. They may be sterilized, for example, by ~iltration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable mediu~ immediately be~ore use.
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 iner~ customary excipient (or W(~2/025~ PCT/US91/05~31 ~ ,7,~

carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegr~ating ag~nts, as for example, agar-agar, calcium 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 ben~onite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol~, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also compriss buffering agents.
Solid compositions of a similar type may also be employed as fillers in ~o~t and hard-~illed gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage ~orms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in this art. They may contain opacifying agents, and can also be of such composition that they ~0 release ~he active compound or compounds in a certain part of the intestinal tract in a delayed manner.
~xamples of embedding oompos~tions which can ~e used are polymeric substances and waxes.

- : . ~, : `: .

W0~2/02534 PCT/US91/05531 The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms ~or oral administration include physiologically acceptable e~ulsions, solutions, ;
suspensions, syrups and elixir~. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubiliz~ng agents and emuls$fiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, l,3-butyleneglycol, . .-dimethylfo:rmamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, ~lavoring and per~umin~ agents~
Suspensions, in addition to the active compounds, may contain suspendin~ agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahy~roxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the lika.
Compositions for rectal administrations are pre~erably suppositories which can be prepared ~y ~ixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body Wo~2/n2s34 PCT/US91/05~31 ~ v - 3~ -temperature and therefore, melt in the rectu~ or vaginal cavity and release the active component.
Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The act$ve component is admixed under sterile conditions with a physiologically acceptable carrier and any needed preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medlum. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present co~po~itions in liposome ~orm can contain, in addition to the lipoxygenase inhibiting compounds o~ the present inven~ion, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
Methods to form liposomes are known in the art. See, for example, ~ethods_in Cell ~ioloov, Ed. by Prescott, Volume XIY, Academic Press, New Yor~, N.Y., p. 33 et seq., (l976).
Actual dosage levels of active ingredient in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular co~position and method of administration.
The selected dosage level therefore depends upon t~e '`~ `' , ' ' ' ~
,.,: ,,, :. : :

W092/02534 PCT/US91/05~31 , . . ~
,.,~'j,J~) I
- 37 - .

desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered t~ a host in single or d$vid2d doses may ~e in amounts, for example, of from about 1 nanomol to about 5 micromols 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 ~or any particular patient will depend upon a variety~of factors including the ~ody weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity o~ the particular disease being treated.
The following examples further illustrate the best mode of carrying out the invention and are not to be construed as limiting of the s;pecification and claims in any way.

EXAMPLE 1: PreP~ration o~ 5~?'-dimeth~xYtri~Yl-3-0'-t-butyldimethyls~Lyl thvmidinç.
Dimethoxytrityl thymidine (5.0 g, 9.2 mmol) and imidaz~le ~1.2 g, 18.4 mmol) were dissolved in 15 ml of anhydrous dimethyl ~ormamide (DMF) and added to tert-butyldimethyl~ilyl chloride (1.7 g, 11.5 mmol).
The reaction mixture was stirred for 4 hours at room temperature, diluted with ethyl acetate and washed with water, saturated sodium chloride and dried with sodium sulfate. A quantitative yield of the title compound was obtained.

:. ''' .. : . : , W092/02534 PCT/US~1/0~31 ~ ~ ., u i 3 EX~MPLE 2: reparation of 3'-Q-t-butyldimethylsilvl thvmidine, 5'-0-dimethoxy-3' 0-t-butyldimethylsilyl thymidine prepared according to the method of Example 1 (O.7 g, 1.1 mmol~ was treated for 1 hour at room temperature with 13 ml of a 3S trichloracetic acid in methylene chloride solution. The reaction mixture was then neutralized with a 5~ (w/v) sodium bicar~onate solution. The organic layer was dried with sodium sul~ate. The title compound was purified by flash chromatography using a 0 to 30~ gradient of ethyl acetate in methylene chloride. The yield of-the-reaction was 85%.

EXAMPLE 3: Preparation of 3'-0-t-butyldilnethylsilyl ~hvmidine ~' aldehvde.
To a well stirred solution of dry ~ethylene chloride at -78-C was added oxalyl chloride (33.0 mmol, 2.88 ml) followed by dropwise addition of DMS0 ~3.12 ml, 4.4 mmol). After 10 minutes, the alcohol (5.6 g, 15.7 mmol), prepa~ed according to the met~od o~ Example 2, in `20.0 ml o~ CH~C12, was add~d dropwise over a period o~ 2 minutes and stirring was continued for 45 minutes. Et3N
(8,1 ml, 58.1 ~mol) was added and stirring continued for another 45 minutes. The reaction mixtur~ was then brought to room temperature and then washed with water ~2 X 10 ml) followed by brine ~10 ml) and dried (Na2SO4). The crude aldehyde was used for the next step.
ExAMæLE 4: Pre~aration of 5'-vinyl-5'-deoxy~3'-t-butyldimethYlsil~l deoxy~hy~idinÇ~
To a solution of methyltriphenyl phosphonium bromide ~O.7 mmol) in dry tetrahydrofuran (THF) at 0~C
was added a solution of sodium bis :" '. "~ -,,', " :

W092/025~ PCT/~S91/0~31 (trimethylsilylamide)(0.6 ~mol~ dropwise. After 30 minutes, a solution of the corresponding 4'-aldehyde in THF was added dropwise under nitrogen. The reaction mixture was stirred for 2 hours, diluted with ethyl acetate, washed with water, ~hen with brine and dried (Na2S0~). The title compound was purified by fl~sh chromatography using 20% ethyl acetate-hexane. The yield was 55-60~.

EXAMPr~ 5: E~paration of 3'-0-t-butYldimethylsilYl-5'-deoxy-5'-hYd~oxvmethyl thymidine, -To a solution of 2M 2-methyl-2-butene (1.6 eq, 1.5 ml, 3 mmol) in 3 ml of anhydrous THF at O-C, 1.6 eqs-of a lM borane-tetrahydrofuran complex t3 ml, 2 mmol) were added slowly under N2.
The solution was stirred for lO minutes followed by the addition of the vinyl thymidine, prepared according to the method o~ Example 4, ~0.7 g, 1.9 mmol) in 5 ml of anhydrous T~F. The reaction mixture was stirred ~or 45 minutes and placed in the re~rigerator for 2 days.
W~rkup was done using a,n aqueous ~olution comprising ~.1 eq of 2M sodium hy~droxide and 3.1 eq of 30% hydrogen peroxide (preferably adding hydrogen peroxide dropwise to the aqueous sodium hydroxlde at O-C
and stirring for 10 minutes). The solution was added slowly through an addition funnel to the reaction mixture at 3'C, stirred for 1 hour, removed from the ice bath, diluted with ethyl acetate, washed with water, satura~ed sodium chloride and dried with sodium sulfate.
The title compound was purified by flash chromatography using a 20-B0% gradient of ethyl acetate in hexane. The yield was 62~.

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' . :`
. . . :

:. : .
... . . ~i, wo g2/02s3~ Pcr/

EXAMPLE 6: ~Feparation of ~'-iodomethYl-$'-deo~x~
3'-O-t-butvldimet~vlsilYl thvmidine.
To a solution of 3'-0-t-butyldimethylsilyl-5'-deoxy-5'-hydroxYmethyl thymidine prepared according to the method of Example 5 (O.3 g, O.9 mmol) in dry acetonitrile (5 ml) and ether (3.4 ml) were added 3 eq of triphenyl phosphine (0.7 g, 2.8 mmol), 4 eq ~f imidazole (O.3 g, 3.7 mmol) and 2.2 eq of iodine (O.5 g, 2.8 mmol). The reaction mixture was stirred for 45 min and the solvent was evaporated. Ethyl acetate was added to the residue and the--residue washed with water, -- ~
saturated sodium chloride and dried with sodium sulfate.
The title compound was purified by flash chromatography using a 30-50% gradient of ethyl acetate in hexane. The yield was 90S.

EXAMPLE 7: Preparation o~ 3'-0-t-butYldimethvls~lvl-5'-deoxY-5'-~hYmildyl me~hvl ~hosphonium iQ~i~Ç~
~o a stirred solution of 5'-iodomethyl-5'-deoxy-3~-0-t-butyldimethysilyl thymidlne prepared according to the method of Example 6 was added iodide t480 mg, 1 mmol) in dry CH3CN ~5 ml) and triphenylphosphine (1.57 g, 6 mmol) and the mixture re~luxed for 12 hours at 90-C. ~he reaction w~s cooled and the solvent was removed. The title compound was puri~ied by flash chromatography using 5% MeOH in C~2Cl2. The product was obtained in 95-96% yield.
EXAMPLE 8: PreParation of 5'-t-butyldimethYlsi 3~-deoxv-3~ .2~-dihYdroxy-3n-pr thvmidine.
Osmium tetraoxide ~OsO~) (4 drops, 2.5 w/v%) in butanol was added to a stirred mixture of 3' (2"-W092/02534 PCT/US91/05~31 ~ 41 -propenyl)-3'-deoxy-5'-0-t-butyldimethylsilyl thymidine prepared according to the procedure described in ~._or~.
.Chem. 1989, 54:2767-2769 tC~X. ~hu et al.) (183 mg, 0.5 mmol) and 4-methylmorpholine N-oxid~ (53 mg, 0045 mmol) in 5.0 ml anhydrous THF at O-C. The react~on ~ixture was then quenched with 10~ agueous sodium metabisulfite (2.0 ml), stirred for 20 minutes, filtered over a pad of silica and diluted with ethyl acetate (25.0 ml). The organic phase was washed with water (5.0 ml) and brine, and then dried with Na~SO~. The solvent was evaporated and the title compound purified by flash chromatography.

EXAMPLE 9: Pre~aration of 5~-o-t-but~ldimethylsi 3'-deoxY-thymid-3'~1-acetaldehYde.
lS Sodium periodate (214 mg, 1 mmol) was added to a stirred solution of the thymidine diol prepared by the method of Example 2 (200 mg, 0.5 mmol) in THF-H20 (4:1 ratio, 5.0 ml). After 1 hour, khe reaction mi~ture was diluted with ethyl acetate (25 ~1), washed with H20 (2 x 5 ml), brine and dried. The title compou~d was purified by flash chromatography with 70% ethyl acetate in h~xane.

EXAMP~E 10: Pre~aration of thYmidine dimers with a three carbon internucleoslde li~kaqe.
The processes of example lOa-lOe are illustrated in Fi~lre 3.
lOa. T~ a stirred suspension of the phosphonium iodide compound prepared according to the method of Example 7 ~241 mg, 0.32~ ~mol) in dry THF (2.0 ml) was added potassium tert-butoxide (0.62 ml, 1.0 M
solution in THF, 0.62 mmol) at 78-C under nitrogen.
After 20 minutes, the 3~-acetaldehyde compound prepared according to the method of Example 9 (80 mg, 0~22 mmol) was added. After 60 ~inutes, tha reaction mixture was : , .

W092/0253~ PCT~US91/0~31 ~ J ~ '1.3 J -~

diluted with ethyl acetate (30 ml), washed with watar (2 x 5 ml) and brine (5 ml) and dried (Na2S0~). The solvent was evaporated and the ol~fin product (Compound 1) was puri~ied by flash chromato~raphy using 70% ethyl S acetate in hexane. The yield was in the r~ngo of 55-60%.
lOb. 10% Pd-C (20 mg) was added to a stirred szlution of Compound 1 ~109 mg) in methanol ~S.0 ml) at 25'C and at 1 atmospheric pressure of hydrogen. After 4 10 hours, the catalyst was filtered over a pad of celite and the solvent was evaporated. Compound 2 was thus recovered and then purified by flash chromatography in 80% ethyl acetate in hexane.
lOc. About 2.8 equivalents of tetra~utyl 15 ammonium fluoride at O'C were added to a stirred solution of Compound 2 ~350 mg) in 5.0 ml of THF. After 3 hours, the solvent was evaporated and Compound 3 was purified by flash chromatography using 10% methanol in CH2C12, lOd. About 0.05 eguivalents of 4-dimethylamino pyridine, 1.4 equivalents of triethylamine and 1.2 equivalents of 4,4'-dimethoxytrityl chloride were added to a stirred solution of Compound 3 (o.6 mmol) in dry pyridine (4.o 25 ml). After 2 hours, t~e reaction mixture was quenched with 2.0 ml of water and then diluted with 2.0 ml of et~yl acetate. The organic phase was separated, washed with brine and dried. Compound 4 was purified by flash chromatography using 5% methanol in methylene chloride.
lOe. About 2.0 equivalents of diisopropyl ethyl amine and 1.0 ml of dry dichlorometh~ne (CH2Cl2) was added to a stirred solution o~ Compound 4 (0.5 mmo~). After 30 minutes, 0.75 equivalents of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was 35 added, drop by drop over a period o~ 20 minute~ and . . ,. .. . - .. :
.
, ,.. , ... , , :
. . , , ,, , . , - ,.
:

:. ::~ .:: :
, .... . .
:

WO~Z/025~ PCT/US91/OS531 stirring continued for another 1 hour. The ~olvent was then evaporated and Compound 5 w~s purified by flash chromatography using ethyl acetate ~containing lS
triethyl amine) under nitrogen atmosphere.
S The processes of steps a-d above re used to make dimers containing three carbon internucleoside linkages in which all three carbons have the formula -CH~-.
Any or all o~ the carbons can be optionally hydroxylated by modifying the process illustrated in Figure 3 as follcws. A drop of 2.5S (w/v) solution of osmium tetraoxide in t-butanol at O-C was added to a ' ~`~'~'' stirred solution of Compound 1 and 4-methyl morpholine N-oxide (~.1 mg) in 0.8 ml of THF. The rsaction mixture was kept at O'C for 24 hours, ~uenchled with an aqueous solution o~ sodium metabisulfite, diluted with ethyl acetate and washed with water and brine.' The solvent was e~aporated and the resulting hydroxylated dimer purified by thin layer chromatography using ethyl acetate a~ an eluent. The hydroxylated dimer is then pxotected and the 5' and ~' ter~inals modi~ied as in steps b-d, above.

EXAMP1E 11: ~re~aration of h~droxYlated three,carbon internu~leoside ~in~aqes.
The thymidine-dimer phosphoramidite compounds produced by steps a-d were used in a modified solld phase phosphoramidite synthetic procedure to make the oligonucleoside sequence~ of Table 1.
The oligodeoxynucleotides were synthesized from the 3' to the 5' terminus.

: . .. :
..: ,: ., .

- ~ . :

W092/02534 PCT/US91/OS~31 4~_ T~le 1 Sequenc ~ç~. Code S' TpTpTpTpTp[TcT]pTpTpTpTpypypT 3' S' TpTpTpTpTpTpTp[TcT]pTpTpypypT 3' 2 5' TpTpTpTpTpTpTpTp[TcT]pypT 3' 3 T= thymidine 1 0 p-- O--P--O
le c= --CH2-C~I~-CH2-Y= tetraethyleneglycol Synthesis then proceeded in accordance with a modifi~d phosphoramidite procedure. The 5'-hydroxyl group of the attached thymidine was reacted with trichloroacetic acid to deprotect: the 5'-hydroxyl group.
Following this deprotection step, the attached thymidine was reacted with the activating a\gent, tetrazole, and a phosphoramidite reagent comprising dimethoxytrityltetraethyleneglycc\lcyanophosphine. The acti~ation step was followed by the capping of unreacted 5'-hydroxyl groups with acetic anhydride and N-methylimidazole. The phosphorous: linkage was then oxidized with iodine in accordance with standard procedures. In sequences l and 2, containing two tetraethyleneglycol (TEG) residues, the deprotecting, activating, capping and oxidizing steps were repeated as described above.
Chain elongation then proceeded via the standard saguential steps o~ deprotection, acti~ation, 3~ c~pping and oxidation with t~e modification t~at a three-carbon linked thymidine dimer, prepared according to the methods of Examples l-9, in the chain was added where desired during an activation step.

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':'~ ":

W~2/02534 PCT/US91/05~31 , .. .
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At the end o~ chain assembly, the thymldine oligomers were removed from the CPG support with concentrated ammonium hydroxide. The solution was then further treated at 55-C for 8 to 15 ~ours to remove all the protecting groups on the exocyclic amines of the bases.

ExAMPm~ 12: Pre~aration o~ 3'-0-acetyl-5'-carbomethoxymethvl-5'-deoxyth~idine About 0.3~ g sodium borohydride was added to a cold (ice bath) stirred, mixture of 3.17 g of 3'-0-acetyl-5'-carbomethoxymethylene-5'-deoxythymidine in 95 ml isopropanol. The mixture was stirred at O~C under nitrogen atmosphere for half an hour, then at room tempQraturQ fo~ an additional ~o~r and one half hours.
The chllled mixture was quenched with 20 ml methanol, followed in 30 minutes ~with 200 ml of distilled water, and then extracted with several portions of ethyl acetate. The combined orqanic extracts were treated with br~ne and dried with anhydrou~ magnesium sulfate. The drying agent was filtered off and the solvent evaporated, yielding the title compound as a residual cllass of 2.7 g.

EXAMPLE 13: Pre~aration of 5'-carbome~hoxyme~hyl~
5'-deoxvth~midine About 10 drops of a 25S (w/v) methanolic solution of sodium methoxide was added to a cold, s~irred solution of 2.~2 g o~ 3'-0-acetyl-6'-carbomethoxymethyl-5~-deoxythymidine, prepared according to the method of Example 12, in about 300 ml of dry (passed throuc3h a bed of neutral alumina) methanol.
The mixtur~ was stirred un~er a nitrogen atmosphere, without replenishing the ice bath, for about 20 hours.
A small amount of cation exchange resin (Bio-Rad AG SOWX8~, was aclded and the ~ixture stirred for 30 .. . ....
. :- .:. . .
:: . . . . .. . . -- ~ .,.

W~2/~253~ PCT/US91/0~31 .~ ~ r, r~ ~ j minutesO The solvent was removed under reduced pressure, yielding a residual glass of 2.1 g, which was treated with warm toluene, and, a~ter cooling, filtered and rinsed out with cyclohexane to yield a crude product as a white solid, 1.64 g.
The title compound was further purified from a trace of starting material by chromatography on silica ~el, eluting with ethyl acetate, then recrystallization from ethyl acetate/hexane to yield white crystals.
EXAMPLE 14: PreParation of 3'-0-t-butvldimethvlsilvl-5'-~2"-hYdroxyethvl!-thvmidine - ~
A~out 19 ml of a lM diisobutylaluminum hydride in tetrahydrofuran (THF) were added to a cold (-40 to -30-C), stirred solution of 1.8~ g of 3'-0-t-butyldimethylsilyl-5~-carboethoxymethyl-5'-deoxythymidine in 40 ml of anhydlrous ~HF, under a nitrogen atmosphere. The reaction temperature was then slowly increased to -20-C.
~he mixture was quench~ed with about 3.5 ml methanol, and the reactlon tempelrature increased to -lO-C. About 18 ml water in 36 ml THF were added ~o the warm mixture and the temperature further increased to 10 C. Most of the T~F was rPmoved, via reduced pressure and the residue diluted with about two volumas of water.
The aqueous phase was extracted several times with ethyl acetate/chloroform. ~he combined extracts were washed with cold 2N hydrochloric acid, and brine, dried with anydrous magnesium sulfate and ~iltered. The sol~ent was removed from ~he filtrate with reduced pressure to yield the title compound (about 1.6 g~.

- . :

:~

W0~2/02534 PCT/US91/05531 EXAMPLE 15: Pre~aration of 3'-O-t-butyldimethylsilvl -5'-r2"-iodoethvl)-5'-deoxYthymidine About 1 g of p-toluenesulfonyl chloride was added to a solution of 1 g of 3'-0-t-butyldimethylsilyl-5'-(2ll-hydroxyethyl)-5'-deoxythymidine, prepared according to the method of Example 14, in 25 to 30 ml of anhydrous pyridine, and the mixture maintained, stoppered, at about 5'C for approximately 19 hours.
The mixture was added to about 200 ml of ice water and extracted several times with ether. The combined organic extracts were washed with cold 2N
- hydrochloric acid, water, and brine. The washed extracts were dried with anhydrous sodium sulfate and filtered. The solvent was removed ~rom the filtrate via reduced pressure to yield a residual glass of 1.24 g of the p-toluenesulfonyl derivative.
About 0.54 g of the p-toluenesulfonyl derivative and 0.38 g sodium iodide were dissolved in 55 ml of dry (molecular sieves, 4A) acetone for three days, followed by the further addition of 0.19 g sodium iodide with stirring for a ~inal day, The reaction mixture w~as filtered and the solvent was evaporated to yield crude product.
The crude product was purified by chromatography on 85 g of silica yel eluting with 25%
ethyl acetate in hexane. The solvent was evaporated to yield 0.4 g of ~he desired 3'-0-t-butyldimethylsilyl-5'(2"-iodoethyl)-5~-deoxythymidine.

EXAMPLE 16: Preparation of 5'-carbomethoxvmethvlene-5~-deoxythY~idine About 10 drops of a 25% sodium methoxide in methanol wer~ added to a stirred solution of 1.5 g of 3'-O-acetyl-5'-carbomethoxymethylene-5'-deoxythymidine in 150 ml of dry methanol (passed through a bed of ; , , : , , ,;
: ` ~ ` ' : : : : , :

: " , WO 92/02534 P(~/US91/05~;31 n n f~ ~/ V

neutral alumina). The mixture was stirred at room temperature under a nitrogen atmosphere for an additional 6 hours.
A small amount of cation exchange resin (Bio-Rad AG-50W-X8) was added to the mixture with stirring for 10 minutes. The solvent was removed under reduced pressure to yield a white solid residue of 1.3 g. The residue was triturated twice with warm toluene, then taken up in hot ethanol, filtered, and chilled to yield the title compound as a white crystalline product, after drying, 0.85 g.

EXAMPLE 17: Pre~aration of 3'-O-t-butYldimeth~lsilYl-5'-(2"-hydroxyethylene~-5'-deoxythymidine A solution of 296 m~ 5~-carbethoxy~ethylene-5'-deoxythymidine was added dropwise under a nitrogen atmosphere to a cold ~ice water bath), stirred solution of 205 mg imidazole and 227 mg t~-butyldimethylsilyl chloride in 1 ml of anhydrous dimethylformamide. After complete addition, the mi~ture W~IS removed ~rom the ice and stirring continued at ambienl: temperature for two hours, then at 35-C for another l:wo hours, and finally at 40-C for half an hour.
The mixture was then quenched with 2 ml of methanol, followed by two to three volumes of water.
The aqueous phase was extracted several times with ethyl acetate. The combined organic extracts were washed with water, saturated bicarbonate solution, and brina, dried with anhydrous magnesium sulfate, and filtered. The solvent was removed from the filtrate via reduced pressure to yield 0.40 g of 3'-O-t-butyldimethylsilylyl-5'-carbomethoxymethylen2-5'-deQxythymidine.
About 4 ml of a lM solution of diisobutylaluminum hydride in tetrahydrofuran was added dropwise to a -30-C to -35~C chilled solution of 0.37 g .
, : .
, . .... : :: :
: : ~

wos2/o2s~ P~T/US91/05531 ,,~ c~
~_ ~ 49 -o~ the 3~-o-t-butyldimethyl5ilyl-5~-carbomethoxymethylene-5'-deoxythymidine dissolved in 10 ml of anhydrous te~rahydrofuran at a temperature below -30-C. After addition was complete, the reaction was stirred under a nitrogen atmosphere for an additional two hours while maintaining the internal temperature in the range of from about -3Q- to about -20-C.
About 0.8 ml o~ methanol was added to the reaction mixture followed by the addition of a solution of 4 ml water in 8 ml of tetrahydrofuran. Most of the more volatile tetrahydro~uran was removed under reduced - --pressure. The aqueous residue was dil~ted-with about twice its volume of water and extracted several tim~s with ethyl acetate. The combined organic extracts were washed with cold lN hydrochloric: acid, and brine, dried with anhydrous magnesium sulfat~! and fil~ered. The filtrate was stripped to yield t:he residual tltle compound, 0.267 g.
A portion of this material was purified to analytical purity by chromatography on silica gel, eluting with 50S ethyl acetate/llexane.

EX~MPLE 18: PreParation of ~hYmidine dimers containinq 2 two carbon - one oxv~en atom ~ 0-C-C-5~) internucleoside Linkaqe 18a. To a stirr~d solution of 5'-0-tritylthymidine is added an equimolar amount of each of a base and 3'-0-t-butyldimethylsilyl-5'-(2n-iodoethyl)-5'-deoxythymidine at 0C. The course of the r~action is monitored'by thin layer chromatography (TL~). After the completion o~ the reaction, the desired dimer is isolate~ and purified ~y ~lash chromatography.
18b. ~o a stirred solution of 3'~0-t-butyldimethylsilyl-5'-(2"-hydroxyethyl)-51-3S deoxythymidine maintained at a te~perature of about -5-C

. ~ . ...
- .:
.. : : -: . ,.:

. .. .
: :. . :
.

WO ~ 02534 PCl`/US91/05531 r~ t ~; v is added 2 equivalents of base, and the solvent is evaporated to dryness. The residus is redissolved in DMF, and an equivalent of 5'-dimethoxytrityl-2',3'-cyclothymidine is added. The reaction mixture i~ heated to about 40-C, and the formation of the d~sired dimer is monitored by TLC. After the completion o~ the reaction, the desired dimer is isolated and purified by flash chromatography.

EXAMPLE 19: De~rotection o~ the 3' end of the dimer of Exam~le 18 The 3'-t-butyldimethylsilyl protecting group of the protected dimers is removed by treatment of a THF
solution of the dimer of Example 18 with 2.8 ~equivalents o~ tetrabutylammonium fluoride at O-C. After the completion o~ the reaction (generally about 3 hours), the solvent is evaporated and the desired dimer is isolated and purified by flash chromatography.

EXAMPLE 20: preparation of a functionallzed dimer unit s~l~able for,~utomated sYnthesis The dimer product o~ Example 19 i5 dissolved in dichloromethane, and 2 equiva].ents of diisopropylethyl amine are added. The mixture is stirred for 30 minutes, followe~ by dropwise addition of 0.75 equivalents of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite over a period of about 20 minutes. The stirring is continued ~or another hour, the solvent is evaporated, and the resulting ~unctionalized dimer is isolated and purified on a flash chromatography column using ethyl acetate under an lnert atmosphere.

W092/02534 PCT/US91/05~31 EXAMPLE 21: Preparation of S~-t-~utyldim~thylsilvl-3'-deoxy-3'-rl".2"-dihydroxv-3"-pro~vl)-thymidine.
O~mium tetraoxide (0504) (4 drops, 2.5~ w/v) in ~utanol was added to a stirred mixture of 3'-(2"-propenyl)-3'-deoxy-5'-O-t-butyldimethylsilyl thymidine (183 mg, 0.5 mmol), prepared according to the literature procedure, and 4-methylmorpholine-N-oxide (53 mg, 0.45 mmol) in 5.0 ml dry THF at 0C. The reaction mixture was then quenched with 10% aqueous sodium metabisulfite (2.0 ml), stirred for 20 minutes, filtered over a pad of silica and-~diluted with ethyl acetate (25.0 ml). The organic phase was washed with water (5.0 ml) and brine, and then dried with Na2SO4. The solvent was evaporated and the title compound purified by flash chromatography.

EXAMPLE 22: Preparation o~ 3'~deoxv-thymicl-3-~1-ace~aldehYde-5'-0~-t-butvldimethylsilylthymidine~
Sodium periodate ~214 Ing ~ 1 mmol) wa~ added to a stirred solution o~ the thymidLn~ diol prepared by the method o~ Example 21 ~200 mg, O.!i mmol) in THF-H20 ~4:1 ratio, 5.0 ml). After 1 hour, the reaction mixture was diluted with ethyl ac~tate ~25.0 ml), washed with H20 t2 x 5.0 ml) and brine and dried. The title compound was purified by flash chromatography with 70% ethyl acetate in hexane.

EXAMPLE 23: PreParation of 5'-o (p-toluenesulfonyl) th~idine~
To a stirred solution of thymidine ~20 g, 82.6 mmol) in dry pyridine (200 ml) was added p-toluenesulfonyl chloride (47.2 g, ~47.6 mmol) at O-C
under nitrogen atmosphere. A~ter 3 hours, the reaction mixture was poured onto ice and extracted with et~yl - , : : . . . : ..
.,, ;, ~ . . ., ~;
.: : . , :, : , :
- :~ . . , :
:.................. :. : : . - .:

W092/02s34 PCT/US91/0~531 ~ ~ ~,,~,3 V s -~

acetate. The solvent was evaporated and the product was crystallized from a mixture of ethyl acetate ~nd methanol. The title ~ompound was a whita crystalline solid obtained in 70-75% yield.

EXAMPLE 24: Pre~aration of 5'-iodo-5'-deoxyt~Ymidine.
To a stirred solution of the thymidine ~osylate (10.55 g, 26.9 mmol) prepared according to the method o~ Example 23 in dry acetone (75 ml) was added sodium iodide (10 g, 66.7 mmol) and the mixture refluxed for 16 hours. The solvent was evaporated and diluted with ethyl acetate. The organic phase was washed with water (2 x ~0 ml) and brine (~0 ml) and dried with sodium sulfate. The title compound was crystallized from methanol as a white crystalline solid in 90-95%
yi~ld.

EXAMPT~ 25: PreParation of 3'~0-t-but~ldimç~hY
5'-~odo-5'-deoxvt;hv~idine.
To a stirred solution of the thymidina iodide ~8~0 g, 24.7 mmol) prepared according to the method of Example 24 in dry DMF, wa5 added imidazole (4.2 g, 61.7 mmol). After 5 minutes tert~butyldimethyl silyl chloride (4.47 g, 29.64 mmol) was added and the mixture stirred for 4 hours. The reacti~n mixture was then diluted with ethyl acetate (250 ml), washed with water ~2 x 100 ml) and brine (50 ml) and then dried with sodium sulfate. The title compound was purified ~y flash c~romatography using 60% ethyl acetate in hexane in 92% yield.

EXA~P~ 26: Pre~aration of 3'-O-t-butvldimet~Ylsi 5'-azido-5'-deoxythYmidine.
To a stirred solution of the 3'-0-t-butyldimethylsilyl-5'-iodo-5'-deoxythymidine ~10.8 g, 20 ... . .
, ' :.' ', .
.
- .
:. : - -: ..

Wo~2/n2534 PCT/US91/0~531 r (~
, ~

mmol) prepared according to the meth~d of Example 25 in dry DMF (50 ml) was added sodium azide (3.9 g, SO mmol) and the mixture heated at O-C for 12 hours. Then the reaction mixture was diluted with ethyl acetate (200 ml) washed with water (2 x SO ml) and brine (SO ml) and then dried with sodium sulfate. The title compound was purified by flash chromatography using 50% ethyl acetate in hexance in 90~ yield.
., 0 EXAMPT.F 27: PreParation of 3'-G-t-butyldimethylsil~l-5'-amino-S~-deoxythvmidine.
To a stirred solution of the thymidine azide (5.0 g, 13.1 mmol) prepared according to the method of Example 26, in MeOH was added 200 mg of 10~ Pd-C und~r ni~rogen atmosphere. The nitrogen gas was then evacuated and replaced by hydrogen. The evacuation and replacement procedure was repeated twice and s~irring was continued under 1 atmospheric pressure of hydrogen ~or 12 hours. The hydrogen was removed and the catalyst ~0 was filtQred over a pad of celit~ and the solvent was removed und~r vacuum. ~he crude produc~ was purifiad by flash chromatography using 5-10% MeOH in CH2Cl2 to give the title compound in 85-87% yield.

EXAMPLE 28: Pre~aration of 3'-azido-3'-deoxY-5'-0-dimethoxvltritvl thvmidine.
To a stirred solution of 3'-azido-3'-deoxythymidine (2.67 g, 10 mmol) in dry pyridine (50 ~1) was added 4-dimethylaminopyridine (61 mg, 0.5 mmol), triethylamine (1.9 ml, 14 mmol) and 4,4'-dimethoxytrityl chloride (4.1 g, 12 mmol) in a sequential order. After 3 hours, water (30 ml~ was added and extracted with et~yl acetate (250 ml). The organic phase was separated, washed wit~ brine ~SOml) and dried with sodium sulfate. The title compound was purified by ;

w092/025~ PCT/US91/05~31 ~lash chromatography using 5S methanol in methylene chloride in 80-85% yield.

EXAMPLE 29: Preparation of 3'-amino-3'-deoxy-5'-o-dimethoxytrityl thvmidine.
To a stirred solut~on of the thymidine azide (3.99 g, 40 mmol), prepared according to the method o~
Example 28, in MeOH was added 200 mg of 10% Pd-C under argon atmosphere. The argon gas was remov~d by vacuum and hydrogen was introduced. This procedure was repeated twice and stirring was continued under 1 atmospher,e of hydrogen pressure for 12 hours. Then hydrogen was removed and the catalyst was filtered over a pad of celite and the solvent was removed under vacuum. The crude product was purified by flash chromatography using 5% MeO~ in methylene chloride to give the title compound in 90-93~ yield.
~H NMR t300 MHz, CDCl3) ~ 7.61 (s, 1 H), 7.60-7.21 (m, 1 H) 6.83-6.87 (m, 3 H), 6.85 (t, J~8.5 Hz, 1 H), 3.80 ~5, 6 H), 3.81-3.73(m, 2 H), 3.53-3.49(~, 1 H), 3.38-3.33(m, 1 H), 2.36-2.33(m, 1 H), ` 2.25-2.20 tm, 1 H), l.51(s, 3 H): IR neat YmaX 3020, 2962, 1697, 1605, 1512, 1246, 1030 cm~.

EXA~IPLE 30: Pre~aration of 5-O'-dimethoxytritvl-3-OI-t-butyldimet~Ylsilyl thvmidine.
Dimethoxytrityl thymidine (5.0 g, 9.2 ~mol) and imidazole (1.2 g, 18.4 m~ol) were dissolved in 15 ml of anhydrous dimethyl formamide (DMF) and added to tert-butyldimethylsilyl chloride (1.7 g, 11.5 m~ol). The reaction ~ixture was stirred for 4 hours at room temperature, diluted with ethyl acetate and washed with water, saturated sodium chloride and dried with sodium ~ulfate. A guantitative yield of the title compound was obtained.

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W0')2/02534 P~/US91/0553t ,~ ,~ . ...
' - 55 -EXAMPTE 31: PreDaration o~ 3'-O-t-butyldimethyLsi ~Y
thymidine.
5'-O-dimethoxytrityl-3'-O-t-butyldimethylsilyl thymidine prepared according to the method of Example 30 (0.7 g, 1.1 mmol) was treated for l hour at roo~
temperature with 13 ml of a 3% solution of trichloracetic acid in methylene chloride. The reaction mixture was then neutral~zed with a 5% (w/v) sodium bicarbonate solution. The organic layer was dried with sodium sulEate. The title compound was purified ~y flash chromatography using a 0 to 30% gradient of ethyl acetate in methylene chloride. The yie}d of ~he reaction was 8S%.

EXAM~LE 32: PreDaration of 3'-O-t-butvldim~th~lsilYl-5'-~arbethox~mçthvlene-5'-deoxvthymidin~.
To a well stirred solution of dry m2thylene-chloride at -78'C was added oxalyl chloride ~33.0 mmol, 2.88 ml) ~ollowed by the dropwise addition of DMS0~3.12 ml, 44 mmol). After 10 minutes, the thymidlne alcohol ~5.6 g, 15.7 mmol), prepared according to the method of Example 31, in 20.0 ml of CH2~12 was added dropwise over a period of 2 minutes and stirring continued for 45 minutes. Et3N (8.1 ml, 58.1 ~mol) was added and stirring continued for another 30 minutes. The reaction mixture was then brouqht to -23-C over a period of 30 minutes. Then carbethoxy methylene triphenylphosphorane ~10.94 g, 31.4 mmol) was added and the reaction mixture stirred for 12 hours at room temperature. The reaction mixture was then diluted with water (2 x 125 ml) and brine (50 ml? and dried (Na2SO~. The crude product was purified by flash chromatography using 20% ethyl acetate - hexane ~ 40% ethyl acetate- hexane to give both the : . ....... . :.- r : ~ : . : ,:,- -- . . : ., : . . . :. .

W0~2/02534 PCT/US91/05~31 r~ r, r~
J ~ ~ rj ~ S6 ~

trans and ~is isomers of the title compound in 3:}
ratio. The combin~d yield was about 72-76%.
Data for trans compound. IR (neat) v~ax 3205, 3180~ 2982~ 2964~ 1698~ 1490~ 1274 cm1; 1H NMR(300 MHZ~
S CDCl3) ~ 7~04 (5~ 1 H), 6.87 (dd. J=15.6 and 5.4 Hz, 1 H), 6.23 (t, J= 6.7 Hz, 1 H), 6.03 (dd, J= 15.6 and 1.6 ~Z~ 1 H), 4.33-4.28 (m, 1 H), 4.14 (q~ J= 71 Hz 2 ~) 4.16-4.12 (m, 1 H) 2.28-2.19 (m, 1 H), 2.09-1.98 (m, 1 H~ 1~87 (S~ 3 H), 1.23 10 (t, J37.1 ~z, 3 H), 0.81 (S~ 9 H) o.o1 (s, 6 H): Calcd for C20H32o6N2si; c, 56.58; H, 7.60; N, 6.60; FoundO C~
56.36; H, 7~30;- N,- 6~60~ - - -EXAMpTT~` 33 Preparation of 3'-0-t-butvldimethYlsilYl-5 ~ -ça~ethQxvmethyl-51 -deoX~th~midine .
To a stirred solution of the unsaturated thymidine ester (4 ~ 24 g, 10 ~mol), prepared according to the method of Example 32, in EtOAc was added 200 mg of 10~ Pd-C under nitrogen atmosphere. The nitrogen gas was re~oved by vacuum and ~ydrogen was introduced. This procedure was r~peated twic~ and stirring was continued under 1 atmospheric pr~ssure o~ hydro~en ~or 16 hours.
Then the catalyst was filtered over a pad of cel~te and the solvent was removed under vacuum. The product was crystallized fro~ a mixture of hexane and et~yl acetate.
The ~itla compound was obtained in 95% yield.
IR(neat) vmax 3180, 2925, 2950, 1696, 1486, 1260, 1240 cml; lH NMR (300 MHZ, CDCl3) ~ 7.20 (Bt 1 H), 6.11 (t, 6.6=Hz, 1 H) 4~07 (q~ J=7.1 HZ, 2 H), 4.03 -3.98 ~m, 1 H), 3.73-7.69 (m, 1 H), 2.51-2~32 (~, 2 H), 2.24-2.15 (m, 1 H), 1.18 It, J=7.1 Hz, 3 H), 0.81 (s, 9 H), 0.01 (s, 6 H), Anal. Calcd for C20H~06N2Si: C, 56.31; H, 8.03; N, 6.57 Found: C, 55.91; H, 7.74; N, 6.50.
.

- . . : ~.
:.~ : . .

W092/025~ PCT/US91/05531 EXAMPLE 34: Pre~aration_of 3~-o-t-butyldimethvlsi 5'-deoxv-thv~id-5-Yl-acetaldehYde.
To a stirred solution of the thymidine ester (3.41 g, 8 mmol), prepared according to the method of Exampl~ 33, in 60 ml of dry CH2Cl2 at -78-C was added DiBAL-H tl6.4 ml, 1.0 M solution in h2xane, 16.4 mmol) dropwise over a period of 3 min. After 20 minutes, thP
reaction mixture was diluted with 300 ml of EtOAc and washed with 50 ml of saturated sodium potassium tartrate solution twice. The organic phase was washed with brine ~25ml) and dried ~Na25O4). The title compound was - - purified by flash chromatography using 50~-70% ethyl~
acetate - hexane in 85-87% yield.

EXAMPLE 35: Preparation of a deoxythvmidine dimer h~vinq a 3'-C-C-N-5' internucleoside llnkaae ~Fiqu~e 3) 35a. To a stirred ~olution of the thymidine amine ~rom Example 27 ~1.07 g, 3 ~mol) and the thymidine aldehyde o~ Example 22 (1.38 g, 3.6 ~mol) ~n 50 ml o~
eth~nol and 10 ml of aqueous bu~'er solution ~pH ~ 5.5, NaH,PO~-NaOH) was addQd a solution of NaCNBH3 in THF ~12 mL, l.O M solution in THF, 12 mmol) dropwisa at 5-C over a period of 1 hour. The reaction mixture was stirred for another 4 hours and diluted with 2.50 ml of ethyl acetate. The reaction mixture was was~ed with water ~2 x 40 ml) and brine (25 ml) and dried tNa2SO4). Compound 1 tFigure 6) was purified by flash chromatography by first eluting with ethyl acetata followed by 5-8~ MeOH-C~2Cl2 in 62-64~ yi~ld.
IH NMR ~300 MHz, ~DCl3) ~ 7.60 ~s, 1 H), 7.19 (s, 1 H), 6.18 (t, J-6.6 Hz, 1 H), 6.08 ~t, J=3.9 Hz, 1 H), 4.29-4.23 (m, 1 H), 4.15-3.98 ~m, 1 H~, 3.91-1.85 (m, 1 H), 3.70-3.78 (m, 2 H), 2.95-2.87 (m, 1 H), 2~84~2r66 (m, 3 H), 2.35-2.05 (m, 5 H), :

` - ~.: . -: : ......................... : ; -:. :: . .. ~ -: :i . ~ . , WO~/02534 PCTfUS9ltO~5~1 1.94 (s, 3 H), 1.93 (s, 3 H), 1.80-1.63 (m, 1 H ), 1.55-1.45 (m, 1 H), 0.93 (s, 9 H), 0.69 (s, 9 H), 0.11 (s, 6 H), 0.07 (s, 6 H).
35b. Compound 1 (Figure 6) (166 mg, 0.23 mmol) was added to a stirred ~olution of trifluoroacetic anhydride ~0.32 ml, 2.3 mmol) and triethyla~ine (0.64 ml, 4.6 mmol) in CH2C12 (5.O ml). After 2 hours, the reaction mixture was quenched with aqueous NaHC03 ~5.0 ml) and diluted with EtOAc (25ml). The organic phase was washed with water (2 x 10 ml), brine (5 ml) and dried (Na2SO4). Compound 2 (Figure 6) was purified by flash chromatography using 7% ~eOH-in CH2Cl2 în 91-93%
yield.
35c. To a stirred solution of Compound 2 (164 mg, 0.2 ~nol) in ~F (4.0 ml) was added tetrabutyl-ammonium fluoride (0.8 mmol) at D'C. After ~ hours, the solvent was evaporated and Compound 3 (Fiqure 6) was purified by flash chromatography using 5%-8% MeOH in CH2Cl2 in 90% yield.
35d. To a stirred ~olution o~ Compound 3 (151 mg, 0.26 mmol) in dry pyridine ~ 3.0 ml) was added 4,4-dimethylaminopyridine ~1.6 mg, 0.0128 mmol) and triethylamine (0.057 ml, 0.42 mmol). After 5 minutes, dimethoxytritylchloride tl21 mg, 0.3~8 mmol) was added ~5 and stirring continued. A~ter 2 hours, the reaction mixture was diluted with ethyl acetate (25 ml) and washed with water (2 x 10 ml), brine (5 ml) and dried (Na2SO~). The crude product was purified by ~lash chromatography using 7% MeOH in C~2Cl2 to give Compound 4 (Figure 3) in 85-87% yield.
35e. Dry diisopropyl ethylamine (0.15 ml, ;
0.67 mmol~ was added to Compound 4 (150 mg, 0.168 mmol) followed by dry CH2Cl~ ~0.5 ml). Then the flask was shaken to dissolve the alcohol and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite ~0.056 mL, 0.25 .;

. ,. . ~, . .

W092/02534 PCT/US91/05~31 'J j~

mmol) was added over a perioA of 20 seconds. After 45 minutes, t~e reaction mixture was quenched with C~30H
tl.0 ml), diluted with EtOAc (50 ml) and Et3N (1.0 ml), washed with 10S aqueous X2CO3 t2 x 5.0 ml), followed by brine (5.0 ml) and dried (Na2SO~). The crude product was purified by flash chromatography using EtOAc to give Compound 5 (Figure 6) in 70-75% yield.

EXAMPLE 36: PreDaration of a deoxythymidine dimer havinq a 3'-N-C-C-5'-internucleoside linka~e !Fi~ure 4).
36a. To a stirred solution of the amine of --~
Example 29 (2.72 g, 5 mmol) and the aldehyde of Example 34 (2.29 g, 6 mmol) in 50 ml of ethanol and 10 ml of 15 aqueous buffer solution (pH - 5.5, NaH2PO~NaOH) was added a solution o~ NaCNBH3 in THF (12ml, 1.0 ~ solution in THF, 12 mmol) dropwise at 5'C over ~ period o~ 1 hour. The reaction mixture was stirred for another 4 hours and then diluted with 250 ml of ethyl acetate.
20 The reaction mixture was washed wit~ water (2 x 60 ml) and brine and driQd ~Na2SO~). Compound 1 (Figure 7) was purifiQd by fl~sh chromatography by first eluting with ethyl acetate followed by 5% MeOH-CH2Cl2. Compound 1 (Figure 7) was obtained in 72-74S yield.
IH NMR (300 MHz, CDCl3) ~ 7.56 (m, 1 H), 7.36-7.34 (m, 2 H), 7.29-7.15 (m, 8 H), 7.03 (s, 1 H), 6.77 (m, 3 H), 6.20 (t, J=6.0 Hz, 1 H), 6.08 (t, J36.7 Hz, 1 H) 4.01-3.97 (m, 2 H), 3.84-3.72 (m, 1 H), 3.72 (s, 6 H), 3.71-3.63 (m, 1 H), 3.48-3.32 30 (m, 2 X), 3.30-3.22 (m, 1 H), 3048-3.32 (m, 2 H), 3.30-3.22 (m, 1 H), 7.52 (m, 2 H), 2.27-2.1~ (m, 3 H), 2.08-1.97 (m, 1 H), 1.83 (s, 3 H), 1.67-1.48 (m, 3 H), 1.43 ts, 3 H), 1-22-1.15 (m, 1 H), 0.82 ~s, 9 H), 0.01 (s, ~

W~9~/02534 PCT/US91/06~31 f~ U ~,~ 'J ;) i i 36b. To a stirred solution of trifluoroacetic anhydride (O.32 ml, 2.3 mmol) and triethylamine (0.64 ml, 4.6 mmol) in CH2Cl2 (5.0 ml) was added Compound 1 (Figure 7) (210 mg, 0.23 mmol). After 2 hours, the reaction was quenched with aqueous NaHCO3 (5.0 ml) and diluted with EtOAc (25 ml). The organic phase was washed with water (2 x 10 ml), brine (5 ml) and dried (Na,SO~). Compound 2 (Figure 7) was purified by flash chromatography using 7S MeOH in CH2C12. Compound 2 was obtained in 89-91% yield.
36c. To a stirred solution of Compound 2 (180 mg, 0.2 mmol) in THF (4.0 ml) was~added tetrabutyla~monium fluoride (0.4 ml, 1.0 M solution in the T~F, 0.4 mmol) at O'C. After 2 hours, the solvent was evaporated and the product was purified by flash chromatography using increasing polarity, 5-8~ MeOH, in CH2Cl2 to give Compound 3 (Figure 7) in 89~ yield.
36d. Dry diisopropyl ethyl amine (0.15 ml, O.67 mmol) was added to Compound 3 (150 mg, 0.168 mmol) followed by thQ addition o~ dry C:H2C11 ~0.5 ml). Then the flask was shaken to dissolva the alcohol and 2--cyanoethyl-N,N-diisopropylchlorophosphora~idite (0.056 ml, 0.25 ~mol) was added over a pariod of 20 seconds.
After 45 minutes the reaotion ~ixture was quenched with CH30H (0.1 ml) and diluted with EtOAc (50 ml) and Et~N
(1.0 ml) and washed with 10~ aqueous K2CO3 (2 x 5.0 ml), and brine (5.0 ml) and dried (Na2SO4). The product was purified by flash chromatography using EtOAc to give Compound 4 in 70-75% yield.
EXAMPLE 37: Svnthesis of deoxYthymidine oliq~ers ~ontaininq a 3'-~-C-C-5' inte_nu~leoside linkaqe.
The thymidine-dimer phosphoramidite compounds produced by steps a-d above were used in a modi~ied . ~ , . . .

.. . . . ..
.

WO~2/0253'l PCT/US91/05~31 solid phase phosphoramidite synthetic procedure to make the oligonucleoside sequences of Table 2.

Table 2 seouence Re. Code 5' TpTpTpTpTpTpTpTp[TnT~pT 3' 4 5' TpTpTpTpTp[TnT]pTpTpTpT 3' 5 5' [TnT]pTpTpTpTpTpTpTpTpT 3' 6 T- thymidine p= --o--P--O--Oe n- 3'-N-C-C-5' The oligodeoxynucleoside ~equenc6~s were synthesized ~rom the 3' to the 5' terminus.
The initial step was the attachment, ~ia a 3'-succina~e linkage, o~ a 5'-dimet~loxytrityl deoxythymidine to a CPG support. The 5'-O-dimQthoxytrityl group o~ the attached thymidine was r~acted with trichloroacetic aci~l ~o deprotect thQ 5'-hydroxyl group.
Chain elongation then proceeded ~ia the standard sequential steps o~ deprotect$on, activation, capping and oxidation with the modification that an -N-C-C- linXed thymidine dimer, prepared a~cording to the methods of Examples 30-37, was added in the chain where desired during an activation step.
At the end of chain assembly, the thymldine oligomers were removed from the CPG support wlth concentrated ammonium hydroxide. The solution was then further treated at 55-C for 8 to 15 hours to remove all the protecting groups on the exocyclic amines o$ the bases.

~ , .
!' , ; ~
:': ' ~ '` " `' ~ ' , . . ' ~ . . '`~ . ''' ' `
. , ' ` ~ .

' ` '; ~ ' W0~2/0253~ PCTtUS91/0~31 ~ r~ 3 ~ rj ~ ;3 EXAMPLE 38: Pre~aration of Tetraethvlene~lycol-terminated ~n~i-RAS onco~ene ~
38a. Preparation of dimethoxytrityltetra-ethyleneglycol (DMTTEG) An excess of tetraethyleneglycol TEG (about 100 ml) was admixed with about 7 ml (5.lg; 40 mmols) of Hunig's base in a round bottom flask. About 3.08g (lO
mmols) of dimethoxytrityl chloride (DMTCl) was added to the TEG admixture and the DMTCl-TEG mixture maintained with constant stirring at room temperature (about 2S~C) for about 8 to 12 hours to ~orm DMTTEG.
~ 38b. Preparation of ~~~~ ~~ ~~~
dimethoxytrityltetraethylene-glycolcyanophosphine (DMTTEGCP).
Six grams of the DMTTEG from step ~a) was admixed with 20 ml of dry dichloromethane. About 6.2 ml of Hunig's base was added to the admixture, followed by the dropwise addition of a chlor~phosphine mixture to form DMTTEGCP. The chlorophosph'Lne mixturs was prepared by dissolving 1.67g of 2-cyanoethyl N,N-diisopropylchlorophosphoramid~te in 5 ml of d~y dichloromethane.
38c. Preparation of T~EG-terminated Anti-RAS
oncogene DNA.
The oligodeoxynucleotides of Table 3 were prepared according to a modified solid phase phosphoramidite method. GAIT, supra. The oligodeoxynucleotides were synthesized from the 3' to the 5' terminus.

' .:' " ', ` ;` :, ~ . ~ . , , W0~2/02534 PCT/US91/05~31 ~ 63 -td TaPle 3 Sequence Ref._ Code 5' X GGA GCT GGT GGC GTA X (A) 3' 7 5' XX GGA GCT GGT GGC GTA XX (A) 3' 8 5' X CCT CGA CCA CCG CAT X (A) 3' 9 5' XX CCT CGA CCA CCG CAT XX rA) 3' lO
5' CCT CGA CCA CCG CAT 3' ll X is TEG
A, C, G ~ T represent the deoxynucleotides adenylic, cytidylic, guanidylic and thymidylic acids, respectively.
Either the nucleoside adenosine (7, 8, 9, lO) or thymidine (ll) was attached to a CPG solid support using a succinate linkage. GAIT, supra. The synthesis o~ ll proceeded in accordance with standard solid phase phosphoramidite procedures. In ~equences 7, 8, 9 and lO, synt~esis proceeded in accorclance with a modi~ied phosphora~idite procedure. The ';' hydroxyl group of the attached adenosine nucleoside wa~; reacted with trichloroacetic acid to deprotec1: the 5' hydroxyl group.
Following this deprotection step 1l the at~ached adenosine nucleoside was reacted with the ~IctiYating agent, tetrazole, and a phosphoramidite reagent comprising D~TTEGCP, prepared by the processes of steps a and b above. The activation step was followed by the capping of unreacted 5' hydroxyl groups with acetic anhydride and N-methylimidazole. The phosphorous lin~age was then oxidized with iodine ~n accordance with standard procedures.
In sequences 8 and lO, containing two TEG
residues, the deprotectin~, activating, capping and oxidizing steps were repe~ted as described above. Chain elongation proceeded via the sequential steps o deprotection, acti~ation, capping and oxid~tion as described above with the modification that the desired ` . :.~ , . ,-W092/n2534 PCT/US91/05531 nucleoside phosphoramidite reagent was substituted for the DMTTEGCP during the activation step. Following attachment of the last desired nucleoside, either one or two ~EG residues were attached at the 5' terminal in a manner analogous to the attachment of TEG at the 3' terminus.
At the end of chain assembly, the DNA strand was removed from the CPG support with concentrated ammonium hydroxide. The solution was then further treated at 55 C for 8 to 15 hours to remove all the protecting groups on the exocyclic amines of the bases.

EXAMPLE 39: Pre~aration of hexaetnvleneqlycol (HEG~-_e,~minated Anti-RAS oncoaene ~A.
Hexaethyleneg~ycol (HEG) terminated anti-RAS
oncogene DNA was prepared accordin~ to the methods of Example 38. HEG was reacted with DMTCl to form DMTHEG.
The DMTHEG was then reacted with a cyanophosphine compoùnd to form DMT~EGCP, which was used in the modified solid phase phosphoramidate synthesis method of Example 38(c) to form HEG-terminated anti-RAS oncogene DNA. The sequences o~ these oligonucleotides are set forth in the following Table 4.
Ta~le 4 sequence 5' X GGA GCT GGT GGC GTA X (A~3' 5' XX GGA GCT GGT GGC GTA XX (A)3' 5' X CCT CGA CCA CCG CAT X (A)3' 5' XX CCT CGA CCA CCG C~T XX (A)3' 5' CCT CGA CCA CCG CAT 3' X is HEG
A, C, G ~ T represent the deoxynucleotides adenylic, cytidylic, guanidylic and thymidylic acids, respectively.

.. , .: , ,~ . :

-WO 92/02534 PCl`/US91/0553 EXAMPT~ 40: ~uclease Resistance of TEG-term1nated ~nti-RAS Oncoqene DNA
The oligonucleotides of Table 4 were dissolved in water. DNA concentrations were then determined by measuring the absorbance of samples at 260 nm (on a Perkin Elmer Lambda 4C Spectrophotom2ter at ambient room temperature) and using calculated extinction coefficients ~method of Cantor and Warsaw, CRC Handbook of BiochemistrY and Molecular 8iolo~v, 3rd. ed. Vol. 1, CRC Press, page 589 (1975)].
The oligonucleotides were incubated for 2 hours at 37~C at a total strand concentration of 6 or 7 ~M in cel]. culture medium containing RPMI 1640: 20 mM N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid), pH 7.4: and 10~ fetal calf serum (FCS) (GIBC0 Laboratories, Grand Island, NY). The FCS was h~at inactivatad at 56'C ror 0.5 hour prior to use. Samples were then placed on ice and deproteinized using five extractions with 24:1 chloro~orm~:isoamyl alcohol.
Samples were either stored frozen at -20'C or immediately loaded onto a refrigl3rated ~4'C) WISP
~Waterq) HPLC autoinjector.
Oligonucleotide hydrolysis was quantitated by determining the amount of disappearance of the parent compound. Oligonucleotides (from the reaction mixture) were separated on an LKB Ultrachrome G$i dual pump c~romatography system equipped with a fixed w~velength detector ~260 nm), and recording integrator, uslng a GenPak FAX ~Waters) anion axchange colu~n equilibrated in Buffar A ~lmM EDTA; lS mM sodi~m phosphate, pH 8.5).
Column temperature was maintained at 60-C using a Waters column ov~n. Fifty ~icroliter sample in~ection volumes were used. The oligonucleotides were eluted using a linear gradient of 0~ to 100~ Buffer B (Buffer A

. ;.

WO~/02534 PCT/US91/05531 ! r ~ U V '~

containing 0.5 M NaCl) over 60 ~inutes. Buffer f}ow rate was 1 mL/min.
Following incubation (2 hrs) in the presence of fetal calf serum-associated exonuclease, no degradation of compounds 7 or 10 was observed (Table 5, see % degradation of major peak). During a similar incubation period, 87.0% and 82.1% of 9 and 8, respectively, remained. In comparison, only 24.7% o~
oligomer 11 remained after the same incubation period.
Table S
SAMPLE ID AREA-MAJOR AREA-MAJOR % DEGRADATION
PEAK/O.O MIN PEAK/2.0 HR MAJOR PE~K
7 0.2325 0.3663 0.0 9 0.3744 0.3258 13.0 8 0.2164 0.1~77 17.9 0.3642 0.3697 0.0 11 1.2861 0.3177 75.3 All four TEG-oligomers were resistant to hydrolysis by the FCS-associated exonucleases. The bis-diTEG-oligomers ~7 and 10) appeared to be completely resistant to hydrolysis. TEG-derivatized oligodeoxynucleotides represent significant improvements over unmodified compounds in terms of resistance to exonuclease hydrolysis.

EXAMPLæ 41: Abilitv of TEG-~ntisense Oli~omers to Inhibit P~otein Expression and Growth in ~uman Tum~r ~ell Lines_and_PRA
Sti~ulation o~ Pe~i~heral Blos~
~mphocytes.
It has been demonstrated by others (Heikkila, R.
et 1.~ ~at~re, 328:~45-449, 1987) that unmodified antisense oligonucleotides directed towards the initiation codon region of the c-~yc oncogene oould inhibit the expression of c-myc protein i~ PHA
stimulated peripheral blood lymphocytes (PBL) resulting W0~2/n2534 PCTIUS91/05~31 _ . 1`!'~
~, J - 67 -in a blocX in the progression o~ cells into th S-phase of the cell cycle. C-myc directed antisense DNA wa~
also 6hown to inhibit the growth of ~L-60 human erytholeukemia cells ~n vitrQ (Wickstrom, E.L., et al., Proc. Natl. Acad. Sci. USA, 85:1028-1032, 1988). The sequences shown in Table 6 were prepared and evaluated by the procedures of Example 41a and 41b.

5' AAC GTT GAG GGG CAT 3' 5' XX AAC GTT GAG GGG CAT XX A 3' (X =-TEG)- ~~~

41a. Comparison of the Effect of Modi~ied (with TEG) and Non-Modified C-MYC Anti~sensQ DNA on the Prcgression of PHA St~mulated P~3L Into the S-Phase of the Cell Cycle.
Human PBLIs were stimulated with P~A ~or 4B hours in the presence or absence of the antisense ol~gonucleotide sequences o~ Table 6. ~he percent of the~population of cells ln each treatment group ln the S-phase of the cell cycle as compared to the nontreated control was determined using standard flow cytometric techniques. The results are shown in Table 7.
TA~ 7 ~o OLIGONUCLEOTIDE CONCENTRATION%CONTROL
_ (~M~ S-PHASE

S' AAC GTT GAG GGG CAT 3~ 30 75 + 6 9 + 10 5' XX AAC GTT GAG GGG CAT XX A 3'30 80 + 4 c 6 -~ , .
..
.. .. : :
:, .. . :
.: . ~ . ..

WO 92/0;!534 PCT'/US91/05531 ~ r, The data show that the presence of TEG at both the ~' and 5' termini does not alter the inhibitory effect of the antisense DNA.

41b. Comparison of the Effect of Modified twith TEG) and Non-Modified C-M~C Antisense DNA on C-MXC
Protein Expression in MOLT-4 Human T-Cell LeuXemia Cells.
Asynchronous exponentially growing Molt-4 cells were incubated for 8 hours in the presence or absence of 60 ~M c-myc directed an~isense DNA. The cells were then ~~-~ incubated for 45 minutes in the presence of ~S-methionine and the content of c-myc protein quantitated using radi.oimmunoprecipitation with a c-myc antibody.
The results are displayed in Table 8.

~REDUCTION
OLIGONUCLEOTIDE CONCENTRATION C-MYC
I~M)PROTEIN
NONE
S' AAC GTT GAG GGG CAT 3' ~0 61.0 ~ 2.6 ~5 5' XX AAC GTT GAG GGG CAT XX A 3' 60 67.9 + 0.7 The TEG cDntaining antisense DNA was slightly more potant than the unmodified antisense DNA.
41c. Comparison of the Effect of Modified ~with TE~) and Unmodi~ied C-MYC ~ntisense DNA to Inhibit the Gro~th of Human CCRF-CEM T-Cell Leukemia Cell Growth in Vitro.
Asynchronous exponentially growing CCRF-CEM cells were incubated for 48 hours in the presence or absence of antisense DNA and then cell num~ers determined in each treatment group. The concentration of antisense . ', . . . . .

, W~2/n2534 PCT/US91/OS~31 , J -,_ - 69 -DNA required to inhibit cell growth by 50~ was then determined IIC50). Both of the modified and non-modified antisense DNAs of Table 5 displayed approximately equivalent (IC50) concentrations of 40~M.
These data demonstrate that the presence of TEG at the 3' and 5' termini of antisense DNA does not affect the ability of such antisense DNA to hybridize with and inhibit the function of target nucleic acids.

EXAMPLE 42: Additional Exonuclease Stable Oliaonucleotides.
The exonuclease stable digonucleotides set forth in Table 9 were prepared according to thP methods of Example 38.

.. , . , : , : . - .: , ~L~
5~ XX A-ACG-TTG-AGG-GGC-ATX-XA 3' XX GCC-CGC-CTC-GGT-CCC-CGC-CCX-XA
XX GGG GCG GAG TTA GGG GCG GCG GGX XA
XX GGG-GAG-GAG-GGA-GGG-GAG-GGA-XXA
XX GGG-GAG-GTG-GGT-GGG-GAG-GGT-XXA
AAG GTT GAG GGG CAT XXA
X AA-CGT-TGA-GGG-GCA-TTX-A
1o XX TTC-GCT-TAC-CAG-AGT=XXA
XX GCG-GGA-GGC-TGC-TGG-XXA
XX GGA-GGC-TGC-TGG-AGC-XXA
XX CAA-GTT-CAT-AGG-TGA-TTG-CTC-XXA
AL-CAC-TCC-TTT-AGC-AAG-XXA
AL-GAA-CGA-TTT-CCT-CAC-XXA
XX CTC-ACT-GCC-GCG-CAT-XXA
-- XX GGG~TCT-TCG-GGC-CAT-XXA - -XX GTC-GAC-CGG-T~C-CAT-XXA
XX TGT-AAC-TGC-TAT-AAA-XXA
XX GTT-CCT-CCT-CTT-TAA-XXA
XX TAC-TGC-CTT-ATA-TTC-XXA
XX TAC-TGA-CTT-ATA-TTT-XXA
XX TTT-ATA-TTC-AGT-CAT-XXA
XX TGG-GGA-GGG-TGG-GGA-GGG-TGG-GGA-AGG-XXA
XX CTT-ATA-TTC-CGT-CAT-XXA
XX TAA-CGC-CTA-TTC-TGC-XXA
XX CGT-CTT-ATC-CGC-AAT-XXA
XX TTG-CTC~TCC-TCT-GTC-XXA
XX CTG-TCT-CCT-CTC-GTT-XXA
XX ATC-TAC-TGG-CTC-CAT-XXA
XX TAC-CTC-GGT-CAT-CTA-XXA
XX ACA-CCC-~AT-TCT-G~A-ATG-GXX-A
XX GGT-AAA-GTC-TTA~ACC-CAC-AXX-A
XX TAC-GGG-GAG-TTG-~CAA-XXA

X is TEG
As will be apparent to those skilled in the art, many variations and modlfications may be made without departing ~rom the spirit and scopç of the present invention.

.- - ~
~: ,; , ,. ;` , , ;. . !

:: ', '' '` ' '. . . `'

Claims (24)

WE CLAIM:

1. A compound comprising an oligonucleoside sequence of from about 6 to about 200 bases having a three atom internucleoside linkage of the formula:
-D-D-D-;
where each D is independently CHR, oxygen or NR6 wherein R is independently hydrogen, OH, SH or NH2, wherein R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D
is oxygen or NR6.

2. The compound according to claim 1 which has a diol at either or both termini.

3. The compound according to claim 1 wherein the diol is tetraethyleneglycol or hexaethyleneglycol.

4. A compound comprising an oligonucleoside sequence of the formula:

where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH, or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;
each W' is independently W or ;

each R1 is independently OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is C1-C12 acyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or modifications thereof;
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197; and q is 0 or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.

5. A compound comprising an oligonucleoside sequence of from about 6 to about 200 bases having the formula:

where each z is independently R' or ;

where w is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH
or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;
each W' is independently W or ;

each R1 is independently OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is C1-C12 acyl;
each R5 is independently hydrogen or C1-C12 alkyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
each e and f is independently an integer from 0 to 50, with the proviso that at least one of e and f be at least 1:
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197; and each m and n is independently an integer from 1 to 200;
each p is independently 2 to 4; And q is 0 or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.

6. A nuclease resistant compound comprising an oligonucleotide sequence of from about 9 to about 200 bases having a diol at either or both termini.

7. A compound according to claim 6 wherein the diol is hexaethyleneglycol or tetraethyleneglycol.

8. A compound comprising an oligonucleotide of the formula:

;

where R is OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl, or NHR4 wherein R4 is C1-C12 acyl;
R1 is hydrogen or C1-C12 alkyl;
oligo (N) is native or modified oligonucleotide sequence of from about 9 to about 200 bases;
each e and f is independently 0 to 50, with the proviso that at least one of e and f be at least 1;
each m and n is independently 1 to 200: and each p is independently 2 to 4.

9. A compound comprising an oligonucleotide of the formula:

;

where R is OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl, or NHR4 wherein R4 is C1-C12 acyl;

oligo N is an oligonucleotide sequence of from about 9 to about 50 bases; and e and f are independently 0 to 50, with the proviso that at least one of e and f be at least one; and and n are independently 0 to 200 with the proviso that at least one of m and n be 1 to 200

10. A method of inhibiting nuclease degradation of compounds comprising preparing oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage of the formula:
-D-D-D-;
where each D is independently CHR, oxygen or NR6 wherein R is independently hydrogen, OH, SH or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6.

11. The method according to claim 10 wherein the oligonucleoside sequences have the formula:

where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH
or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;

each W' is independently W or ;

each R1 is independently OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is C1-C12 acyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or modifications thereof;
j is an integer from 1 to about 200:
k is 0 or an integer from 1 to about 197; and q is 0 or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.

12. A method of stabilizing nucleotide or oligonucleoside sequences comprising attaching a diol to either or both termini of said nucleotide or oligonucleoside sequence.

13. A method according to claim 12 wherein a trityldiolcyanophosphine is used to protect the 5' terminus of said compound.

14. A composition useful for inhibiting gene expression comprising a physiologically acceptable carrier and a compound comprising oligonucleoside sequences of from about 6 to about 200 bases having a non-phosphate containing three atom internucleoside linkage of the formula:
-D-D-D-;
where each D is independently CHR, oxygen or NR6 wherein R is independently hydrogen, OH, SH or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6.

15. The composition according to claim 14 wherein the oligonucleoside sequence has the formula:

where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH
or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;

each W' is independently W or ;

each R1 is independently OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is C1-C12 acyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or modifications thereof;
j is an integer from 1 to about 200;
k is 0 or an integer from 1 to about 197; and q is 0 or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.

16. A composition useful for inhibiting gene expression comprising a nuclease resistant compound comprising an oligonucleotide sequence of from about 9 to about 200 bases having a diol at either or both termini and a physiologically acceptable carrier.

17. A composition according to claim 16 wherein the diol is hexaethyleneglycol or tetraethyleneglycol.

18. A composition according to claim 16 wherein the compound comprises an oligonucleotide of the formula:

;

where R is OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl, or NHR4 wherein R4 is C1-C12 acyl:
R1 is hydrogen or C1-C12 alkyl;
oligo (N) is native or modified oligonucleotide sequence of from about 9 to about 200 bases;
each e and f is independently 0 to 50, with the proviso that at least one of e and f be at least 1;
each m and n is independently 1 to 200; and each p is independently 2 to 4.

19. A method of inhibiting gene expression in a mammal in need of such treatment comprising administering to said mammal an effective amount of a compound comprising an oligonucleoside sequence of from about 6 to about 200 bases having a three atom internucleoside linkage of the formula:
-D-D-D-;
where each D is independently CHR, oxygen or NR6 wherein R is independently hydrogen, OH, SH or NH2, wherein R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D
is oxygen or NR6.

20. The method according to claim 19 wherein the oligonucleoside sequence has the formula:

where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH
or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;
each W' is independently W or ;

each R1 is independently OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl or NHR4 wherein R4 is C1-C12 acyl;
each y is independently H or OH;
each B is independently adenine, cytosine, guanine, thymine, uracil or modifications thereof;
j is an integer from 1 to about 200;
X is 0 or an integer from 1 to about 197; and q is 0 or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.

21. The method according to claim 20 wherein the compound has a diol at either or both termini.

22. A method according to claim 19 wherein the compound comprises an oligonucleotide of the formula ;

where R is OH, SH, NR2R3 wherein R2 and R3 are independently hydrogen or C1-C6 alkyl, or NHR4 wherein R4 is C1-C12 acyl;
R1 is hydrogen or C1-C12 alkyl;
oligo (N) is native or modified oligonucleotide sequence of from about 9 to about 200 bases;
each e and f is independently 0 to 50, with the proviso that at least one of e and f are at least 1;
each m and n is independently 1 to 200; and each p is independently 2 to 4.

23. A compound comprising a nucleoside dimer of the formula:

where W is -D-D-D- wherein each D is independently CHR, oxygen or NR6, wherein R is independently hydrogen, OH, SH
or NH2, R6 is hydrogen or C1-C2 alkyl, with the proviso that only one D is oxygen or NR6;
each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
R7 is OH, t-butyldimethylsilyloxy or a phosphoramidite;
and R8 is OH, a protecting group or t-butyldimethylsilyloxy.

24. A compound of the formula:
5' Xn CCT CGA CCA CCG CAT Xn (A) 3';
where X is tetraethyleneglycol; and n is 1 or 2.