NZ724764B2 - Oligonucleotide compositions and methods of making the same - Google Patents

Abstract

The present disclosure provides a solid phase method of making oligonucleotides via sequential coupling cycles including at least one coupling of a dinucleotide dimer subunit to a free 3'-terminal group of a growing chain. The oligonucleotides include at least two nucleoside subunits joined by a N3'?P5' phosphoramidate linkage. The method may include the steps of (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'-protected amino-dinucleotide-5'-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3'?P5' phosphoramidite linkage; and (c) oxidizing (e.g., sulfurizing) the linkage. The compositions produced by the subject methods may include a reduced amount of one or more (N-x) oligonucleotide products. Also provided are pharmaceutical compositions including the subject oligonucleotide compositions. ?P5' phosphoramidate linkage. The method may include the steps of (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'-protected amino-dinucleotide-5'-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3'?P5' phosphoramidite linkage; and (c) oxidizing (e.g., sulfurizing) the linkage. The compositions produced by the subject methods may include a reduced amount of one or more (N-x) oligonucleotide products. Also provided are pharmaceutical compositions including the subject oligonucleotide compositions.

Description

OLIGONUCLEOTIDE COMPOSITIONS AND METHODS OF MAKING THE SAME CROSS REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing dates of U.S. provisional application serial No. 61/987,396, filed May 1, 2014, and U.S. provisional application serial No. 62/151,909 filed April 23, 2015 (attorney reference number 185/002X), the disclosures of which are herein incorporated by reference.

INTRODUCTION Nucleic acid polymer chemistry has played a role in many developing technologies in the pharmaceutical, diagnostic, and analytical , and more particularly in the subfields of antisense and anti—gene therapeutics, combinatorial chemistry, branched DNA signal ication, and array—based DNA diagnostics and analysis. Some of this polymer chemistry has been directed to improving the binding strength, specificity, and se resistance of natural nucleic acid polymers, such as DNA. e c acid (PNAs), phosphorothioate, methylphosphonate and phosphoramidate intemucleoside linkages are examples of some polymer chemistries that have been applied to oligonucleotides to provide for one or more desirable properties such as se resistance, cellular uptake and solubility.

Oligonucleotide N3'—>P5' phosphoramidates can form stable duplexes with complementary DNA and RNA strands, as well as stable triplexes with DNA es, and are resistant to nucleases. Oligonucleotide N3'—>P5' thiophosphoramidates have found use as potent antisense agents both in vitro and in vivo. For example, oligonucleotide containing nds that inhibit rase activity can be used to treat telomerase—mediated disorders, such as cancer, since cancer cells express telomerase activity and normal human c cells do not possess telomerase activity at biologically relevant levels. As such, methods of preparing and isolating such oligonucleotides are of interest.

SUMMARY The present disclosure provides a solid phase method of making oligonucleotides via sequential coupling cycles including at least one ng of a eotide dimer subunit to a 2015/028327 free 3’--terminal group (e.g., a 3’x-hydroxyi or 3“--amino group) of a growing chain. The subject methods include making oligonucleotides where at least two of the nucleoside subunits are joined by a N3’—>P5’ phosphoramidate subunit e. The method may include the steps of (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'—protected amino—dinucleotide—S'—phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside 5' phosphoramidite linkage; and (c) oxidizing the linkage. In some cases, ing the linkage include sulfurizing to produce an internucleoside N3'—>P5' thiophosphoramidate linkage.

Aspects of the present disclosure include oligonucleotide compositions produced by the subject methods that include a reduced amount of one or more (N—X) ucleotide products. In some cases, the reduced amount is less than (1.9 X N) parts to 100 by weight of one or more (N—X) products relative to N product. Oligonucleotides prepared ing to the subject methods include an oligonucleotide haVing a sequence of N nucleoside ts complementary to the RNA component of human telomerase, wherein at least two of the nucleoside subunits are joined by a N3’—>P5’ thiophosphoramidate inter—subunit linkage. Also provided are pharmaceutical compositions including the subject oligonucleotide compositions.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B show an HPLC chromatogram (A) and “P NMR spectra (B) for a TA dimer thiophosphoramidate (compound 7e, Scheme 1). s 2A and 2B show an HPLC togram (A) and “P NMR spectra (B) for a AA dimer thiophosphoramidate (compound 7a, Scheme 1).

Figures 3A and 3B show an HPLC chromatogram (A) and “P NMR spectra (B) for a GG dimer thiophosphoramidate (compound 7c, Scheme 1).

Figures 4A and 4B show an HPLC chromatogram (A) and “P NMR spectra (B) for a GT dimer thiophosphoramidate (compound 7d, Scheme 1).

Figures 5A and 5B show an HPLC chromatogram (A) and “P NMR spectra (B) for a GA dimer thiophosphoramidate (compound 7b, Scheme 1).

Figures 6A and 6B show LCMS traces for dimer amidates TA, AA, GA, GT and GG.

Figure 7 shows an HPLC chromatogram of the product of a 140 umole scale synthesis of imetelstat using a monomer coupling strategy.

Figure 8 shows an HPLC chromatogram of the product of a 140 umole scale synthesis of imetelstat using a dimer block coupling strategy.

TIONS The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

As used herein, the terms polynucleotide and oligonucleotide are used hangeably. Whenever an oligonucleotide is represented by a sequence. of letters, such as ”ATGUCCTG,” it is understood that the nucleotides are in 51...}3“ order from left to right and that ”A” denotes deoxyadenosine. ”C” denotes deoxyeytidine. ”G” denotes uanosine, ”T” denotes thymidine, and ”U” denotes deoxyur‘idihe, unless otherwise noted.

As used herein, oside" includes the natural nucleosides, including 2'—deoxy and roxyl forms, e. g. as described in Kornberg and Baker, DNA Replication, 2nd Ed.

(Freeman, San Francisco, 1992). gs" in reference to nucleosides includes synthetic nucleosides having ed base moieties and/or modified sugar moieties, e. g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e. g. stability, icity, or the like, such as disclosed by Uhlmann and Peyman (Chemical Reviews, 90:543—584, 1990). In some embodiments, a nucleoside or nucleoside analog es a 3’—hydroxyl group or a 3’— amino group.

The terms "base" and obase” are used interchangeably and defined herein to include (i) conventional DNA and RNA bases (uracil, thymine, adenine, guanine, and cytosine), and (ii) modified bases or base analogs (e. g., 5—methyl—cytosine, 5—bromouracil, or inosine). A base analog is a chemical whose molecular structure mimics that of a conventional DNA or RNA base.

As used herein, "pyrimidine" means the pyrimidines occurring in natural nucleosides, including cytosine, thymine, and uracil, and common analogs thereof, such as those ning oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, halo, and like, tuents. The term as used herein further includes pyrimidines with common protection groups attached, such as N4 — lcytosine. Further common pyrimidine protection groups are sed by Beaucage and Iyer Tetrahedron 48: 2223—2311 (1992).

As used herein, "purine" means the purines occurring in natural sides, including adenine, guanine, and hypoxanthine, and common analogs thereof, such as those containing oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, halo, and like, substituents.

The term as used herein r includes s with common protection groups attached, such as zoylguanine, NZ—isobutyrylguanine, N6—benzoyladenine, and the like. Further common purine tion groups are disclosed by Beaucage and Iyer Tetrahedron 48: 2223—2311 (1992).

As used herein, the term "—protected—" as a component of a chemical name refers to art— recognized protection groups for a particular moiety of a compound, e. g. "5'—protected— hydroxyl" in reference to a side includes triphenylmethyl (i.e., trityl), p— anisyldiphenylmethyl (i.e., monomethoxytrityl or MMT), di—p—anisylphenylmethyl (i.e., dimethoxytrityl or DMT), and the like; and a protected nucleobase in reference to a nucleobase including a heteroatom protected with a group such as a dimethylaminoformamidine (DMF), benzoyl (Bz), isobutyryl, and the like. Art—recognized tion groups include those described in the following references: Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Amamath and Broom, Chemical Reviews, 77:183—217, 1977; Pon et al., Biotechniques, 6:768—775, 1988; Ohtsuka et al, Nicleic Acids Research, 10:6553—6570, 1982; Eckstein, editor, Oligonucleotides. and Analogues: A Practical Approach (IRL Press, Oxford, 1991), Greene and Wuts, Protective Groups in Organic Synthesis, Second Edition, (John Wiley & Sons, New York, 1991), Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987), Beaucage and Iyer edron 48: 2223—2311 (1992), and like references.

As used , "oligonucleotide N3'—>P5' phosphoramidate" means an oligomer, usually linear, of nucleoside ts linked by at least one N3'—>P5' phosphoramidate linkage.

In general terms, the nucleoside subunits comprise nucleosides or side analogs, but may also comprise more l moieties haVing compatible chemistry, such as abasic sugars and other hydrocarbon moieties, such as described in the following references: Newton et al., Nucleic Acids Research, 21: 1155—1162 (1993); n et al, J. Am. Chem. Soc., 114: 7976—7982 (1992); Jaschke et al, Tetrahedron Letters, 34: 4 (1992); Ma et al., International application PCT/CA92/00423; Zon et al., International application PCT/US90/06630; Durand et al., Nucleic 2015/028327 Acids Research, 18: 6353—6359 ; Salunkhe et al., J. Am. Chem. Soc., 114: 772 (1992); and the like. In some instances, the term means an oligonucleotide wherein all intemucleosidic es are replaced by N3'—>P5' phosphoramidate linkages, i.e. the term comprehends lly as well as fully "amidated" oligomers. In some instances, it means an oligonucleotide wherein all the intemucleosidic es are replaced by N3'—>P5' phosphoramidate linkages and wherein the nucleo side subunits are the natural nucleosides or analogs thereof. A subject oligonucleotide N3'—>P5' phosphoramidate in which every linkage is an N3'—>P5' phosphoramidate linkage ("fully amidated") may be imbedded in or ed to other oligonucleotides or polynucleotides to form a larger er which is "partially amidated." A subject oligonucleotide N3'—>P5' oramidate may include any convenient 3’ and/or 5’ al groups. In some embodiments, the oligonucleotide N3'—>P5' phosphoramidate includes a 3’—hydroxyl terminal group or a 3’—amino terminal group.

As used herein, the terms hate” and “phosphate group” are meant to encompass a thiophosphate group and an oxophosphate group.

As used herein, the term "phosphoramidite amino group" refers to the amino group, —— NR4R5, attached to the phosphorus atom of a phosphoramidite group, and the term horamidite nitrogen" refers to the nitrogen atom of the phosphoramidite amino group.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups haVing from 1 to carbon atoms and such as 1 to 6 carbon atoms (e. g., “an alkyl of 1 to 6 carbons atoms”), or 1 to 5 (e.g., “an alkyl of 1 to 5 carbons atoms”), or 1 to 4 (e.g., “an alkyl of 1 to 4 carbons atoms”), or 1 to 3 carbon atoms (e.g., “an alkyl of 1 to 3 carbons atoms”). This term es, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3—), ethyl (CH3CH2—), n— propyl (CH3CH2CH2—), isopropyl ((CH3)2CH—), n—butyl (CH3CH2CH2CH2—), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2—), and neopentyl ((CH3)3CCH2—).

The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as —O—, —N—, —S—, —S(O)n— (where n is 0 to 2), —NR— (where R is hydrogen or alkyl) and haVing from 1 to 5 substituents selected from the group consisting of alkoxy, substituted , cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, aryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO—aryl, —SO—heteroaryl, —SOZ— alkyl, —SOZ—aryl, —SOZ—heteroaryl, and —NRaRb, wherein Ra and Rb may be the same or different and are chosen from hydrogen, optionally substituted alkyl, lkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. In some instances, tituted alkyl” refers to an alkyl group as defined herein having from 1 to 5 substituents selected from the group consisting of alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, sulfonamido, and —NRaRb, n Ra and Rb may be the same or different and are chosen from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably haVing from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight—chained or ed, and which are optionally interrupted with one or more groups selected from —O—, —NR10—, —NR10C(O)—, —C(O)NR10— and the like. This term includes, by way of e, methylene (—CH2—), ethylene (—CHZCH2—), n—propylene (—CHZCHZCH2—), opylene (—CHZCH(CH3)—), (—C(CH3)2CH2CH2—), (—C(CH3)2CH2C(O)—), (—C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like.

“Substituted alkylene” refers to an alkylene group haVing from 1 to 3 hydrogens replaced with tuents as bed for carbons in the definition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as defined herein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R’NHR’: where R, is alkyl group as d herein and R” is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups —alkylene—aryl and —substituted alkylene—aryl where alkylene, tuted alkylene and aryl are defined herein.

“Alkoxy” refers to the group —O—alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, y, ethoxy, n—propoxy, isopropoxy, n—butoxy, t—butoxy, sec— butoxy, n—pentoxy, and the like. The term “alkoxy” also refers to the groups l—O—, cycloalkyl—O—, cycloalkenyl—O—, and alkynyl—O—, where alkenyl, cycloalkyl, lkenyl, and alkynyl are as defined herein.

The term ituted alkoxy” refers to the groups substituted alkyl—O—, substituted alkenyl—O—, substituted cycloalkyl—O—, substituted cycloalkenyl—O—, and substituted alkynyl—O— where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH—alkoxy, wherein alkoxy is defined herein.

The term “haloalkoxy” refers to the groups alkyl—O— wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

The term “haloalkyl” refers to a substituted alkyl group as bed above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group.

Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups —alkylene—O—alkyl, alkylene—O—substituted alkyl, substituted alkylene—O—alkyl, and substituted alkylene—O—substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group —alkylene—S—alkyl, alkylene—S— substituted alkyl, substituted alkylene—S—alkyl and substituted alkylene—S—substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched arbyl groups haVing from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and haVing at least 1 and preferably from 1 to 2 sites of double bond ration. This term includes, by way of e, bi—Vinyl, allyl, and en—l—yl. ed within this term are the cis and trans isomers or es of these isomers.

The term ituted alkenyl” refers to an alkenyl group as defined herein haVing from 1 to 5 tuents, or from 1 to 3 substituents, selected from , substituted , cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, cyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO— substituted alkyl, —SO—aryl, —SO—heteroaryl, —SOg—alkyl, —SOz—substituted alkyl, —SOz—aryl and — SOz—heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and ably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (—CECH), and propargyl (—CHZCECH).

The term “substituted alkynyl” refers to an alkynyl group as defined herein haVing from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, tuted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, yloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, aryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO— substituted alkyl, —SO—aryl, —SO—heteroaryl, —SOg—alkyl, —SOz—substituted alkyl, —SOg—aryl, and — SOZ—heteroaryl.

“Alkynyloxy” refers to the group ynyl, wherein alkynyl is as defined herein. loxy es, by way of example, ethynyloxy, propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl—C(O)—, substituted alkyl—C(O)—, alkenyl— C(O)—, substituted alkenyl—C(O)—, alkynyl—C(O)—, substituted alkynyl—C(O)—, lkyl—C(O)—, substituted cycloalkyl—C(O)—, cycloalkenyl—C(O)—, substituted cycloalkenyl—C(O)—, aryl—C(O)—, substituted aryl—C(O)—, heteroaryl—C(O)—, substituted heteroaryl—C(O)—, heterocyclyl—C(O)—, and substituted heterocyclyl—C(O)—, n alkyl, substituted alkyl, alkenyl, substituted alkenyl, l, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cyclic, and tuted cyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)— “Acylamino” refers to the groups —NR20C(O)alkyl, —NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, —NR20C(O)substituted cycloalkyl, — NR20C(O)cycloalkenyl, —NR20C(O)substituted cycloalkenyl, —NR20C(O)alkenyl, — O)substituted alkenyl, —NR20C(O)alkynyl, —NR20C(O)substituted alkynyl, —NR20C(O)aryl, (O)substituted aryl, —NR20C(O)heteroaryl, —NR20C(O)substituted heteroaryl, —NR20C(O)heterocyclic, and —NR20C(O)substituted heterocyclic, n R20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, tuted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined . carbonyl” or the term “aminoacyl” refers to the group —C(O)NR21R22, wherein R21 and R22 independently are ed from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted lkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted cyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. carbonylamino” refers to the group —NR21C(O)NR22R23 where R21, R22, and R23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where each R is independently en, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl—C(O)O—, substituted alkyl—C(O)O—, cycloalkyl—C(O)O—, substituted cycloalkyl—C(O)O—, aryl—C(O)O—, heteroaryl—C(O)O—, and heterocyclyl—C(O)O— wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein. sulfonyl” refers to the group 21R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound o to form a heterocyclic or substituted heterocyclic group and alkyl, WO 68310 2015/028327 substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

“Sulfonylamino” refers to the group —NRZISOZR22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, lkenyl, tuted lkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the atoms bound thereto to form a cyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted l, alkynyl, substituted alkynyl, lkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and tuted heterocyclic are as defined herein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms haVing a single ring (such as is present in a phenyl group) or a ring system haVing multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and l) which condensed rings may or may not be ic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, lkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted lkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, yloxy, thioheteroaryloxy, kyl, —SO—substituted alkyl, —SO—aryl, —SO—heteroaryl, —SOZ—alkyl, —SOZ—substituted alkyl, —SOZ—aryl, —SOZ—heteroaryl and trihalomethyl. In such cases, an aryl group that is substituted with from 1 to 5 substituents (e.g., as described herein) is referred to as a “substituted aryl”.

“Aryloxy” refers to the group —O—aryl, wherein aryl is as defined herein, ing, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH2.

The term “substituted amino” refers to the group —NRR where each R is ndently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, tuted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl ed that at least one R is not hydrogen.

The term ” refers to the group —N3.

“Carboxyl,” xy” or “carboxylate” refers to —COZH or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or xylalkyl” refers to the groups —C(O)O—alkyl, —C(O)O—substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, —C(O)O—aryl, —substituted aryl, —C(O)O—cycloalkyl, —C(O)O—substituted cycloalkyl, —C(O)O—cycloalkenyl, —C(O)O—substituted cycloalkenyl, —C(O)O—heteroaryl, —C(O)O—substituted heteroaryl, —C(O)O—heterocyclic, and —C(O)O—substituted heterocyclic, wherein alkyl, tuted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted cyclic are as defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups —O—C(O)O— alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O- C(O)O—cycloalkyl, —O—C(O)O—substituted cycloalkyl, —O—C(O)O—cycloalkenyl, —O—C(O)O— substituted cycloalkenyl, —O—C(O)O—heteroaryl, —O—C(O)O—substituted heteroaryl, )O— heterocyclic, and —O—C(O)O—substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, tuted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cyclic, and substituted heterocyclic are as defined herein. ” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable lkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups haVing from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, tuted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, lkenyl, substituted cycloalkenyl, acyl, ino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, teroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, aryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO—substituted alkyl, —SO—aryl, —SO—heteroaryl, lkyl, —SOz—substituted alkyl, ryl and eteroaryl.

“Cycloalkenyl” refers to non—aromatic cyclic alkyl groups of from 3 to 10 carbon atoms haVing single or multiple rings and haVing at least one double bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups haVing from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, tuted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, noacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, aryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO—substituted alkyl, —SO—aryl, SO—heteroaryl, —SOg—alkyl, —SOz—substituted alkyl, —SOz—aryl and eteroaryl.

“Cycloalkynyl” refers to non—aromatic cycloalkyl groups of from 5 to 10 carbon atoms haVing single or multiple rings and haVing at least one triple bond.

“Cycloalkoxy” refers to —O—cycloalkyl.

“Cycloalkenyloxy” refers to —O—cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and l to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, nyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for e as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is ic and at least one ring within the ring system is aromatic that the point of attachment is through an atom of an aromatic ring. In , provided certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N—oxide (N—>O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, enyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with l to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, tuted alkoxy, substituted alkenyl, substituted l, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, l, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, aryloxy, cyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted koxy, thioaryloxy, thioheteroaryloxy, —SO—alkyl, —SO—substituted alkyl, —SO—aryl, —SO—heteroaryl, —SOZ— alkyl, —SOZ—substituted alkyl, ryl and —SOZ—heteroaryl, and trihalomethyl. In such cases, a heteroaryl group that is substituted with from 1 to 5 substituents (e. g., as described herein) is referred to as a “substituted heteroaryl”.

The term “heteroaralkyl” refers to the groups —alkylene—heteroaryl where ne and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O—heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group haVing a single ring or le condensed rings, including fused bridged and spiro ring systems, and haVing from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, , or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, ed that the point of attachment is through the non—aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are ally oxidized to provide for the N—oxide, —S(O)—, or —SOZ— moieties.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, zine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, l,2,3,4— tetrahydroisoquinoline, 4,5 ,6,7—tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, ene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), l,l— dioxothiomorpholinyl, dinyl, pyrrolidine, tetrahydrofuranyl, and the like.

Unless otherwise constrained by the tion for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with l to 5, or from 1 to 3 tuents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, tuted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, yl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, y, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO—alkyl, —SO—substituted alkyl, —SO—aryl, —SO—heteroaryl, —SOZ—alkyl, — SOg—substituted alkyl, —SOg—aryl, —SOg—heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group erocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic—S—.

The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —N02.

“OX0” refers to the atom (=0).

“Sulfonyl” refers to the group kyl, SOg—substituted alkyl, SOg—alkenyl, SOZ— substituted alkenyl, SOg—cycloalkyl, bstituted cylcoalkyl, SOz—cycloalkenyl, SOZ— substituted lkenyl, SOZ—aryl, SOZ—substituted aryl, SOZ—heteroaryl, SOZ—substituted heteroaryl, SOz—heterocyclic, and bstituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, tuted l, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, tuted aryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl—SOT, —SOT, and 4—methylphenyl—SOZ—.

“Sulfonyloxy” refers to the group —OSOg—alkyl, OSOg—substituted alkyl, OSOZ— alkenyl, OSOZ—substituted alkenyl, OSOZ—cycloalkyl, OSOZ—substituted cylcoalkyl, OSOZ— cycloalkenyl, OSOZ—substituted cylcoalkenyl, OSOZ—aryl, ubstituted aryl, OSOZ— heteroaryl, OSOg—substituted heteroaryl, OSOg—heterocyclic, and 0802 tuted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein. ” refers to the group —SH. o” or the term “thioketo” refers to the atom (28).

“Alkylthio” or the term “thioalkoxy” refers to the group —S—alkyl, wherein alkyl is as defined herein. In certain ments, sulfur may be oxidized to —S(O)—. The sulfoxide may exist as one or more stereoisomers.

The term “substituted thioalkoxy” refers to the group —S—substituted alkyl.

The term “thioaryloxy” refers to the group aryl—S— wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl—S— wherein the heteroaryl group is as defined herein including optionally tuted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl—S— wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.

In addition to the sure herein, the term ituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be ed with =0, =NR70, =N—OR70, 2N2 or :8) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, —R60, halo, =0, —OR70, —SR70, —NR80R80, trihalomethyl, —CN, —OCN, —SCN, —NO, -N02, 2N2, —N3, -SOZR70, -SOZO’ M", —SOZOR70, —OSOZR70, —osozoM+, R70, —P(O)(O’)2(M+)2, —P(O)(OR70)O’ M", —P(O)(OR70) 2, 70, —C(S)R70, —C(NR70)R70, ’ M", —C(O)OR70, -C(S)OR70, —C(O)NRSOR80, —C(NR70)NR80R80, —OC(O)R70, —OC(S)R70, -OC(O)O 'M+, —OC(O)OR70, -OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70CO[ M+, 02R70, —NR70C(S)OR70, —NR70C(O)NR80R80, —NR70C(NR70)R70 and —NR70C(NR70)NRSORSO, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two RSO’s, taken together with the nitrogen atom to which they are bonded, form a 5—, 6— or 7—membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms ed from the group consisting of O, N and S, of which N may have —H or C1—C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K", Na+, Li+; an ammonium ion, such as +N(R60)4; or an alkaline earth ion, such as [Ca2+]05, [Mg2+]0.5, or [Ba2+]0.5 (“subscript 0.5 means that one of the counter ions for such nt alkali earth ions can be an ionized form of a nd of the invention and the other a counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized nd of the invention can serve as the counter ion for such divalent alkali earth ions). As specific es, —NR80R80 is meant to include —NH2, —NH—alkyl, N—pyrrolidinyl, N—piperazinyl, 4N—methyl—piperazin—l—yl and N— morpholinyl.

In addition to the sure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O'M+, —0R7°, —SR70, —S’M+, —NR80R80, trihalomethyl, —CF3, —CN, —OCN, —SCN, —NO, —N02, —N3, —SOZR70, so; M", —sogR70, 70, —oso;M+, —osogR70, —P03'2(M+)2, —P(O)(OR70)O’ M.‘, —P(O)(OR70)2, —C(O)R70, —C(S)R70, —C(NR70)R70, co; M.', -C02R70, -C(S)OR70, RSOR80, —C(NR70)NR80R80, —OC(O)R70, -OC(S)R70, oco; M.', —OC02R70, -OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70CO[ M.', —NR70C02R70, —NR70C(S)OR70, —NR70C(O)NR80R80, —NR70C(NR70)R70 and —NR70C(NR70)NRSORSO, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not —O'M+, —OR70, —SR70, or —S’M+.

In addition to the groups sed with respect to the individual terms , substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O'M+, -OR70, —SR70, —S'M+, —NR80R80, trihalomethyl, —CF3, —CN, —NO, -N02, —S(O)2R70, —S(O)20'M+, —S(O)20R70, -OS(O)2R70, 2 0'M+, -OS(O)20R70, —P(O)(O')2(M+)2, —P(O)(OR70)O'M+, —P(O)(OR70)(OR70), —C(O)R70, —C(S)R7 0, —C(NR70)R70, R70, —C(S)OR70, —C(O)NRSOR80, 0)NR80R80, —OC(O)R70, —OC(S)R7 0, —OC(O)OR70, -OC(S)OR70, —NR70C(O)R70, —NR70C(S)R70, —NR70C(O)OR70, —NR70C(S)OR70, — NR70C(O)NR80R80, (NR70)R70 and —NR7OC(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined.

In addition to the disclosure herein, in a n embodiment, a group that is substituted has 1, 2, 3, or 4 tuents, l, 2, or 3 substituents, l or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with r substituents to themselves (e. g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a tuted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the m number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl—(substituted aryl)—substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the 2015/028327 adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)—(alkyl)—O—C(O)—.

As to any of the groups disclosed herein which contain one or more substituents, it is tood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non—feasible. In addition, the subject compounds include all stereochemical s g from the substitution of these compounds.

The term “pharmaceutically able salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or c acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, and the like; and when the molecule contains a basic onality, salts of c or inorganic acids, such as hydrochloride, and the like. Pharmaceutically acceptable salts of st include, but are not limited to, aluminium, um, arginine, barium, benzathine, calcium, cholinate, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, tromethamine, N—methylglucamine, N,N’—dibenzylethylene—diamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, diisopropylamine, diisopropylethylamine, triethylamine and triethanolamine salts.

The term “salt thereof’ means a nd formed when a proton of an acid is ed by a , such as a metal cation or an c cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. Salts of interest include, but are not limited to, aluminium, ammonium, arginine, barium, benzathine, calcium, cesium, cholinate, ethylenediamine, lithium, magnesium, meglumine, procaine, N—methylglucamine, piperazine, potassium, sodium, hamine, zinc, N,N’—dibenzylethylene—diamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, diisopropylethylamine, triethylamine and triethanolamine salts. It is understood that for any of the oligonucleotide structures ed herein that include a backbone of intemucleoside linkages, such oligonucleotides may also include any convenient salt forms. In some embodiments, acidic forms of the intemucleoside linkages are depicted for simplicity. In some ces, the salt of the subject compound is a monovalent cation salt. In certain ces, the salt of the subject compound is a divalent cation salt. In some instances, the salt of the subject compound is a ent cation salt. “Solvate” refers to a complex formed by combination of solvent les with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N—dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have same atomic tivity but different atomic arrangement in space. isomers include cis—trans isomers, E and Z isomers, enantiomers, and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol—keto and enamine tautomers, —NH—P(=S)(OH)—O— and —NH—P(=O)(SH)—O—, or the tautomeric forms of heteroaryl groups containing a —N=C(H)—NH— ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric arrangements of the groups described herein are possible. For example, it is understood that an ucleotide described by the following structure: Nko—E—oH H E o=§>—SH psnpsnpsrpsrpsmsmsms—G G G T T A G A c A IOAI also encompasses the following structure showing one possible alternate tautomeric ement of linkage groups: s=§>—o+ s=§>—GT Olnpsrpsnpsnpsrpsrpsmsmsms—G G G T T A G A c A IOAI Where “nps” represents a thiophosphoramidate linkage (—NH—P(=O)(SH)—O— or —NH— P(=S)(OH)—O—) connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside. It is understood that all tautomeric forms of a t compound are encompassed by a structure Where one possible tautomeric ement of the groups of the compound is described, even if not specifically indicated. Any convenient tautomeric arrangement of the groups of the subject compounds may be utilized in describing the compounds.

It will be appreciated that the term “or a salt or solvate or stereoisomer thereof” is ed to include all permutations of salts, solvates and isomers, such as a e of a pharmaceutically acceptable salt of a stereoisomer of subject compound. It is understood that the term “or a salt thereof” is intended to include all ations of salts. It is understood that the term “or a pharmaceutically acceptable salt thereof” is intended to include all permutations of salts. It is understood that the term “or a solvate thereof” is intended to include all permutations of solvates. It is understood that the term “or a stereoisomer thereof” is ed to include all permutations of stereoisomers. It is understood that the term “or a tautomer thereof” is intended to include all ations of tautomers. Thus for example it follows that it is intended to include a e of a pharmaceutically acceptable salt of a tautomer of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically ive amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other , cause the tumor to shrink or decrease the growth rate of the tumor.

“Patient” refers to human and non—human subjects, especially mammalian subjects.

The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal cularly a human) that includes: (a) ting the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the e or medical condition, such as, eliminating or causing regression of the disease or medical condition in a t; (c) suppressing the e or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.

As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound lly . “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.

As used herein, the term “substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is lly ated.

The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

Before the present invention is further described, it is to be understood that this ion is not limited to particular ments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, for y, described in the context of separate embodiments, may also be provided in ation in a single embodiment. Conversely, s features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub— ation. All ations of the embodiments pertaining to the invention are specifically embraced by the t ion and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub— combinations of the various embodiments and elements thereof (e. g., elements of the chemical groups listed in the ments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub—combination was individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ry skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, methods and als of interest are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended , the singular forms “a,” “an,” and “the” include plural nts unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is ed to serve as antecedent basis for use of such exclusive terminology as “solely,77 4‘only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of te embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub— ation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the t application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be ent from the actual publication dates which may need to be independently confirmed.

ED DESCRIPTION As summarized above, the present disclosure provides a solid phase method of preparing ucleotides via sequential coupling cycles including the ng of a dinucleotide dimer to a free 3’terminal group (eg, a rox§/l or 3’—amino group) of a growing chain. in general terms the synthesis ds from the S’s-terminal to the 3"--terminal of a target ueleotide ce and htcludes at least one coupling of a dinucleotide dinner. The dimer may be coupled to the free 3’ terminal group of a growing chain via any convenient Chemistry. In some cases, the dimer is a 3’-protected—dinucleotide—5'—phosphoramidite dimer, Where the dinucleotide may e any convenient inter—nucleoside linkage. The oligonucleotide may include one or more phosphoramidate inter—subunit linkages (e.g., an oxo— phosphoramidate or thiophosphoramidate e).

In some embodiments, the oligonucleotide includes a sequence of nucleoside subunits containing at least one subunit defined by the formula: HN R3 RO/ \o—§ Where B is a , a protected purine, a pyrimidine or a ted pyrimidine, or an analog thereof; X is O or S; R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, a phosphate protecting group; and R3 is selected from the group consisting of hydrogen, O—RZ, and halogen, wherein R2 is H, an alkyl, a substituted alkyl (e.g., — (CH2)nW(CH2)mH, Where n is between 1—10, m is between 0—10 and W is O, S, or NH) or a hydroxyl protecting group. It is understood that some of the oligonucleotides including a subunit described by the formula above may also exist in a salt form. Such forms in so far as they may exist, are intended to be included Within the scope of the present disclosure.

The subject methods provide for a reduced number of coupling cycles relative to methods involving only nucleoside monomer subunit couplings and provide for reduced amounts of non—target oligonucleotide ts of the synthesis. The retrosynthetic gy utilized for ing a target oligonucleotide sequence may be selected depending on a variety of factors, such as the length and sequence of the target oligonucleotide so as to minimize the amounts of particular non—target oligonucleotide products of the synthesis.

In some embodiments, the subject methods provide for the preparation of compositions that have a reduced amount of one or more (N—X) ts ve to a target oligonucleotide of interest.

In certain embodiments, any of the compositions described herein that have a reduced amount of one or more (N—X) ts relative to a target oligonucleotide of interest are unpurified.

As used herein, the term “(N—X) product” (where X is an integer from 1 to N—l and N is the number of nucleoside residues in a target ucleotide), refers to a non—target oligonucleotide produced during the subject methods of preparation that lacks X nucleoside residues by comparison with the sequence of a target oligonucleotide of N residues in length.

The target oligonucleotide is the product which the subject method of preparation is ed to produce. As such, a (N—l) t is a non—target oligonucleotide that lacks any one nucleoside residue out of the sequence of the target oligonucleotide. As such, in some cases, the term “(N— 1) t” refers to a variety of rget oligonucleotide products, each of which lack one nucleoside residue by comparison to the sequence of the target oligonucleotide. Similarly, the term “(N—X) product” refers to a variety of non—target oligonucleotide products, each of which lack X nucleoside residues by comparison to the sequence of the target oligonucleotide. For eXample, a (N—2) product is a rget oligonucleotide that lacks any two nucleoside residues out of the sequence of the target ucleotide. In some cases the X residues are contiguous to each other ve to the target oligo nucleotide sequence. In other cases, the X residues are discontiguous to each other relative to the target oligo nucleotide sequence. The X side residues may be lacking from any location of the target sequence and may be produced from unreacted 3’—terminal groups during a ng cycle. The (N—X) products of the subject methods may include one or more further modifications that derive from the subject methods of synthesis, e.g., a partial deprotection modification, loss of a nucleobase (e. g., depurination), capping of a terminal group, derivatization via a synthesis reagent (e. g., phenylacetylation by a sulfurization reagent), and the like. A variety of modified oligonucleotides are le depending on the chemistry of oligonucleotide synthesis and reagent utilized. Unless indicated otherwise, all such modifications are meant to be encompassed by the term (N—X) product.

In some embodiments, the subject methods result in the reduction of one or more non—target products of oligonucleotide sis selected from a partially protected product or a partially protected (N—X) product, e. g., an oligonucleotide product including one or more nucleobase ting groups. In the t oligonucleotide compositions, the target oligonucleotide sequence may be more readily ed or purified from other oligonucleotide— containing products of the method, e. g., (N—X) products and ts lacking a nucleobase.

Embodiments of the subject methods and compositions are described in more detail in the sections below.

METHODS OF MAKING OLIGONUCLEOTIDES ] The present disclosure es a method of preparing an oligonucleotide. The subject s may include at least one coupling of a dinueelotide dimer to the free 3" terminal group of a growing oligonucleotide chain. Any convenient oligonucleotide synthesis methods and chemistries may be utilized in the subject methods of preparation. Giigonueleotide synthesis chemistries and methods of interest that may be d for use in the subject methods include, but are not limited to, phosphoramidite, sphonate, phosphodiester, hotriester, phosphite triester, and those described by Fearon et al. in US. 5,824,793, the diselsoure of which is herein incorporated by reference in its entirety. The oligonucleotide components of the invention compounds may be synthesized by adapting conventional protocols for the type of chemistry selected. Methods of interest for the synthesis of oligonucleotides having N3’—>P5’ phosphoramidate chemistries include, but are not limited to, those methods described in McCurdy et al., (1997) Tetrahedron Letters, 38:207—210 and Pongracz & Gryaznov, (1999) edron Letters, 49:7661—7664.

An oligonucleotide of interest may be prepared using the subject methods via sequential couplings starting from the 5’—terrninal and ding to the 3’—terminal of the target oligonucleotide sequence. The Sawterminal ntieleoside suhunit may be attached to any convenient solid support via an al linking group or 5’—terniinal group. Then, subunit. couplings to the growing oligonucleotide Chain may he achieved using either dimer phosphoramidites or monomer phosphoramidites. atively, the 5’-terininal dinuoleotide suhunit may be attached to any convenient solid support via an optional linking group or 5"--terminal group. Once the first subunit (e. g., monomer or dimer subunit) is attached to the solid support, the subunit may be deprotected to produce a free, immobilized 3’—terminal group. in some eases, the method includes coupling a support hound 3’—terminal group with a 3’—proteeted~dinueleotide~5'— 2015/028327 oramidite dimer. In certain ments, the S’s-terminal group is a S’s-hydroxyl group.

In certain embodiments, the 3’—terrninal group is a 3’—arnino group.

In some instances, the method includes the steps of: (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'—pr0tected amino— dinucleotide thiophosphoramidate 0r phosphoramidite—S'—phosphoramidite dimer in the presence of a nucleophilic catalyst to form an intemucleoside N3'—>P5' phosphoramidite linkage; and (c) oxidizing the e.

The target oligonucleotide sequence may be synthesized using a retrosynthetic strategy that es sequentially coupling of both dimer and monomer subunits to the 3’terminal group of the g ucleotide chain. As such, in some embodiments, the method r includes the steps of: (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'—protected aminonucleoside—5'—phosph0ramidite monomer in the presence of a nucleophilic catalyst to form an intemucleoside N3'—>P5' phosphoramidite linkage; and (c) oxidizing the linkage to produce a 5' oramidate linkage.

As used herein, the term “N3'—>P5' phosphoramidite linkage” refers to the phosphorus (III) intermediate of the N3'—>P5' phosphoramidate linkage. In general terms, an N3'—>P5' phosphoramidate linkage is formed by oxidizing an N3'—>P5' phosphoramidite linkage to a orus (V) product (e. g., a N3'—>P5' phosphoramidate linkage that may include an 0x0 (P20) or a thio (P=S) group). In some cases, the oxidizing step may be described as sulfurizing the N3'—>P5' phosphoramidite linkage to produce a N3'—>P5' thiophosphoramidate linkage.

As used herein, "N3'—>P5' phosphoramidate", N3' phosphoramidate" and horamidate” refer to an cleosidic subunit linkage described by the formula: 3’-NH—P(=X)(OR)—O—5’ or a tautomer thereof, wherein the 3’ and 5’ refer to the carbon atoms of the sugar moieties of consecutive nucleosides which are connected by way of the linkage, and wherein R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, or a phosphate protecting group, and X is a chalcogen, such as oxygen or sulfur. It is understood that, when R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, or a phosphate protecting group, some of the intemucleosidic subunit linkages described by the formula above may also exist in a salt form.

Such forms in so far as they may exist, are intended to be included within the scope of the present disclosure. In some cases, when X is sulfur, the phosphoramidate may be refered to as a thiophosphoramidate. In some cases, when X is oxygen, the “phosphoramidate” may be refered to as an “oxophosphoramidate”. In some cases, when R is a phosphate protecting group it may be an alkyl, an l, an aryl, an aralkyl, a cycloalkyl, or a substituted version thereof. In some cases, R is a phosphate protecting group containing 10 or less carbon atoms. In certain instances, when R is a phosphate ting group it is an alkyl having from 1 to 6 carbon atoms; an electron—withdrawing B—substituted ethyl (e. g., B—trihalomethyl—, B—cyano—, o—, or B—nitro— substituted ethyl); an electron—withdrawing substituted phenyl (e. g., halo—, sulfo—, , or nitro—, substituted phenyl); or an electron—withdrawing substituted phenylethyl. In some embodiments, when R is a phosphate protecting group it is methyl, B—cyanoethyl, or 4— nitrophenylethyl. In certain embodiments, R is hydrogen, , or B—cyanoethyl. on— withdrawing substituents of interest include, but are not limited to, halo, cyano, nitro, sulfo, or mono—, di—, or trihalomethyl, and the like. Halogen atom substituents are usually fluoro, chloro, bromo, or iodo; and in some instances, they are fluoro or . "Electron—withdrawing" s the tendency of a substituent to attract valence ons of the molecule of which it is a part, i.e. it is electronegative, e. g. March, Advanced Organic Chemistry, pgs. 16—18 (John Wiley, New York, 1985). Guidance for selecting a phosphate protecting group is provided in Beaucage and Iyer, Tetrahedron 48: 2223—2311 (1992). For convenience, nucleotide oramidates are mes indicated herein by a subscripted "np" or "pn" for N3'—>P5' phosphoramidates and P3'—>N5' phosphoramidates, respectively. Thus, "UnpU" is a dinucleotide in which a 3'— aminouridine and a uridine are linked by an N3'—>P5' phosphoramidate linkage. When the linkage is an oxo—phosphoramidate, the nucleotide oxo—phosphoramidate is mes indicated herein by a subscripted "npo" or "opn" for N3'—>P5' phosphoramidates and P3'—>N5' phosphoramidates, respectively. Similarly, nucleotide thiophosphoramidates are mes indicated herein by a subscripted "nps" or "spn" for N3'—>P5' thiophosphoramidates and P3'—>N5' thiophosphoramidates, respectively. rly, 2'—fluoro substituents are indicated by a cripted "f". Thus, ”UfinpU” is a dinucleotide in which the 5'—most 3'—amino—2'—fluorouridine is linked to a uridine by an N3'—>P5' phosphoramidate linkage. A single leading subscripted p indicates a 5' monophosphate, and a single trailing subscripted "n" indicates a 3'—amino group.

WO 68310 In some instances, the internucleoside subunit linkage is described by the formula: 3’-NH—P(=X)(OR)—O—5’ or a er thereof, wherein the 3’ and 5’ refer to the carbon atoms of the sugar moieties of consecutive sides which are connected by way of the linkage, and where R is hydrogen and X is oxygen or sulfur. It is understood that for any of the oligonucleotides described herein that e such an ucleoside linkage, such oligonucleotides may also e any convenient salt forms of the linkage. As such, the ucleoside linkage may be in a salt form that es any convenient counterion.

Any convenient protecting group gies may be utilized in the subject methods to protect the base, phosphoramidite, phosphoramidate, 5’, 2’ and/or 3’ groups. ting groups of interest include, but are not limited to, those protecting groups described by Ohkubo et al., Org. Lett., 2010, 12 (ll), pp 2496—2499; and Beaucage and Iyer, Tetrahedron 48: 2223—2311 (1992).

As used , the term “phosphate protecting group” refers to a protecting group that may be attached to a phosphorus—containing intersubunit linkage of an oligonucleotide.

When present, a phosphate protecting group may prevent (i.e., block) reaction of the phosphorus— containing linkage at the location where the phosphate protecting group is attached. Any convenient phosphorus—containing intersubunit linkages (e. g., P(III) and P(V) linkages) may be protected by the subject phosphate protecting groups, including, but not d to, phosphoramidite, oxophosphoramidate, thiophosphoramidate, phosphate ester, thiophosphate ester, phosphodiester linkages and the like. The phosphate protecting group may be attached to an available oxygen atom of the phosphorus—containing intersubunit linkage. Any convenient protecting groups may be utilized as a phosphate protecting group. Phosphate protecting groups of interest include, but are not d to, an alkyl, an alkenyl, an aryl, an aralkyl, a cycloalkyl, or a substituted n thereof, such as an alkyl having from 1 to 6 carbon atoms, such as an electron—withdrawing B—substituted ethyl (e. g., B—trihalomethyl—, o—, B—sulfo—, or B—nitro— substituted ethyl); an electron—withdrawing substituted phenyl (e. g., halo—, sulfo—, cyano—, or nitro—, substituted phenyl); or an electron—withdrawing substituted phenylethyl, methyl, [3— cyanoethyl, or 4—nitrophenylethyl. In certain embodiments, phosphate protecting group is methyl, or B—cyanoethyl. Electron—withdrawing substituents of interest include, but are not limited to, halo (e. g., chloro or fluoro), cyano, nitro, sulfo, or mono—, di—, or trihalomethyl, and the like.

The minal group of the growing oligonucleotide chain may include a 3’— hydroxyl, a 3’—amino group or a protected version thereof. Any convenient hydroxyl and/or amino protecting groups may be utilized at the 3’—terminal group during ucleotide synthesis. In some embodiments, the 3’terminal group is a protected 3’—amino group and the method includes deprotecting or removing the protecting group to produce a free 3’amino group.

As used herein, the term "free amino group" in nce to the monomers and dimers means an amino group available for reacting with the phosphoramidite group of an incoming monomer or dimer. In some embodiments, a free amino group is a primary amine.

After the deprotection (e. g., detritylation) step, the amino group may be in the form of a salt (e. g., the salt of a conjugate base of the acid used for detritylation). This salt optionally may be neutralized with a basic solution such as 2% triethylamine or pyridine in acetonitrile after the detritylation step.

In some embodiments, the minal group is a protected 3’—hydroxyl group and the method es deprotecting or removing the ting group to produce a free 3’—hydroxyl group. In some embodiments, the minal group is a protected 3’—amino group and the method includes deprotecting or removing the protecting group to produce a free 3’—amino group. The protected 3’—amino or 3’—hydroxyl group may be protected with a trityl protecting group. In certain embodiments, the trityl protecting group is triphenylmethyl (Tr, Pth—). In certain embodiments, the trityl protecting group is 4,4’—dimethoxytrityl (DMT).

Deprotection of the 3’—terminal amino or hydroxyl group may be achieved using any convenient methods. Methods of st include, but are not limited to, those s bed by Beaucage and Iyer, Tetrahedron 48: 2223—2311 (1992). In some cases, deprotection of the protected 3' amino group of a terminal nucleoside includes detritylation to produce a free 3’terminal group, e. g., acid—catalyzed detritylation.

In general, the dimer or monomer subunit oramidites include a protected 3’—hydroxyl or 3’—amino group that is the same as the 3’terminal group of the terminal nucleoside attached to the solid support. 3’—Protection of the incoming subunit phosphoramidites prevents undesirable polymerization of the chain.

Any convenient solid phase supports may be used in the subject methods. Solid supports of interest e, but are not d to, microparticles made of controlled pore glass (CPG), highly cross—linked polystyrene (e. g., hase HL 400 or GE Primer 350), acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, such as those disclosed in the following exemplary references: Meth. Enzymol., n A, pages11—147, vol.44 (Academic Press, New York, 1976); U.S. Pat. Nos. 814; 4,413,070; and 4,046;720; and Pon, Chapter 19, in Agrawal, , s in Molecular Biology, Vol.20, (Humana Press, Totowa, N.J., 1993). Further supports of interest include polystyrene beads; polystyrene grafted with polyethylene glycol (e. g., TentaGelTM, Rapp Polymere, Tubingen Germany); and the like.

Selection of the support characteristics, such as material, porosity, size, shape, and the like, and the type of g moiety employed s on a variety of factors, such as protection groups employed, length of final product, quantity of final t, and the like. Exemplary linking moieties are disclosed in Pon et al, Biotechniques, 6:768—775 (1988); Webb U.S. Pat. No. 4,659,774; Barany et al, ational patent application PCT/US91/06103; Brown et al, J. Chem.

Soc. Commun., 1989: 891—893; Damha et al, Nucleic Acids Research, 18: 3813—3821(1990); Beattie et al, Clinical try, 39: 719—722 (1993); Maskos and Southern, Nucleic Acids Research, 20: 1679—1684 (1992); and the like.

In some embodiments, the solid supports that find use in the subject methods include CPG and polystyrene grafted with polyethylene glycol and possessing a terminal amino group (e. g., TentaGel—NHZ TM, Rapp Polymere, Tubingen Germany). The aminopropyl group may be used as a spacer between CPG and the nucleoside linkage. In some cases, the linkage to the 5'—hydroxyl of the first nucleoside is a succinyl group which provides a base—labile ester linkage that may be cleaved after synthesis with aqueous a.

Following deprotection, the support—bound nucleoside is capable of reacting with a dimer or monomer t phosphoramidite to form an intemucleoside linkage. It is understood that the support—bound side may refer to a single e attached to a solid support or may refer to the terminal residue of an oligonucleotide chain that is attached to the support.

Any convenient coupling chemistry, coupling reagents and methods may be utilized in the subject methods. Considerable guidance in making selections concerning coupling conditions, protecting groups, solid phase supports, linking groups, deprotection reagents, reagents to cleave products from solid phase ts, purification of product, and the like, in the t of the subject methods can be found in literature, e. g. Gait, editor, Oligonucleotide sis: A Practical ch (IRL Press, Oxford, 1984); Amamath and Broom, Chemical 2015/028327 Reviews, Vol. 77, pgs. 183—217 (1977); Pon et al, Biotechniques, Vol. 6, pgs. 768—775 (1988); Ohtsuka et al, Nucleic Acids Research, Vol. 10, pgs. 6553—6570 (1982); Eckstein, editor Oligonucleotides. and Analogues: A Practical ch (IRL Press, Oxford, 1991), Greene and Wuts “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987), Beaucage and Iyer, Tetrahedron 48: 2223—2311 (1992), and like references.

The coupling step of the subject methods may be carried out in the temperature range of —20 to 200 degrees Centigrade. In some instances, the reaction is carried out at ambient temperature (about 15—30 degrees Centigrade). The reaction may be performed by adding a on of the phosphoramidite dimer or monomer and a solution of an activator (or a solution containing the phosphoramidite dimer or monomer and the activator) to the reaction vessel containing the free amino group of an (oligo)nucleotide covalently attached to a solid support.

Generally, activators of interest include nucleophilic catalysts that displace the more stable phosphoramidite amino group to form a highly ve (and less stable) intermediate which, in turn, reacts with the free 3' amino group of a solid ted oligonucleotide N3'—>P5' oramidate. The mixture is then mixed by such methods as mechanical vortexing, sparging with an inert gas, etc. Altemately, the solution(s) of dimer or monomer and activator can be made to flow through a reaction vessel (or column) containing the solid supported (oligo)nucleotide with a free 3'—terminal group. The monomer and the activator either can be premixed, mixed in the valve—block of a suitable synthesizer, mixed in a pre—activation vessel and pre—equilibrated if desired, or they can be added tely to the reaction vessel.

Activators of interest that may be utilized in the subject s include, but are not limited to, tetrazole, 5—(ethylthio)tetrazole, 5—(4—nitrophenyl)tetrazole, 5—(2—thiophene) tetrazole, le, pyridinium chloride, and the like, e. g. activating agents as described by Beaucage and Iyer edron 48: 2223—2311 (1992); Berner et al, Nucleic Acids Research, 17: 853—864 (1989); Benson, Chem. Rev. 41: 1—61 (1947). As used herein, the term "tetrazole activator" refers to activators which are tetrazole or derivatives of ole. In some ments, the activator is tetrazole. Convenient solvents e, but are not limited to, acetonitrile, tetrahydrofuran, methylene chloride, and the like. Care may be exercised to use dry (free from water) dimer or monomer, activator, and solvent for the ng step and for the solvent used to wash the solid support ately before the coupling step.

After ng, the unreacted 3'—amino groups of the support—bound growing chain of the oligonucleotide may be optionally capped with a convenient capping agent before the next deprotection step (e.g., detritylation step) to render them inert to subsequent coupling steps. This capping step may improve the HPLC profile of the preparation to make purification more facile, and may also improve the overall yield of product. g ts useful in the subject s include electrophilic ts such as acetic anhydride and isobutyric anhydride, acid chlorides such as adamantyl carbonyl chloride, pivaoyl chloride, and the like, ocyanates, chloroformates, etc. Also useful are phosphoramidites in conjunction with an activator and followed by oxidation, and H—phosphonate salts such as triethylammonium isopropyl—H—phosphonate used in conjunction with an acid chloride such as l chloride or adamantyl carbonyl chloride.

In some embodiments, the method includes oxidizing an internucleoside N3'—>P5' phosphoramidite linkage. As used herein, the terms "oxidize, II II oxidation, II 4‘oxidizing”, and the like, in reference to a phosphorus—containing ucleosidic linkage means a process or treatment for converting the phosphorus atom of the linkage from a phosphorus (III) form to a orus (V) form. ion of the internucleotide linkages may be performed at any convenient point in the synthesis using any convenient methods. In some embodiments, ion is performed in a stepwise manner, e. g., during every coupling cycle. In other embodiments, oxidation of multiple internucleotide linkages is performed at the end of the synthesis. In some instances, oxidizing a N3'—>P5' phosphoramidite linkage (e. g., using an iodine/water based oxidizing agent) produces an oxo—phosphoramidate e. In other instances, oxidizing a N3'—>P5' phosphoramidite linkage includes sulfurization to produce a thiophosphoramidate linkage. Sulfurization may be performed using any convenient methods. ization methods of interest include those described by Gryazonov et al., 018015 the disclosure of which is herein incorporated by reference in its entirety. Sulfurizing agents for use in the invention include elemental sulfur, thiuram disulfides such as tetraethyl thiuram disulfide, acyl disulfides such as phenacyldisulfide, phosphinothioyl disulfides such as S— TetraTM, and oxo—3H—l,2—benzodithiol—3—one. In some embodiments, sulfurization may be performed using elemental sulfur (S 8). In certain embodiments, sulfurization may be performed using Beaucage reagent, using methods as described by Iyer et al., J. Organic Chemistry 55:4693—4699, 1990.

Oxidizing agents which are useful in the method include iodine, chlorine, bromine, peracids such as m—chlorobenzoic acid, hydroperoxides such as t—butylhydroperoxide, ethyl hydroperoxide, methyl hydroperoxide and the like, ozone, mixed acyl—sulfinic anhydrides such as 3H—2,l—benzoxathiolan—3—one—l—oxide, salts of persulfates such as sodium, ammonium, and tetrabutylammonium persulfate and the like, roxysulfates such as M, sodium and/or other hypochlorites, peroxides such as diethyl peroxide or bis(trimethylsilyl)peroxide, or hydrogen de or non aqueous hydrogen peroxide equivalents such as urea/hydrogen de complex, etc. Other useful oxidizing agents which may be used to convert phosphorus (III) to orus (V) are described in Beaucage and Iyer Tetrahedron 48: 2223—2311 (1992).

In some cases, the oxidizing or izing agent may have a cy to undergo an undesired Arbuzov side reaction in parallel with the desired oxidation (Beaucage and Iyer, cited above). The Arbuzov side reaction can lead to a deprotected phosphoramidate which is le to the acidic conditions of subsequent detritylation steps, and result in oligonucleotide fragmentation. In certain embodiments, hydrogen peroxide is used as the oxidizing agent to mimimize the Arbuzov side reaction. In certain embodiments, oxidation includes contacting the oligonucleotide with a solution of 15% hydrogen peroxide, 3.5% water, 20% pyridine, and 75% THF.

In some embodiments, the method includes the steps of: (a) deprotecting a protected 3' amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) reacting the free 3' amino group with either: (i) a 3'—protected amino—dinucleotide oramidate—5'—phosphoramidite dimer; (ii) a 3'—protected aminonucleoside—5'—phosphoramidite monomer; in the presence of a nucleophilic catalyst to form an intemucleoside N3'—>P5' phosphoramidite linkage; (c) ing the linkage; and (d) repeating steps (a) through (c) until the polynucleotide is synthesized, n the repeating steps (a) through (c) ses performing step (b)(i) at least once.

In some embodiments, the repeating steps (a) through (c) comprises performing step (b)(i) twice or more. In certain embodiments, the repeating steps (a) through (c) comprises performing step (b)(i) 3 times or more, such as 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 15 times or more, 20 times or more, or even 30 times or more. In certain embodiments, the repeating steps (a) through (c) comprises performing step (b)(i) at every coupling step. In certain embodiments, the repeating steps (a) through (c) comprises performing step (b)(i) at every coupling step except one. In certain embodiments, the repeating steps (a) h (c) comprises performing step (b)(ii) once and only once. In certain embodiments, the repeating steps (a) through (c) comprises performing step (b)(ii) twice and only twice.

As described herein, it is understood that the term phosphoramidate linkage is meant to encompass both oxo—phosphoramidate and thiophosphoramidate linkages (e. g., as depicted in a I). In certain embodiments of the method, oxidizing the intemucleoside 5' phosphoramidite linkage produces an osphoramidate linkage. In some embodiments of the method, oxidizing the internucleoside N3'—>P5' phosphoramidite linkage includes sulfurization to produce a thiophosphoramidate e.

In some embodiments of the method, the oligonucleotide is described by a (I): Z_ _L O B HN R3 /P\ B R0 0 R6 R3 Formula (I) wherein: each B is independently a , a protected purine, a pyrimidine or a ted pyrimidine, or an analog thereof; each X is independently oxygen or ; each R3 is hydrogen, fluoro, hydroxyl, an alkoxy, a substituted alkoxy or a protected hydroxyl; R6 is amino, hydroxyl, a protected amino, a protected hydroxy, —O—L—Z or —NH—L— each L is ndently an optional linker; each Z is independently H, a lipid, a support, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl or a phosphate protecting group; and n is an integer of 1 to 1000. When R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl or a phosphate protecting group, it is understood that some of the oligonucleotides of a (I), may also exist in a salt form. Such forms in so far as they may eXist, are intended to be included Within the scope of the present disclosure.

In some embodiments of a (I), each R3 is hydrogen. In some embodiments of Formula (I), each R3 is fluoro. In some embodiments of Formula (I), each R3 is hydroxyl.

In some embodiments of Formula (I), R6 is amino. In n embodiments of Formula (I), R6 is hydroxyl.

In some embodiments of Formula (I), each R is hydrogen. It is understood that when R is hydrogen, the phosphate linkage may be charged under aqueous conditions, such as physiological conditions. As such, it is understood that oligonucleotides of Formula (I) may also e any convenient salt forms of the linkage. As such, the internucleoside linkage of Formula (I) may be in a salt form that includes any convenient counterion. In some embodiments of Formula (I), each R is an alkyl or a substituted alkyl. In some embodiments of Formula (I), each R is an aryl or a tuted aryl. In some embodiments of Formula (I), each R is a phosphate ting group.

In some embodiments of Formula (I), Z is H. In some embodiments of Formula (I), Z is a lipid (e. g., as described herein). In certain cases, the lipid is a fatty acid (e. g., as described herein). In some embodiments of Formula (I), Z is a support. In some embodiments of a (I), Z is a carrier. In some embodiments of a (I), Z is an oligonucleotide. In some ments of Formula (I), Z is a polymer. In certain cases, the polymer is a PEG. In some embodiments of Formula (I), Z is a polypeptide. In some ments of Formula (I), Z is a detectable label. In some embodiments of Formula (I), Z is a tag.

In some embodiments of Formula (I), L is absent.

In some embodiments, each B is independently selected from A, C, G, T and U or a protected version thereof.

In certain embodiments of Formula (I), n is an integer of between 1 and 500, such as n 1 and 100, between 1 and 75, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 15, between 1 and 10, or between 4 and 10. In certain ments, n is an integer of between 1 and 100, such as between 5 and 50, between 10 and 50, between 10 and 40, n 10 and 30, between 10 and 25 , n 10 and 20, between 12 and 18, or between 12 and 16. In certain ments, n is 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25.

In certain embodiments of the method, the oligonucleotide comprises a sequence of nucleoside subunits complementary to the RNA component of human telomerase, and wherein at least two of the nucleoside subunits are joined by a N3’—>P5’ phosphoramidate inter— subunit linkage.

In some ments of the method, the oligonucleotide includes a sequence of between 3 and 50 nucleoside contiguous subunits complementary to the RNA ent of human telomerase, such as between 5 and 40, between 10 and 40, between 10 and 30, between and 25, between 10 and 20, between 12 and 18, or between 12 and 16 nucleoside ts. In certain embodiments, the oligonucleotide includes a ce of 10 or more contiguous nucleoside subunits complementary to the RNA component of human telomerase. In certain embodiments, the oligonucleotide includes a sequence of 7 or more uous nucleoside subunits, such as 7, 8, 9, 10, ll, l2, l3, l4, l5, 16 or 17 contiguous nucleoside subunits. In certain embodiments, the oligonucleotide includes a sequence of between 11 and 18, such as between 11 and 16 contiguous nucleoside subunits complementary to the RNA component of human telomerase.

In some instances of the method, the N3’—>P5’ thiophosphoramidate inter—subunit linkage is described by the following structure: P(S)(OR)—O—5’ where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a tuted aryl and a phosphate protecting group. It is tood that, when R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, some of the internucleoside subunit linkages described by the formula above may also exist in a salt form. Such forms in so far as they may exist, are intended to be ed Within the scope of the t disclosure.

In some instances of the method, the N3’—>P5’ thiophosphoramidate inter—subunit linkage is described by the following structure: 3’—NH—P(S)(OR)—O—5’ Where R is hydrogen. It is understood that for any of the oligonucleotides described herein that includes such an inter—subunit linkage, such oligonucleotides may also include any convenient salt forms of the linkage. As such, the inter—subunit linkage may be in a salt form that includes any convenient counterion.

In some embodiments of the method, the oligonucleotide includes the sequence TAGGGTTAGACAA (SEQ ID NO:3). In certain ments, all of the internucleotide inter— t es of the TAGGGTTAGACAA (SEQ ID NO:3) ce are N3'—> PS' oramidate inter—subunit linkages. In certain instances, all of the N3'—> PS' phosphoramidate inter—subunit linkages of the sequence are N3'—> PS' thiophosphoramidate inter—subunit linkages (e. g., nps linkages). In certain instances, all of the N3'—> PS' phosphoramidate inter—subunit linkages of the sequence are N3'—> PS' oxo—phosphoramidate inter—subunit linkages (e. g., np linkages).

In some embodiments of the method, the polynucleotide includes a 3’—amino or a 3’—hydroxyl terminal group. In certain embodiments of the method, the polynucleotide includes a 3’—amino terminal group. In certain embodiments of the method, the polynucleotide includes a 3 ’ —hydroxyl al group.

In some embodiments of the , the oligonucleotide is described by the structure: “£0 i! o T o {5! O=|L-SH (ID—l A O=|!’-SH «'3—[Gnp5GnpsenpsTnpsTnpsAnpsG"psAnpscnpsAnpsi1 A Where “nps” represents a thiophosphoramidate linkage (e.g., —NH—P(=O)(SH)—O— or a tautomer thereof), connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside.

] It is understood that all embodiments referring to an oligonucleotide are also applicable to the salt forms of said oligonucleotide.

In some ments of the method, the oligonucleotide is described by the structure: H OH i? —O T O=|i’-SH O=|i’-SH O_[GnpsGnpsGnpsTnpsTnpsAnpsansAnpanpsAnps] I A or a salt thereof; WO 68310 Where “nps” represents a thiophosphoramidate linkage (e. g., —NH—P(=O)(SH)—O— or a tautomer thereof, or a salt thereof), connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent side. In certain embodiments, the ition includes a pharmaceutically acceptable salt of the compound. In certain instances, the composition includes a sodium salt of the compound. In certain embodiments, the composition includes a divalent cation salt of the compound, such as a magnesium salt of the nd. In certain embodiments, the composition includes a trivalent cation salt of the compound, such as an aluminium salt of the compound.

In certain embodiments of the method, the oligonucleotide is described by the following structure, Where each M“ is independently hydrogen or any convenient counterion of a salt, each X is ndently l, 2 or 3 and n is an integer from 5 to 13, such as 5, 6, 7, 8, 9, 10, ll, 12 or 13, such as n is 13: HJIH \N <’ I} S—P-O N o N NH c: b o HN NH <’ IA S=I?-O N o N NH2 0- kg 0 HIT] NH <’ IA S=I?-O N o N NH2 o— 79 o HN NH <’ IA S=E-O N o N NH2 0- 0 HN \(k/gNH S=I?-O o N o o— p 0 HN \(k/gNH S=I?-O o N o 0' p NH2 _' (/N |\NA S—P-O N O N O o- b ,N NH HN < IA S=fi’-O N o N NH 0- 7;) N42 HN (IN |\N s=F"—o N 0 N’J C3 NH2 NH | I NAG s=|ID—o O o— 79 NH ' (IN |\NA s=E—o o N N o— b NH2 “3” «N I)” S=FI’—O N O N/ 0— L7 NH2 (M )n In certain instances, each X is 1. In certain instances, each X is independently l or 2. In certain instances, each X is ndently l or 3. In certain instances, M“ is hydrogen.

In certain embodiments of the method, the oligonucleotide is bed by the following structure and may include any convenient cationic counterions of a salt: ] In certain embodiments of the method, the oligonucleotide is described by the structure: H'F NH <’ | A s=fi>—o N o N NH2 0' p o S=Ff-O N o N’ NH2 0' p O s=fi>—o N o N/ NH2 o- 7;) o HN \ffigNH S=E>—o o N o o- p o Ht 1'0)“: S=E>—o o N o O' p NH2 N \ “H“ </ | j“ s=fi>—o o N N/ o 0- 7Q + N S=E’-O N o N NH2 0' jg NH2 a \ HN «N | j“ s=fi>—o o N ”NH 0' b 2 NH I \i 3:}:3—0 N O o- b NH2 Na+ N \ N NH <’ | 4 WO 68310 2015/028327 In certain embodiments of the method, the C11 nucleotide e of the TAGGGTTAGACAA (SEQ ID NO:3) sequence derives from a 3'—protected aminonucleoside—5'— phosphoramidite monomer. By “derives from” is meant that the e of interest is introduced during synthesis via a particular subunit. In certain ces, the T1 to A10, A12 and A13 residues of the TAGGGTTAGACAA (SEQ ID NO:3) sequence derive from 3'—protected amino— dinucleotide thiophosphoramidate—5'—phosphoramidite dimers.

In some cases, the method includes sequential coupling of the following 3'— protected amino—dinucleotide thiophosphoramidate—S'—phosphoramidite dimers and 3'—protected aminonucleoside—5'—phosphoramidite monomer to a terminal group of a solid phase support: TA, GG, GT, TA, GA, C and AA. It is understood that for simplicity, a protected phosphoramidite subunit that finds use in couplings of the subject methods may be depicted via the symbols X1 or XIXZ, where X1 and X2 are independently any convenient nucleosides linked via any convenient intemucleoside linkage (e. g., as described ). Any convenient synthetic strategies may be utilized in the subject methods. Some strategies of interest are shown below to demonstrate how the preparation of an oligonucleotide target sequence may be allocated to particular dimer and/or r subunits.

] Exemplary retrosynthetic strategies represented by the following lists of sequential dimer and/or monomer ts are provided for an exemplary target oligonucleotide sequence TAGGGTTAGACAA (SEQ ID NO:3). It is understood that this list of strategies is not exhaustive, and may be adapted for application to any convenient target oligonucleotide synthesis. In some embodiments, the method includes sequential coupling of one of the following series of 3'—protected amino—dinucleotide thiophosphoramidate—S'—phosphoramidite dimers and/or 3'—protected aminonucleoside—5'—phosphoramidite monomers to a terminal group of a solid phase support: TA, G, G, G, T, T, A, G, A, C, A, A ] T, AG, G, G, T, T, A, G, A, C, A, A T, A, GG, G, T, T, A, G, A, C, A, A T, A, G, GG, T, T, A, G, A, C, A, A T, A, G, G, GT, T, A, G, A, C, A, A T, A, G, G, G, TT, A, G, A, C, A, A T, A, G, G, G, T, TA, G, A, C, A, A ] T,AH(},CL(}jT,T,AILIA,CL[\VA T,AH(},CL(},T,1)[\,ChA,CL[\VA T,AH(},CL(},T,1)[\,CLIACL[\VA T,AH(},CL(},T,1)[\,CLIA,CH\VA T,AH(},CL(},T,1)[\,CLIA,CLZXA ] 13%,CKL(3,T,T§[\,CLIA,CL[\VA TA,CLCH3,T,T,A,(L1A,C,A”As TA,CL(L(3T,T,A,(L1A,C,A”As TA,CL(L(3,TT,A,(L1A,C,A”As TAH(},CL(L'FJTAH(LIA,CL[\VA 13%,ChCL(3/T,T,AILIA,CL[\VA TA,G3G;CLT§T§A,CLA,C,A,A TA,G;G;G;T,THAJ3,AC,A,A TA,CL(3,G;T,T,A,(L1A,Cfig[\ TA,CL(L(3,T,TVA,G3A,C,Afis TVAGgGCLTET,A,G3A,C,A,A TVAGgGgGTQT,A,G3A,C,A,A TVAGgGgGgTT,A,G3A,C,A,A TVAGgGgGgT,TA,G3A,C,A,A T,AG;G,G;T,TVAG;A,C,A,A ] T,AG;G,G;T,T,A,GAWC,A,A T,AG;G,G;T,T,A,G;AC,A,A T,ACL(L(3,T,T,A,CLZ&(:A,A T,ACL(L(3,T,T,A,CLZ&(11AA T,A,G(L(3T,T,AWCLzA,C,A,A T,A,CKL(3,TT,AWCLzA,C,A,A T,A,CKL(3,T,TA,CLZ&(11A,A T,A,GCLCLT§T,AG;A,C,A,A L(3,T,T,A,CLA,C,A,A ] T,A,GCLCLT§T,A,G3AC,A,A T,A,CKL(3,T,T,A,CLZ&(:A,A T,AHCK3,CL'T,T,AHCLIA,CLZXA T,AH(},CK}jTT,AHCLIA,CL[\VA T,AH(},CK},T,13\,CLIA,CL[\VA T,AH(},CK}jT,T,AILIA,CL[\VA T,AH(},CK},T,T)[\,ChA,CL/\VA T,AH(},CK},T,T)[\,CLIACL[\VA T,AH(},CK},T,T)[\,CLIA,CH\VA ,CK},T,T)[\,CLIA,CL[XA T,AH(},CL(3T,13\,CLIA,CL[\VA T,AH(},CL(}T,T,AILIA,CL[\VA T,AH(},CL(3T,T)[\,Cfl\,CL/\VA T,AH(},CL(3T,T)[\,Chz\CL/\VA T,AH(},CL(3T,T)[\,Chz\,C¥\VA T,AH(},CL(3T,T)[\,Chz\,CL[XA T,AH(},CL(}jTT,AILIA,CL[\VA T,AH(},CL(}jTT,AHCLA,CL[\VA T,AH(},CL(}jTT,AHCLIACLZXVA T,AH(},CL(}jTT,AHCLIA,CH\VA T,AH(},CL(}jTT,AHCLIA,CLZXA ] ,CL(},T,13\,CLA,CL[\VA ,CL(},T,13\,CLIACLZXVA T,AH(},CL(},T,13\,CLIA,CH\VA T,AH(},CL(},T,13\,CLIA,CL[XA ] T,AH(},CL(}jT,T,AILIACLZXVA T,AH(},CL(}jT,T,AILIA,CH\VA ,CL(}jT,T,AILIA,CLZXA T,AH(},CL(},T,T)[\,ChA,C¥\VA T,AH(},CL(},T,T)[\,ChA,CL[XA TA,GG;GT}T,A,G;A,C,A,A TA,GG;G;TT,A,G;A,C,A,A TA,GG;G;T,TA,G3A,C,A,A TA, GG, G, T, T, AG, A, C, A, A TA, GG, G, T, T, A, GA, C, A, A TA,GG,GfLT§A,G,AC,A,A TA, GG, G, T, T, A, G, A, CA, A TA, GG, G, T, T, A, G, A, C, AA G,TT,A,G,A,C,A,A TA,G,GG,T,TA,G,A,C,A,A TA, G, GG, T, T, AG, A, C, A, A TA, G, GG, T, T, A, GA, C, A, A TA,G,GGXLT§A,G,AC,A,A ] TA, G, GG, T, T, A, G, A, CA, A TA,G,G(LT§T,A,G,A,C,AA,aQ TA, GG, GT, TA, G, A, C, A, A TA, GG, GT, T, AG, A, C, A, A TA,GG,GT§T,A,GA,C,A,A TA,GG,GT§T,A,G,AC,A,A TA,GG,GT§T,A,G,A,CA,A GT)T,A,G,A,C,AA,aC TA,GG,GT)TA,GA,C,A,A TA,GG,GT)TA,G,AC,A,A TA,GG,GT)TA,G,A,CA,A TA, GG, GT, TA, G, A, C, AA, etc TA,G,GG,TTHAG,AC,A,A TA, G, GG, TT, AG, A, CA, A TA, G, GG, TT, AG, A, C, AA TA, G, G, GT, TA, GA, CA, A ] TA, G, G, GT, TA, GA, C, AA TA, G, G, GT, TA, GA, CA, A TA,G,G,G,TTNAG,AC,AA TA, G, GG, T, TA, GA, CA, A TA, G, GG, T, TA, GA, C, AA TA, G, GG, T, TA, G, AC, AA, etc T, A, G, GG, TT, AG, AC, AA T, A, GG, G, TT, AG, AC, AA T, AG, G, G, TT, AG, AC, AA TA, G, G, G, TT, AG, AC, AA T, AG, G, GT, T, AG, AC, AA, etc T, AG, GG, T, T, AG, AC, AA, etc T, AG, GG, TT, A, G, AC, AA, etc T, AG, GG, TT, AG, A, C, AA, etc T, AG, GG, TT, AG, AC, A, A T, AG, GG, TT, AG, AC, AA TA, G, GG, TT, AG, AC, AA TA, GG, G, TT, AG, AC, AA TA, GG, GT, T, AG, AC, AA TA, GG, GT, TA, G, AC, AA ] TA, GG, GT, TA, GA, C, AA or TA, GG, GT, TA, GA, CA, A.

In some embodiments, the method includes sequential coupling of a series of 3'— protected amino—dinucleotide thiophosphoramidate—S'—phosphoramidite dimers and/or 3'— protected aminonucleoside—5'—phosphoramidite monomers to a terminal group of a solid phase support, where at least the final coupling of the synthesis is a dimer coupling. In certain ments, the second—to—last coupling and the final coupling are dimer couplings. In certain cases, when N is even, the method includes N/2 dimer couplings. In n ces, when N is even, the method includes N/2—l dimer couplings. In certain instances, when N is even, the method includes N/2—2 dimer couplings. In certain instances, when N is even, the method es N/2—3 dimer couplings. In certain instances, when N is even, the method includes N/2—4 dimer couplings. In n instances, when N is even, the method includes N/2—5 dimer couplings. In certain cases, when N is odd, the method includes N/2—l dimer couplings. In certain ces, when N is odd, the method includes N/2—2 dimer ngs. In certain instances, when N is odd, the method includes N/2—3 dimer couplings. In certain instances, when N is odd, the method includes N/2—4 dimer couplings. In certain instances, when N is odd, the 2015/028327 method es N/2—5 dimer couplings. In certain instances, when N is odd, the method includes N/2—6 dimer couplings. For example, a sequential coupling of the following series of 3'—protected amino—dinucleotide thiophosphoramidate—5'—phosphoramidite dimers and/or 3'—protected aminonucleoside—5'—phosphoramidite monomers to a terminal group of a solid phase support: T, A, G, G, G, T, T, A, G, A, C, AA T, A, G, G, G, T, T, A, G, AC, AA T, A, G, G, G, T, T, A, GA, C, AA ] T, A, G, G, G, T, T, AG, A, C, AA T, A, G, G, G, T, TA, G, A, C, AA T, A, G, G, G, TT, A, G, A, C, AA T, A, G, G, GT, T, A, G, A, C, AA T, A, G, GG, T, T, A, G, A, C, AA T, A, GG, G, T, T, A, G, A, C, AA T, AG, G, G, T, T, A, G, A, C, AA TA, G, G, G, T, T, A, G, A, C, AA, etc T, A, G, G, G, T, T, AG, AC, AA T, A, G, G, G, TT, AG, AC, AA T, A, G, GG, TT, AG, AC, AA T, AG, GG, TT, AG, AC, AA TA, G, GG, TT, AG, AC, AA TA, GG, G, TT, AG, AC, AA TA, GG, GT, T, AG, AC, AA TA, GG, GT, TA, G, AC, AA TA, GG, GT, TA, GA, C, AA.

In some embodiments of the method, the 3'—protected dinucleotide thiophosphoramidate—5'—phosphoramidite dimer is described by the formula XIXZ, Where X1 and X2 are independently selected from a ted adenine, a protected ne, a protected guanine, thymine and uracil.

Lipid modified oligonucleotides A variety of synthetic approaches can be used to conjugate a lipid moiety L' to the ucleotide, depending on the nature of the e selected, including the approaches described in Mishra et al., (1995) Biochemica et Biophysica Acta, 1264:229—237, Shea et al., (1990) Nucleic Acids Res. 18:3777—3783, and Rump et al., (1998) j. Chem. 9:341—349.

The synthesis of compounds in which the lipid moiety is ated at the 5’ or 3’ terminus of the oligonucleotide can be achieved h use of suitable functional groups at the appropriate terminus, in some cases an amino group, which can be reacted with carboxylic acids, acid chlorides, anhydrides and active esters. Thiol groups may also be used as functional groups (see Kupihar et al., (2001) anic and Medicinal Chemistry 9: 1241—1247). Both amino— and thiol— modifiers of different chain lengths are commercially available for oligonucleotide synthesis. Oligonucleotides having N3’—>P5’ phosphoramidate (e. g., N3’—>P5’ thiophosphoramidate) linkages contain no groups (rather than 3’—hydroxy found in most conventional ucleotide chemistries), and hence these oligonucleotides provide a unique unity for conjugating lipid groups to the 3’—end of the ucleotide. s approaches can be used to attach lipid groups to the termini of oligonucleotides with the N3’—>P5’ phosphoramidate (e. g., N3’—>P5’ osphoramidate) chemistry (see e. g., 3—palmitoylamido—l—O—(4,4’—dimethoxytrityl)—2—O—succinyl propanediol linker of Table 2). For attachment to the 3’ terminus, the conjugated compounds can be synthesized by reacting the free 3’—amino group of the fully protected solid support bound oligonucleotide with the corresponding acid anhydride followed by deprotection with ammonia and purification. Alternatively, coupling of carboxylic acids of lipids to the free 3’—amino group of the support bound ucleotide using coupling agents such as carbodiimides, HBTU or 2— chloro— l—methylpyridinium iodide can be used to conjugate the lipid groups. These two methods form an amide bond between the lipid and the oligonucleotide. Lipids may also be attached to the oligonucleotide chain using a phosphoramidite derivative of the lipid coupled to the oligonucleotides during chain elongation. This approach yields a phosphoramidate (e. g., thiophosphoramidate) linkage connecting the lipid and the oligonucleotide (exemplified by propyl—palmitoyl and 2—hydroxy—propyl—palmitoyl compounds). Still another approach involves reaction of the free 3’—amino group of the fully protected support bound ucleotide with a suitable lipid aldehyde, followed by reduction with sodium cyanoborohydride, which produces an amine linkage.

WO 68310 For attachment to the 5’ terminus, the oligonucleotide can be synthesized using a modified, lipid—containing solid support, followed by synthesis of the oligonucleotide in the 5’ to 3’ direction as described in Pongracz & Gryaznov (1999). An example of the ed support is provided below. In the instance where n=l4, the fatty acid is palmitic acid: on of 3—amino— l,2—propanediol with palmitoyl chloride, followed by dimethoxytritylation and ylation provided the intermediate used for coupling to the solid support. R may be long chain alkyl amine controlled pore glass. 4/12O 1-120, , , o E ii /<"H\ c CPL KHz A’ \ ,./ ~ ACE: \ ) it own ' .‘1 DIMERS USEFUL FOR MAKING UCLEOTIDES In some embodiments of the method of making an oligonucleotide, the method includes contacting a support—bound free 3'—terminal group (e. g., a 3'—hydroxyl or 3'—amino group) with a dinucleotide dimer subunit to form an inter—subunit linkage. In general, the dinucleotide dimer is tected and includes a 5’—group e of coupling with the 3’— terminal group. In some embodiments, the dinucleotide dimer includes a 5’—phosphoramidite.

The dinucleotide dimer may include a 3’—protected amino group or a 3’—protected hydroxyl group. In some embodiments, the dinucleotide is decribed by the formula XIXZ, where X1 and X2 are independently any convenient sides (e. g., A, C, G, T or U or a ted version thereof) linked via any convenient internucleoside linkage (e. g., as described herein). The dinucleotide may include any convenient internucleoside linkage between the two nucleosides. intemucleoside linkages of interest that find use in the. dinucleotide dimers include, but are not limited to, a phosphodiester, a phosphotriester, a inethylphosphonate, a phosphoramidate (e.g., a thiophospht‘naniida‘te) and a phosphorothioa‘te e. in some cases. the dinucieotide dimer is a 3"protected-{iinucleotide--5'— phosphoramidite dimer, or a synthetic precursor f, where the dinucleotide is decribed by the formula XIXZ, Where X1 and X2 are independently selected from A, C, G, T and U or a protected version thereof, and Where X1 and X2 are linked Via a phosphodiester, a phosphotriester, a methylphosphonate, a phosphoramidate (eg, a thiophosphoramidate) or a phosphorothioate e, or a protected n thereof.

In some embodiments of the method of making an oligonucleotide, the method es contacting a support—bound free 3'—amino group with a 3'—protected dinucleotide phosphoramidate—5'—phosphoramidite dimer to form an intemucleoside N3'—>P5' phosphoramidite linkage. Any convenient 3'—protected amino—dinucleotide phosphoramidate—5'— phosphoramidite dimer, or synthetic precursors thereof, may find use in the subject s. In some cases, the dimer may be represented by the one of the following sequences: AA, AC, AG, AT, AU, CA, CC, CG, CT or CU, GA, GC, GG, GT or GU, TA or UA, TC or UC, TG or UG and TT or UU. In some cases, the dimer includes protected 2’—hydroxyl groups.

In certain embodiments, the dinucleotide dimer is a dinucleotide thiophosphoramidate compound described by Formula (II): Formula (II) n B1 and B2 are each independently a purine, a protected purine, a dine or a protected pyrimidine, or an analog thereof; R11 is hydrogen, a protecting group or a phosphoramidite group; R12 is hydrogen or a protecting group; and R13 is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl or a protecting group. In some cases, B1 and/or B2 e a nucleobase protecting group. It is understood that, when R13 is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl or a protecting group, some of the eotide dimers described by Formula (II) may also exist in a salt form. Such forms in so far as they may eXist, are intended to be included Within the scope of the present disclosure.

In some embodiments of Formula (II), R11 is hydrogen. In some ments of Formula (II), R11 is a protecting group. Any convenient protecting groups may find use in the subject dimers of Formula (II). In some ments of Formula (II), R11 is a levulinate—based protecting group. In some embodiments of Formula (II), R11 is a levulinate protecting group (i.e., —COCH2CH2COCH3). In some embodiments of Formula (II), R11 is a 5’—phosphoramidite group.

] In some embodiments of Formula (II), R12 is hydrogen. In some embodiments of Formula (II), R12 is a ting group. In certain embodiments, R12 is a trityl group (e. g., a triphenylmethyl (Trt), a monomethoxytrityl (MMT), or a dimethoxytrityl (DMT)). In some embodiments of Formula (II), R12 is a Trt protecting group.

In some embodiments of Formula (II), R12 is a photocleavable protecting group.

Any convenient photocleavable protecting groups may find use in the preparation of the subject dinucleotide dimers and synthetic precursors thereof. In some embodiments of a (II), R12 is a substituted pixyl ting group, such as a nitro, fluoro, methyl, trifluoromethyl, and/or y—substituted pixyl protecting group. In some embodiments of Formula (II), R12 is a pixyl protecting group (i.e., a 9—(9—phenyl)xanthenyl).

] In some embodiments of Formula (II), R11 is a levunyl protecting group and R12 is a trityl protecting group.

In some embodiments of Formula (II), R13 is hydrogen. In some embodiments of Formula (II), R13 is a protecting group. In certain embodiments, R13 is a 2—cyano—ethyl group.

In certain embodiments, the 3'—protected amino—dinucleotide phosphoramidate—5'— phosphoramidite dimer is described by Formula (III): Formula (111) wherein B1 and B2 are each independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof. In some cases, B1 and/or B2 e a nucleobase protecting group.

] In certain embodiments, the 3'—protected amino—dinucleotide phosphoramidate—5'— phosphoramidite dimer is described by Formula (111): Formula (IV) wherein B1 and B2 are each ndently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog f; and R18 is a trityl protecting group (such as a Trt, a DMT or a MMT) or a pixyl protecting group.

In some embodiments of Formulae (II) or (III), B1 and B2 are each independently selected from a protected adenine, a protected cytosine, a protected e, thymine and uracil.

In some embodiments of Formulae (II) or (III), B1 and B2 are each independently ed from A(Bz), A(DMF), C(Bz), utyryl), T and U. In some embodiments of Formulae (II) or (III), B1 is A(Bz). In some embodiments of Formulae (II) or (III), B1 is A(DMF). In some embodiments of Formulae (II) or (III), B1 is C(Bz). In some embodiments of Formulae (II) or (III), B1 is G(isobutyryl). In some embodiments of Formulae (II) or (III), B1 is T or U. In some embodiments of Formulae (II) or (III), B2 is A(Bz) or A(DMF). In some embodiments of Formulae (II) or (III), B2 is C(Bz). In some embodiments of Formulae (II) or (III), B2 is G(isobutyryl). In some embodiments of Formulae (II) or (III), B2 is T or U.

In some embodiments of ae (II) or (III), B1 is A(Bz) or A(DMF) and B2 is A(Bz) or A(DMF). In some embodiments of Formulae (II) or (III), B1 is A(Bz) or A(DMF) and B2 is C(Bz). In some embodiments of Formulae (II) or (III), B1 is A(Bz) or A(DMF) and B2 is G(isobutyryl). In some embodiments of Formulae (II) or (III), B1 is A(Bz) or A(DMF) and B2 is T or U.

In some embodiments of Formulae (II) or (III), B1 is C(Bz) and B2 is A(Bz) or A(DMF). In some embodiments of Formulae (II) or (III), B1 is C(Bz) and B2 is C(Bz). In some embodiments of Formulae (II) or (III), B1 is C(Bz) and B2 is utyryl). In some embodiments of Formulae (II) or (III), B1 is C(Bz) and B2 is T or U.

In some embodiments of Formulae (II) or (III), B1 is G(isobutyryl) and B2 is A(Bz) or A(DMF). In some embodiments of ae (II) or (III), B1 is G(isobutyryl) and B2 is C(Bz). In some embodiments of Formulae (II) or (III), B1 is utyryl) and B2 is G(isobutyryl). In some embodiments of Formulae (II) or (III), B1 is G(isobutyryl) and B2 is T or In some embodiments of Formulae (II) or (III), B1 is T or U and B2 is A(Bz) or A(DMF). In some embodiments of Formulae (II) or (III), B1 is T or U and B2 is C(Bz). In some embodiments of Formulae (II) or (III), B1 is T or U and B2 is G(isobutyryl). In some embodiments of ae (II) or (III), B1 is T or U and B2 is T or U.It is understood that any of the embodiments of Formulae (II) or (III) described herein, can also be applied to Formula (IV).

] Any of the dimers described herein may be adapted for use in the subject methods. The subject dimers may be prepared ing to any convenient methods from any convenient nucleoside monomers. Nucleoside monomers of interest that find use in the preparation of the subject nucleoside dimers e, but are not limited to, monomers l6, 17, 12 and 13 which are depicted in the synthetic schemes disclosed herein. Dinucleotide dimers of interest include osphitylated dimers that find use in the preparation of the subject phosphitylated dinucleotide dimers, such as dimers 18 and 19 which find use in the preparation of phosphitylated dinucleotide dimers such as 20, or dimer 14 which finds use in the preparation of phosphitylated dinucleotide dimers such as 15.

] In some embodiments, the dimers of Formulae (III) and (IV) are prepared Via the method depicted in the following scheme: WO 68310 R\16 O B1 HOWO B1 Protect 5'-OH Om, Hrf HZN‘ R16 1 H N\R17 | Couple nucleoside e 8 RJO 82 Sulfurlzatlon HN: O/VCN Deprotect | A ¢ CN HN S=P-O/\/ —’ I CN I —’ \/ O Phosphitylation | 0 I32 HN‘: S R17 HN\ Where B1 and B2 are each independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; R15 is hydrogen or an amino protecting group; R17 is an amino protecting group; and R16 is a hydroxyl protecting group. In certain embodiments, R15 is hydrogen. In certain embodiments of monomer 16, R16 is a silyl. In certain embodiments of monomer 16, R16 is TBDMS (tert—butyldimethylsilyl). In certain embodiments of monomer 17, R17 is a trityl (Trt). In certain embodiments of monomer 17, R17 is a monomethoxytrityl (MMT).

In n embodiments of r 17, R17 is a dimethoxytrityl (DMT). In certain embodiments of monomer 17, R17 is a pixyl. In certain embodiments of dimers 18—20, R17 is a trityl (Trt). In certain embodiments of dimers 18—20, R17 is a monomethoxytrityl (MMT). In certain embodiments of dimers 18—20, R17 is a dimethoxytrityl (DMT). In certain embodiments of dimers 18—20, R17 is a pixyl.

In some embodiments, the dimers of Formulae (III) and (IV) are prepared Via the method depicted in the following scheme, Where the monomer 13 is prepared from 11 Via monomer 12 and d with a nucleoside e to produce dimers 14 which is converted to dimer 15: 0 I31 0 82 Flo/\g Protect NHz HOW nate Protection : —> 5 —> H2N‘ “N 11 ‘R13 12 WOO/19,81Deprotect at 3-NH2 H| /\/CN NR13 S O Couple nucleoside amidite | Sulfurization O HN‘: O/\/CN A ' ,P\ 0 81 Deprotect at 5'-OH N Ow A HN‘~ Phosphitylation S=|l’—O/\/CN HN‘: Where B1 and B2 are each independently a purine, a protected , a pyrimidine or a protected pyrimidine, or an analog thereof; and R13 and R14 are each independently a protecting group. In certain embodiments of monomers 12 and 13, R13 is a trityl. In certain embodiments of monomers 12 and 13, R13 is a pixyl. In certain embodiments of dimers 14 and 15, R14 is a trityl.

In certain embodiments of dimers 14 and 15, R14 is a oxytrityl. In certain embodiments of dimers 14 and 15, R14 is a monomethoxytrityl. In certain embodiments of dimers 14 and 15, R14 is a pixyl.

Monomers of st that find use in preparation of the subject dinuc1eotide dimers according to the methods described herein include, but are not limited to: O/\/CN O/\/CN N’ ”(I 1 I A o HN‘: N,P\O/\<—7/B O A ‘ g o MeO O o o NOWB NOWo B 0 o C HN HN O Q We R O 0 R where B is a purine, a protected purine, a pyrimidine or a ted pyrimidine, or an analog thereof and R is hydrogen or an alkyl (e. g., methyl) or a halogen (e. g., bromo). In certain cases, B is selected from A(Bz), , T, A(DMF), C(Bz), or U.

OLIGONUCLEOTIDE COMPOSITIONS In addition to a target oligonucleotide, a variety of non—target oligonucleotide synthesis products may be produced during oligonucleotide sis. Minor products that may be present in oligonucleotide preparations include, but are not limited to, deletion ts (e. g., products g one or more nucleoside residues), products that include one or more protecting groups, terminated products (e. g., products that include a capped oligonucleotide chain), products that lack one or more nucleobases, products that include partially oxidized phosphoramidite linkages and products that include partially sulfurized linkages. As used herein, target oligonucleotide refers to an oligonucleotide sequence of interest, which is the target product of the method of preparation. As used herein, the terms “non—target product” and “minor product” are used hangeably and refer to any ucleotide—containing product that is not the target product, and which may occur during and after the cycles of the target oligonucleotide synthesis.

The subject methods provide for compositions that include an improved purity of target oligonucleotide. In some embodiments, the composition includes 50% or more by weight of the target oligonucleotide, such as about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or even about 95% or more by weight of the target oligonucleotide. In certain embodiments, the composition es 50% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 55% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 60% or more by weight of the target oligonucleotide. In n embodiments, the composition includes 65% or more by weight of the target oligonucleotide. In certain ments, the ition es 70% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 75% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 80% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 85% or more by weight of the target oligonucleotide. In certain embodiments, the WO 68310 composition includes 90% or more by weight of the target oligonucleotide. In certain embodiments, the composition includes 95% or more by weight of the target oligonucleotide.

In some embodiments, the subject methods provide for a coupling efficiency of 95% or more, such as 96% or more, 97% or more, 98% or more, or even 98% or more.

In some embodiments, the subject methods provide for a mean coupling efficiency that is 0.5% or more, such as 0.75% or more, 1.0% or more, 1.25% or more, 1.5% or more, 1.75% or more, 2.0% or more, 2.5% or more, or even 3.0% or more, than the mean coupling efficiency of a l synthesis performed using only monomer subunits. In certain ments, the subject methods provided for a 96% or greater coupling efficiency. In certain ments, the subject methods provides for a coupling efficiency that is 2% or greater than the coupling efficiency of a control synthesis performed using only monomer subunits.

After synthesis, the subject compositions may undergo one or more purification steps (e. g., HPLC chromatography, affinity tography, ion exchange chromatography, gel filtration, etc.), e. g., to remove one or more minor products from the target oligonucleotide. It is understood that, in the subject compositions, the reduced amounts of minor products and/or increased amount of target oligonucleotide provided by the subject methods of preparation may refer to such amounts and purities obtained immediately post synthesis and before any further purification or separation steps (e. g., HPLC chromatography) have been performed. As such, in some cases, the subject compositions may be referred to as synthesis preparations, e.g., fied synthesis ations. By unpurified is meant that no chromatography purification steps have been performed on the composition. Chromatography purification refers to any convenient purification method that includes absorption of target polynucleotide to a tography t and subsequent elution of the target polynucleotide. In some cases, chromatography purification refers to reverse phase chromatography purification.

] The t methods provide for compositions including a d amount of one or more minor ts. By reduced amount is meant that the amount by weight of the minor product in the composition relative to the target oligonucleotide is reduced relative to a control synthesis, e. g., a synthesis where the oligonucleotide is ed using only monomer couplings.

In some embodiments, the reduced amount of minor product is about 20% or less of the amount by weight of the target oligonucleotide, such as about 15% or less, about 10% or less, or about % or less of the amount by weight of the target oligonucleotide. In certain embodiments, the reduced amount of minor product is 20% or less of the amount by weight of the target oligonucleotide, such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, % or less, 4% or less, 3% or less, 2% or less, or even 1% or less of the amount by weight of the target oligonucleotide. In certain embodiments, the minor product is a (N—X) product.

The subject methods of preparation may provide for compositions having a reduced amount of one or more (N—X) products ve to a target oligonucleotide of st, where X is an integer from 1 to N—1 and N is the number of nucleoside residues in the target oligonucleotide. As such, (N—1) product may refer to any and all oligonucleotide products that lack any one nucleotide residue in comparison to a target oligonucleotide (e. g, a N product). As such, a (N—2) t refers to any and all oligonucleotide products that lack any two nucleotide residues in comparison to a target oligonucleotide (e. g, a N product). In certain embodiments, the minor product is a (N—1) product. In certain embodiments, the minor product is a (N—2) t.

In certain embodiments, the minor product is a (N—3) product. In certain ments, the minor product is a (N—4) product. In certain ments, the minor product is a (N—5) product. In certain embodiments, the minor product is a (N—6) product. In n ments, the minor product is a (N—7) product.

In n embodiments, any of the compositions described herein that have a reduced amount of one or more (N—X) products relative to a target oligonucleotide of interest are unpurified.

In some embodiments, the subject compositions include a low ratio of (N—1) product to target oligonucleotide product. In some cases, the low ratio is less than (2.0 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide, where N refers to the number of nucleotide residues in the target oligonucleotide sequence. In certain embodiments, the ratio is less than (1.9 X N) parts to 100 parts by weight of (N—1) t relative to target oligonucleotide, such as less than (1.8 X N) parts to 100, less than (1.7 X N) parts to 100, less than (1.6 X N) parts to 100, less than (1.5 X N) parts to 100, less than (1.4 X N) parts to 100, less than (1.3 X N) parts to 100, less than (1.2 X N) parts to 100, less than (1.1 X N) parts to 100, less than (1.0 X N) parts to 100, less than (0.9 X N) parts to 100, less than (0.8 X N) parts to 100, less than (0.7 X N) parts to 100, less than (0.6 X N) parts to 100, less than (0.5 X N) parts to 100, less than (0.4 X N) parts to 100, less than (0.3 X N) parts to 100, less than (0.2 X N) parts to 100, or even less than (0.1 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (1.5 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (1.2 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (1.0 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (0.5 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide.

In some ments, the subject itions include a low ratio of (N—2) product to target oligonucleotide product. In some cases, the low ratio is less than (2.0 X N) parts to 100 parts by weight of (N—2) t ve to target oligonucleotide, where N refers to the number of nucleotide residues in the target oligonucleotide sequence. In certain embodiments, the ratio is less than (1.9 X N) parts to 100 parts by weight of (N—2) product relative to target oligonucleotide, such as less than (1.8 X N) parts to 100, less than (1.7 X N) parts to 100, less than (1.6 X N) parts to 100, less than (1.5 X N) parts to 100, less than (1.4 X N) parts to 100, less than (1.3 X N) parts to 100, less than (1.2 X N) parts to 100, less than (1.1 X N) parts to 100, less than (1.0 X N) parts to 100, less than (0.9 X N) parts to 100, less than (0.8 X N) parts to 100, less than (0.7 X N) parts to 100, less than (0.6 X N) parts to 100, less than (0.5 X N) parts to 100, less than (0.4 X N) parts to 100, less than (0.3 X N) parts to 100, less than (0.2 X N) parts to 100, or even less than (0.1 X N) parts to 100 parts by weight of (N—2) product relative to target oligonucleotide. In certain embodiments, the t itions e a low ratio of less than (1.5 X N) parts to 100 parts by weight of (N—2) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (1.2 X N) parts to 100 parts by weight of (N—2) product relative to target oligonucleotide. In certain embodiments, the subject compositions e a low ratio of less than (1.0 X N) parts to 100 parts by weight of (N—1) product relative to target oligonucleotide. In certain embodiments, the subject compositions include a low ratio of less than (0.5 X N) parts to 100 parts by weight of (N—2) product ve to target oligonucleotide.

In some embodiments, the subject compositions include (N—1) product in an amount of 20% or less of the total non—target oligonucleotides in the ition, such as 15% or less, 10% or less or even 5% or less of the total non—target oligonucleotides.

Any of a wide variety of oligonucleotide compositions can be prepared using the methods described herein. A variety of classes and types of ucleotides are of interest for preparation using the subject methods (e. g., as described herein). Oligonucleotides suitable for preparation according to the subject methods e, but are not limited to, anti—sense oligonucleotides, RNA oligonucelotides, siRNA oligonucleotides, RNAi oligonucleotides, DNA aptamers, micro RNA,and the like.

Oligonucleotides complementary to RNA component of Telomerase Aspects of the disclosure include compounds and compositions including oligonucleotides complementary to the RNA component of human telomerase, and methods for making the same. The compounds may inhibit telomerase activity in cells with a high potency and have cellular uptake characteristics.

As summarized above, the subject methods provide for reduced amounts of non— target oligonucleotide products of the synthesis. In certain cases, the subject methods provide for increase amounts of target oligonucleotide product of the synthesis. In some ments, the subject methods provide for the preparation of compositions that have a reduced amount of one or more (N—X) ts relative to a target oligonucleotide of interest. Table 1 sets forth amounts of interest of some non—target oligonucleotide products.

] In certain embodiments, any of the compositions bed herein that have a d amount of one or more (N—X) products ve to a target oligonucleotide of interest are unpurified.

] Table 1. Levels of oligonucleotide products in compositions of interest. The subject compositions may include one or more of the following components at one of the levels indicated in Table l.

Product % of composition old Amounts Range relative to Range relative to (by weight) relative to target target (by weight) target (by weight) (by weight) Oligos imetelstat target 50% or more, N/A N/A N/A 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 2015/028327 80% or more, 85% or more, 90% or more, 95% or more (N-1) products less than 11 % less than (1.9 X N) from about (0.1 X from about 1 to (including less than 10 % parts to 100, less than N) to about (0.5 X about 20 parts in derivatives thereof less than 9 % (1.8 X N) parts to 100 N) parts in 100, 100, from about 1 such as Phenylacetyl less than 8 % less than (1.7 X N) from about (0.1 X to about 10 parts in and iBu derivatives) less than 7 % parts to 100, less than N) to about (0.4 X 100, from about 1 (e.g., post peak 1 (N- less than 6 % (1.6 X N) parts to 100, N) parts in 100, to about 8 parts in 1) product) less than 5 % less than (1.5 X N) from about (0.2 X 100, from about 1 less than 4 % parts to 100, less than N) to about (0.3 X to about 6 parts in less than 3 % (1.4 X N) parts to 100, N) parts in 100, 100, from about 1 less than 2 % less than (1.3 X N) to about 5 parts in less than 1 % parts to 100, less than about (0.1 X N) 100, from about 2 less than 0.5 % (1.2 X N) parts to 100, parts in 100, about to about 4 parts in less than (1.1 X N) (0.2 X N) parts in 100, parts to 100, less than 100, about (0.3 X (0.9 X N) parts to 100, N) parts in 100, about 1parts in less than (0.8 X N) about (0.4 X N) 100, about 2 parts parts to 100, less than parts in 100, about in 100, about 3 (0.7 X N) parts to 100, (0.5 X N) parts in parts in 100, about less than (0.6 X N) 100, 4 parts in 100, parts to 100, about 5 parts in less than (0.5 X N) 100 parts to 100, less than (0.4 X N) parts to 100, less than 1 part in less than (0.3 X N) 4, less than 1 part parts to 100, less than in 5, less than 1 (0.2 X N) parts to 100, part in 6, less than less than (0.1 X N) 1 part in 7, less parts to 100 than 1 part in 8, less than 1 part in 9, less than 1 part in 10, less than 1 part in 20, less than 1 part in 25, less than 1 part in 100 (N-2) and (N-3) 4% or more at least (1.0 X N) parts from about (1.0 X from about 5 to products individually 6% or more to 100, at least (1.5 X N) to about (5.0 X about 50 parts in or combined 8% or more N) parts to 100, at N) parts in 100, 100, from about 10 (including 10% or more least (2.0 X N) parts to from about (2.0 X to about 50 parts in derivatives thereof 12% or more 100, at least (2.5 X N) N) to about (5.0 X 100, from about 20 such as Phenylacetyl 14% or more parts to 100, at least N) parts in 100, to about 50 parts in and iBu derivatives) 16% or more (3.0 X N) parts to 100, from about (2.5 X 100, from about 30 (e. g., Post Peaks 18% or more at least (3.3 X N) parts N) to about (4.0 X to about 50 parts in 2+3+4, or Post Peaks 20% or more to 100 N) parts in 100, 100, from about 5 3+4, or post Peak 2, 25% or more from about (3.0 X to about 40 parts in 3 or 4) less than (3.3 X N) N) to about (4.0 X 100, from about 5 less than 25% parts to 100, less than N) parts in 100, to about 30 parts in less than 20% (3.0 X N) parts to 100, from about (3.0 X 100, from about 5 less than 18% less than (2.5 X N) N) to about (3.5 X to about 20 parts in less than 16% parts to 100, less than N) parts in 100 100, from about 10 less than 14% (2.0 X N) parts to 100, to about 20 parts in less than 12% less than (1.5 X N) about (1.0 X N) 100 less than 10% parts to 100, less than parts in 100, about (1.0 X N) parts to 100 (1.5 X N) parts in about 10 parts in 100, about (2.0 X 100, about 15 parts N) parts in 100, in 100, about 20 about (2.5 X N) parts in 100, about parts in 100, about 25 parts in 100, (3.0 X N) parts in about 30 parts in 100, about (3.3 X 100, about 35 parts N) parts in 100, in 100, about 40 about (3.5 X N) parts in 100, about parts in 100 45 parts in 100, about 50 parts in at least 5 parts in 100, at least 10 parts in 100, at least 12 parts in 100, at least 14 parts in 100, at least 15 parts in 100, at least 20 parts in 100, at least 30 parts in 100, at least 40 parts in 100 Total rget 45% or less, less than (8.5 X N) from about (0.4 X from about 5 to oligonucleotides 40% or less, parts to 100, less than N) to about (5.0 X about 50 parts in % or less, (8.0 X N) parts to 100, N) parts in 100, 100, from about 10 % or less, less than (7.5 X N) from about (0.8 X to about 50 parts in % or less, parts to 100, less than N) to about (4.0 X 100, from about 20 % or less (7.0 X N) parts to 100, N) parts in 100, to about 50 parts in less than (6.5 X N) from about (1.6 X 100, from about 20 parts to 100, less than N) to about (4.0 X to about 40 parts in (6.0 X N) parts to 100, N) parts in 100, 100, from about 20 less than (5.5 X N) from about (1.6 X to about 30 parts in parts to 100, less than N) to about (2.5 X 100, (5.0 X N) parts to 100, N) parts in 100 less than (4.5 X N) about 25 parts in parts to 100, less than about (1.9 X N) 100 (4.0 X N) parts to 100, parts to 100 less than (3.5 X N) at least 10 parts in parts to 100, less than at least (1.0 X N) 100, at least 15 (3.0 X N) parts to 100, parts per 100, at parts in 100, at less than (2.5 X N) least (1.5 X N) least 20 parts in parts to 100, less than parts per 100, at 100, at least 25 (2.0 X N) parts to 100, least (2.0 X N) parts in 100, less than (1.5 X N) parts per 100 parts to 100, less than less than 40 parts (1.0 X N) parts to 100 in 100, less than 30 parts in 100, less than 25 parts in 100, less than 20 parts in 100, less than 15 parts in 2015/028327 In certain embodiments, the composition has less than (2.0 X N) parts to 100 parts by weight of (N— 1) product relative to a compound, wherein the compound includes a polynucleotide having a sequence of N nucleoside subunits complementary to the RNA component of human telomerase, wherein at least two of the side subunits are joined by a N3’—>P5’ thiophosphoramidate inter—subunit linkage. In certain embodiments, the ratio is less than (1.9 X N) parts to 100 parts by weight of (N—l) product relative to N product, such as less 100 parts by weight of (N—l) product relative to N product.

In some embodiments, the composition has less than 1 part in 4 by weight of a (N—l) product relative to a compound (such as, less than 1 part in 5, less than 1 part in 6, less than 1 part in 7, less than 1 part in 8, less than 1 part in 9, less than 1 part in 10, less than 1 part in 15, less than 1 part in 20, less than 1 part in 25, less than 1 part in 50, less than 1 part in 100 by weight of a (N—l) product relative to a compound), wherein the nd ses a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase, n at least two of the nucleoside ts are joined by a N3’—>P5’ thiophosphoramidate or oxophosphoramidate inter—subunit linkage. In certain embodiments, the polynucleotide has a sequence of 10 or more nucleoside ts complementary to the RNA component of human telomerase, such as 10, ll, 12, l3, 14, 15, l6, 17, 18, 19, 20 or more nucleoside subunits.

In certain instances, the polynucleotide es a sequence of 13 or more nucleoside subunits complementary to the RNA component of human telomerase, such as 15 or more, 20 or more, 30 or more, 50 or more nucleoside subunits complementary to the RNA component of human telomerase.

] In certain embodiments, the polynucleotide includes a ce of 7 or more nucleoside subunits complementary to the RNA component of human telomerase, such as 7, 8, 9, , ll, 12, l3, l4, l5, 16 or 17 nucleoside subunits complementary to the RNA component of human telomerase. In n embodiments, the polynucleotide includes a sequence of nucleoside subunits complementary to the RNA component of human telomerase of between 11 and 18, such as n 11 and 16 contiguous nucleoside ts complementary to the RNA component of human telomerase.

In some embodiments, the cleotide includes between 3 and 50 contiguous nucleoside subunits complementary to the RNA ent of human telomerase, such as between 5 and 40, between 10 and 40, between 10 and 30, between 10 and 25 between 10 and , or between 12 and 15 nucleoside subunits. In certain embodiments, the oligonucleotide includes a ce of 10 or more contiguous nucleoside subunits mentary to the RNA component of human telomerase. In certain ments, the composition has less than 1 part in by weight of a (N—l) product relative to the compound. In certain embodiments, the composition has less than 1 part in 20 by weight of a (N—l) product relative to the compound. In certain embodiments, the composition has less than 1 part in 25 by weight of a (N—l) product relative to the compound. In certain embodiments, the composition has less than 1 part in 30 by weight of a (N—l) product relative to the compound. In certain ments, the composition has less than 1 part in 50 by weight of a (N—l) product relative to the compound.

In some embodiments, the composition has less that 1 part in 4 by weight of any (N—X) product relative to the compound, such as less than 1 part in 5, less than 1 part in 6, less than 1 part in 7, less than 1 part in 8, less than 1 part in 9, less than 1 part in 10, less than 1 part in 20, less than 1 part in 25, less than 1 part in 30, or even less than 1 part in 50 by weight, of any (N—X) product relative to the compound.

In some embodiments, the ition has less that 40 part in 100 by total weight of (N—X) polynucleotide—containing products relative to the compound, such as less than 35 parts in 100, less than 30 parst in 100, less than 25 parts in 100, less than 20 parts in 100, or even less than 15 parts in 100 by weight, of (N—X) polynucleotide—containing products relative to the compound.

In some embodiments, the composition has at least 5 parts in 100 by weight of (N—2) and (N—3) products relative to the compound, such as, at least 10 parts in 100 by weight, at least 12 parts in 100 by weight, at least 14 parts in 100 by weight, at least 15 parts in 100 by weight, at least 20 parts in 100 by weight, at least 30 parts in 100 by , or at least 40 parts in 100 by weight of (N—2) and (N—3) products relative to the compound.

In some embodiments, the composition has the following e of (N—X) polynucleotide—containing products: less that 1 part in 4 by weight of a (N—l) product relative to the N product; and at least 10 parts in 100 by weight of (N—2) and (N—3) products relative to the N product.

In certain embodiments, the oligonucleotide N product comprises a 3’—terminal nucleoside subunit that is absent in the (N—l) product.

] The oligonucleotide nd may be described by the formula: O—(X'-L')Il where 0 represents the oligonucleotide ing a sequence of nucleoside subunits complementary to the RNA component of human telomerase, X' is an optional linker group, L' represents the lipid moiety and n is an integer from 1—5.

Design of the nds therefore requires the selection of two entities, O and L', and the determination of the structural linkage(s) between these entities, which may involve the optional linker group X'.

In some embodiments, the oligonucleotide compound may be described by the formula: O—(X'-L')Il where 0 represents the oligonucleotide including a sequence of side subunits mentary to the RNA component of human rase, X' is an optional linker group, L' represents the lipid moiety and n is 1, such as an oligonucleotide of Formula (I), or a salt f, wherein in Formula (I), Z is the lipid moiety, L is the optional linker and the B groups correspond to the sequence of nucleoside subunits mentary to the RNA component of human telomerase.

The oligonucleotide component 0 may be regarded as the “effector” component of the compound in that it is this component that effects inhibition of the telomerase enzyme by binding to the RNA component of telomerase. Thus, the sequence of O is selected such that it includes a region that is complementary to the ce of the telomerase RNA, which is shown in SEQ ID NO:1 The region that is complementary to the telomerase RNA ent may in theory be targeted to any portion of the telomerase RNA, but particular regions of the telomerase RNA are preferred target for inhibitory oligonucleotides. One preferred target region is the region spanning nucleotides 30—67 of SEQ ID NO: 1, which includes the “template region,” an ll nucleotide region of sequence 5’—CUAACCCUAAC—3’ (SEQ ID NO: 2) that spans nucleotide 46—56 of SEQ ID NO: I. The template region functions to specify the sequence of the telomeric repeats that telomerase adds to the chromosome ends and is essential to the actiVity of the telomerase enzyme (see Chen at al., Cell 100:503—514, 2000; Kim et al., Proc. Natl. Acad. Sci., USA 98(14):7982—7987, 2001). Compounds of the invention that contain an oligonucleotide moiety sing a ce complementary to all or part of the template region are thus ularly preferred. Another red target region is the region spanning nucleotides 137— 179 of hTR (see Pruzan et al, Nucl. Acids Research, 30:559—5 88, 2002). Within this region, the sequence spanning 141—153 is a preferred target. PCT publication WO 98/28442 describes the use of oligonucleotides of at least 7 nucleotides in length to inhibit telomerase, where the oligonucleotides are designed to be mentary to accessible portions of the hTR sequence outside of the template region, including nucleotides 137—196, 290—319, and 350—380 of hTR.

] The region of O that is ed to the hTR sequence is preferably exactly complementary to the corresponding hTR sequence. While mismatches may be tolerated in certain ces, they are expected to decrease the specificity and actiVity of the resultant oligonucleotide conjugate. In particular embodiments, the base sequence of the oligonucleotide O is thus selected to include a sequence of at least 5 nucleotides exactly complementary to the telomerase RNA, and enhanced rase inhibition may be obtained if increasing lengths of complementary ce are employed, such as at least 8, at least 10, at least 12, at least 13 or at least 15 nucleotides exactly mentary to the telomerase RNA. In other embodiments, the sequence of the oligonucleotide includes a sequence of from at least 5 to 20, from at least 8 to , from at least 10 to 20 or from at least 10 to 15 nucleotides exactly complementary to the telomerase RNA sequence. l telomerase inhibitory actiVity may be obtained when the full length of the oligonucleotide O is selected to be complementary to the telomerase RNA.

However, it is not ary that the full length of the oligonucleotide component be exactly complementary to the target sequence, and the oligonucleotide sequence may include regions that are not complementary to the target sequence. Such regions may be added, for example, to confer other properties on the nd, such as sequences that facilitate purification. If the oligonucleotide component 0 is to include regions that are not complementary to the target sequence, such regions may be positioned at one or both of the 5’ or 3’ termini. In instances WO 68310 where the region of exact complementarity is targeted to the template region, effective telomerase inhibition may be achieved with a short (5—8 nucleotide) region of exact complementarity to which a telomerase—like (G—rich) sequence is joined at the 5’ end. ary sequences that are complementary to the human telomerase RNA and which may be included as part of the oligonucleotide component 0, or which may be used as the entire oligonucleotide component 0 include the following: hTR complementary sequences (regions of Oligonucleotide sequence SEQ ID NO:1 of US. ation 2012329858) GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG AUUU UUUGUCUAAC CCUAACUGAG AAGGGCGUAG GCGCCGUGCU UUUGCUCCCC GCGCGCUGUU UUUCUCGCUG ACUUUCAGCG GGCGGAAAAG CCUCGGCCUG UCCA CCGUUCAUUC UAGAGCAAAC AAAAAAUGUC AGCUGCUGGC CCGUUCGCCC CUCCCGGGGA CCUGCGGCGG GUCGCCUGCC CAGCCCCCGA ACCCCGCCUG GAGGCCGCGG UCGGCCCGGG GCUUCUCCGG AGGCACCCAC UGCCACCGCG AAGAGUUGGG CUCUGUCAGC UCUC UCGGGGGCGA GGGCGAGGUU CAGGCCUUUC AGGCCGCAGG AAGAGGAACG GAGCGAGUCC CCGCGCGCGG CGCGAUUCCC UGAGCUGUGG GACGUGCACC CAGGACUCGG CUCACACAUG C (SEQ ID NO: 1) GCTCTAGAATGAACGGTGGAAGGCGGCAGG 137—166 (SEQ ID NO: 6) GGCGGCAGG 137—151 (SEQ ID NO: 7) GGAAGGCGGCAGG 137—149 (SEQ ID NO: 8) GTGGAAGGCGGCA 139—151 (SEQ ID NO: 9) ] GTGGAAGGCGG 141—151 (SEQ ID NO: 10) CGGTGGAAGGCGG 141—153 (SEQ ID NO: 11) ACGGTGGAAGGCG 4 (SEQ ID NO: 12) AACGGTGGAAGGCGGC 143—155 (SEQ ID NO: 13) ATGAACGGTGGAAGGCGG 144—158 (SEQ ID NO: 14) ACATTTTTTGTTTGCTCTAG 160-179 (SEQ ID NO: 15) TAGACAA 42-54 (SEQ ID NO: 3) GTTAGGGTTAG 46—56 (SEQ ID NO: 4) GTTAGGGTTAGAC 44—56 (SEQ ID NO: 16) GTTAGGGTTAGACAA 42-56 (SEQ ID NO: 17) GGGTTAGAC 44-52 CAGTTAGGG 50—58 CCCTTCTCAGTT 54—65 (SEQ ID NO: 18) CGCCCTTCTCAG 56—67 (SEQ ID NO: 19) ] In some embodiments, the polynucleotide comprises a sequence ed from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4); TAGGGTTAGACAA (SEQ ID NO:3); and CAGTTAGGGTTAG (SEQ ID NO:5).

The choice of the type of nucleoside linkages used in the synthesis of the 0 component may be made from any of the ble oligonucleotide tries, ing but not d to, phosphodiester, phosphotriester, methylphosphonate, P3’—>N5’ phosphoramidate, N3’—>P5’ phosphoramidate, N3’—>P5’ thiophosphoramidate, and phosphorothioate linkages.

In some embodiments, the oligonucleotide component 0 has at least one N3’—>P5’ phosphoramidate (e.g.,N3’—>P5’ thiophosphoramidate) linkage. In certain embodiments, the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3’—>P5’ phosphoramidate inter—subunit linkages. In certain cases, the N3’—>P5’ phosphoramidate inter—subunit linkages are N3’—>P5’ thiophosphoramidate inter—subunit linkages. In certain cases, the N3’—>P5’ phosphoramidate inter—subunit linkages are N3’—>P5’ oxo—phosphoramidate inter—subunit linkages.

In certain cases, the N3’—>P5’ thiophosphoramidate inter—subunit linkage has the following structure: P(S)(OR)—O—5’ Where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group. It is understood that some of the oligonucleotide ents 0 including an subunit linkage bed by the formula above Where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, may also exist in a salt form. Such forms in so far as they may eXist, are intended to be included Within the scope of the present disclosure.

In some instances, the N3’—>P5’ thiophosphoramidate inter—subunit linkage is bed by the following structure: 3’—NH—P(S)(OR)—O—5 ’ where R is hydrogen. It is understood that for any of the oligonucleotide components 0 described herein that include such an inter—subunit linkage, such oligonucleotide components 0 may also include any convenient salt forms of the linkage. As such, the inter—subunit linkage may be in a salt form that includes any ient counterion.

The compounds of the invention are more ive in producing telomerase inhibition in cells than ponding oligonucleotides that are not conjugated to lipid components. The lipid component L' is believed to function to enhance cellular uptake of the compound, particularly in facilitating passage through the cellular membrane. While the mechanism by which this occurs has not been fully elucidated, one possibility is that the lipid component may facilitate binding of the nd to the cell membrane as either a single molecule, or an aggregate (micellar) form, with uent internalization. However, understanding of the e mechanism is not required for the invention to be utilized.

The lipid ent may be any lipid or lipid derivative that provides enhanced cellular uptake compared to the unmodified oligonucleotide. Preferred lipids are hydrocarbons, fats (e. g., glycerides, fatty acids and fatty acid derivatives, such as fatty amides) and sterols.

Where the lipid component is a hydrocarbons, the L' component may be a substituted or unsubstituted cyclic hydrocarbon or an tic straight chain or branched hydrocarbon, which may be saturated or unsaturated. Preferred examples are straight chain unbranched arbons that are fully saturated or polyunsaturated. The length of the arbon chain may vary from C2—C30, but optimal telomerase inhibition may be obtained with carbon chains that are C8—C22.

Preferred es of saturated hydrocarbons es) are listed below: atic name / Carbon chain Tetradecane C14H30 Pentadecane C15H32 Hexadecane C16H34 Heptadecane C17H36 Octadecane C18H38 Nonadecane C19H40 Eicosane C20H42 Mono— and poly—unsaturated forms (alkenes and polyenes, such as alkadienes and alkatrienes) of hydrocarbons may also be selected, with compounds having one to three double bonds being preferred, although compound having more double bonds may be employed.

Alkynes (containing one or more triple bonds) and alkenynes (triple bond(s) and double bond(s)) may also b ed.

Substituted forms of arbons may be employed in the compounds of the ion, with substituent groups that are inert in vivo and in vitro being preferred. A particularly preferred substituent is fluorine. Exemplary generic structures of polyfluorinated hydrocarbons include: CF3(CF2)n—(CH2)m— Where m is at least 1, preferably at least 2, and n=l— , such as fluorotridecane: 2)9(CH2)3; and CH3(CH2)a(CF2)b(CH2)C— Where a, b and c are independently 1—30.

Other suitable lipid components include simple fatty acids and fatty acid tives, glycerides and more complex lipids such as sterols, for example terol. Fatty acids and their derivatives may be fully saturated or mono— or poly—unsaturated. The length of the carbon chain may vary from C2—C30, but optimal telomerase inhibition may be obtained with carbon chains that are C8—C22. Preferred examples of saturated fatty acids are listed below: ] Systematic name /Trivial name / Carbon chain Tetradecanoic myristic 14:0 Hexadecanoic palmitic 16:0 Octadecanoic stearic 18:0 ] Eicosanoic arachidic 20:0 ] Mono— and poly—unsaturated forms of fatty acids may also be employed, with compounds having one to three double bonds being preferred, although compounds having more double bonds may also be employed. Examples of common mono— and poly—unsaturated fatty acids that may be employed include: Systematic name / Trivial name / Carbon chain Cis—9—hexadecanoic palmitoleic 16: 1(n—7) Cis—6—octadecanoic petroselinic 18:1 (n—l2) Cis—9—octadecanoic oleic 18:1 (n—9) 9,12—octadecadienoic linoleic 18:2 (n—6) 6,9,12—octadecatrienoic gamma—linoleic 18:3 (n—6) 9,12,15—octadecatrienoic alpha—linoleic 18:3 (n—3) ,14—eicosatetraenoic arachidonic 20:4 (n—6) Fatty acids with one or more triple bonds in the carbon chain, as well as branched fatty acids may also be employed in the compounds of the invention. Substituted forms of fatty acids may be employed in the compounds of the invention. As with the hydrocarbon groups, substituent groups that are inert in vivo and in vitro are preferred, with fluorine being a particularly preferred. Exemplary generic structures of polyfluorinated derivatives of fatty acids suitable for use in the invention are: CF3(CF2)n—(CH2)mCO— where m is at least 1, preferably at least 2, and , and CH3(CH2)a(CF2)b(CH2)CCO— where a, b and c are independently l— In some cases, between one and five L' ents (n=l—5) are covalently linked to the 0 component, optionally via a linker. More usually 1 or two L'components are utilized (n=l or 2). Where more than one L' component is linked to the 0 component, each L' component is independently selected.

It will be appreciated that compounds of the invention described as having a specified hydrocarbon as the L' moiety and compounds described as having a specified fatty acid (with the same number of carbon atoms as the specified hydrocarbon) are closely related and differ in ure only in the nature of the bond that joins the L' moiety to the ucleotide, which in turn is a result of the synthesis procedure used to produce the compound. For example, and as described in more detail below, when compounds are synthesized having the L' moiety conjugated to the 3’—amino terminus of an oligonucleotide (having phosphoramidate or thiophosphoramidate internucleoside linkages), the use of the aldehyde form of a fatty acid (a fatty aldehyde) as the ng material results in the ion of an amine linkage between the lipid chain and the oligonucleotide, such that the lipid group appears as a hydrocarbon. In contrast, use of the ylic acid, acid anhydride or acid chloride forms of the same fatty acid results in the formation of an amide linkage, such that the lipid group appears as a fatly acid derivative, specifically in this instance a fatty amide (as noted in the definitions section above, for the sake of simplicity, the term “fatty acid” when bing the conjugated L' group is used broadly herein to include fatty acid derivatives, including fatty amides). This is illustrated in the ing schematics which depict the 3’—amino terminus of a phosphoramidate oligonucleotide joined to a C14 lipid component. In tic A, L' is tetradecanoic acid tic acid), in which the connection between L' and 0 groups is an amide. In schematic B, L' is tetradecane, and the connection between the L' and 0 groups is an amine.

Schematic A \ Schema tic B The linkage between the O and L' components may be a direct linkage, or may be via an optional linker moiety, e. g., x' or optional linker L of Formula (I). The linker group may serve to facilitate the chemical synthesis of the compounds. Whether or not a linker group is used to mediate the conjugation of the O and L' ents, there are le sites on the oligonucleotide component 0 to which the L' component(s) may be conveniently conjugated.

Suitable linkage points include the 5’ and 3’ termini, one or more sugar rings, the intemucleoside ne and the nucleobases of the ucleotide. In some cases, the L' moiety is attached to the 3’ or 5’ terminus of the oligonucleotide.

If the L' component is to be attached to the 3’ us, the attachment may be directly to the 3’ substituent, which in the case of the preferred phosphoramidate and thiophosphoramidate oligonucleotides is the 3’—amino group, and in other instances, such as conventional phosphodiester oligonucleotides, is a 3—hydroxy group. Alternatively, the L' moiety may be linked via a 3’—linked phosphate group, in which a hexadecane hydrocarbon is linked to the 3’ phosphate of a thiophosphoramidate oligonucleotide through an O—alkyl . If the L' moiety is to be linked to the 5’ terminus, it may be attached through a 5’—linked phosphate group.

Attachment to a base on the O moiety may through any suitable atom, for example to the N2 amino group of guanosine. Where n>l such that a plurality of lipid moieties is to be attached to the 0 component, the dually selected L' ents may be ed at any suitable site(s). For example, one L' group may be attached to each terminus, various L' groups may be attached to the bases, or two or more L' groups may be attached at one terminus.

The optional linker component x' may be used to join the O and L' components of the compounds. It is understood that the optional linker (e. g., x', or L of Formula (1)) may be attached to the polynucleotide (e. g., 0) through a terminal phosphate group, e. g., a ked or a ’—linked phosphate group. If a linker is to be employed, it is incorporated into the synthesis procedures as described herein. Examples of suitable linker groups include amino glycerol and O—alkyl glycerol—type linkers which respectively can be depicted by the c structures: \\\~,/'1:CH2]m [CHZ]H\\;/ R ' wherein R’=H, OH, NHZ or SH; Y=O, S or NR; R=H, an alkyl or a substituted alkyl; and n and m are independently integers between 1—18.

Specific examples of le linkers are the aminoglycerol linker in which R’=OH, Y=O, and m and n are each 1: the inoglycerol linker, in which R’=OH, Y=NH, and m and n are each 1: \\N,/Y\N// H H and the O—alkyl glycerol linker in which R=H: WOA[/\-:3—- Exemplary lipid—modified oligonucleotides that may be prepared according to the t methods include those compounds bed in Figure l (e.g., Figures lA—lDD) of US.

Application 20329858 to Gryaznov et al “Modified oligonucleotides for telomerase inhibition”, the disclosure of which is herein incorporated by reference in its ty.

In certain embodiments, the composition includes a compound described by the structure: H OH i? NWO—E’—O T o=F|>—SH O=|i’-SH O—[GnpsGnpsGnpsTnpsTnpsAnpsansAnpanpsAnps] I A Where “nps” represents a thiophosphoramidate linkage (e. g., —NH—P(=O)(SH)—O—), connecting the bon of one nucleoside to the 5'—carbon of the adjacent nucleoside.

It is understood that all embodiments referring to a compound are also applicable to the salt forms of said compound.

In certain embodiments, the composition includes a compound bed by the structure: H OH i? NWO—E’—O T o=F|>—SH O=|i’-SH O—[GnpsGnpsGnpsTnpsTnpsAnpsansAnpanpsAnps] I A or a salt thereof; Where “nps” represents a thiophosphoramidate linkage (e. g., —NH—P(=O)(SH)—O— or a tautomer thereof, or a salt thereof), connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside. In certain embodiments, the composition includes a pharmaceutically acceptable salt of the nd. In certain instances, the ition includes a sodium salt of the compound. In n embodiments, the composition includes a divalent cation salt of the compound, such as a magnesium salt of the compound. In n embodiments, the composition includes a trivalent cation salt of the compound, such as an aluminium salt of the compound.

In certain embodiments, the composition es an oligonucleotide described by the following structure, Where each M“ is independently hydrogen or any convenient counterion of a salt, each X is independently l, 2 or 3 and n is an integer from 5 to 13, such as 5, 6, 7, 8, 9, , ll, 12 or 13, such as n is 13: H'i‘H <’ | ,J S=P'O N O N NH (-3 i )1 o H'F NH <’ | * s=Fg—o N o N’ NH2 o— ‘97 o H.“ «N I i“ S=E’-O N o N’ NH2 0- kg 0 HI «N | 3:“ s=fi>—o N o N/ NH2 0' b 0 HI 1%”: s=Fg—o o N o o— p 0 HI 1%“: S=E’-O o N o o- p NH2 ““ «N I)” s=Fg—o o N N’ o o- b HN <’ | A s=I?—o N o N NH o— ‘97 NH, HN «N I)“ S=P-O C3. :0)N N’ NH | l NAG O O o— ‘97 MHz N \ NH < I j S=FI"O O N N O NH2 W «N I)“ S=E’-O o N N/ o- b NH2 (M )n In certain instances, each X is 1. In certain instances, each X is independently l or 2. In certain instances, each X is independently l or 3. In certain instances, M“ is en.

In certain embodiments, the composition includes an oligonucleotide described by the following structure and may include any convenient cationic counterions of a salt: H [\IJH \N <’ | A S=P-O N o N NH (5 o O i j N H'fl NH <’ | A s=fi>—o N o N NH2 0- p o HI? NH <’ | A S=E’-O N o N NH2 0- b o HI? NH (’N | A S=E-O o N NH2 0' s j 0 HN \(figNH s=fi>—o o N o O' s 7 O HN \fL/gNH s=E>—o o N o 0' p NH2 N” «N ‘N S=E’-O o N N/J o HN <,N | A S=E’-O N o N NH 0— 79 NE W «N I)“ S=P-O (-3. i0)N N/ NH | s—Fg—o NAG o- NH2 N” «N | )N S=P-O O N N/ ('3' K 7 NH2 N \ N NH <’ | J s—Fg—o N o N o- b In certain embodiments, the composition es a compound described by the structure: OH NH \N <’ | J S—P-O o N N NH (5 o O + i j s=fi>—o N o N/ NH2 0' b o Na+ N HRH NH <’ | A s=fi>—o N o N NH2 0' p o s=fi>—o N o N NH2 0' s 7 O I \kaNH s=fi>—o o N o 0— 7Q o HI 1%)“: s=fi>—o o N o o- NH2 N \ “H“ </ I j“ S=Fl>_—O o N N’ o O K 7 s=fi>—o N o N NH2 0- 7;) MHZ a \ HN «N I j“ S=P-O o N N/ (5. i j NH2 3:}:3—0 N O o- p NH2 Na+ \ NH «N I j“ s=fi>—o o N N/ 0' b NH2 NH </N I \)N 8—9—0 0 N N/ 0- 797 Also provided are compound active pharmaceutical ingredient itions including an oligonucleotide—containing compound. As used herein, an active pharmaceutical ingredient refers to a composition that is produced using the subject s of preparation, where the ition may optionally be subjected to one or more further purification steps post synthesis. In general, an active pharmaceutical ingredient is a composition suitable for formulation into a pharmaceutical composition. In some cases, the compound active pharmaceutical ingredient composition is not purified post synthesis, such that the oligonucleotide—containing components of the composition reflect those products ed during oligonucleotide synthesis.

In some embodiments, the compound active pharmaceutical ingredient has less than 9% by weight of a (N—l) product, wherein the compound comprises a cleotide having a ce of 10 or more nucleoside subunits complementary to the RNA component of human telomerase, wherein at least two of the side subunits are joined by a N3’—>P5’ thiophosphoramidate or oxophosphoramidate inter—subunit linkage (e. g., as described ).

In some embodiments, the compound active pharmaceutical ingredient has less than 9% by weight of a (N—l) product, wherein the compound or a pharmaceutically acceptable salt thereof comprises a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase, wherein at least two of the nucleoside subunits are joined by a 5’ thiophosphoramidate or oxophosphoramidate inter— t linkage (e. g., as described herein).

In some embodiments of the compound active pharmaceutical ingredient, the nucleoside subunits complementary to the RNA component of human rase are all joined by N3’—>P5’ thiophosphoramidate inter—subunit linkages.

In some embodiments of the compound active pharmaceutical ingredient, the N3’—>P5’ thiophosphoramidate inter—subunit e has the following structure: 3’—NH—P(S)(OR)—O—5’ where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group. When R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, it is understood that some of the inter—subunit es bed by the formula above may also exist in a salt form. Such forms in so far as they may eXist, are intended to be included within the scope of the present disclosure. 2015/028327 In some embodiments of the compound active pharmaceutical ingredient, the N3’—>P5’ thiophosphoramidate inter—subunit linkage has the following structure: 3’—NH—P(S)(OR)—O—5’ Where R is hydrogen. It is understood that for any of the compound active pharmaceutical ingredients described herein that include such an inter— t linkage, such compound active pharmaceutical ingredient may also include any convenient pharmaceutically acceptable salt forms of the linkage. As such, the inter—subunit linkage may be in a pharmaceutically acceptable salt form that includes any ient counterion of the salt.

In some embodiments of the compound active pharmaceutical ingredient, the polynucleotide comprises between 10 and 50 contiguous nucleoside ts complementary to the RNA component of human telomerase (e. g., as bed herein).

In some embodiments of the compound active pharmaceutical ingredient, the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4); TAGGGTTAGACAA (SEQ ID NO:3); and CAGTTAGGGTTAG (SEQ ID NO:5).

In some ments of the compound active pharmaceutical ient, the polynucleotide es a 3’amino or a 3’—hydroxyl al group. In certain embodiments of the compound active pharmaceutical ingredient, the polynucleotide includes a 3’amino terminal group. In certain embodiments of the compound active pharmaceutical ingredient, the polynucleotide includes a 3’—hydroxyl terminal group.

In some embodiments of the compound active pharmaceutical ingredient, the compound has the structure: WO 68310 “£0 E o T o {5! o=F|>—SH (ID—l A o=F|>-SH c'>—[Gnp5Gnpse"psTnpsTnpsAnpsansAnpscnpsAnpsi A wherein “nps” represents a thiophosphoramidate linkage —NH—P(=O)(SH)—O—, ting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside.

It is understood that all embodiments referring to a compound active pharmaceutical ingredient are also applicable to the salt forms of said compound active pharmaceutical ingredient.

In some embodiments of the compound active pharmaceutical ingredient, the compound has the structure: H OH II WO—P—O T a. —| o I O=|L-SH c'>—[Gnp5Gnpse"psTnpsTnpsAnpsansAnpscnpsAnpsi A or a pharmaceutically able salt thereof; wherein “nps” represents a thiophosphoramidate linkage —NH—P(=O)(SH)—O— (or a tautomer thereof or a pharmaceutically acceptable salt thereof, as described herein), connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside. In certain embodiments of the compound active pharmaceutical ingredient, the composition includes a sodium salt of the compound. In n embodiments, the composition includes a divalent cation salt of the compound, such as a magnesium salt of the compound. In certain embodiments, the composition includes a trivalent cation salt of the nd, such as an aluminium salt of the compound.

In certain embodiments of the compound active ceutical ingredient, the compound is bed by the following structure, Where each M“ is independently hydrogen or any ient rion of a salt, each X is independently l, 2 or 3 and n is an integer from 5 to 13, such as 5, 6, 7, 8, 9,10,11,12 or 13, such as n is 13: H'IH </ | ,j‘ S=P'O O N N NH (-3 i y o H'F NH <’ | * s=Fg—o N o N NH2 0- kg 0 H'.“ «N I 3:“ S=E-O N o N’ NH2 0- kg 0 HN </N | 3:“ S=E-O N o N/ NH2 0- If) 0 HI 1*)”: S=E’-O o N o o- p 0 HI I i” S=E-O o N o 0' b NH2 _' <,N |\NA S—E-O N o N o ,“ NH HN < | A s=fi>—o N o N NH2 0 NH2 HN «N I)“’ 860=P- O N NNH2 NH | S=P'O N/go o NH2 NH «N I)“ S=E-O o N N’ O NH2 NH «N I)” S=E’-O o N N’ o- b NH2 (M )n In certain ces, each X is 1. In certain instances, each X is independently l or 2. In certain instances, each X is independently l or 3. In certain instances, M“ is hydrogen.

In certain embodiments of the compound active pharmaceutical ient, the compound is described by the following structure and may include any convenient cationic counterions of a salt: H_'IH \N </ I 4 S—P-O o N N NH (5 o O k j N “N </ I i“ S—E-O N o N/ NH2 0- kg 0 “N «N I 1” S=E’-O N o N/ NH2 0- b o HN NH _|_ <N I A S—E’O o N NH2 0- kg 0 HN TipNH S=E’-O o N o o- p o HN \(tgNH s=fi>—o o N o 0' p NH2 ‘“ «N |\N S=Il3-O N O N/J O 0' L7 ,N NH HN < | A S=E’-O N o N NH2 0- 7;) MHZ “N «N I)”’ S=P-O (-3 :0)N NNH2 NH | S:FI>—O N’go 0- b NH2 NH «N I)” S—Fg—o N o N/ 0— 7Q NH2 N” «N I)” S=E’-O N o N/ In some embodiments of the nd active pharmaceutical ingredient, the compound is described by the structure: OH [\IJH \N <’ | J S=P‘O O N N NH ('3 K 7 O O + s=fi>—o N o N/ NH2 0- p o S=|f’-O N o N/ NH2 0- p o S=|f’-O N o N/ NH2 0- 7;) o Hi 1%”: s=fi>—o o N o O' s 7 O Hi I i“ S=|f’-O o N o 0' p NH2 N \ I“ < I j“ S=FI>:O O N N/ + {—7 N S=|f’-O N o N NH2 0- lg NH2 a \ HN «N I j“ S=|?-O i jo N N/ o- NH2 + \ SzFI)—O N O 0' b NH2 Na+ \ N“ «N I j“ S=|f’-O o N N/ o— b NH2 NH </N I \JN S=E’-O o N N/ o- b ] In some embodiments, the compound active ceutical ingredient has less that 9% by weight of the (N—l) product, such as less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by weight, less than 2% by weight, or even less than 1% by weight of the (N—l) product. In certain embodiments, the compound active pharmaceutical ingredient has less that 5 % by weight of the (N—l) product. In certain embodiments, the compound active pharmaceutical ingredient has less that 2 % by weight of the (N—l) product.

In some embodiments, the active pharmaceutical ingredient has less that 9 % of any (N—X) product, such as less than 8% by , less than 7% by weight, less than 6% by weight, less than 5% by weight, less than 4% by weight, less than 3% by , less than 2% by weight, or even less than 1% by weight of any (N—X) product.

In some embodiments, the compound active pharmaceutical ingredient has less that 9 % by weight in total of (N—X) polynucleotide—containing products, such as less than 8% by weight, less than 7% by weight, less than 6% by weight, less than 5% by , less than 4% by weight, less than 3% by weight, less than 2% by weight, or even less than 1% by weight in total of (N—X) polynucleotide—containing products.

In some embodiments, the compound active pharmaceutical ingredient has the following profile of (N—X) polynucleotide—containing products: less that 1 part in 4 by weight of a (N—l) product relative to the N product; and at least 10 parts in 100 by weight of (N—2) and (N—3) ts relative to the N product.

FORMULATIONS Also provided are pharmaceutical compositions that include an oligonucleotide composition (e. g., as described ). The oligonucleotide compositions (e. g., as described herein) can also be formulated as a ceutical composition for inhibition of transcription or translation in a cell in a disease condition d to overeXpression of the target gene.

In some embodiments, the pharmaceutical composition includes an ucleotide ition (e.g., as described herein) formulated in a pharmaceutically acceptable excipient. In certain embodiments, the oligonucleotide composition is a compound active ceutical ingredient having less than 9 % by weight of a (N—l) product, wherein the compound comprises a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA ent of human telomerase, wherein at least two of the nucleoside subunits are joined by a N3’—>P5’ thiophosphoramidate inter—subunit linkage. 2015/028327 The present invention provides compounds that can specifically and potently inhibit telomerase activity, and which may therefore be used to inhibit the proliferation of telomerase— positive cells, such as tumor cells. A very wide variety of cancer cells have been shown to be telomerase—positive, including cells from cancer of the skin, connective tissue, adipose, , lung, stomach, pancreas, ovary, cerviX, uterus, kidney, bladder, colon, prostate, central nervous system (CNS), retina and hematologic tumors (such as myeloma, ia and lymphoma).

Cancers of st include, but are not limited to, myelofibrosis, thrombocythemia, myelodysplasic syndrome and myelogenous leukemia.

The subject compounds can be used to treat hematologic malignancies and myeloproliferative disorders, including but not limited to, essential thrombocythemia (ET), themia vera (PV) c myelogenous leukemia (CML), myelofibrosis (MF), chronic neutrophilic leukemia, chronic eosinophilic leukemia, and acute myelogenous leukemia (AML).

The subject compounds can be used to treat myelodysplastic syndromes, which e such disease as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with ineage dysplasia, refractory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia (CMML). The subject compounds can be used to treat hematological diseases, such as those described in PCT patent application No. PCT/US 13/070437 filed November 15, 2013, the sure of which is incorporated herein by reference in its entirety.

Accordingly, the compounds provided herein are broadly useful in treating a wide range of malignancies. More importantly, the compounds of the present ion can be effective in providing treatments that discriminate between malignant and normal cells to a high degree, avoiding many of the deleterious side—effects present with most current herapeutic regimens which rely on agents that kill dividing cells indiscriminately. er, the compounds of the invention are more potent than lent unconjugated oligonucleotides, which means that they can be administered at lower doses, providing enhanced safety and significant reductions in cost of treatment. One aspect of the invention therefore is a method of treating cancer in a patient, comprising administering to the patient a therapeutically effective dose of a compound of the present invention. rase tors, including compounds of the invention, may be employed in conjunction with other cancer ent approaches, including surgical removal of primary tumors, chemotherapeutic agents and radiation treatment. Hence, the invention relates to compounds and compositions provided herein for use as a medicament. The invention also relates to compounds and compositions provided herein for use in treating or preventing any one of the malignancies mentioned hereinbefore.

For therapeutic application, a compound of the invention is formulated in a therapeutically ive amount with a pharmaceutically acceptable carrier. One or more invention nds (for example, having different L' or 0 ents) may be included in any given formulation. The pharmaceutical carrier may be solid or liquid. Liquid carriers can be used in the preparation of solutions, emulsions, suspensions and pressurized compositions. The compounds are dissolved or suspended in a pharmaceutically acceptable liquid excipient.

Suitable examples of liquid carriers for parenteral administration of the oligonucleotides preparations include water (which may contain ves, e.g., cellulose derivatives, ably sodium carboxymethyl cellulose solution), phosphate buffered saline solution (PBS), alcohols (including monohydric alcohols and polyhydric alcohols, e. g., glycols) and their derivatives, and oils (e. g., fractionated coconut oil and arachis oil). The liquid carrier can contain other suitable pharmaceutical additives including, but not limited to, the following: solubilizers, suspending agents, emulsifiers, buffers, thickening agents, colors, viscosity regulators, preservatives, stabilizers and osmolarity regulators.

For parenteral administration of the compounds, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile carriers are useful in sterile liquid form compositions for eral administration.

Sterile liquid pharmaceutical itions, solutions or suspensions can be utilized by, for example, intraperitoneal injection, subcutaneous injection, intravenously, or lly.

The oligonucleotides can also be stered intravascularly or via a vascular stent.

The liquid carrier for pressurized compositions can be a nated hydrocarbon or other pharmaceutically acceptable propellant. Such pressurized compositions may also be lipid ulated for delivery via inhalation. For administration by asal or intrabronchial tion or insufflation, the oligonucleotides may be formulated into an aqueous or partially aqueous solution, which can then be ed in the form of an aerosol.

The nds may be administered topically as a solution, cream, or , by ation with pharmaceutically acceptable vehicles containing the active compound.

The pharmaceutical compositions of this invention may be orally administered in any acceptable dosage including, but not limited to, formulations in capsules, tablets, powders or granules, and as suspensions or solutions in water or non—aqueous media. Pharmaceutical compositions and/or formulations comprising the ucleotides of the present invention may include carriers, ants, diluents, thickeners, flavoring agents, emulsifiers, dispersing aids or binders. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, may also be added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending . If desired, certain sweetening, ng or coloring agents may also be added.

While the compounds of the invention have superior characteristics for cellular and tissue penetration, they may be formulated to provide even greater benefit, for example in liposome carriers. The use of liposomes to facilitate cellular uptake is described, for example, in U.S. Pat. No. 4,897,355 and U.S. Pat. No. 4,394,448. us publications describe the formulation and preparation of liposomes. The compounds can also be formulated by mixing with additional penetration enhancers, such as unconjugated forms of the lipid moieties described above, including fatty acids and their derivatives. es include oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. oleoyl—rac—glycerol), dilaurin, caprylic acid, arichidonic acid, glyceryl l—monocaprate, l—dodecylazacycloheptan—2—one, acylcamitines, acylcholines, mono— and cerides and physiologically acceptable salts f (i.e., oleate, laurate, caprate, ate, palmitate, stearate, linoleate, etc.).

Complex formulations comprising one or more penetration enhancing agents may be used. For example, bile salts may be used in combination with fatty acids to make complex ations. Exemplary combinations include chenodeoxycholic acid (CDCA), generally used at concentrations of about 0.5 to 2%, combined with sodium e or sodium laurate, lly used at concentrations of about 0.5 to 5%.

Pharmaceutical compositions and/or formulations sing the oligonucleotides of the present ion may also include chelating agents, surfactants and non—surfactants.

Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e. g., sodium salicylate, 5—methoxysalicylate and nilate), N—acyl derivatives of collagen, laureth—9 and N—amino acyl tives of beta—diketones (enamines).

Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene—9—lauryl ether and polyoxyethylene—20—cetyl ether; and perfluorochemical emulsions, such as FC—43. Non— surfactants include, for example, unsaturated cyclic ureas, l—alkyl— and l—alkenylazacyclo— alkanone tives, and non—steroidal anti—inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone.

Thus, in another aspect of the invention, there is provided a method of formulating a pharmaceutical composition, the method comprising providing a compound as described herein, and combining the compound with a pharmaceutically acceptable excipient. Preferably the compound is provided at pharmaceutical , as defined below. The method may further comprise adding to the compound, either before or after the addition of the excipient, a penetration ing agent.

The ceutical composition may comply with pharmaceutical purity standards.

In some cases, for use as an active ingredient in a pharmaceutical ation, a subject compound is purified away from reactive or potentially immunogenic ents present in the mixture in which they are ed..

The pharmaceutical composition may be aliquoted and packaged in either single dose or dose units. The dosage requirements for treatment with the oligonucleotide compound vary with the particular itions employed, the route of administration, the severity of the symptoms ted, the form of the compound and the particular subject being treated.

Pharmaceutical compositions of the invention can be administered to a subject in a formulation and in an amount effective to achieve a clinically desirable result. For the treatment of cancer, desirable results include reduction in tumor mass (as determined by palpation or imaging; e. g., by radiography, radionucleotide scan, CAT scan, or MRI), reduction in the rate of tumor growth, reduction in the rate of metastasis ion (as ined e.g., by histochemical analysis of biopsy specimens), reduction in biochemical markers ding general markers such as ESR, and tumor—specific markers such as serum PSA), and improvement in quality of life (as determined by clinical assessment, e. g., Karnofsky score), increased time to progression, disease— free survival and overall survival.

The amount of compound per dose and the number of doses required to achieve such effects will vary depending on many factors including the disease indication, characteristics of the patient being treated and the mode of administration. In some instances, the formulation and route of administration will provide a local concentration at the disease site of between 1 uM and 1 nM of the compound.

In general, the compounds are administered at a concentration that affords effective results without causing any harmful or deleterious side effects. Such a concentration can be achieved by stration of either a single unit dose, or by the administration of the dose divided into convenient subunits at suitable als throughout the day.

UTILITY The methods and compositions of the ion, e.g., as described above, find use in a variety of applications. Applications of interest e, but are not d to: therapeutic applications, diagnostic applications, ch applications, and ing applications, as ed in greater detail below.

The subject compounds find use in a variety of therapeutic applications. In some embodiments, the methods of producing an oligonucleotide are applied to prepare oligonucleotides that provide for a therapeutic benefit. The types of diseases which are ble using the compositions of the present invention are limitless. For example the compositions may be used for treatment of a number of genetic diseases. In some embodiments, the subject methods and itions have antisense ations. In some embodiments, the subject methods and compositions have antigene applications. In certain embodiments, the subject s and compositions have telomerase inhibition applications, such as those described in U.S. Patent 6,835,826, and U.S. Publication 20120329858, the disclosures of which are herein incorporated by reference in their entirety.

The subject compounds and methods find use in a variety of diagnostic applications, including but not limited to, the development of clinical diagnostics, e. g., in vitro diagnostics or in vivo tumor imaging agents. Such applications are useful in diagnosing or confirming diagnosis of a disease condition, or susceptibility thereto. The s are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with the disease.

EXAMPLES The following examples are put forth so as to e those of ordinary skill in the art with a complete disclosure and ption of how to make and use the present invention, 2015/028327 and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. s have been made to ensure accuracy with respect to s used (e. g. amounts, ature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in s Celsius, and pressure is at or near atmospheric. By “average” is meant the arithmetic mean. Standard abbreviations may be used, e. g., bp, base ); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, ); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(1y); and the like.

General Synthetic Procedures Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e. g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley—Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative c is, Fourth Edition, New York: Longman, 1978).

] Compounds as described herein can be purified by any purification protocol known in the art, including tography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e. g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J.

Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer— Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, “Protective Groups in c Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, ctive Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der schen Chemie”, Houben—Weyl, 4th edition, Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.—D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine”, Verlag , Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: ccharide and Derivate”, Georg Thieme , Stuttgart 1974. The ting groups may be removed at a convenient subsequent stage using methods known from the art.

The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. A y of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

EXAMPLE 1 Sflthesis of imetelstat sodium using dimeric phosphoramidites.

Imetelstat sodium is synthesized using a solid support (Controlled pore glass or polymeric solid support) and monomer phosphoramidites such as ABZ or Admf, C, G”311 and T amidites in the following sequence: GGGTTAGACAA—NH2—3’ (SEQ ID NO:3) where R = Lipid linker group Table 2: Structure of the Amidites and Solid Su ort Abbreviated Description Structure Name Amidite Admf 3’ T—ritylamino——N6— dimethylformamidino— 2’ ,3 —dideoxyadenosine— ’ ——(2cyanoethyl)—N,N— diisopropyl Phosphoramidite WO 68310 Abbreviated Description Structure Name Amidite Admf 3’- (MMT) Monomethoxytritylamino— N6—dimethy1f0rmamidino— 2’ deoxyadenosine— ’—(2—cyanoethy1)—N,N— diisopropyl Phosphoramidite 3’—(Dimethy1— substituted Pixy1)amin0—N6— dimethylformamidino— 2’ ,3’—dideoxyadenosine— ’ —(2—cyanoethy1)—N,N— diisopropyl Phosphoramidite 3’—Dimethoxytritylamino— N6—dimethy1f0rmamidino— 2’ ,3’—dideoxyadenosine— ’—(2—cyanoethy1)—N,N— diisopropyl Phosphoramidite Abbreviated Description ure Name Amidite ABZ 3’—Trity1amino—N6— benzoyl—Z’ ,3 ’ - dideoxyadenosine—S ’ —(2— cyanoethy1)—N,N— diisopropyl Phosphoramidite Amidite C(Bz) 3’— Tritylamino—N— benzoyl—Z’ ,3 ’ - dideoxycytidine 5’—(2— cyanoethy1)— N,N— diisopropylphosphoramidi ty1amino—N2— isobutyryl—Z’ , 3 ’— dideoxyguanosine—S ’ —(2— cyanoethy1)—N,N— diisopropyl Phosphoramidite iated Description Structure Name Amidite T 3’—Tritylamino—3’— deoxythymidine 5’—(2— cyanoethyl)—N,N— diisopropylphosphoramidi oyl— 3—palmitoylamido— l —O— aminoglycerol (4,4’—dimethoxytrityl)—2— —solid support O—succinyl propanediol Controlled Pore Glass Support NittoPhaseHL 3—palmitoylamido— 1'0" Palmitoyl 400 (4,4’-dimethoxytrityll) - O—succmyl propaned101 Polymeric 0—Polymeric solid t , Solid Support ric Solid Support, :0}:W The imetelstat backbone is NPS which is similar to starting phosphoramidites and therefore the coupling efficiency is approximately 92%. Utilization of dimer phosphoramidites allows fewer coupling steps which can lead to higher yield and purity at the intermediate stage after synthesis. The following dimer oramidites were prepared as shown below using a method as described in synthetic scheme 1: TA, AA, GA, GG and GT.

The synthesis of the dimer phosphoramidites required three monomer amidates (4a to 4c, scheme 1) and three 5’—TBDMS—3’ amino nucleoside intermediates (3a to 3c, scheme 1) for A, G and T nucleosides. TBDMS is tert—butyldimethylsilyl. The intermediates (3a to 3c, scheme 1) were prepared from two kinds of starting materials, 5’—OH—3’—NH—Tr—2’—deoxy—N—benzoyl adenosine (1a), 3’—NH—Tr—2’—deoxy—N—isobutyryl guanosine (1b), and 3’—amino— thymidine (2). Tr or Trt refer to trityl.

The 5’—hydroxyl group of 1a and 1b were protected with TBDMS groups using t— butyldimethylsilyl chloride and imidazole in DMF (N,N—dimethylformamide), and then the trityl groups at the 3’—amino positions were deprotected by the treatment with acetic acid in water.

The resulting intermediates, 3a to 3c were coupled with the corresponding amidates, 4a to 4c, using benzylmercaptotetrazole (BMT) as an activator in dimethylformamide and the subsequent sulfurization (P 111 to P V) was performed using xanthane hydride and pyridine (scheme 1). In general, the sulfurization reaction was ted easily. The outcomes of the coupling reactions varied depending on moisture, reaction time, and equivalency of amidates. Anhydrous conditions using nitrogen or argon gas and a quick coupling reaction was desirable since a longer reaction time lead to more side products such as P (V) oxidation products. The P (111) intermediates of the dimer have different stabilities. The TA intermediate was stable enough to monitor the on completion by TLC and HPLC. Other P (111) intermediates were not stable enough to tr the coupling reaction and reaction completion was checked after the sulfurization was completed (scheme 1). P(V) s are more stable for dimers AA, GA, GG and GT. For TA dimer (5e), 1.3 equivalent of amidate (4c) was used for the coupling and the other four dimers (5a~5d) required approximately 3 lent of amidates (4a and 4b) (scheme 1). Amidate rs 4a—4c are prepared by ng s described in U.S. Patent ,859,233.

Scheme 1. S nthetic Scheme of Dimer Amidates (P—reagent is cyanoethoxy—bis(N,N—diisopropylamino)phosphine) I, L no 1% $12 W “3‘1 (fitt. s: z,—" — {a :5: s; rat-mason gypsum; ow £3 Lg . » fl _2‘ »_ u ‘— ' —’ —3.‘ In» :53 mm: m: ‘1- [w] [m I] K :53. 8: «w- """ 1‘ an anagram fie. . 11 8 #4:. 33,5}. mm {1 its. 31mm 3—»:‘ r” ‘* ~ 3} first}; my ~' —a~ ,3" NB:. .

T it} Emmam h‘yrflfifie, mfidfim . 2 Ni} “km\N 3*) g3. 3%:th . "a; rename. émsdama our; 3' 35‘ Exam ml; 3:. 3‘»? £1 52’»? Hp ,3 i E“ {3-H u mamas “0‘4 “*3 No L T o E? » ,. ,. t .m m: t m ass: rm E S HF 93‘ mm.-~. .v- .c , eel—$28 §}_?L:S P»Rea§§ara§, BEST, MW!, 3‘3 a? $3-?23 B? —» V G AC.” :"—F‘ C! I fl £0a C ‘t. x1 Q. :3 i‘.—f ' m m: M . .t 53 N1; ‘lyuxj _«—\ NH = NH x: “F" NH Sn," gu‘ \. L:f—~\K .-' \ 3:3 \\ t K’ ; ; x‘ i) \a ; ' 1Q j 5‘: — ‘5 — H \— 'H in— <_: ’5‘f"\ 5‘591“ a? x, Ln ng R: u fa MW: 33mm ?b. fitmfimfi 53:213.“ TC. Eixalmfi E33373“- ?6. Exam: 8233”!” 3?.“ 8‘3sz 34-33%}: The TBDMS protecting group at 5’—hydroxyl group was deprotected using HF°pyridine in acetonitrile and the final phosphitylation was performed with phosphitylating reagent in the presence of BMT and N—methylimidazole (NMI) to make the dimer sophoroamidates, 7a to 7e (Scheme 1). The final products (7) and three intermediates (3, 5, 6) were purified by column chromatography. The step and l yields of reactions with quantities of final amidates obtained are listed in Table 3. Summary of analysis s for the five dimer amidates are shown in Table 4.

Table 3. Yields of Dimer sis _,TBDMS_ 5 :TBDMS_ Dimer 3’—NH— Dimer Overall 3 —Am1no 3 —NH—Tr— (Quant't1 y) T . r D'1mer am1'da et y1eld ( 0/)0 Nucleoside Dimer (2T9Ag) 58% 91% 71% 51% 19.1% 58 % 82 % 70 % 47 % 15.6 % ( 1.7 g) 58 % (3.4 g) 58 % 67 % 57 % 38 % 8.4 % ( 1.9 g) 58 % 99 % 42 % 38 % 9.2 % (1-8 g) Table 4. Summary of Dimer Analysis LCMS Amount (C2110) (g) 148.281(s), l48.l93(s), 73.811(s), 1169.4 73.723(s), 72.981(s), (s) (1169.23) ' l48.262(m), 74.034(m), 72.774(d), 1282.5 72.267(d) (1282.35) ' 148.156(m), 74.244(s), 73.993(s), 1268.5 (Na) 72.912(s), 72.76l(s) (1246.32) 148.159(m), 73.993(s), 73.811(s), 1151.4 73.295(s), 73.100(s) (1151.22) 148.168(m), 148.0ll(s), 74.175(s), 1264.5 GA 962% 3'4 73.942(s), 73.170(s), 72.906(s) (1264.33) Sflthesis Procedure of Dimer Thiophosphoroamidates 1) ation of 5’—TBDMS—3’—amino nucleoside (for Adenosine and Guanosine). a) Dissolve 3’—NH—Tr—2’—deoxynucleoside (l.0eq) and imidazole (5.0eq) in DMF and heat to 60°C. b) Add TBDMSCl (l.2eq) to the heating solution then stir for 1 hr at 60°C. c) Add saturated aqueous NaHC03 solution to reaction mixture then extract with ethyl acetate. d) The organic layer is washed by saturated aqueous NaHC03 solution and brine solution. e) Add anhydrous NaZSO4 to the separated organic layer for drying then . f) The filtrate is concentrated. g) Add 80% aqueous acetic acid solution to the concentrated reaction mixture then stir for 1 hour at t temperature. h) Remove the product solid by filtration then add saturated aqueous NaHC03 solution to the filtrate then extract by ethyl acetate four times. i) The organic layer is dried over anhydrous NaZSO4 then d the solid by filtration. j) The filtrate is concentrated then purified by column chromatography (Eluent: Ethyl acetate: Methanol=9:l 9 5:1). k) 5’—TBDMS—3’—amino—2’—daoxynucleoside is obtained as White solid. 2] Preparation of MS—3’—amino side [for Thymidine] a) Dissolve 5’—OH—3’—amino—2’—deoxynucleoside (l.0eq) and imidazole (5.0eq) in DMF and heat up to 60°C. b) Add TBDMSCl (l.2eq) to the heating on then stirred for 1 hr at 60°C. c) Add saturated aqueous NaHC03 solution to reaction mixture then extract with ethyl acetate four times. d) Add anhydrous NaZSO4 to the organic layer for drying and . e) The filtrate was concentrated. f) The concentrated crude mixture is ed by column chromatography (Eluent: Ethyl acetate: Methanol=lS:l 9 5:1). g) 5’—TBDMS—3’—amino thymidine is obtained as a White solid. 3] Preparation of 5’—TBDMS—3’—NH—Tr dimer a) To remove the moisture, 5’—TBDMS—3’—amino nucleoside (l.0eq) and EMT (benzylmercaptotetrazole, l.0~5.0eq) are azeotroped by itrile three times then dissolved in DMF at ambient temperature under N2 here. b) Add monomer amidate (3.0eq) in DMF (using minimum amount to dissolve the monomer amidate) to the reaction solution by drop wise then stir for 1 hour at ambient temperature under nitrogen atmosphere. Monomer e is prepared according to methods bed in U.S. Patent 233. c) Add xanthane hydride (2.0eq) and ne (4.0eq) to the reaction solution then stir for 1 hour at ambient temperature under en atmosphere. d) Add saturated aqueous NaHC03 solution to reaction mixture then extract with ethyl acetate. e) The aqueous layer is extracted with ethyl acetate. f) The separated organic layers are combined and then washed by saturated aqueous NaHC03 solution and brine solution. g) Add anhydrous NaZSO4 to the organic layer for drying and filter, then the filtrate is concentrated. h) The concentrated crude e is ed by column chromatography (Eluent: ethyl acetate: methanol=l.5:l 9 EA only). i) 5’—TBDMS—3’—NH—Tr dimer is obtained as a pale yellow solid. 4] Preparation of 5’—OH—3’—NH—Tr dimer a) Dissolve 5’—TBDMS—3’—NH—Tr dimer (l.0eq) in ACN (20mL) under nitrogen atmosphere and then add HF— pyridine solution with ng at ambient temperature for 1.5 hours. b) Add saturated aqueous NaHC03 on to reaction mixture then extract with ethyl acetate. c) The separated organic layer is washed by saturated aqueous NaHC03 solution and brine solution. d) Add anhydrous NaZSO4 to the organic layer for drying and filtering then the filtrate is concentrated. e) The concentrated crude mixture is purified by column chromatography (Eluent: ethyl acetate, methanol, methylene chloride vent) f) 5’—OH—3’—NH—Tr dimer is obtained as a white solid. 5] Preparation of Dimer phosphorothioamidate (Dimer amidate] A) To remove any moisture, 5’—Hydroxy—3’—NH—Tr dimer is azeotroped by acetonitrile three times then dissolved in ACN at ambient temperatureunder nitrogen atmosphere. b) Add BMT (l.3eq), NMI (N—Methyl imidazole, 0.3eq) and phosphitylation reagent (2.0eq) to the reaction solution then stir for 1 hour at ambient temperature. c) Add saturated aqueous NaHC03 solution to reaction mixture then extract with ethyl acetate. d) The separated organic layer is washed by brine solution. e) Add anhydrous NaZSO4 to the organic layer for drying and ing, then the filtrate is concentrated. ] f) Dissolve concentrated reaction mixture in methylene chloride (lOmL) then add hexane to itate the solid. g) Decant the upper solution layer to remove excess phosphitylation reagent. (Repeat decantation process 5 times). h) The remaining solid is purified by column chromatography (Eluent: ethyl acetate, acetone, methylene chloride vent) ] i) Dimer is obtained as a white solid.

Imetelstat Synthesis Utilizing Dimer Amidates Five dimer amidates were used in place of monomer amidates as the building blocks for the synthesis of imetelstat and the results were compared with the s obtained from the amidates of monomer. For the coupling of the C nuceloside into imetelstat, the r ng clock was used as depicted in the sequence below. The synthesis was performed at a 140 umole scale using an Akta Oligopilot 100.

S’R-TA GG GT TAQ C fi—NHz 3’ (SEQ ID NO: 3) ] Dimer amidates were used as building blocks to make imetelstat. Using the reagents and synthesis parameters listed in Tables 5A and 5B, the five dimer amidates (AA, TA, GG, GA, and GT) and one monomer amidate (C), as shown above, are coupled to make the imetelstat sequence on low—loading CPG (PALM 0051, 64.6 umol/g). The coupling time is 500 sec and the equivalency of the es were used. After the solid—phase synthesis, the support is treated with ethanolic ammonium solution (NH4OHzEtOH=3:1(v/v)) at 65°C for 15 hours. The crude product is isolated by evaporation of solvents and ed by UV spectroscopy and HPLC.

Table 5. Exemplary Synthesis Parameters (A) and Reagent Composition (B) for oligonucleotide Synthesis. ACN is acetonitrile. DCA is dichloroacetic acid. PADS is phenylacetyl disulfide. ETT is 5—Ethylthio—1H—Tetrazole A B 3:. 3:33; ”“3 Reagent Name Composition :: g F} Deblock 5% DCA in e Amidite as “3 0.2M in ACN 333 Activator 0.5M ETT in ACN ’ 7:3: ‘73 Thiolation 0.2M PADS in ACN:LTD=l:l Cap A 20% NMI on ACN Cap B IBUA:LTD:ACN=l:l:8 E5313 DEA 20% DEA in ACN 33.13? 5:} 3 53:: 3‘58 '3 531‘ Using an Akta Oligopilot 100, synthesis runs on a 140 umole scale were ted using the monomer block method and the dimer block block . The synthesis conditions for the synthesis runs were similar to those listed in Table 5A—B.

Table 6. tic ters for 140 umole scale Synthesis (AKTA Oligopilot 100) Imetelstat stat Parameters sis using Synthesis using Monomers Dimers CT (min) 3 min (2nd 6 min) Deblock (5% DCA in e) Linear flow (cm/hr) 0.1M, 2.5eq 0.1M, 2.5eq Amidate (last 2: 3.0eq) (last AA: 3.0eq) Activator 0.5M ETT (AmidatezActivator, 4:6) Coupling 1st Coupling double coupling CT for Flow through 1.8 min (min) CT for Recycle (min) 1.8 min (1st: 4 min) Thiolation 5.27 min (0.1M PADS in AN:LTD=9: 1) Linear flow (cm/hr) 80 cm/hr CT (min) 1 min (1st: 2 min) Capping (Cap A: 20% NMI in AN, CV 1 CV (1st: 2 CV) CapB: IBUAzLTDzAN 212128) Linear flow (cm/hr) 120 cm/hr (20% DEA in AN) Linear flow (cm/hr) Analysis of oligonucleotides by HPLC—MS showed that the FLP (full length product) purity was improved significantly when the five dimer blocks were used for synthesis, giving 72% purity by HPLC as summarized in Tables 7 and 8. The crude oligo prepared using the monomer blocks showed only 45% FLP purity. Further, the total OD (optical density) was increased by more than double from 5,299 to 11,623 affording the crude yield of 3.34 g/mmol.

The (N—l) product level and the PO content were decreased to 2.4% from 11.2% and to 5% from %, respectively.

An advantage of using dimer blocks includes that the production time is shortened and the amounts of solvents used during the solid—phase synthesis are reduced.

Table 7. Analysis Result for 140 umole Scale Synthesis Imetelstat Synthesis Imetelstat Synthesis Attributes using Monomer using Dimer Amidate Amidate FLP 44.4 0/0 74.0 % HPLC eakl ”'0 % <N-1> product UV Weight (mg) g/mmol LC/MS The synthesis of five dimer amidates was completed successfully with the yields of 9% to 19% from 5’—hydroxy—3’—amino nucleoside or 5’—hydroxy—3’—tritylamino nucleoside giving 1.7 gram to 3.4 gram. Optimization of reaction ions for each step was not studied extensively. The dimers block syntheses of imetelstat were conducted on a 140 umol scale and the results were compared with the data obtained from synthesis using monomer amidates. The dimer blocks strategy for preparation of stat was shown to provide substantial improvements because the purity and yield were improved icantly, e. g., on a 140 umol scale (HPLC Purity: dimer 74.0% (Figure 8), monomer 44.4% (Figure 7), Crude yield by tal optical density): dimer 468 mg, r 213 mg). In addition a lower amount of npo linkagewas generated since there were fewer coupling steps in the synthesis using dimers.

Coupling efficiency for the dimer (140 umole scale Synthesis) shows that the dimer synthesis had 96% coupling efficiency whereas the monomer synthesis is at 94%. Since there were only seven coupling for the dimer the FLP for dimer was at 71.6% which is close to the theoretically ated Full Length Product at 72% and the monomer with 13 couplings reported a FLP of 45.6% vs the theoretically predicted at 44%.

Table 8. Analysis of Results for 140 umole Scale Synthesis Products of Retention % area Products of dimer Retention % area dimer monomer synthesis time (min) monomer synthesis time (min) synthesis synthesis target 38.2 44.4 target 37.9 74.0 Post Peak 39.8 11.0 Post Peak 1 39.8 2.5 N-l (N-C) N-l (N-G) Post Peak 2 . . Post Peak 2 N-2+iBu, N-2, N-2+iBu, N-2, N-G+Phen lacet l N-G+Phen lacet 1 Post Peak 3 42.9 Post Peak 3 42.4 N-2+Phenylacetyl, N-2+Phenylacetyl, N-3 (N-A-A-C) N-3 -C) N-3+Phenylacetyl N-3+Phenylacetyl oligonucleotides 54.7 oligonucleotides “+Phenylacetyl” denotes a product derived from reaction with an oxidation reagent Imetelstat synthesis utilizing fewer coupling steps provides for both Full Length Product Purity and Yield that are substantially higher. Resolution of impurities es easier purification of stat where there are less amounts of minor products closely running near the main peak in HPLC to e itions having higher purity of imetelstat. This improvement is desirable for lower cost of goods for manufacture of imetelstat sodium, e.g., the cost of goods can be 30—40% less when implemented at manufacturing scale.

Scheme 2. Synthetic Scheme of GA Dimer Amidate 85g of TBAG was prepared from 300 g of APG2 according to the methods described herein Via the steps shown in Scheme 2.

] Scheme 3. Synthetic Scheme of AA Dimer Amidate N):m um _l t r o kWt W] N ][ | \ [i if 3“ IL“ \' ..., K , \‘ u N 8- NW mN | "‘ “T HS—L ‘5’" .n moms W‘" .o i" ”\‘N “W. \ . , memes ’3‘ J _. ". ll ll N x” mi NH b :‘z—‘g: !.-:\ aye—q, .-’=\ 0 ‘WN=NW' $45? “:3 {KL/5) Vwf Nat—“N. .('=\ k0 ‘J_ \ e59 ’ 4f ' _—." ""\ if L] LJ _ i, \x v» I n Qty“ mm mam: TWA-2 mm a {I} to we ‘ \1 Wk “W as mffl‘ fl] NW 135?} [I N ,L, HajfiJ L ‘\ N.‘ [10.14 LI\;:::W ’ J' ,.

I K in l N l ,1 N \N w 3|) To N N “rams-o 0 , we fix] THE-A u ,1 it TE [if ”a“ M “if” —. M“? —» W7 WW“? ga—§=s —» \ " , f 3 3‘1”? N“ N Jays "n’ ‘9", o—Ea ‘er M: LEO ‘W. Nc’ ' a" W NC 7 W D‘Wi Q_\\V{ Misfzk NR NH .;:W"_'\~ ,=\' In _\ \=N~"‘ 1&4," 'X:W‘ “—2)" ‘N=’_[—’“N_' .35" - (x LQ-‘n ch’” 6%?» LEI” mama mm 32mm 430 g of TBAPAl was obtained from 800g of the crude APAl (purity: 46%) according to the methods described herein Via the steps shown in Scheme 3.

Scheme 4. Synthesis of TA Dimer Amidate.

O B O HOW B Protect NH2 HOW l Protection : —> 5 —> H2N HN\ Pixyl 1: B = T 2: B = T O T De 0 rotect at 3'—NH 5 : —> \ CN HN\P_ _ _ /\/ Couple with A(DMF) amidite 34" O my I Sulfurization ane Hyd) O 3 vAdmf HN‘: O/\/CN A ' ,P\ 0 T Deprotect at 5'-OH N OW Phosph'lty a Ionl t' H'If S=F|’-O/\/CN HN‘: TA dimer amidate (5) has been prepared according to the methods described herein Via the steps shown in Scheme 4 at scales of synthesis from 100mg to lg.

WO 68310 Scheme 5. Cou lin and ization durin Dimer Amidate s nthesis 0 OX 0 INlHO O + NFLOHAgNWN/fi ETTACN o HZN‘ \<N HNTr o i” #OAQ‘\‘ N O O HN\ I—_N " O N N ,P~ / \\\ NC\/\O 0m ‘N N\ x N Q/ / Table 9. ng and Sulfurization during Dimer e synthesis Starting Mol. Eq. of reagents Solvent type Reaction Pdt. Yield Analysis Material and time/ Weight Quantity Amount temp. l l ETT (1.0 eq), Acetonitrile R 300mg LCMS Xantane hydride (2.06q), (5.0 mL) (crude) 00 mg T for 3+2 Pyridine (1.5 mL) 2 l 0.4M ETT (2.0 mL), neat R 350mg LCMS Xantane hydride (1.26q), (crude) 00 mg T for 3+2 Pyridine (2.0 mL) A variety of nucleoside monomers were prepared according to the methods described herein which find use in the preparation of the dimer compounds.

Scheme 6: S nthesis of Levulinate rotected monomers O NOHHNOGAO/B HO/\<_7’B Pg—CI mAgB—> Deprotect o B ~ —~ ”OW H2l\f ne HN Levunyl gp S —20°C for 16h Pg HN\ 33' Pg: Trg 1 35% 23: Pg: Tr Pg 2b: Pg = TMS 3b: P9 = TMS Bsse tion Phosphytilation O/\/CN | —. XN/KWB Scheme 7: S nthesis of a bis—DMF A amidite NH2 N¢\N/ «(bf/2f”A </Nf”| DMF- DMA O N N/) HO —> HO DMF £- H2N 86% N\ \\N/ N¢\T/ Phosphitylation O/\/CN N \ N reagent A </ I ,F|>\ 0 N N/J DCM / DMF A 63% NC Scheme 8: S nthetic Scheme for MMT DMT and Pix 1 Monomers A es : NH2 NH2 OAGfN/NfNlN/J Protect 3'-NH2 O/\<:7/<N/NfNlN/J DMF - DMA H H Pyridine 5 DMF HZN HN PG: MMT, DMT, Pixyl N¢\T/ N¢\N/ <':I[l:TN I \ N \\N Phosph'l ' O/A\V/CN / Ity atlon I < | O N A HOW N reagent N’P\O/\<:7/N N s DCI //k\ 5 HN DCM HN PG PG PG: MMT, DMT, Pixyl Amidite Admf 3’- (MMT) Monomethoxytritylamino— N6—dimethy1f0rmamidino— 2’ ,3’—dide0xyadenosine— yanoethy1)—N,N— diisopropyl Phosphoramidite e Admf 3’-(dimethy1—substituted (pixyl) Pixy1)amin0—N6— dimethylformamidino— 2’ ,3’—dide0xyadenosine— ’—(2—cyanoethy1)—N,N— diisopropyl Phosphoramidite Amidite Admf 3’—Dimethoxytritylamino— (DMT) N6—dimethylformamidino— 2’ ,3’—dideoxyadenosine— ’—(2—cyanoethyl)—N,N— diisopropyl Phosphoramidite While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be tuted without departing from the true spirit and scope of the invention. In addition, many cations may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended .

EMBODIMENTS The present disclosure provides a composition having less than 1 part in 4 by weight of a (N—l) product ve to a compound or a salt thereof, where the compound includes a polynucleotide having a sequence of 10 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3’—>P5’ phosphoramidate inter—subunit linkage. In some embodiments of the composition, the N3’—>P5’ phosphoramidate inter—subunit linkage is a N3’—>P5’ thiophosphoramidate inter—subunit linkage having the structure: P(S)(OR)— 0—5’ where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a tuted aryl and a ate ting group, or a salt thereof.

In some embodiments of the composition, the nd includes a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase. In some embodiments of the composition, the polynucleotide includes a sequence comprising 13 or more side subunits complementary to the RNA component of human telomerase. In some embodiments of the ition, the polynucleotide includes between 3 and 50 contiguous nucleoside subunits complementary to the RNA WO 68310 component of human telomerase. In some ments of the composition, the nucleoside subunits complementary to the RNA component of human telomerase are all joined by 5’ phosphoramidate inter—subunit linkages. In some embodiments of the ition, the polynucleotide includes a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5). In some embodiments of the composition, the polynucleotide includes a 3’amino or a 3’ hydroxyl terminal group.

In some ments of the composition, the nd has the structure: H OH j? NWO—?—O T O=|i’-SH O=|i’-SH O_[GnpanpanpsTnpsTnpsAnpanpsAnpanpsAnps] I A or a salt thereof; Where “nps” represents a thiophosphoramidate linkage —NH—P(=O)(SH)— 0—, connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside. In some embodiments of the composition, the salt is a pharmaceutically acceptable salt.

In some embodiments of the composition, the compound has the structure: OH “I“ < I ,j“ S=P'O N O N NH (5 i j o H'il NH <’ IA S=fi’-O N o N NH2 o— 7;) o HI“ </ | i“ s=fi>—o N o N NH2 0- kg 0 HN <’ I 1H S=|?-O N o N NH2 0- S 7 0 HI fl: S=fi’-O o N o o— p o HI W: S=E-O o N o o- p NH2 _' (/N |\NA S—E-O o N N o HN </ I 1H S=I?-O N o N NH O' p NH2 HN (/N |\N s=F'>—o N N4 (-3. i0) NH2 NH | S—E-O N’J§O o- NH2 I (/N |\NA S=E-O N O NH2 I‘H </N I)“ S=FI"O O N N/ o- b NH2 (M )n wherein each M“ is independently en or a counterion of a salt, each X is independently l, 2 or 3 and n is an integer from 5 to 13. In certain instances, M“ is hydrogen.

In some embodiments of the composition, the compound has the structure: OH NH \N <’ | J S—P-O o N N NH (5 o o + i j s=fi>—o N o N/ NH2 0' b o s=fi>—o N o N/ NH2 0' p o s=fi>—o N o N/ NH2 0- kg 0 HN NH I \fig s=fi>—o o N o o— p o HI fl: s=fi>—o o N o o- NH2 N \ “H“ </ I j“ s=fi>—o o N N’ o- ; 3 o s=fi>—o N o N NH2 0- lg MHZ a «N \ HN I j“ s=fi>—o o N N/ O' b NH2 3:}:3—0 N O o- p NH2 Na+ \ NH «N I j“ s=fi>—o o N N/ O' K 7 NH2 NH </N I \)N 8—9—0 0 N N/ o- 797 In some embodiments, the composition has less than 1 part in 6 by weight of a (N—l) product relative to the compound. In some ments, the composition has less than l part in 10 by weight of a (N—l) product relative to the compound. In some embodiments, the composition has less than 1 part in 20 by weight of a (N—l) product relative to the compound. In some embodiments, the composition has less that 1 part in 4 by weight of any (N—X) product ve to the compound. In some embodiments, the composition has less that 40 part in 100 by total weight of (N—X) cleotide—containing products relative to the compound. In some embodiments, the ition has the following profile of (N—X) polynucleotide—containing ts: less that 1 part in 4 by weight of a (N—l) product relative to the compound; at least 10 parts in 100 by weight of (N—2) and (N—3) products relative to the compound.

The t disclosure provides a compound active ceutical ingredient having less than ll % by weight of a (N—l) product, where the compound or a ceutically acceptable salt thereof includes a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase, where at least two of the nucleoside subunits are joined by a N3’—>P5’ phosphoramidate inter—subunit linkage.

In some embodiments of the compound active pharmaceutical ingredient, the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3’—>P5’ thiophosphoramidate inter—subunit linkages. In some embodiments of the compound active pharmaceutical ingredient, the 5’ phosphoramidate inter—subunit linkage is a N3’—>P5’ thiophosphoramidate inter—subunit linkage having the structure: 3’—NH—P(S)(OR)— 0—5’ where R is selected from the group consisting of hydrogen, an alkyl a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, or a pharmaceutically acceptable salt thereof.

In some embodiments of the compound active pharmaceutical ingredient, the polynucleotide includes between 10 and 50 contiguous nucleoside subunits complementary to the RNA component of human telomerase. In some embodiments of the compound active pharmaceutical ient, the cleotide includes a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4); TAGGGTTAGACAA (SEQ ID NO:3); and CAGTTAGGGTTAG (SEQ ID NO:5). In some embodiments of the compound active pharmaceutical ingredient, the polynucleotide includes a 3’amino or a 3’—hydroxyl terminal group.

In some embodiments of the compound active ceutical ient, the compound has the structure: “£0 E o T o {5! (ID—l A o=F|>—SH c'>—[Gnp5GnpsenpsTnpsTnpsAnpsansAnpscnpsAnpsi or a pharmaceutically able salt thereof; Where “nps” represents a thiophosphoramidate linkage —NH—P(=O)(SH)—O—, connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside.

In some embodiments of the compound active pharmaceutical ingredient, the compound has the structure: OH “I“ < I ,j“ S=P'O N O N NH (5 i j o HI}! NH <’ IA S=fi’-O N o N NH2 o— 7;) o HI“ </ | i“ s=fi>—o N o N NH2 0- kg 0 HN <’ I 1H S=|?-O N o N NH2 0- 7;) 0 HI fl: S=fi’-O o N o o- s 7 0 HI W: S=E-O o N o O' p NH2 _' (/N |\NA S—E-O o N N o HN </ I 1H S=I?-O N o N NH O' p NH2 HN (/N |\N s=F'>—o N N4 (-3. i0) NH2 NH | S—E-O N’gO o- NH2 I (/N |\NA S=E-O N O N O NH2 I‘H </N I)“ S=FI"O O N N/ o- b NH2 (Mx+)n Where each M“ is independently hydrogen or a counterion of a pharmaceutically acceptable salt, each X is independently l, 2 or 3 and n is an integer from 5 to 13. In certain instances, M“ is hydrogen.

] In some embodiments of the nd active pharmaceutical ingredient, the compound has the structure: OH NH \N <’ | J S—P-O o N N NH (5 o O + i j s=fi>—o N o N/ NH2 0' b o s=fi>—o N o N/ NH2 0' p o s=fi>—o N o N/ NH2 0- kg 0 HN NH I \fig s=fi>—o o N o 0— 7Q o HI 1%)“: s=fi>—o o N o o- NH2 N \ “H“ </ I j“ S=Fl>_—O o N N’ o O K 7 s=fi>—o N o N NH2 0- 7;) MHZ a \ HN «N I j“ s=fi>—o o N N/ O' b NH2 3:}:3—0 N O o- p NH2 Na+ \ NH «N I j“ s=fi>—o o N N/ 0' b NH2 NH </N I \)N 8—9—0 0 N N/ 0- 797 ] In some embodiments, the compound active pharmaceutical ingredient has less that 9 % by weight of the (N—l) product. In some embodiments, the compound active ceutical ingredient has less that 5 % by weight of the (N—l) product. In some embodiments, the compound active pharmaceutical ingredient has less that ll % of any (N—x) product. In some embodiments, the compound active pharmaceutical ingredient has less that 45 % by weight in total of (N—x) polynucleotide—containing products. In some embodiments, the compound active pharmaceutical ingredient has the ing profile of (N—x) polynucleotide— containing products:less that 5 % by weight of a (N—l) product; and at least 10 % by weight of (N—2) and (N—3) ts.

Also provided is a ceutical composition including a composition (e. g., of any one of the embodiments described herein) formulated in a pharmaceutically acceptable excipient. Also provided is a pharmaceutical ition ing a nd active pharmaceutical ingredient (e. g., of any one of the embodiments described herein) formulated in a pharmaceutically acceptable excipient.

The t sure provides a method of synthesizing a polynucleotide. In some embodiments, the method includes the steps of: (a) deprotecting the protected 3' amino group of a terminal nucleoside attached to a solid phase t, said deprotecting forming a free 3' amino group; (b) ting the free 3' amino group with a 3'—protected amino—dinucleotide phosphoramidate—5'—phosphoramidite dimer in the presence of a nucleophilic catalyst to form an intemucleoside N3'—>P5' phosphoramidite linkage; and (c) oxidizing the linkage.

In some embodiments, the method further includes: (a) deprotecting the protected 3' amino group of a terminal nucleoside ed to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'—protected aminonucleoside—5'—phosphoramidite monomer in the presence of a nucleophilic catalyst to form an internucleoside N3'—>P5' phosphoramidite linkage; and (c) oxidizing the linkage. In some embodiments of the method, the oxidizing the linkage includes sulfurization to produce a thiophosphoramidate linkage. In some embodiments of the method, the oxidizing the linkage es an oxophosphoramidate linkage.

In some embodiments of the method, the 3'—protected amino—dinucleotide phosphoramidate—5'—phosphoramidite dimer has the formula: wherein X is O or S and B1 and B2 are each independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof. In some embodiments of the method, the B1 and B2 are each independently selected from ted adenine, protected cytosine, protected guanine, thymine and uracil. In some embodiments of the , the B1 and B2 are each independently selected from A(Bz), A(DMF), C(Bz), G(isobutyryl), T and U. In some embodiments of the method, X is S.

In some ments of the method, the polynucleotide is of the formula: Z_ _L O B HN R3 \ /X /P\ B R0 0 R6 R3 where: each B is independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or ; each R3 is hydrogen, fluoro, or hydroxyl, an alkoxy, a substituted alkoxy or a protected hydroxyl; L is an al linker; Z is H, a lipid, a support, a carrier, an oligonucleotide, a PEG, a polypeptide, a detectable label, or a tag; R6 is amino, hydroxyl, a protected amino, a ted hydroxy, —O—L—Z or —NH— L—Z; R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, or a ate protecting group; and n is an integer of l to 1000; or a salt thereof; and the method comprises the steps of: (a) deprotecting a protected 3' amino group of a terminal nucleoside attached to a solid phase support, said ecting forming a free 3' amino group; (b) reacting the free 3' amino group with either: (i) a 3'—protected amino—dinucleotide phosphoramidate—5'—phosphoramidite dimer; or (ii) a 3'—protected aminonucleoside—5'—phosphoramidite monomer; in the ce of a nucleophilic catalyst to form an internucleoside N3'—>P5' phosphoramidite linkage; (c) oxidizing the linkage; and (d) repeating steps (a) h (c) until the polynucleotide is synthesized, wherein the repeating steps (a) through (c) comprises performing step (b)(i) at least once.

In some embodiments of the method, the oxidizing the linkage comprises ization to produce a thiophosphoramidate linkage. In some embodiments of the , the oxidizing the linkage produces an oxophosphoramidate linkage. In some embodiments of the method, the polynucleotide comprises a sequence of nucleoside ts complementary to the RNA component of human telomerase, and wherein at least two of the side ts are joined by a N3’—>P5’ phosphoramidate subunit linkage. In some embodiments of the method, the N3’—>P5’ phosphoramidate inter—subunit linkage is a N3’—>P5’ thiophosphoramidate inter— subunit linkage haVing the structure: 3’—NH—P(S)(OR)—O—5’ where R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a tuted aryl and a phosphate protecting group, or a salt thereof.

In some embodiments of the method, the polynucleotide includes the sequence TAGGGTTAGACAA. In some embodiments of the method, all of the intemucleotide inter— subunit linkages of the TAGGGTTAGACAA sequence are N3'—> PS' phosphoramidate inter— subunit linkages. In some embodiments of the method, polynucleotide has the structure: WO 68310 H OH ” NW0_ _I? O T O=|r_SH O=|i’-SH O_[GnpsGnpsGnpsTnpsTnpsAnpsansAnpanpsAnps] I A or a salt thereof; Where “nps” represents a thiophosphoramidate linkage —NH—P(=O)(SH)— 0—, connecting the 3'—carbon of one nucleoside to the 5'—carbon of the adjacent nucleoside.

In some embodiments of the method, the polynucleotide has the structure: H'i‘H \N <’ I/J S=P—O O N N NH ('3 O O 1: N HI}! NH <’ IA S=I?-O N o N’ NH2 0 kg 0 Hr </N I i“ S=I?-O N o N/ NH2 0- b 0 Hr </N I i“ S=I?-O N o N/ NH2 0- b 0 Hi fl: s=E—o o N o o- p 0 Hi fl: s=E—o o N o 0' p NH2 “H“ «N P“ S=fi’-O N o N’J o O. b ”N <’ IA S=I?-O N o N NH 0— lg NHz H'fl </N I)”’ 86-0=P- —| O N NNH2 NH | 1 N’J§O 8—9—0 0 o NH2 “3“ «N I)“ s=E—o o N N/ o— b NHz “H“ «N I)” S=|?-O N O N’ o— b ”“2 (M )n n each M“ is independently hydrogen or a counterion of a pharmaceutically acceptable salt, each X is independently l, 2 or 3 and n is an integer from 5 to 13. In certain instances, M“ is hydrogen.

In some embodiments of the method, the polynucleotide has the structure: OH NH \N <’ | J S—P-O o N N NH (5 o o + i j s=fi>—o N o N/ NH2 0' b o s=fi>—o N o N/ NH2 0' p o s=fi>—o N o N/ NH2 0- kg 0 HN NH I \fig s=fi>—o o N o 0— 7Q o HI fl: s=fi>—o o N o o- NH2 N \ “H“ </ I j“ s=fi>—o o N N’ o- ; 3 o HN <’ | 1H s=fi>—o N o N NH2 0- 7;) MHZ a \ HN «N I j“ s=fi>—o o N N/ o- i j NH2 S=Fl’-O N O o- p NH2 Na+ \ NH «N I j“ s=fi>—o o N N/ 0' b NH2 NH </N I \)N S—Ffi-O o N N/ 0- 797 In some embodiments of the method, the C11 nucleotide e of the TAGGGTTAGACAA sequence derives from a 3'—pr0tected aminonucleoside—5'— phosphoramidite monomer. In some embodiments, the method includes sequential coupling of the following tected amino—dinucleotide thiophosphoramidate—5'—phosphoramidite dimers TA, GG, GT, TA, GA and AA and 3'—protected ucleoside—5'—phosphoramidite r C to the solid phase support. In some embodiments of the method, the 3'—protected amino— dinucleotide phosphoramidite—5'—phosphoramidite dimer is bed by the formula XIXZ, wherein X1 and X2 are independently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil. In some embodiments of the method, the 3'—protected aminonucleoside—5'—phosphoramidite dimer is ed from ted adenine, protected cytosine, protected guanine, thymine and uracil.

The present sure provides a dinucleotide thiophosphoramidate compound described by Formula (II): Formula (II) wherein: B1 and B2 are each independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; R11 is hydrogen, a protecting group or a phosphoramidite group; and R12 and R13 are each independently hydrogen or a protecting group; or a salt thereof.

In some embodiments of the compound, B1 and B2 are each ndently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil. In some embodiments of the nd, B1 and B2 are each independently selected from A(Bz), A(DMF), C(Bz), utyryl), T and U. In some embodiments of the compound, R11 is a 5’— phosphoramidite; R12 is a protecting group and R13 is a protecting group. In some embodiments of the compound, B1 is A(Bz) or A(DMF) and B2 is A(Bz) or A(DMF). In some embodiments of WO 68310 the compound, B1 is A(Bz) or A(DMF) and B2 is C(Bz). In some embodiments of the compound, B1 is A(Bz) or A(DMF) and B2 is G(isobutyry1). In some embodiments of the compound, B1 is A(Bz) or A(DMF) and B2 is T. In some embodiments of the compound, B1 is A(Bz) or A(DMF) and B2 is U. In some embodiments of the compound, B1 is C(Bz) and B2 is A(Bz) or A(DMF). In some embodiments of the compound, B1 is C(Bz) and B2 is C(Bz). In some embodiments of the compound, B1 is C(Bz) and B2 is G(isobutyry1). In some embodiments of the compound, B1 is C(Bz) and B2 is T. In some embodiments of the nd, B1 is C(Bz) and B2 is U. In some embodiments of the nd, B1 is G(isobutyry1) and B2 is A(Bz) or A(DMF). In some embodiments of the compound, B1 is G(isobutyry1) and B2 is C(Bz). In some embodiments of the compound, B1 is G(isobutyry1) and B2 is G(isobutyry1). In some embodiments of the compound, B1 is G(isobutyry1) and B2 is T. In some embodiments of the compound, B1 is G(isobutyry1) and B2 is U. In some embodiments of the compound, B1 is T or U and B2 is A(Bz) or A(DMF). In some embodiments of the compound, B1 is T or U and B2 is C(Bz). In some embodiments of the compound, B1 is T or U and B2 is G(isobutyry1). In some embodiments of the compound, B1 is T or U and B2 is T. In some ments of the compound, B1 is T or U and B2 is U.

All possible combinations of the above—indicated embodiments are considered to be ed Within the scope of this invention.

Claims (45)

WHAT IS CLAIMED IS:

1. A method of synthesizing a cleotide having at least two nucleoside subunits joined by a ′ oxophosphoramidate or N3′→P5′ thiophosphoramidate inter-subunit linkage, the method comprising the steps of: (a) deprotecting a protected 3' amino group of a al protected 3’ amino nucleoside attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'-protected amino-dinucleotide phosphoramidate-5'-phosphoramidite dimer in the ce of a nucleophilic catalyst to form an internucleoside N3'→P5' phosphoramidite linkage; and (c) oxidizing the linkage to produce a N3′→P5′ oxophosphoramidate or N3′→P5′ thiophosphoramidate inter-subunit linkage.

2. The method of claim 1, further comprising: (a) deprotecting a protected 3' amino group of a terminal protected 3’amino side attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) contacting the free 3' amino group with a 3'-protected aminonucleoside-5'- phosphoramidite monomer in the ce of a nucleophilic catalyst to form an internucleoside N3'→P5' phosphoramidite e; and (c) oxidizing the linkage.

3. The method of claim 1 or 2, wherein oxidizing the linkage comprises sulfurization to produce a N3'→P5' thiophosphoramidate linkage.

4. The method of claim 1 and 2, wherein oxidizing the linkage produces an N3'→P5' oxophosphoramidate e.

5. The method of any one of claims 1-4, wherein the 3'-protected amino-dinucleotide phosphoramidate-5'-phosphoramidite dimer has the formula: wherein X is O or S and B1 and B2 are each independently a purine, a ted purine, a pyrimidine or a protected pyrimidine, or an analog thereof.

6. The method of claim 5, n the B1 and B2 are each independently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil.

7. The method of claim 6, wherein the B1 and B2 are each independently selected from A(Bz), A(DMF), C(Bz), G(isobutyryl), T and U.

8. The method of any one of claims 5-7, wherein X is S.

9. The method of any one of claims 1-8, wherein the polynucleotide is of the formula: each B is independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is hydrogen, fluoro, or hydroxyl, an alkoxy, a substituted alkoxy or a protected hydroxyl; L is an optional linker; Z is H, a lipid, a support, a carrier, an ucleotide, a PEG, a ptide, a detectable label, or a tag; R6 is amino, hydroxyl, a protected amino, a protected hydroxy, -O-L-Z or –NH-L-Z; R is hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, or a phosphate protecting group; and n is an integer of 1 to 1000; or a salt thereof; and the method comprises the steps of: (a) deprotecting a protected 3' amino group of a al side attached to a solid phase support, said deprotecting forming a free 3' amino group; (b) reacting the free 3' amino group with either: (i) a 3'-protected amino-dinucleotide phosphoramidate-5'-phosphoramidite dimer; or (ii) a 3'-protected aminonucleoside-5'-phosphoramidite monomer; in the presence of a nucleophilic catalyst to form an internucleoside N3'→P5' phosphoramidite linkage; (c) oxidizing the linkage; and (d) ing steps (a) through (c) until the polynucleotide is synthesized, wherein the repeating steps (a) through (c) comprises performing step (b)(i) at least once.

10. The method of claim 9, wherein oxidizing the linkage ses sulfurization to produce a N3'→P5' thiophosphoramidate linkage.

11. The method of claim 9, wherein oxidizing the e produces an N3'→P5' oxophosphoramidate linkage.

12. The method of any one of claims 8-11, wherein the polynucleotide comprises a sequence of nucleoside subunits, and wherein at least two of the nucleoside subunits are joined by a N3′→P5′ oxophosphoramidate or N3′→P5′ thiophosphoramidate inter-subunit linkage.

13. The method of claim 12, wherein the N3′→P5′ sphoramidate or N3′→P5′ thiophosphoramidate inter-subunit e is a N3′→P5′ thiophosphoramidate inter-subunit linkage having the structure: 3′—NH—P(S)(OR)—O—5’ wherein R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate ting group, or a salt thereof.

14. The method of any one of claims 9-12, wherein the polynucleotide comprises the sequence TAGGGTTAGACAA.

15. The method of claim 14, wherein all of the internucleotide subunit linkages of the TAGGGTTAGACAA sequence are N3'→ P5' oxophosphoramidate or N3′→P5′ thiophosphoramidate inter-subunit linkages.

16. The method of any one of claims 1-15, n the polynucleotide has the structure: H OH N O P O T O SH O P SH O A O P SH O [GnpsGnpsGnpsTnpsTnpsAnpsGnpsAnpsCnpsAnps] A or a salt thereof; n “nps” represents a thiophosphoramidate linkage —NH—P(═O)(SH)—O—, connecting the 3'-carbon of one nucleoside to the 5'-carbon of the adjacent nucleoside.

17. The method of any one of claims 1-15, wherein the polynucleotide has the structure: (Mx+)n wherein each Mx+ is independently hydrogen or a counterion of a pharmaceutically acceptable salt, each x of Mx+ is independently 1, 2 or 3 and n is an integer from 5 to 13.

18. The method of claim 17, wherein the cleotide has the structure:

19. The method of claim 14, wherein the C11 nucleotide residue of the TAGGGTTAGACAA ce derives from a 3'-protected aminonucleoside-5'- phosphoramidite monomer.

20. The method of claim 14, n the method comprises sequential coupling of the following 3'-protected amino-dinucleotide thiophosphoramidate-5'-phosphoramidite dimers TA, GG, GT, TA, GA and AA and 3'-protected aminonucleoside-5'-phosphoramidite monomer C to the solid phase t.

21. The method of any one of claims 1 to 5, wherein the 3'-protected amino-dinucleotide phosphoramidite-5'-phosphoramidite dimer is described by the formula X1X2, wherein X1 and X2 are independently selected from protected adenine, protected cytosine, ted guanine, thymine and uracil.

22. The method of claim 1, wherein the 3'-protected aminonucleoside-5'-phosphoramidite dimer is selected from protected adenine, protected cytosine, ted guanine, e and uracil.

23. A dinucleotide thiophosphoramidate compound described by Formula (II): Formula (II) wherein: B1 and B2 are each independently a purine, a ted purine, a pyrimidine or a protected pyrimidine, or an analog thereof; R11 is a phosphoramidite group; and R12 and R13 are each independently a protecting group; or a salt thereof.

24. The compound of claim 23, wherein B1 and B2 are each independently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil.

25. The compound of claim 24, wherein B1 and B2 are each independently ed from A(Bz), A(DMF), C(Bz), G(isobutyryl), T and U.

26. The compound of claim 25, wherein B1 is A(Bz) or A(DMF) and B2 is A(Bz) or A(DMF).

27. The compound of claim 25, wherein B1 is A(Bz) or A(DMF) and B2 is C(Bz).

28. The compound of claim 25, wherein B1 is A(Bz) or A(DMF) and B2 is G(isobutyryl).

29. The compound of claim 25, n B1 is A(Bz) or A(DMF) and B2 is T.

30. The compound of claim 25, wherein B1 is A(Bz) or A(DMF) and B2 is U.

31. The compound of claim 25, wherein B1 is C(Bz) and B2 is A(Bz) or A(DMF).

32. The compound of claim 25, wherein B1 is C(Bz) and B2 is C(Bz).

33. The compound of claim 25, wherein B1 is C(Bz) and B2 is G(isobutyryl).

34. The nd of claim 25, n B1 is C(Bz) and B2 is T.

35. The compound of claim 25, wherein B1 is C(Bz) and B2 is U.

36. The compound of claim 25, wherein B1 is G(isobutyryl) and B2 is A(Bz) or A(DMF).

37. The compound of claim 25, wherein B1 is G(isobutyryl) and B2 is C(Bz).

38. The compound of claim 25, wherein B1 is G(isobutyryl) and B2 is G(isobutyryl).

39. The nd of claim 25, wherein B1 is G(isobutyryl) and B2 is T.

40. The compound of claim 25, wherein B1 is G(isobutyryl) and B2 is U.

41. The compound of claim 25, wherein B1 is T or U and B2 is A(Bz) or A(DMF).

42. The compound of claim 25, wherein B1 is T or U and B2 is C(Bz).

43. The nd of claim 25, wherein B1 is T or U and B2 is G(isobutyryl).

44. The compound of claim 25, wherein B1 is T or U and B2 is T.

45. The compound of claim 25, wherein B1 is T or U and B2 is U.