US009278987B2

(12) United States Patent (10) Patent No.: US 9.278,987 B2 Hanson et al. (45) Date of Patent: Mar. 8, 2016

(54) FUNCTIONALLY-MODIFIED 5,386,023 A 1/1995 Sanghviet al. OLGONUCLEOTDES AND SUBUNITS 5,457,191. A 10/1995 Cook et al.

THEREOF 3.685.- WW A '83 SARnetUCO a 5,506,351 A 4/1996 McGee (71) Applicant: Sarepta Therapeutics, Inc., Corvallis, 5,521,063 A 5/1996 Summerton et al. OR (US) 5,521,302 A 5, 1996 Cook 5,539,082 A 7/1996 Nielsen et al. (72) Inventors: Gunnar J. Hanson, Bothell, WA (US); 5,541,307 A 7/1996 Cook et al. Dwightg D. Weller,:- Corvallis,y OR (US);s 5,554,7465,571,902 A 11/19969, 1996 Ravikumar et al. Bao Zhong Cai, Corvallis, OR (US); 5,576,302 A 1 1/1996 Cook et al. Ming Zhou, Coppell, TX (US) 5,578,718 A 1 1/1996 Cook et al. 5,580,767 A 12/1996 Cowsert et al. (73) Assignee: Sarepta Therapeutics, Inc., Corvallis, 5,587,361. A 12/1996 Cook et al.

OR (US) 35;- w A 32; OOK a c 5,599,797 A 2f1997 Cook et al. (*) Notice: Subject to any disclaimer, the term of this 5,602,240 A 2f1997 De Mesmaeker et al. patent is extended or adjusted under 35 5,608,046 A 3, 1997 Cook et al. U.S.C. 154(b) by 0 days. 5,610,289 A 3, 1997 Cook et al. 5,698,685 A 12/1997 Summerton et al. 5,702,891 A 12/1997 Kolberg et al. (21) Appl. No.: 14/358,992 5,734,039 A 3/1998 Calabretta et al. 5,749,847 A 5/1998 Zewert et al. (22) PCT Filed: Nov. 15, 2012 5,801,154 A 9, 1998 Baracchini et al. 5,892,023 A 4/1999 Pirotzky et al. (86). PCT No.: PCT/US2O12/065350 5,955,318 A 9, 1999 Simons et al. 6,030,954 A 2/2000 Wu et al. S371 (c)(1), 6,060,456 A 5/2000 Arnold, Jr. et al. (2) Date: May 16, 2014 (Continued)

PCT Pub. Date: May 23, 2013 l al ay 25, JP 5-504563 A 7, 1993 (65) Prior Publication Data JP 2002-167441 A 6, 2002 US 2014/033OOO6A1 Nov. 6, 2014 (Continued) OTHER PUBLICATIONS Related U.S. Application Data Agrawal et al., “Oligodeoxynucleoside phosphoramidates and (60) Provisional application No. 61/561,806, filed on Nov. phosphorothioates as inhibitors of human immunodeficiency virus.” 18, 2011. Proc. Natl. Acad. Sci. USA 85:7079-7083, 1988. Agrawal et al., “Site-specific excision from RNA by RNase H and (51) Int. Cl. mixed-phosphate-backbone oligodeoxynucleotides.” Proc. Natl. C07F 9/6558 (2006.01) Acad. Sci. USA 87: 1401-1405, 1990. C7F 9/6584 (2006.01) Akhtar et al., “Interactions of antisense DNA oligonucleotide analogs C7F 9/656 (2006.01) with phospholipid membranes (liposomes).” Nucleic Acids Research 19(20)5551-5559, 1991. C7H 2L/00 (2006.01) Anderson et al., “Inhibition of Human Cytomegalovirus Immediate (52) U.S. Cl. Early Gene Expression by an Antisense Oligonucleotide Comple CPC ...... C07F 9/65583 (2013.01); C07F 9/65586 mentary to Immediate-Early RNA.” Antimicrobial Agents and Che (2013.01); C07F 9/65616 (2013.01); C07F motherapy 40(9)-2004-2011, Sep.1996. 9/65846 (2013.01); C07H 21/00 (2013.01) (Continued) (58) Field of Classification Search USPC ...... 540/467,542:544/123, 82 See application file for complete search history. Primary Examiner — Kristin Vajda (74) Attorney, Agent, or Firm — Seed IP Law Group PLLC (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 5,034,506 A 7, 1991 Summerton et al. Functionally-modified oligonucleotide analogues compris 5,138,045 A 8, 1992 Cook et al. ing modified intersubunit linkages and/or modified 3' and/or 5,142,047 A 8, 1992 Summerton et al. 5'-end groups are provided. The disclosed compounds are 5,166,315 A 11, 1992 Summerton et al. 5,185,444 A 2, 1993 Summerton et al. useful for the treatment of diseases where inhibition of pro 5,212.295 A 5, 1993 Cook tein expression or correction of aberrant mRNA splice prod 5,217.866 A 6, 1993 Summerton et al. ucts produces beneficial therapeutic effects. 5,218, 105 A 6, 1993 Cook et al. 5,223,168 A 6, 1993 Holt 5,378,825 A 1/1995 Cook et al. 16 Claims, 4 Drawing Sheets US 9.278,987 B2 Page 2

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(56) References Cited Nielsen, "Peptide nucleic acids as antibacterial agents via the antisense principle.” Exp. Opin. Invest. Drugs 10(2):331-341, 2001. OTHER PUBLICATIONS Nielsen, "Peptide nucleic acids: on the road to new gene therapeutic drugs.” Pharmacol. Toxicol. 86(1):3-7, 2000. GenBank Accession No. Y 1 1275.1, retrieved Jul. 15, 2010, from Nielsen et al., “Sequence-selective recognition of DNA by strand http://www.ncbi.nlm.nih.gov/nuccore/4127812. 4 pages. displacement with a thymine-substituted polyamide.” Science GenBank Accession No. AB01 1549, retrieved Jul. 15, 2010, from http://www.ncbi.nlm.nih.gov/nuccore/4589740. 35 pages. 254(5037): 1497-1500, 1991. Gerdes et al., “Experimental Determination and System Level Analy Nikaido, “Transport across the bacterial outer membrane.” J sis of Essential Genes in Escherichia coli MG 1655.” Journal of Bioenerg Biomembr 25(6):581-589, 1993. Bacteriology 185(19):5673-5684, Oct. 2003. 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FIGURE 3 U.S. Patent Mar. 8, 2016 Sheet 4 of 4 US 9.278,987 B2

US 9,278,987 B2 1. 2 FUNCTIONALLY MODIFIED Several groups have reported the synthesis of positively OLGONUCLEOTDES AND SUBUNITS charged oligonucleotides (Bailey, C. P. et al. Nucleic Acids Res. 26(21):4860-7 (1998); Micklefield, J., Curr, Med, Chem, THEREOF 8(10): 1157-79 (2001); Egli, M. et al., Biochemistry 44(25): 9045-57 (2005)). For example, a class of guanidinium linked STATEMENT REGARDING SEQUENCE 5 nucleosides (designated DNG), formed by replacement of the LISTING phosphate linkages in DNA and RNA by achiral guanidino groups, has been reported (Dempcy, R. O. et al., Proc. Nat'll The sequence listing associated with this application is Acad. Sci. USA 91 (17):7864-8 (1994): Dempcy, R. O. et al., provided in text format in lieu of a paper copy, and is hereby Proc. Nat'l Acad. Sci. USA 93(9):4326-30 (1996); Barawkar, incorporated by reference into the specification. The name of 10 D. A. et al., Proc. Nat'l Acad. Sci. USA 95(19): 11047-52 (1998); Linkletter, B. A. et al., Nucleic Acids Res. 29(11): the text file containing the sequence listing is 120178 2370-6 (2001)). Oligomers linked with positively charged 498USPCSEQUENCELISTING..txt. The text file is methylated thiourea linkages have also been reported (Arya, about 13 KB, was created on May 16, 2014, and is being D. P. et al., Proc. Nat 'l Acad. Sci. USA 96(8): 4384-9 (1999)). submitted electronically via EFS-web. Replacement of Some of these linkages with neutral urea 15 linkages has been reported to reduce the tendency of Such BACKGROUND OF THE INVENTION positively charged oligomers towards non-sequence-specific binding (Linkletter, B. A. et al., Bioorg. Med. Chem. 8(8): 1893-901 (2000)). Morpholino oligomers containing (1-pip 1. Technical Field erazino) phosphinylideneoxy and (1-(4-(c)-guanidino-al The present invention is generally related to oligonucle kanoyl))-piperazino) phosphinylideneoxy linkages have otide compounds (oligomers) useful as antisense compounds, been described previously (see e.g., WO2008036127). and more particularly to oligomer compounds comprising Although significant progress has been made, there modified intersubunit linkages and/or terminal groups, and remains a need in the art for oligonucleotide analogues with the use of Such oligomer compounds in antisense applica improved antisense orantigene performance. Such improved tions. antisense or antigene performance includes; stronger affinity 2. Description of the Related Art 25 for DNA and RNA without compromising sequence selectiv Antisense oligomers are generally designed to bind in a ity; improved pharmacokinetics and tissue distribution; sequence specific manner to DNA or RNA regions and alter improved cellular delivery and reliable and controllable in the expression of disease-causing proteins. Requirements for vivo distribution. Successful implementation of antisense therapeutics include BRIEF SUMMARY OF THE INVENTION (a) stability in vivo, (b) sufficient membrane permeability and 30 cellular uptake, and (c) a good balance of binding affinity and Compounds of the present invention address these issues sequence specificity, Many oligonucleotide analogues have and provide improvements over existing antisense molecules been developed in which the phosphodiester linkages of in the art. Modification of the intersubunit linkages and/or native DNA are replaced by other linkages that are resistant to conjugation of terminal moieties to the 5' and/or 3' terminus nuclease degradation (see, e.g., Barawkar, D. A. et al., Proc. of an oligonucleotide analogue, for example a morpholino Natl Acad. Sci. USA95(19): 11047-52 (1998); Linkletter, B. 35 oligonucleotide, results in an antisense oligomer having Supe A. et al., Nucleic Acids Res. 29(11):2370-6 (2001); Mickle rior properties. For example, in certain embodiments the dis field, J., Curr. Med. Chem.8(10):1157-79 (2001)). Antisense closed oligomers have enhanced cell delivery, potency, and/or oligonucleotides having other various backbone modifica tissue distribution compared to other oligonucleotide ana tions have also been prepared (Crooke, S.T., Antisense Drug logues and/or can be effectively delivered to the target organs. Technology. Principles, Strategies, and Applications, New 40 These superior properties give rise to favorable therapeutic York, Marcel Dekker (2001); Micklefield, J., Curr. Med. indices, reduced clinical dosing, and lower cost of goods. Chem. 8(10):1157-79 (2001); Crooke, S.T., Antisense Drug In one embodiment, described herein are compounds com Technology, Boca Raton, CRC Press (2008)). In addition, prising a backbone, the backbone comprising a sequence of oligonucleotides have been modified by peptide conjugation morpholino ring structures joined by intersubunit linkages, in order to enhance cellular uptake (Moulton, H. M. et al., the intersubunit linkages joining a 3'-end of one morpholino Bioconjug Chem 15(2):290-9 (2004); Nelson, M. H. et al., 45 ring structure to a 5'-end of an adjacent morpholino ring Bioconjug. Chem. 16(4):959-66 (2005); Moulton, H. M. et structure, wherein each morpholino ring structure is bound to al., Biochim Biophys Acta (2010)). a base-pairing moiety, Such that the compound can bind in a The performance of Such nucleic acid analogues as anti sequence-specific manner to a target nucleic acid. sense or antigene drugs has been hampered by certain char In one aspect is a compound having the structure of For acteristics of the various analogues. For example, analogues 50 with negatively charged linkages, including phosphorothio mula (I): ate-linked analogues, Suffer from considerable electrostatic repulsion between the negative charges of the oligomer and (I) the DNA or RNA target. The phosphorothioates also exhibit G5 non-specific binding to other cellular components such as 55 proteins. These attributes limit the therapeutic effectiveness of antisense oligomers comprised of native RNA, native DNA, and negatively charged analogues (Crooke, S.T., Anti w=1-x sense Drug Technology. Principles, Strategies, and Applica Y tions, New York, Marcel Dekker (2001); Crooke, S.T., Anti sense Drug Technology, Boca Raton, CRC Press (2008)). The 60 nonionic methylphosphonate-linked oligonucleotide ana logues can be transported into cells by passive diffusion and/ N r or fluid phase endocytosis, but their use is hampered by ste YR 12 reoisomeric complexity and poor solubility (Crooke, S. T., pi Antisense Drug Technology. Principles, Strategies, and 65 Applications, New York, Marcel Dekker (2001); Micklefield, R13 J. Curr, Med, Chem, 8(10):1157-79 (2001)). or a salt or isomer thereof,

US 9,278,987 B2 10 L1 is selected from: ence to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with par ++ ticularity in the appended claims. A better understanding of the features and advantages of the present invention will be 10 obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the prin ciples of the invention are utilized, and the accompanying +--- drawings of which: FIG. 1 illustrates preparation of a linker for solid-phase 15 synthesis; FIG. 2 illustrates preparation of a solid support for oligo +--- mer synthesis; FIG. 3 illustrates solid phase synthesis of oligomers; and FIG. 4 illustrates cleavage of oligomer from Solid Support. + (x + DETAILED DESCRIPTION OF THE INVENTION Definitions 25 In the following description, certain specific details are set ++ forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the invention may be practiced without these details. In 30 other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which fol low, the word “comprise' and variations thereof, such as, --O-- 35 “comprises and “comprising are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” wherein Further, headings provided herein are for convenience only each Q' and Q are each selected from a bond, O or and do not interpret the Scope or meaning of the claimed N(R) ; invention. each E is independently selected from optionally sub 40 Reference throughout this specification to “one embodi stituted aryl or optionally substituted heteroaryl; ment” or “an embodiment’ means that a particular feature, each E is independently an optionally substituted nitro structure or characteristic described in connection with the gen containing heteroaryl; embodiment is included in at least one embodiment. Thus, the each L and L are each independently a bond, option appearances of the phrases "in one embodiment” or “in an ally substituted C-C alkyl, or optionally substituted 45 embodiment in various places throughout this specification heteroalkyl; and are not necessarily all referring to the same embodiment. m, p, q, S. and t are each independently 1-4. Furthermore, the particular features, structures, or character In another embodiment, the present disclosure provides a istics may be combined in any Suitable manner in one or more method of inhibiting production of a protein, the method embodiments. Also, as used in this specification and the comprising exposing a nucleic acid encoding the protein to an 50 appended claims, the singular forms “a,” “an and “the oligomer of the present disclosure. include plural referents unless the content clearly dictates In another embodiment, the disclosure is directed to a otherwise. It should also be noted that the term 'or' is gen method of treating a disease in a subject, the method com erally employed in its sense including “and/or unless the prising administering atherapeutically effective amount of an content clearly dictates otherwise. oligomer. Methods of making the oligomers and methods for 55 The terms below, as used herein, have the following mean their use are also provided. ings, unless indicated otherwise: These and other aspects of the invention will be apparent upon reference to the following detailed description. To this “Amino” refers to the —NH radical. end, various references are set forth herein which describe in “Cyano” or “nitrile” refers to the CN radical. more detail certain background information, procedures, 60 “Hydroxy” or “hydroxyl refers to the –OH radical. compounds and/or compositions, and are each hereby incor “Imino” refers to the –NH Substituent. porated by reference in their entirety. “Guanidinyl refers to the NHC(=NH)NH, substituent. INCORPORATION BY REFERENCE “Amidinyl refers to the C(-NH)NH substituent. 65 “Nitro” refers to the NO radical. All publications, patents, and patent applications men “Oxo’ refers to the –O Substituent. tioned in this specification are herein incorporated by refer “Thioxo’ refers to the –S Substituent. US 9,278,987 B2 11 12 “Cholate” or “CA” refers to the following structure: through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specifi cation, an alkylene chain may be optionally Substituted as described below. "Alkoxy” refers to a radical of the formula—OR, where R. is an alkyl radical as defined. Unless stated otherwise specifi 10 cally in the specification, an alkoxy group may be optionally substituted as described below. “Alkoxyalkyl refers to a radical of the formula—ROR where R is an alkyl radical as defined and where R, is an alkylene radical as defined. Unless stated otherwise specifi 15 cally in the specification, an alkoxyalkyl group may be optionally substituted as described below. "Deoxycholate' or “dCA refers to the following structure: “Alkoxyalkylcarbonyl refers to a radical of the formula —C(=O)ROR where R is an alkyl radical as defined and

where R, is an alkylene radical as defined. Unless stated otherwise specifically in the specification, analkoxyalkylcar bonyl group may be optionally substituted as described below. Alkylcarbonyl refers to a radical of the formula 25 —C(=O)R where R is an alkyl radical as defined above. Unless Stated otherwise specifically in the specification, an alkylcarbonyl group may be optionally Substituted as described below. Alkyloxycarbonyl refers to a radical of the formula 30 —C(=O)CR, where R is an alkyl radical as defined. Unless stated otherwise specifically in the specification, an alkyloxy carbonyl group may be optionally substituted as described below. Representative alkyloxycarbonyl groups include, but Alkyl refers to a straight or branched hydrocarbon chain 35 are not limited to C(=O)CCHCH-OH, -C(=O) radical which is Saturated or unsaturated (i.e., contains one or OCHCHOCHCH-OH, —C(=O) more double and/or triple bonds), having from one to thirty OCHCHOCHCHOCHCH-OH, —C(=O) carbon atoms, and which is attached to the rest of the mol OCHCHOCH, C(=O)CCHCHOCHCHOCH or ecule by a single bond. Alkyls comprising any number of C(=O)CCHCHOCHCHOCHCHOCH, carbon atoms from 1 to 30 are included. An alkyl comprising 40 “Heteroalkylene' refers to an alkylene radical as described up to 30 carbon atoms is referred to as a C-C alkyl, like above where one or more carbon atoms of the alkylene is wise, for example, an alkyl comprising up to 12 carbonatoms replaced with a O, N or S atom. Unless stated otherwise is a C-C alkyl. Alkyls (and other moieties defined herein) specifically in the specification, the heteroalkylene group comprising other numbers of carbon atoms are represented may be optionally substituted as described below. Represen similarily. Alkyl groups include, but are not limited to, C-Co 45 tative heteroalkylene groups include, but are not limited to alkyl, C-Co alkyl, C-C alkyl, C-Co alkyl, C-C alkyl, –OCHCHO , –OCHCHOCHCHO , O C-C alkyl, C-C alkyl, C-C alkyl, C-C alkyl, C-Cs –OCHCHOCHCHOCHCHO alkyl, C-C alkyl and C-Cs alkyl. Representative alkyl Alkylamino” refers to a radical of the formula—NHR, or groups include, but are not limited to, methyl, ethyl, n-propyl. —NRR, where each Ris, independently, an alkyl radical as 1-methylethyl (iso-propyl), n-butyl, i-butyl, S-butyl, n-pentyl, 50 defined above. Unless stated otherwise specifically in the 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, specification, an alkylamino group may be optionally substi ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-di tuted as described below. enyl, ethynyl, propynyl, but-2-ynyl, but-3-ynyl, pentynyl, Amidyl refers to a radical of the formula —N(H)C hexynyl, and the like. Unless stated otherwise specifically in (=O)—R, where R is an alkyl or aryl radical as defined the specification, an alkyl group may be optionally Substi 55 herein. Unless stated otherwise specifically in the specifica tuted as described below. tion, an amidyl group may be optionally Substituted as Alkylene' or “alkylene chain” refers to a straight or described below. branched divalent hydrocarbon chain linking the rest of the Amidinylalkyl refers a radical of the formula—R C molecule to a radical group. Alkylenes may be saturated or (—NH)NH where R is an alkylene radical as defined above. unsaturated (i.e., contains one or more double and/or triple 60 Unless Stated otherwise specifically in the specification, an bonds). Representative alkylenes include, but are not limited amidinylalkyl group may be optionally Substituted as to, C-C alkylene, C-C alkylene, C-C alkylene, C-C, described below. alkylene, C-C alkylene, C-C alkylene, Calkylene. Rep Amidinylalkylcarbonyl refers a radical of the formula resentative alkylene groups include, but are not limited to, —C(=O)R C(=NH)NH where R is an alkylene radical methylene, ethylene, propylene, n-butylene, ethenylene, pro 65 as defined above. Unless stated otherwise specifically in the penylene, n-butenylene, propynylene, n-butynylene, and the specification, an amidinylalkylcarbonyl group may be like. The alkylene chain is attached to the rest of the molecule optionally substituted as described below. US 9,278,987 B2 13 14 Aminoalkyl refers to a radical of the formula —R stated specifically in the specification, a cycloalkyl group NRR, where R, is an alkylene radical as defined above, and may be optionally substituted. each R is independently a hydrogen or an alkyl radical. “Cycloalkylalkyl refers to a radical of the formula—R.R. “Thioalkyl refers to a radical of the formula—SR, where where R, is an alkylene chain as defined above and R is a R is an alkyl radical as defined above. Unless stated other cycloalkyl radical as defined above. Unless stated otherwise wise specifically in the specification, a thioalkyl group may specifically in the specification, a cycloalkylalkyl group may be optionally substituted. be optionally substituted. Aryl refers to a radical derived from a hydrocarbon ring “Cycloalkylcarbonyl refers to a radical of the formula system comprising hydrogen, 6 to 30 carbon atoms and at —C(=O)R where R is a cycloalkyl radical as defined 10 above. Unless stated otherwise specifically in the specifica least one aromatic ring. The aryl radical may be a monocyclic, tion, a cycloalkylcarbonyl group may be optionally Substi bicyclic, tricyclic or tetracyclic ring system, which may tuted. include fused or bridged ring systems. Aryl radicals include, Cycloalkyloxycarbonyl refers to a radical of the formula but are not limited to, aryl radicals derived from the hydro —C(=O)CR where R is a cycloalkyl radical as defined carbon ring systems of aceanthrylene, acenaphthylene, 15 above. Unless stated otherwise specifically in the specifica acephenanthrylene, anthracene, aZulene, benzene, chrysene, tion, a cycloalkyloxycarbonyl group may be optionally Sub fluoranthene, fluorene, as-indacene, S-indacene, indane, stituted. indene, naphthalene, phenalene, phenanthrene, pleiadene, “Fused’ refers to any ring structure described herein which pyrene, and triphenylene. Unless stated otherwise specifi is fused to an existing ring structure. When the fused ring is a cally in the specification, the term “aryl' or the prefix "ar heterocyclyl ring or a heteroaryl ring, any carbonatom on the (such as in “aralkyl) is meant to include aryl radicals that are existing ring structure which becomes part of the fused het optionally substituted. erocyclyl ring or the fused heteroaryl ring may be replaced "Aralkyl” refers to a radical of the formula —R. R. with a nitrogen atom. where R, is an alkylene chain as defined above and R is one “Guanidinylalkyl refers a radical of the formula—R or more aryl radicals as defined above. Examples of aralkyl 25 NHC(=NH)NH where R is an alkylene radical as defined include, but are not limited to, benzyl, diphenylmethyl, trityl above. Unless stated otherwise specifically in the specifica and the like. Unless stated otherwise specifically in the speci tion, aguanidinylalkyl group may be optionally Substituted as fication, an aralkyl group may be optionally Substituted. described below. Arylcarbonyl refers to a radical of the formula—C(=O) “Guanidinylalkylcarbonyl refers a radical of the formula R where R is one or more aryl radicals as defined above, for 30 —C(=O)R NHC(=NH)NH, where R is an alkylene example, phenyl. Unless stated otherwise specifically in the radical as defined above. Unless stated otherwise specifically specification, an arylcarbonyl group may be optionally sub in the specification, a guanidinylalkylcarbonyl group may be stituted. optionally substituted as described below. Aryloxycarbonyl refers to a radical of the formula "Halo’ or “halogen refers to bromo, chloro, fluoro or —C(=O)CR, where R is one or more aryl radicals as 35 iodo. defined above, for example, phenyl. Unless stated otherwise "Haloalkyl refers to an alkyl radical, as defined above, that specifically in the specification, an aryloxycarbonyl group is substituted by one or more halo radicals, as defined above, may be optionally substituted. e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichlo Aralkylcarbonyl refers to a radical of the formula romethyl, 2.2.2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2- —C(=O)R. R., where R is an alkylene chain as defined 40 fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated above and R is one or more aryl radicals as defined above, for otherwise specifically in the specification, a haloalkyl group example, phenyl. Unless stated otherwise specifically in the may be optionally substituted. specification, an aralkylcarbonyl group may be optionally “Perhalo' or “perfluoro' refers to a moiety in which each substituted. hydrogen atom has been replaced by a halo atom or fluorine Aralkyloxycarbonyl refers to a radical of the formula 45 atom, respectively. —C(=O)CR, R where R, is an alkylene chain as defined “Heterocyclyl or "heterocyclic ring refers to a stable 3 above and R is one or more aryl radicals as defined above, for to 24-membered non-aromatic ring radical comprising 2 to 23 example, phenyl. Unless stated otherwise specifically in the carbonatoms and from one to 8 heteroatoms selected from the specification, anaralkyloxycarbonyl group may be optionally group consisting of nitrogen, oxygen, phosphorous and Sul substituted. 50 fur. Unless stated otherwise specifically in the specification, Aryloxy' refers to a radical of the formula—OR where the heterocyclyl radical may be a monocyclic, bicyclic, tricy R is one or more aryl radicals as defined above, for example, clic or tetracyclic ring system, which may include fused or phenyl. Unless stated otherwise specifically in the specifica bridged ring systems; and the nitrogen, carbon or Sulfur atoms tion, an arylcarbonyl group may be optionally Substituted. in the heterocyclyl radical may be optionally oxidized; the “Cycloalkyl refers to a stable, non-aromatic, monocyclic 55 nitrogenatom may be optionally quaternized; and the hetero or polycyclic carbocyclic ring, which may include fused or cyclyl radical may be partially or fully saturated. Examples of bridged ring systems, which is saturated or unsaturated, and such heterocyclyl radicals include, but are not limited to, attached to the rest of the molecule by a single bond. Repre dioxolanyl, thienyl 1.3dithianyl, decahydroisoquinolyl, sentative cycloalkyls include, but are not limited to, cycloak imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidi lyls having from three to fifteen carbon atoms, from three to 60 nyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, ten carbon atoms, from three to eight carbon atoms, from 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, three to six carbon atoms, from three to five carbon atoms, or oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrro three to four carbon atoms. Monocyclic cycloalkyl radicals lidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahy include, for example, cyclopropyl, cyclobutyl, cyclopentyl, drofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thia cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals 65 morpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo include, for example, adamantyl, norbornyl, decalinyl, and thiomorpholinyl, 12-crown-4,15-crown-5,18-crown-6.21 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise crown-7. aza-18-crown-6, diaza-18-crown-6, aza-21-crown US 9,278,987 B2 15 16 7, and diaza-21-crown-7. Unless stated otherwise specifically —OC(=O)NRR —OR —SR, -SOR. -SO.R. in the specification, a heterocyclyl group may be optionally OSO.R. S.O.OR =NSO.R. and -SO.NRR, substituted. “Substituted also means any of the above groups in which “Heteroaryl” refers to a 5- to 14-membered ring system one or more hydrogen atoms are replaced with —C(=O)R. radical comprising hydrogen atoms, one to thirteen carbon C(=O)CR —CHSO.R. —CHSO.NRR, SH, atoms, one to six heteroatoms selected from the group con —SR, or—SSR. In the foregoing, RandR, are the same or sisting of nitrogen, oxygen, phosphorous and Sulfur, and at different and independently hydrogen, alkyl, alkoxy, alky least one aromatic ring. For purposes of this invention, the lamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl radical may be a monocyclic, bicyclic, tricyclic or haloalkyl, heterocyclyl N-heterocyclyl, heterocyclylalkyl, tetracyclic ring system, which may include fused or bridged 10 heteroaryl, N-heteroaryl and/or heteroarylalkyl. In addition, ring systems; and the nitrogen, carbon or Sulfur atoms in the each of the foregoing Substituents may also be optionally heteroaryl radical may be optionally oxidized; the nitrogen substituted with one or more of the above substituents. Fur atom may be optionally quaternized. Examples include, but thermore, any of the above groups may be substituted to are not limited to, azepinyl, acridinyl, benzimidazolyl, ben include one or more internal oxygen or Sulfur atoms. For Zothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, ben 15 example, an alkyl group may be substituted with one or more Zooxazolyl, benzothiazolyl, benzothiadiazolyl, benzob.1, internal oxygen atoms to form an ether or polyether group. 4 dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl. Similarily, an alkyl group may be substituted with one or benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl. more internal sulfur atoms to form a thioether, disulfide, etc. benzopyranonyl, benzofuranyl, benzofuranonyl, benzothie Amidyl moieties may be substituted with up to 2 halo atoms, nyl (benzothiophenyl), benzotriazolyl, benzo4.6imidazol, while other groups above may be substituted with one or more 2-alpyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, diben halo atoms. With the exception of alkyl groups, all other Zothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, groups may also be substituted with amino or monoalk indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindoli lyamino. With the exception of alkyl and alkylcarbonyl nyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, Oxa groups, all other groups may also be substituted with guanidi diazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridi 25 nyl or amidynyl. Optional substitutents for any of the above nyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, groups also include arylphosphoryl, for example—RP(Ar) 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl phenazinyl, phe wherein Ra is an alkylene and Ar is aryl moiety, for example nothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, phenyl. pyrrolyl pyrazolyl pyridinyl, pyrazinyl, pyrimidinyl, The terms “antisense oligomer' or “antisense compound pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinu 30 are used interchangeably and refer to a sequence of subunits, clidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thia each having a base carried on a backbone subunit composed diazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., of ribose or other pentose sugar or morpholino group, and thienyl). Unless stated otherwise specifically in the specifi where the backbone groups are linked by intersubunit link cation, a heteroaryl group may be optionally Substituted. ages that allow the bases in the compound to hybridize to a All the above groups may be either substituted or unsub 35 target sequence in a nucleic acid (typically an RNA) by Wat stituted. The term “substituted as used herein means any of son-Crick base pairing, to form a nucleic acid:oligomer het the above groups (i.e., alkyl, alkylene, alkoxy, alkoxyalkyl, eroduplex within the target sequence. The oligomer may have alkylcarbonyl, alkyloxycarbonyl, alkylamino, amidyl, amidi exact sequence complementarity to the target sequence or nylalkyl, amidinylalkylcarbonyl, aminoalkyl, aryl, aralkyl, near complementarity. Such antisense oligomers are arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl, aralkyloxy 40 designed to block or inhibit translation of the mRNA contain carbonyl, aryloxy, cycloalkyl, cycloalkylalkyl, cycloalkylcar ing the target sequence, and may be said to be "directed to a bonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, sequence with which it hybridizes. guanidinylalkyl, guanidinylalkylcarbonyl, haloalkyl, hetero A “morpholino oligomer' or “PMO' refers to a polymeric cyclyl and/or heteroaryl), may be further functionalized molecule having a backbone which supports bases capable of wherein at least one hydrogen atom is replaced by a bond to 45 hydrogen bonding to typical polynucleotides, wherein the a non-hydrogen atom Substituent. Unless stated specifically polymer lacks a pentose Sugar backbone moiety, and more in the specification, a Substituted group may include one or specifically a ribose backbone linked by phosphodiester more substituents selected from: Oxo, —COH, nitrile, nitro, bonds which is typical of nucleotides and nucleosides, but hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl, alky instead contains a ring nitrogen with coupling through the lcarbonyl, alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl, ary 50 ring nitrogen. An exemplarymorpholino' oligomer com loxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy, prises morpholino subunit structures linked together by (thio) cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl, cycloalkyla phosphoramidate or (thio)phosphorodiamidate linkages, lkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl, het joining the morpholino nitrogen of one subunit to the 5' exo eroaryl, dialkylamines, arylamines, alkylarylamines, diary cyclic carbon of an adjacent Subunit, each Subunit comprising lamines, trialkylammonium (—NR), N-oxides, imides, and 55 a purine or pyrimidine base-pairing moiety effective to bind, enamines; a silicon atom in groups such as trialkylsilyl by base-specific hydrogen bonding, to a base in a polynucle groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, triar otide. Morpholino oligomers (including antisense oligomers) ylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for are detailed, for example, in U.S. Pat. Nos. 5,698,685: 5,217, example, trifluoromethyl or trifluoromethoxy. “Substituted 866; 5,142,047; 5,034,506; 5,166,315; 5,185,444; 5,521,063: also means any of the above groups in which one or more 60 5,506,337 and pending U.S. patent application Ser. Nos. hydrogen atoms are replaced by a higher-order bond (e.g., a 12/271,036: 12/271,040; and PCT publication number double- or triple-bond) to a heteroatom such as oxygen in WO/2009/064471 all of which are incorporated herein by oXo, carbonyl, carboxyl, and ester groups; and nitrogen in reference in their entirety. groups such as imines, OXimes, hydrazones, and nitriles. For “PMO-- refers to phosphorodiamidate morpholino oligo example, “substituted includes any of the above groups in 65 mers comprising any number of (1-piperazino)phosphi which one or more hydrogen atoms are replaced with nylideneoxy, (1-(4-(c)-guanidino-alkanoyl))-piperazino) NRC(=O)NRR, NRC(=O)CR - NRSO.R. phosphinylideneoxy linkages that have been described US 9,278,987 B2 17 18 previously (see e.g., PCT publication WO/2008/036127 when placed together, e.g., from opposite ends of the analog, which is incorporated herein by reference in its entirety. constitute sequence that spans the target sequence. “PMO-X' refers to phosphorodiamidate morpholino oli Target and targeting sequences are described as “comple gomers disclosed herein. mentary to one another when hybridization occurs in an A phosphoramidate group comprises phosphorus having antiparallel configuration. A targeting sequence may have three attached oxygenatoms and one attached nitrogenatom, “near or “substantial complementarity to the target while a “phosphorodiamidate group comprises phosphorus sequence and still function for the purpose of the presently having two attached oxygenatoms and two attached nitrogen described methods, that is, still be “complementary.” Prefer atOmS. ably, the oligonucleotide analog compounds employed in the “Thiophosphoramidate' or “thiophosphorodiamidate' 10 linkages are phosphoramidate or phosphorodiamidate link presently described methods have at most one mismatch with ages, respectively, wherein one oxygen atom, typically the the target sequence out of 10 nucleotides, and preferably at oxygen pendant to the backbone, is replaced with Sulfur. most one mismatch out of 20. Alternatively, the antisense “Intersubunit linkage” refers to the linkage connecting two oligomers employed have at least 90% sequence homology, morpholino subunits. 15 and preferably at least 95% sequence homology, with the “Charged”, “uncharged”, “cationic' and “anionic” as used exemplary targeting sequences as designated herein. For pur herein refer to the predominant state of a chemical moiety at poses of complementary binding to an RNA target, and as near-neutral pH, e.g., about 6 to 8. For example, the term may discussed below, a guanine base may be complementary to refer to the predominant state of the chemical moiety at physi either a cytosineor uracil RNA base. ological pH, that is, about 7.4. A "heteroduplex’ refers to a duplex between an oligoncu “Lower alkyl refers to an alkyl radical of one to six carbon leotide analog and the complementary portion of a target atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-bu RNA. A “nuclease-resistant heteroduplex’ refers to a hetero tyl, isoamyl. n-pentyl, and isopentyl. In certain embodiments, duplex formed by the binding of an antisense oligomer to its a “lower alkyl group has one to four carbon atoms. In other complementary target, Such that the heteroduplex is Substan embodiments a “lower alkyl group has one to two carbon 25 tially resistant to in vivo degradation by intracellular and atoms; i.e. methyl or ethyl. Analogously, “lower alkenyl extracellular nucleases, such as RNAse H, which are capable refers to an alkenyl radical of two to six, preferably three or of cutting double-stranded RNA/RNA or RNA/DNA com four, carbon atoms, as exemplified by allyl and butenyl. plexes. A “non-interfering substituent is one that does not An agent is “actively taken up by mammalian cells' when adversely affect the ability of an antisense oligomer as 30 the agent can enter the cell by a mechanism other than passive described herein to bind to its intended target. Such substitu diffusion across the cell membrane. The agent may be trans ents include small and/or relatively non-polar groups such as ported, for example, by “active transport, referring to trans methyl, ethyl, methoxy, ethoxy, or fluoro. port of agents across a mammalian cell membrane by e.g. an An oligonucleotide or antisense oligomer “specifically ATP-dependent transport mechanism, or by “facilitated hybridizes to a target polynucleotide if the oligomer hybrid 35 transport, referring to transport of antisense agents across izes to the target under physiological conditions, with a Tm the cell membrane by a transport mechanism that requires greater than 37°C., greater than 45°C., preferably at least 50° binding of the agent to a transport protein, which then facili C., and typically 60° C.-80° C. or higher. The “Tm” of an tates passage of the bound agent across the membrane. oligomer is the temperature at which 50% hybridizes to a The terms “modulating expression' and/or “antisense complementary polynucleotide. Tm is determined under 40 activity” refer to the ability of an antisense oligomer to either standard conditions in physiological saline, as described, for enhance or, more typically, reduce the expression of a given example, in Miyada et al., Methods Enzymol. 154:94-107 protein, by interfering with the expression or translation of (1987). Such hybridization may occur with “near or “sub RNA. In the case of reduced protein expression, the antisense stantial” complementary of the antisense oligomer to the oligomer may directly block expression of a given gene, or target sequence, as well as with exact complementarity. 45 contribute to the accelerated breakdown of the RNA tran Polynucleotides are described as “complementary' to one scribed from that gene. Morpholino oligomers as described another when hybridization occurs in an antiparallel configu herein are believed to act via the former (steric blocking) ration between two single-stranded polynucleotides. mechanism. Preferred antisense targets for steric blocking Complementarity (the degree that one polynucleotide is oligomers include the ATG start codon region, splice sites, complementary with another) is quantifiable in terms of the 50 regions closely adjacent to splice sites, and 5'-untranslated proportion of bases in opposing strands that are expected to region of mRNA, although other regions have been Success form hydrogen bonds with each other, according to generally fully targeted using morpholino oligomers. accepted base-pairing rules. An "amino acid subunit is preferably an O-amino acid A first sequence is an “antisense sequence' with respect to residue (—CO CHR NH ); it may also be a B- or other a second sequence if a polynucleotide whose sequence is the 55 amino acid residue (e.g. —CO CHCHR NH ), where first sequence specifically binds to, or specifically hybridizes R is an amino acid side chain. with, the second polynucleotide sequence under physiologi The term “naturally occurring amino acid refers to an cal conditions. amino acid present in proteins found in nature. The term The term “targeting sequence' is the sequence in the oli “non-natural amino acids’ refers to those amino acids not gonucleotide analog that is complementary (meaning, in 60 present in proteins found in nature; examples include beta addition, Substantially complementary) to the target sequence alanine (B-Ala) and 6-aminohexanoic acid (Ahk). in the RNA genome. The entire sequence, or only a portion, of An “effective amount’ or “therapeutically effective the analog compound may be complementary to the target amount refers to an amount of antisense oligomer adminis sequence. For example, in an analog having 20 bases, only tered to a mammalian Subject, either as a single dose or as part 12-14 may be targeting sequences. Typically, the targeting 65 of a series of doses, which is effective to produce a desired sequence is formed of contiguous bases in the analog, but may therapeutic effect, typically by inhibiting translation of a alternatively be formed of non-contiguous sequences that selected target nucleic acid sequence. US 9,278,987 B2 19 20 “Treatment of an individual (e.g. a mammal. Such as a human) or a cell is any type of intervention used in an attempt (I) to alter the natural course of the individual or cell. Treatment G5 includes, but is not limited to, administration of a pharmaceu tical composition, and may be performed either prophylacti cally or Subsequent to the initiation of a pathologic event or 5 w=1-x contact with an etiologic agent. Y A “tautomer refers to a proton shift from one atom of a O B molecule to another atom of the same molecule. The com pounds presented herein may exist as tautomers. Tautomers 10 r are compounds that are interconvertible by migration of a N hydrogenatom, accompanied by a Switch of a single bond and YR 12 adjacent double bond. In bonding arrangements where tau pi tomerization is possible, a chemical equilibrium of the tau R13 tomers will exist. All tautomeric forms of the compounds 15 disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, Solvent, and pH. Some examples of tautomeric interconver sions include: or a salt or isomer thereof, X re- vs.OH x H NH res- w (2.

V N H

N H N N N. ss N - 2 \, s-s 2 \, N N z NH NN N^ NN M H N HNN/ NS/

Antisense Oligomers wherein: 50 n is an integer from 1 to 50: A. Oligomers with Modified Intersubunit Linkages G is halogen, OH, alkoxy, OSO,(alkyl), OSO,(aryl), or As noted above, one embodiment of the present disclosure is directed to oligomers comprising novel intersubunit link ages. In some embodiments, the oligomers have higher affin ity for DNA and RNA than do the corresponding unmodified 55 oligomers and demonstrate improved cell delivery, potency, and/or tissue distribution properties compared to oligomers r having other intersubunit linkages. The structural features and properties of the various linkage types and oligomers are 60 described in more detail in the following discussion. -- each B is an independently selected base pair moiety; Applicants have found that enhancement of antisense each Y is independently O or NR': optionally, R'' and activity, biodistribution and/or other desirable properties can X8e are bonded together form a ring: be optimized by preparing oligomers having various inter 65 each W is independently S or O: Subunit linkages. In one aspect is a compound having the Z is -(L)-(R'), -(L)-(L)-(R'); or (L)-(L'?)- structure of Formula (I): (R'7);

US 9,278,987 B2 27 28 -continued embodiments is a compound of Formula (I), wherein n is 1. c) G is halogen, Wis O.Y is O. R' is an optionally substituted p triphenylmethyl group, and X is X4. In some embodiments is a compound of Formula (I), wherein n is 1, G is halogen, W is O, Y is O. R' is an optionally substituted triphenylmethyl g group, and X is X5. In some embodiments is a compound of d) Formula (I), whereinn is 1, G is halogen, Wis O.Y is O. R' p S is an optionally substituted triphenylmethyl group, and X is X6. In some embodiments is a compound of Formula (I), g 10 wherein n is 1, G is halogen, W is O, Y is O. R' is an e) optionally substituted triphenylmethyl group, and X is X7. In p Some embodiments is a compound of Formula (I), wherein n is 1, G is halogen, W is O, Y is O. R' is an optionally substituted triphenylmethyl group, and X is X8. -- g s 15 In another embodiment is a compound of Formula (I) f) wherein G is -(L')-(L')-(R'). In another embodiment is a compound of Formula (I) wherein G is -(L')-(R'). In another embodiment is a compound of Formula (I) wherein ----O-- , and G is -(L)-(L'?)-(R17). g) In another embodiment is a compound of Formula (I) ---O-- wherein n is an integer from 1 to 35 and G is 25 wherein each Q' and Q are each selected from a bond, O or N(R) ; r each E' is independently selected from optionally sub stituted aryl or optionally substituted heteroaryl; 30 each E is independently an optionally substituted nitro -- gen containing heteroaryl: each L and L are each independently a bond, option In another embodiment is a compound of Formula (I) wherein ally substituted C-C alkyl, or optionally substituted n is an integer from 1 to 35 and W is O. In another embodi heteroalkyl; and 35 m, p, q, S. and t are each independently 1-4. ment is a compound of Formula (I) wherein n is an integer In some embodiments of Formula (I), Zis-(L')-(R'). In from 1 to 35 and Y is O. In another embodiment is a com other embodiments, Z is -(L')-(L)-(R'). In another pound of Formula (I) wherein n is an integer from 1 to 35, G embodiments, Z is -(L)-(R'). In a further embodiment of Formula (I), each Y is independently O, NH, or NR''. In a 40 further embodiment of the aforementioned embodiments, each W is O. In yet a further embodiment, each R" is inde pendently hydrogen or alkyl. In a further embodiment, each R’ is independently C-C alkyl. In yet a further embodi ment, each X is independently selected from X1,X2, X3, X4, 45 X5, X6, X7, or X8. r In one embodiment is a compound of Formula (I), wherein Z is -(L')-(R'), each Y is independently O, NH, or NR', each Wis O, each R" is independently hydrogen oralkyl, each -- R’ is independently C-C alkyl, each X is independently 50 selected from X1,X2, X3, X4, X5, X6, X7, or X8 and n is an each Wis O., each Y is O, and each X is independently selected integer from 1 to 35. from X1, X2, X3, X4, X5, X6, X7, or X8. In another embodiment is a compound of Formula (I), In another embodiment is a compound of Formula (I) wherein n is 1, Z is -(L')-(R'), each Y is independently O, wherein n is an integer from 1 to 35, G is NH, or NR', each Wis O, each R' is independently hydrogen 55 or alkyl, each R is independently C-C alkyl, and X is selected from X2, X3, X4, X5, X6, X7, or X8. In some embodiments of Formula (I) whereinn is 1, G is halogen. In another embodiment, Wis O. In another embodiment, Y is O. In another embodiment, R' is aralkyl. In another embodi 60 ment R' is an optionally substituted triphenylmethyl group. r In some embodiments is a compound of Formula (I), wherein n is 1, G is halogen, W is O, Y is O. R' is an optionally substituted triphenylmethyl group, and X is X2. In -- Some embodiments is a compound of Formula (I), wherein n 65 is 1, G is halogen, W is O, Y is O. R' is an optionally each W is O, each Y is O, and at least one X is X2. In some substituted triphenylmethyl group, and X is X3. In some embodiments, at least one X2 is selected from —O-alkylene US 9,278,987 B2 29 30 CO.H. —O-alkylene-CHN, N(R')-alkylene-COH, and alkenylene-COH, -L1-CO-alkenylene-CHN, -L1-CO —N(R)-alkylene-CHN. In some embodiments, at least one arylene-COH, and -L1-CO-arylene-CHN, wherein L1 is X2 is —O-alkylene-CO2H. In some embodiments, at least one X2 is —O-alkylene-CHN. In some embodiments, at least one X2 is N(R)-alkylene-COH. In some embodi R6 ments, at least one X2 is N(R)-alkylene-CHN. In some embodiments, at least one X2 is selected from —O-alkylene COH, -O-alkylene-CHN - N(R)-alkylene-COH, and —N(R)-alkylene-CHN, wherein -alkylene- is —CH2—, —CH2CH2—, or —CH2CH2CH2—. In further embodi 10 and —alkylene- is —CH2—, —CHCH , or ments, at least one X2 is selected from —N(H)CHCO.H. —CH2CH2CH2—. - N(CH)CHCOH, - N(CHCH)CHCOH, -N(H) In another embodiment is a compound of Formula (I) CHCH-COH, and —N(CH)CH-CHCO2H. In some wherein n is an integer from 1 to 35, G is embodiments, at least one X2 is N(H)CHCO2H. In some 15 embodiments, at least one X2 is —N(CH)CHCO2H. In some embodiments, at least one X2 is N(CH2CH) CHCO2H. In some embodiments, at least one X2 is N(H) CHCH-COH. In some embodiments, at least one X2 is —N(CH)CH-CHCO2H. In further embodiments, at least r one X2 is selected from -L1-CO-alkylene-COH, -L1-CO alkylene-CHN, -L1-CO-alkenylene-COH, -L1-CO-alk enylene-CHN, -L1-CO-arylene-COH, and -L1-CO -- arylene-CHN. In some embodiments, at least one X2 is -L1-CO-alkylene-COH. In some embodiments, at least one 25 X2 is -L1-CO-alkylene-CHN. In some embodiments, at each W is O, each Y is O, at least one X is X2 and X2 is least one X2 is -L1-CO-alkenylene-CO2H. In some embodi selected from: ments, at least one X2 is -L1-CO-alkenylene-CHN. In some embodiments, at least one X2 is -L1-CO-arylene-COH. In some embodiments at least one X2 is L1-CO-arylene-CHN 30 In further embodiments, at least one X2 is selected from -L1-CO-alkylene-COH, -L1-CO-alkylene-CHN, -L1-CO -- N O alkenylene-COH, -L1-CO-alkenylene-CHN, -L1-CO arylene-COH, and -L1-CO-arylene-CHN, wherein L1 is 35 -- N O

40 In yet further embodiments, at least one X2 is selected from -L1-CO-alkylene-COH, -L1-CO-alkylene-CHN, -L1-CO alkenylene-COH, -L1-CO-alkenylene-CHN -L1-CO arylene-COH, and -L1-CO-arylene-CHN, wherein L1 is 45 +OR O

50 and -alkylene- is —CH2—, —CH2CH2—, or —CH2CH2CH2—. In another embodiment, at least one X2 is selected from -L1-CO-alkylene-COH, -L1-CO-alkylene CHN, -L1-CO-alkenylene-COH, -L1-CO-alkenylene 55 +OR CHN, -L1-CO-arylene-COH, and -L1-CO-arylene-CHN In some embodiments, at least one X2 is wherein L1 is

60 -- N O

65 In another embodiment, at least one X2 is selected from -L1-CO-alkylene-COH, -L1-CO-alkylene-CHN, -L1-CO US 9,278,987 B2 31 32 In some embodiments, at least one X2 is -continued

A V O N N -- N N O

10 N N1 In some embodiments, at least one X2 is

15 O O OH. O O O)- V

In some embodiments, at least one X2 is 25

30 In some embodiments, at least one X2 is --O- 35 -- O

In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 40

In some embodiments, at least one X2 is r 45 -- -- \ /N O 50 each W is O, each Y is O, at least one X is X2 and X2 is selected from: N NH. 55 N 1 -- O In some embodiments, at least one X2 is

65 C - c. US 9,278,987 B2 33 34 In some embodiments, at least one X2 is

SN

and -alkylene is —CH2—, —CH2CH2—, or —CHCHCH . In another embodiment is a compound of Formula (I) 10 In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is wherein n is an integer from 1 to 35, G is r 15 r -- -- each W is O, each Y is O, at least one X is X2, and X2 is selected from-L1-CONH-alkylene-COH, -L1-CONH-alky each W is O, each Y is O, at least one X is X2 and X2 is lene-CHN - L1-CONH-arylene-COH, and -L1-CONH 25 selected from: arylene-CHN. In some embodiments, X2 is -L1-CONH alkylene-COH. In some embodiments, X2 is -L1-CONH alkylene-CHN. In some embodiments, X2 is -L1-CONH arylene-COH. In some embodiments, X2 is -L1-CONH / \ arylene-CHN. In another embodiment, X2 is selected from 30 -L1-CONH-alkylene-COH, -L1-CONH-alkylene-CHN -- \ /N-( N -L1-CONH-arylene-COH, and -L1-CONH-arylene-CHN wherein L1 is

35 / \ O --O-- -- \ /N-( N O 40 \ { In another embodiment, X2 is selected from -L1-CONH alkylene-COH, -L1-CONH-alkylene-CHN -L1-CONH arylene-COH, and -L1-CONH-arylene-CHN, wherein L1 45 --O- \ / --( )-- 50 and -alkylene- is —CH2—, —CH2CH2—, or —CHCHCH . In another embodiment, X2 is selected from -L1-CONH-alkylene-COH, -L1-CONH-alkylene CHN, -L1-CONH-arylene-COH, and -L1-CONH-arylene --O- 55 CHN, wherein L1 is In some embodiments, at least one X2 is

60

In another embodiment, X2 is selected from -L1-CONH alkylene-COH, -L1-CONH-alkylene-CHN -L1-CONH 65 +C O arylene-COH, and -L1-CONH-arylene-CHN, wherein L1 is US 9,278,987 B2 35 36 In some embodiments, at least one X2 is

O In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is OH. 10 In some embodiments, at least one X2 is ... or -- 2O each W is O, each Y is O, at least one X is X2 and X2 is selected from: In some embodiments, at least one X2 is

O 25

OH. () O O In another embodiment is a compound of Formula (I) is N Y \e wherein n is an integer from 1 to 35, G is w

Z5 O B, 40 N N \; N NYN s -- 45 each W is O, each Y is O, at least one X is X2, and X2 is selected from -L1-SO-alkylene-COH, -L1-SO-alkylene CHN, -L1-SO-arylene-COH, and -L1-SO-arylene- 50 CHN. In some embodiments, X2 is -L1-SO-alkylene COH. In some embodiments, X2 is L1-SO-alkylene CHN. In some embodiments, X2 is -L1-SO-arylene-COH. In some embodiments, X2 is -L1-SO-arylene-CHN. In another embodiment, X2 is selected from-L1-SO-alkylene- 55 COH, -L1-SO-alkylene-CHN -L1-SO-arylene-COH, and -L1-SO-arylene-CHN, wherein L1 is

--O--\ / 60

In another embodiment, X2 is selected from -L1-SO-alky- 65 lene-COH, -L1-SO-alkylene-CHN, -L1-SO-arylene COH, and -L1-SO-arylene-CHN, wherein L1 is US 9,278,987 B2 37 38 -continued In some embodiments, at least one X2 is O

OH, and O O OS!f 5 OSl K) ( NO)- NHM -- O)- NHM OH. 10 In some embodiments, at least one X2 is N1 YN \ 11 N of NN OS!f 15 S. V : NO)- NHM In some embodiments, at least one X2 is In some embodiments, at least one X2 is O OH. V O 25 N 'N S1 OSlO -- O)- NHA 30 OH. O In some embodiments, at least one X2 is

In some embodiments, at least one X2 is 35 N1 YN. \ Ni? V O N N. Y.2 OSlO 40 --O- EN In another embodiment is a compound of Formula (I) N \; 45 wherein n is an integer from 1 to 35, G is NYN In some embodiments, at least one X2 is 50 r

55 -- each W is O, each Y is O, at least one X is X2, and X2 is selected from -L1-alkylene-COH, -L1-alkylene-CHN -L1-arylene-COH, and -L1-arylene-CHN. In some 60 embodiments, X2 is -L1-alkylene-COH. In some embodi ments, X2 is -L1-alkylene-CHN. In some embodiments, X2 is -L1-arylene-CO2H. In some embodiments, X2 is -L1 arylene-CHN. In some embodiments, X2 is selected from -L1-alkylene-COH, -L1-alkylene-CHN, -L1-arylene 65 COH, and -L1-arylene-CHN. In another embodiment, X2 is selected from -L1-alkylene-COH, -L1-alkylene-CHN -L1-arylene-COH, and -L1-arylene-CHN, wherein L1 is US 9,278,987 B2 39 40 -continued

In another embodiment, X2 is selected from -L1-alkylene COH, -L1-alkylene-CHN -L1-arylene-COH, and -L1 arylene-CHN, wherein L1 is --O s --O-- and -alkylene- is —CH2—, —CH2CH2—, or —CHCHCH . In another embodiment, X2 is selected from -L1-alkylene-COH, -L1-alkylene-CHN, L1-arylene COH, and -L1-arylene-CHN, wherein L1 is OH, and --O- In another embodiment, X2 is selected from -L1-alkylene COH, -L1-alkylene-CHN, -L1-arylene-COH, and -L1 30 O)- V arylene-CHN, wherein L1 is 35 In some embodiments, at least one X2 is --O- --O and -alkylene- is —CH2—, —CH2CH2—, or —CH2CH2CH2—. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is In some embodiments, at least one X2 is r -- In some embodiments, at least one X2 is each W is O, each Y is O, at least one X is X2 and X2 is selected from: 60 -- N

--O 65 US 9,278,987 B2 41 42 In some embodiments, at least one X2 is -continued N N 2 N. NH. f --O-(S RN 10 In some embodiments, at least one X2 is In some embodiments, at least one X2 is

15 --O- OH. --O-(\ / )-(OH. In some embodiments, at least one X2 is In some embodiments, at least one X2 is

OH. 25 N N \ /

In some embodiments, at least one X2 is --O-( O 30

In another embodiment is a compound of Formula (I) OH. wherein n is an integer from 1 to 35, G is 35

40 r In some embodiments, at least one X2 is

-- 45 s NH. N each W is O, each Y is O, at least one X is X2 and X2 is le N selected from:

50

55 In any of the aforementioned embodiments of Formula (I) wherein at least one X is X2 is another embodiment wherein O n is an integer from 30-35. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least 60 one X is X2, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X2, n is an integer from 20-24. In further embodiments of any of the aforementioned embodi ments of Formula (I) wherein at least one X is X2, n is an 65 integer from 15-19. In further embodiments of the aforemen tioned embodiments of Formula (I) wherein at least one X is X2, n is an integer from 10-14. In further embodiments of the US 9,278,987 B2 43 44 aforementioned embodiments of Formula (I) wherein at least -continued one X is X2, n is an integer from 5-9. In yet further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X2, n is an integer from 1-4. In another embodiment is a compound of Formula (I) 5 O-( --O- wherein n is an integer from 1 to 35, G is O)- CN, and r 10 O)- ( 15 -- In some embodiments, at least one X is X3 and X3 is each W is O, each Y is O, and at least one X is X3. In further embodiments at least one X3 is selected from -L1-alkyl and -L1-heterocyclyl. In some embodiments at least one X3 is -L1-alkyl. In yet another embodiment, at least one X3 is --\ /Y -L1-alkyl and L1 is In some embodiments, at least one X is X3 and X3 is 25

In another embodiment, at least one X3 is-L1-alkyl and L1 is 30

In some embodiments, at least one X is X3 and X3 is --O- 35 In another embodiment, at least one X3 is-L1-alkyl and L1 is --O-( 40 --O – In come embodiments, at least one X is X3 and X3 is In a further embodiment of the aforementioned embodiments wherein at least one X is X3, alkyl is methyl, ethyl, propyl. 45 and isopropyl. In another embodiment, alkyl is CF. In another embodiment, alkyl is CN. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 50 In some embodiments, at least one X is X3 and X3 is

r 55 -- each W is O, each Y is O, at least one X is X3 and X3 is In some embodiments, at least one X is X3 and X3 is selected from: 60

N s-/ -- \ /N \ / 65 --O-( US 9,278,987 B2 45 46 In another embodiment is a compound of Formula (I) each W is O, each Y is O, at least one X is X3 and X3 is wherein n is an integer from 1 to 35, G is selected from: r 5 --O-O. -- 10 each W is O, each Y is O, at least one X is X3, and X3 is 15 -L1-heterocyclyl. In yet another embodiment, at least one X3 is -L1-heterocyclyl and L1 is

and

In another embodiment, at least one X3 is -L1-heterocyclyl 25 and L1 is

In some embodiments, at least one X is X3 and X3 is

30 --O-O. In another embodiment, at least one X3 is -L1-heterocyclyl 35 and L1 is In some embodiments, at least one X is X3 and X3 is

--O-- 40

In another embodiment of the aforementioned embodiments In some embodiments, at least one X is X3 and X3 is wherein at least one X is X3, heterocyclyl is pyrrolidine, 45 tetrahydrofuran, piperidine, morpholine, piperazine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, pyridine, and pyrimidine. In a further embodiment, heterocy clyl is pyrrolidine. In a further embodiment, heterocyclyl is -- \ / -O piperidine. In a further embodiment, heterocyclyl is morpho 50 line. In a further embodiment, heterocyclyl is piperazine. In a further embodiment, heterocyclyl is pyridine. In a further In some embodiments, at least one X is X3 and X3 is embodiment, heterocyclyl is pyrimidine. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 55 r In some embodiments, at least one X is X3 and X3 is -- 65

US 9,278,987 B2 49 50 In some embodiments, at least one X is X3 and X3 is In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

H N 1n-N. 5 H

In some embodiments, at least one X is X3 and X3 is r 10

H -- N 1n-N. each W is O, each Y is O, at least one X is X3, and X3 is 15 selected from -L1-CNH NH, -L1-alkylene-CNH NH, -L1-arylene-CNH NH, -L1-alkylene-N(R'). -L1 arylene-N(R'). -L1-alkylene-N(R), -L1-arylene-N(R), In some embodiments, at least one X is X3 and X3 is -L1-alkylene-heterocyclyl, -L1-arylene-heterocyclyl, -L1 alkylene-N(O)(R), -L1-arylene-N(O)(R), -L1-alkylene NH CNH NH, and -L1-arylene-NH CNH-NH. In a further embodiment, at least one X3 is selected from -L1 N 1n-N. arylene-CNH NH, -L1-arylene-N(R'). -L1-arylene-N H (R), -L1-arylene-heterocyclyl, -L1-arylene-N(O)(R), and -L1-arylene-NH CNH NH. In some embodiments, at least one X3 is -L1-arylene-CNH NH. In some embodi 25 ments, at least one X3 is -L1-arylene-N(R'). In some In some embodiments, at least one X is X3 and X3 is embodiments, at least one X3 is -L1-arylene-N(R). In some embodiments, at least one X3 is -L1-arylene-heterocyclyl. In Some embodiments, at least one X3 is -L1-arylene N(O)(R). In some embodiments, at least one X3 is -L1 1N1 N. 30 arylene-NH CNH NH. In a further embodiment of the N aforementioned embodiments, L1 is

In some embodiments, at least one X is X3 and X3 is 35

1. N H In yet a further embodiment of the aforementioned embodi 1-n 40 ments, L1 is

In some embodiments, at least one X is X3 and X3 is R6 45 1-n 1. In another embodiment is a compound of Formula (I) 50 wherein n is an integer from 1 to 35, G is In some embodiments, at least one X is X3 and X3 is

1N-1N1 55 r H / -- In some embodiments, at least one X is X3 and X3 is 60 each W is O, each Y is O, at least one X is X3, and X3 is selected from -L1-CNH NH, -L1-alkylene-CNH NH -L1-alkylene-N(R'). -L1-alkylene-N(R), -L1-alkylene heterocyclyl, -L1-alkylene-N(O)(R), and -L1-alkylene 1N1-1 NH CNHI NH2. In a further embodiment, at least one X3 / 65 is selected from -L1-CNH NH, -L1-alkylene-CNH NH, and -L1-alkylene-NH CNH NH2. In yet a further embodiment, at least one X3 is selected from -L1-alkylene US 9,278,987 B2 51 52 N(R'). -L1-alkylene-N(R), -L1-alkylene-heterocyclyl, In some embodiments, at least one X3 is and -L1-alkylene-N(O)(R). In some embodiments, at least one X3 is -L1-CNH NH. In some embodiments, at least one X3 is -L1-alkylene-CNH NH. In some embodiments, at least one X3 is -L1-alkylene-NH CNH NH. In some 5 embodiments, at least one X3 is -L1-alkylene-N(R'). In some embodiments, at least one X3 is -L1-alkylene-N(R). In some embodiments, at least one X3 is -L1-alkylene-het erocyclyl. In some embodiments, at least one X3 is -L1 alkylene-N(O)(R). In a further embodiment of the afore 10 In some embodiments, at least one X3 is mentioned embodiments, L1 is NH -- \ /N

In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is In another embodiment of the aforementioned embodiments,

25 r --O- -- 30 In yet a further embodiment of the aforementioned embodi each W is O, each Y is O, at least one X is X3 and X3 is ments, -alkylene- is —CH2CH2— or —CH2CH2CH2—. In selected from Some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. In some embodiments of the aforementioned embodiments, -alkylene- is 35 —CHCHCH . In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

40

r 45 -- 50 each W is O, each Y is O, at least one X is X3 and X3 is selected from ()--- 55 60 -- N/ \, NH \ / NH2. 65 US 9,278,987 B2 53 54 -continued In some embodiments, at least one X3 is

V /N NH 5 -- N O)- ) - 10 NH s In some embodiments, at least one X3 is N NH

NN / , and 15 --O- O)-( 2O \- ) In some embodiments, at least one X3 is In some embodiments, at least one X3 is 25 --O- -- \ /N \- 30 In some embodiments, at least one X3 is

In some embodiments, at least one X3 is 35 /-, --O- -- N / \ .S. 40 \ , NH )

In some embodiments, at least one X3 is In some embodiments, at least one X3 is --O-)- 45 50 \- In some embodiments, at least one X3 is - N N \ / \ { 55 In some embodiments, at least one X3 is In some embodiments, at least one X3 is 60 +O-N / |C 65 /

US 9,278,987 B2 59 60 NH CNHI NH2. In some embodiments, at least one X3 is -continued -L1-CO-alkylene-CNH NH. In some embodiments, at least one X3 is -L1-CO-alkylene-N(R'). In some embodi ments, at least one X3 is -L1-CO-alkylene-N(R). In some -- embodiments, at least one X3 is -L1-CO-alkylene-heterocy 5 clyl. In some embodiments, at least one X3 is -L1-CO-alky OJN-Null NH2 lene-N(O)(R). In some embodiments, at least one X3 is N -L1-CO-alkylene-NH CNH-NH. In a further embodi H NH2. ment of the aforementioned embodiments, L1 is 10 In some embodiments, at least one X3 is

--O-- 15

In another embodiment of the aforementioned embodiments, L1 is

--O- 25

30 In yet a further embodiment of the aforementioned embodi ments, -alkylene- is —CH2—, —CH2CH2—, In another embodiment is a compound of Formula (I) —CH2CHCH . —CHCHCHCH , and wherein n is an integer from 1 to 35, G is —CH2CH2CH2CHCH-. In some embodiments of the aforementioned embodiments, -alkylene- is —CH2—. In 35 Some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. In some embodiments of the aforementioned embodiments, -alkylene- is —CHCHCH . In some embodiments of the aforemen tioned embodiments, -alkylene- is —CH2CH2CH2CH2—. In 40 r Some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2CH2CH2CH2—. In another embodiment is a compound of Formula (I) -- wherein n is an integer from 1 to 35, G is 45 each W is O, each Y is O, at least one X is X3, and X3 is selected from -L1-CO-alkylene-CNH-NH -L1-CONH alkylene-CNH NH, -L1-CO-alkylene-N(R'). -L1 CONH-alkylene-N(R'). -L1-CO-alkylene-N(R), -L1 50 CONH-alkylene-N(R) -L1-CO-alkylene-heterocyclyl, -L1-CONH-alkylene-heterocyclyl, -L1-CO-alkylene-N(O) r (R), -L1-CONH-alkylene-N(O)(R), -L1-CO-alkylene NH CNH-NH and -L1-CONH-alkylene-NH-CNH NH. In a further embodiment, at least one X3 is selected -- from -L1-CO-alkylene-CNH NH, -L1-CO-alkylene-N (R'). -L1-CO-alkylene-N(R)-L1-CO-alkylene-heterocy each W is O, each Y is O, at least one X is X3 and X3 is clyl, -L1-CO-alkylene-N(O)(R), and -L1-CO-alkylene selected from NH CNHI NH2. In some embodiments, at least one X3 is -L1-CO-alkylene-CNH NH. In some embodiments, at 60 least one X3 is -L1-CO-alkylene-N(R'). In some embodi ments, at least one X3 is-L1-CO-alkenylene-N(R). In some O embodiments, at least one X3 is -L1-CO-alkenylene-hetero N N HN NH d 8. cyclyl. In some embodiments, at least one X3 is -L1-CO \ / y-ll 65 alkenylene-N(O)(R). In some embodiments, at least oneX3 NH is -L1-CO-alkenylene-NH CNHI NH. In a further embodiment of the aforementioned embodiments, L1 is US 9,278,987 B2 61 62 -continued

5 --O-O\-

In another embodiment of the aforementioned embodiments, In some embodiments, at least one X3 is

10

--O- 15 In some embodiments, at least one X3 is In yet a further embodiment of the aforementioned embodi ments, -alkylene- is —CH2CH2—, —CH2CH2CH2—, —CH2CH2CHCH , and —CH2CH2CH2CH2CH2—. In Some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. In some embodiments of the aforementioned embodiments, -alkylene- is --O- —CH2CH2CH2—. In some embodiments of the aforemen tioned embodiments, -alkylene- is —CH2CH2CH2CH2—. In Some embodiments of the aforementioned embodiments, 25 In some embodiments, at least one X3 is -alkylene- is —CH2CH2CH2CH2CH2—. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 30 --O-

In some embodiments, at least one X3 is r 35 -- 40 each W is O, each Y is O, at least one X is X3, and X3 is --O- selected from -L1-N(R'). -L1-N(R), -L1-N(O)(R), and -L1-NH CNH NH. In some embodiments, at least one X3 is -L1-N(R'). In some embodiments, at least one X3 is In some embodiments, at least one X3 is -L1-N(R). In some embodiments, at least one X3 is -L1-N 45 (O)(R). In some embodiments, at least one X3 is-L1-NH CNH NH2. In further embodiments at least one X3 is selected from: --O-( 50 In some embodiments, at least one X3 is / \ 4. s --O-O. 60 In yet further embodiments at least one X3 is selected from

65 \ O-(\,s and --O-(- - US 9,278,987 B2 63 64 -continued In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is N N --OY) 5 In some embodiments, at least one X3 is r 10 --O-(- -- each Wis O, each Y is O, at least one X is X4, and at least one 15 X4 is selected from N(R')-aryl, - N(R')-alkylene-aryl, - N(R)-alkylene-N(R)-aryl, - N(R)-alkylene-N(R')CO In some embodiments, at least one X3 is aryl, - N(R')-alkylene-N(R')SO,-aryl, and N(R')-alky lene-N(R')CH-aryl. In a further embodiment, at least one X4 is N(R)-aryl. In yet a further embodiment, at least one --OA) X4 is

In any of the aforementioned embodiments of Formula (I) 25 wherein at least one X is X3 is another embodiment wherein n is an integer from 30-35. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least y and y one X is X3, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) 30 wherein at least one X is X3, n is an integer from 20-24. In In some embodiments, at least one X4 is further embodiments of any of the aforementioned embodi ments of Formula (I) wherein at least one X is X3, n is an integer from 15-19. In further embodiments of the aforemen tioned embodiments of Formula (I) wherein at least one X is 35 X3, n is an integer from 10-14. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least one X is X3, n is an integer from 5-9. In yet further embodi y ments of the aforementioned embodiments of Formula (I) 40 wherein at least one X is X3, n is an integer from 1-4. In another embodiment the X3 group is provided in a In some embodiments, at least one X4 is protected form. In another embodiment the protected form comprises a protected nitrogen. In another embodiment the protected nitrogen is protected with a trifluoroacetyl group. 45 In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is y 50

In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is r 55 --

60 each W is O, each Y is O, and at least one X is X4. In further r embodiments at least one X4 is selected from —O-alkylene aryl, - N(R)-aryl, - N(R)-alkylene-aryl, - N(R)-alky lene-N(R')-aryl, - N(R)-alkylene-N(R')CO-aryl, -- —N(R)-alkylene-N(R')SO-aryl, and N(R)-alkylene-N 65 (R')CH2-aryl. In a further embodiment, at least one X4 is each Wis O, each Y is O, at least one X is X4, and at least one —O-alkylene-aryl. X4 is N(R)-alkylene-aryl. In further embodiments, -alky US 9,278,987 B2 65 66 lene- is —CH2—, —CH(CH)—, —CH2CH2—, and In some embodiments, at least one X4 is —CH2CHCH-. In some embodiments, -alkylene- is —CH2—. In some embodiments, -alkylene- is —CH (CH)—. In some embodiments, -alkylene- is —CH2CH2—. In some embodiments, -alkylene- is —CH2CH2CH2—. In a 5 further embodiment, at least one X4 is selected from: - C 10 N ro-ro In some embodiments, at least one X4 is 15 N to ho -ho In some embodiments, at least one X4 is

C-C 25

N ro , and 30 In some embodiments, at least one X4 is

35 --O In some embodiments, at least one X4 is In some embodiments, at least one X4 is 40

+rc 45 --O In another embodiment is a compound of Formula (I) In some embodiments, at least one X4 is wherein n is an integer from 1 to 35, G is 50

thro 55 r In some embodiments, at least one X4 is -- 60 each Wis O, each Y is O, at least one X is X4, and at least one X4 is selected from N(R)-alkylene-N(R)-aryl, and - N(R)-alkylene-N(R')CH2-aryl. In some embodiments, at least one X4 is N(R)-alkylene-N(R)-aryl. In some +ric 65 embodiments, at least one X4 is N(R')-alkylene-N(R') CH-aryl. In further embodiments, at least one X4 is selected from US 9,278,987 B2 67 68 In some embodiments, at least one X4 is

and

10 In a further embodiment of the aforementioned embodi ments, -alkylene- is selected from —CHCH . —CH2CH2CH2—, —CH2CH2CHCH . and —CHCHCHCHCH-. In some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. 15 In some embodiments of the aforementioned embodiments, r -alkylene- is —CH2CHCH . In some embodiments of the aforementioned embodiments, -alkylene- 1S In some embodiments, at least one X4 is —CH2CH2CHCH . In some embodiments of the afore mentioned embodiments, -alkylene- is —CH2CH2CH2CHCH-. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is ro 25 In some embodiments, at least one X4 is r 30 -- each Wis O, each Y is O, at least one X is X4, and at least one lu) 35 X4 is selected from -L1-CO-alkylene-aryl, -L1-CO-alk enylene-aryl, -L1-CO-arylene-aryl, -L1-CONH-alkylene ~ aryl, -L1-CONH-arylene-aryl, -L1-SO-alkylene-aryl, -L1 SO-arylene-aryl, -L1-CO-aryl, -L1-SO-aryl, -L1-CO In yet a further embodiment, at least one X4 is selected from alkylene-P(aryl), and -L1-SO-alkylene-P(aryl). In a -N(R)-alkylene-N(R')CO-aryl and N(R)-alkylene-N 40 further embodiment, at least one X4 is -L1-SO-alkylene-P (R')SO-aryl. In further embodiments, at least one X4 is (aryl). In a further embodiment, at least one X4 is -L1-CO selected from alkylene-P(aryl). In a further embodiment, at least one X4 is selected from -L1-CO-alkylene-aryl, -L1-CO-alkenylene aryl, -L1-CO-arylene-aryl, -L1-CONH-alkylene-aryl, -L1 45 CONH-arylene-aryl, -L1-SO-alkylene-aryl, -L1-SO arylene-aryl, -L1-CO-aryl, and -L1-SO-aryl. In a further embodiment, at least one X4 is selected from-L1-SO-alky lene-aryl, -L1-SO-arylene-aryl, and -L1-SO-aryl. In some embodiments, at least one X4 is -L1-SO-alkylene-aryl. In 50 Some embodiments, at least one X4 is -L1-SO-arylene-aryl. In some embodiments, at least one X4 is -L1-SO-aryl. In yet a further embodiment, at least one X4 is selected from -L1 CO-alkylene-aryl, -L1-CO-alkenylene-aryl, -L1-CO arylene-aryl, -L1-CONH-alkylene-aryl, -L1-CONH 55 arylene-aryl, and -L1-CO-aryl. In a further embodiment, at least one X4 is selected from -L1-CONH-alkylene-aryland -L1-CONH-arylene-aryl. In some embodiments, at least one In some embodiments, at least one X4 is X4 is -L1-CONH-alkylene-aryl. In some embodiments, at least one X4 is -L1-CONH-arylene-aryl. In yet a further 60 embodiment, at least one X4 is selected from -L1-CO-alky lene-aryl, -L1-CO-alkenylene-aryl, -L1-CO-arylene-aryl, and -L1-CO-aryl. In some embodiments, at least one X4 is -L1-CO-alkylene-aryl. In some embodiments, at least one X4 is -L1-CO-alkenylene-aryl. In some embodiments, at least 65 one X4 is -L1-CO-arylene-aryl. In some embodiments, at --O least one X4 is -L1-CO-aryl. In a further embodiment of the aforementioned embodiments, L1 is US 9,278,987 B2 69 70 In some embodiments, at least one X4 is

5

In another embodiment of the aforementioned embodiments, L1 is C - 2-- 10

15 In a further embodiment, at least one X4 is selected from

In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

r 25 -- 30 each W is O., each Y is O. at least one X is X4, and X4 is selected from

35

-- N O and 40

In some embodiments, at least one X4 is

C - 2--

In another embodiment is a compound of Formula (I) In some embodiments, at least one X4 is 55 wherein n is an integer from 1 to 35, G is 60 r 65 --

US 9,278,987 B2 73 74 -continued In some embodiments, at least one X4 is

Cl, and 5 -- \ /N O \

10 C O /

In some embodiments, at least one X4 is 15 O CF.

Cl -- /\ \,/ O 'y, In some embodiments, at least one X4 is

25 In some embodiments, at least one X4 is

O 30 --O\ / O CF3. In some embodiments, at least one X4 is O-o- 35 In some embodiments, at least one X4 is N N O / Y, \ / 40 -- \ /N O C

In some embodiments, at least one X4 is ( )—K) 45 C.

In some embodiments, at least one X4 is -- /\ \,/ O / 50

-- \ / N O CF In some embodiments, at least one X4 is 55

In some embodiments, at least one X4 is O -- \ /N 60

65 tC, C US 9,278,987 B2 75 76 In some embodiments, at least one X4 is In some embodiments, at least one X4 is +O- O N 10 O o-CF3.

In some embodiments, at least one X4 is 15

25 In some embodiments, at least one X4 is toro, In some embodiments, at least one X4 is 30

tor, 35

40 to rol In some embodiments, at least one X4 is In a further embodiment, at least one X4 is selected from

45

toro, 50

55 In some embodiments, at least one X4 is

and

60

65 US 9,278,987 B2 77 78 In some embodiments, at least one X4 is -continued

5

10 In some embodiments, at least one X4 is

In some embodiments, at least one X4 is 15 --O-( ) In some embodiments, at least one X4 is

In some embodiments, at least one X4 is -- \ / -( )-so 25 In some embodiments, at least one X4 is

|CO 30 In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 35 In any of the aforementioned embodiments of Formula (I) wherein at least one X is X4 is another embodiment wherein n is an integer from 30-35. In further embodiments of the 40 aforementioned embodiments of Formula (I) wherein at least r one X is X4, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X4, n is an integer from 20-24. In -- further embodiments of any of the aforementioned embodi 45 ments of Formula (I) wherein at least one X is X4, n is an each Wis O, each Y is O, at least one X is X4, and at least one integer from 15-19. In further embodiments of the aforemen X4 is selected from-L1-alkylene-aryl, -L1-arylene-aryl, -L1 tioned embodiments of Formula (I) wherein at least one X is aryl, and -L1-alkylene-P(aryl). In some embodiments, at X4, n is an integer from 10-14. In further embodiments of the least one X4 is -L1-alkylene-aryl. In some embodiments, at aforementioned embodiments of Formula (I) wherein at least 50 one X is X4, n is an integer from 5-9. In yet further embodi least one X4 is -L1-arylene-aryl. In some embodiments, at ments of the aforementioned embodiments of Formula (I) least one X4 is -L1-aryl. In some embodiments, at least one wherein at least one X is X4, n is an integer from 1-4. X4 is -L1-alkylene-P(aryl). In a further embodiment, at least In another embodiment is a compound of Formula (I) one X4 is selected from wherein n is an integer from 1 to 35, G is 55

--O-( ) s 60 r -- 65 --O-(\ / )- s and each W is O, each Y is O, and at least one X is X5. In further embodiments at least one X5 is selected from —O-alkylene US 9,278,987 B2 79 80 heteroaryl, - N(R)-alkylene-heteroaryl, - N(R)-alkylene In another embodiment is a compound of Formula (I) N(R)-heteroaryl, - N(R)-alkylene-N(R')CO-heteroaryl, wherein n is an integer from 1 to 35, G is —N(R)-alkylene-N(R')SO-heteroaryl, and N(R)-alky lene-N(R')CH-heteroaryl. In yet further embodiments, at least one X5 is selected from N(R)-alkylene-heteroaryl, 5 —N(R)-alkylene-N(R)-heteroaryl, - N(R')-alkylene-N (R')CO-heteroaryl, - N(R)-alkylene-N(R')SO-het eroaryl, and —N(R)-alkylene-N(R')CH-heteroaryl. In another embodiment is a compound of Formula (I) 10 r wherein n is an integer from 1 to 35, G is --

15 each Wis O, each Y is O, at least one X is X5, and at least one X5 is selected from-L1-heteroaryl, -L1-alkylene-heteroaryl, -L1-arylene-heteroaryl, -L1-CO-alkylene-heteroaryl, -L1 r CO-alkenylene-heteroaryl, -L1-CO-arylene-heteroaryl, -L1 CONH-alkylene-heteroaryl, -L1-CONH-arylene-heteroaryl, -- -L1-SO-alkylene-heteroaryl, -L1-SO-arylene-heteroaryl. In a further embodiment at least one X5 is selected from each Wis O, each Y is O, at least one X is X5, and at least one -L1-SO-alkylene-heteroaryl and -L1-SO-arylene-het X5 is N(R')-alkylene-heteroaryl. Inafurther embodiment, eroaryl. In some embodiments, at least one X5 is -L1-SO at least one X5 is —O-alkylene-heteroaryl. In yet further 25 alkylene-heteroaryl. In some embodiments, at least one X5 is embodiments, -alkylene- is —CH2—, —CH(CH)—, -L1-SO-arylene-heteroaryl. In a further embodiment at least —CH2CH2—, and —CH2CH2CH2—. In some embodi one X5 is selected from -L1-CONH-alkylene-heteroaryland ments, -alkylene- is —CH2—. In some embodiments, -alky -L1-CONH-arylene-heteroaryl. In some embodiments, at lene- is —CH(CH)—. In some embodiments, -alkylene- is least one X5 is -L1-CONH-alkylene-heteroaryl. In some —CH2CH2—. In some embodiments, -alkylene- is 30 embodiments, at least one X5 is -L1-CONH-arylene-het eroaryl. In a further embodiment at least one X5 is selected —CHCHCH from -L1-CO-alkylene-heteroaryl, -L1-CO-alkenylene-het In another embodiment is a compound of Formula (I) eroaryl, and -L1-CO-arylene-heteroaryl. In some embodi wherein n is an integer from 1 to 35, G is ments, at least one X5 is -L1-CO-alkylene-heteroaryl. In 35 some embodiments, at least one X5 is -L1-CO-alkenylene heteroaryl. In some embodiments, at least one X5 is -L1-CO alkenylene-heteroaryl. In a further embodiment of the afore mentioned embodiments, L1 is r 40 -- --O-- each Wis O, each Y is O, at least one X is X5, and at least one 45 X5 is selected from N(R)-alkylene-heteroaryl, - N(R)- In another embodiment of the aforementioned embodiments, aylkylene-N(R)-heteroaryl, - N(R)-alkylene-N(R')CO L1 is heteroaryl, - N(R')-alkylene-N(R')SO-heteroaryl, and —N(R)-alkylene-N(R')CH-heteroaryl. In some embodi ments, at least one X5 is N(R')-alkylene-heteroaryl. In 50 some embodiments, at least one X5 is N(R)-alkylene-N (R)-heteroaryl. In some embodiments, at least one X5 is —N(R)-alkylene-N(R')CO-heteroaryl. In some embodi --O- ments, at least one X5 is N(R') - alkylene-N(R')SO,- 55 heteroaryl. In some embodiments, at least oneX5 is N(R)- alkylene-N(R')CH-heteroaryl. In a further embodiment of In yet a further embodiment of the aforementioned embodi the aforementioned embodiments, -alkylene- is selected from ments, -alkylene- is selected from —CH2—, —CH(CH)—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2—, and —CH2CH2CH2—. In some embodiments and —CH2CH2CH2CH2CH2—. In some embodiments of the 60 of the aforementioned embodiments, -alkylene- is —CH2—. aforementioned embodiments, -alkylene- is —CHCH-. In some embodiments of the aforementioned embodiments, In some embodiments of the aforementioned embodiments, -alkylene- is —CH(CH)—. In some embodiments of the -alkylene-is—CHCHCH . In some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. aforementioned embodiments, -alkylene- is In some embodiments of the aforementioned embodiments, —CH2CH2CHCH-. In some embodiments of the afore 65 -alkylene- is —CH2CHCH-. In some embodiments, at mentioned embodiments, -alkylene- is least one X5 is -L1-heteroaryl. In a further embodiment, at least one X5 is selected from US 9,278,987 B2 82 In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

5

10 r --

15 each Wis O, each Y is O, at least one X is X5, and at least one X5 is selected from-L1-alkylene-heteroaryland-L1-arylene heteroaryl. In some embodiments at least one X5 is -L1 arylene-heteroaryl. In some embodiments at least one X5 is -L1-alkylene-heteroaryl. In a further embodiment, at least one X5 is selected from

25 In some embodiments, at least one X is X5 and X5 is

N 30 N\ /N-( \

In some embodiments at least one X5 is 35

40

RN

45 In some embodiments at least one X5 is --O- ( ) RN . 55 In some embodiments at least one X5 is In some embodiments at least one X5 is

60

tC) 65 O US 9,278,987 B2 83 84 In some embodiments at least one X5 is (CHCH), OCH, -N(R')-arylene-O(CH-CH-),OH, and N(R"-arylene-O(CHCH)OCH. In further embodi ments, at least one X6 is selected from —O-alkylene-O (CHCH), OH and —O-alkylene-O(CHCH), OCH. In some embodiments, at least one X6 is —O-alkylene-O (CHCH), OH. In some embodiments, at least one X6 is —O-alkylene-O(CHCH)OCH. In further embodiments, at least one X6 is selected from N(R)-alkylene-O (CHCH),OH, -N (R')-alkylene-O(CH-CH-),OCH, N(R)-arylene-O(CHCH), OH, and N(R)-arylene-O 10 (CHCH)OCH. In yet further embodiments, at least one X6 is selected from N(R)-alkylene-O(CHCH), OH and In some embodiments at least one X5 is - N(R)-alkylene-O(CH2CH), OCHs. In some embodi ments, at least one X6 is N(R)-alkylene-O(CH CH), OH. In some embodiments, at least oneX6 is N(R')- 15 alkylene-O(CHCH)OCH. In further embodiments, at least one X6 is selected from N(R')-arylene-O(CH, CH), OH and N(R)-arylene O(CH.QH)OCH, In some embodiments, at least one X6 is —N(R')-arylene-O (CHCH), OH. In some embodiments, at least one X6 is - N(R')-arylene-O(CHCH), OCHs. In yet a further (C) embodiment of the aforementioned embodiments, -alkylene is selected from —CH2CH2—, —CH2CH2CH2—, —CHCHCHCH , and —CHCHCHCHCH-. In In some embodiments at least one X5 is Some embodiments of the aforementioned embodiments, 25 -alkylene- is —CHCH-. In some embodiments of the aforementioned embodiments, -alkylene- is —CHCHCH-. In some embodiments of the aforemen tioned embodiments, -alkylene- is —CH2CH2CH2CH2—. In Some embodiments of the aforementioned embodiments, 30 -alkylene- is —CH2CH2CH2CH2—. s y In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

In any of the aforementioned embodiments of Formula (I) 35 wherein at least one X is X5 is another embodiment wherein n is an integer from 30-35. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least one X is X5, n is an integer from 25-29. In further embodi r ments of the aforementioned embodiments of Formula (I) 40 wherein at least one X is X5, n is an integer from 20-24. In further embodiments of any of the aforementioned -- embodiments of Formula (I) wherein at least one X is X5, n is an integer from 15-19. In further embodiments of the afore each W is O, each Y is O, at least one X is X6, and X6 is mentioned embodiments of Formula (I) wherein at least one 45 selected from-L1-alkylene-(OCH2CH), OH, -L1-CO-alky X is X5, n is an integer from 10-14. In further embodiments of lene-O(CHCH), OH, -L1-CO-alkylene-O(CH2CH), the aforementioned embodiments of Formula (I) wherein at OCH -L1-SO-alkylene-O(CHCH), OH, -L1-SO-alky least one X is X5, n is an integer from 5-9. In yet further lene-O(CHCH), OCH -L1-CO-arylene-O(CH2CH), embodiments of the aforementioned embodiments of For OH, -L1-CO-arylene-O(CHCH), OCH -L1-SO mula (I) wherein at least one X is X5, n is an integer from 1-4. 50 arylene-O(CH2CH), OH, and -L1-SO-arylene-O In another embodiment is a compound of Formula (I) (CHCH)OCH. In a further embodiment, at least one X6 wherein n is an integer from 1 to 35, G is is selected from -L1-SO-alkylene-O(CH2CH), OH, -L1 SO-alkylene-O(CH2CH), OCH, -L1-SO-arylene-O (CHCH), OH, and -L1-SO-arylene-O(CHCH)OCH. 55 In yet a further embodiment, at least one X6 is selected from -L1-SO-alkylene-O(CHCH), OH and -L1-SO-alkylene O(CHCH)OCH. In some embodiments, at least one X6 is -L1-SO-alkylene-O(CH2CH), OH. In some embodiments, r at least one X6 is -L1-SO-alkylene-O(CH2CH)OCH. In a 60 further embodiment, at least one X6 is selected from -L1 SO-arylene-O(CHCH), OH and -L1-SO-arylene-O -- (CHCH)OCH. In some embodiments, at least one X6 is -L1-SO-arylene-O(CHCH), OH. In some embodiments, each W is O, each Y is O, and at least one X is X6. In further at least one X6 is -L1-SO-arylene-O(CHCH)OCH. In a embodiments at least one X6 is selected from —O-alkylene 65 further embodiment, at least one X6 is selected from -L1 O(CHCH),OH, —O-alkylene-O(CHCH)OCH, alkylene-(OCH2CH), OH, -L1-CO-alkylene-O(CH2CH), —N(R)-alkylene-O(CHCH.),OH, -N(R')-alkylene-O OH, -L1-CO-alkylene-O (CHCH), OCH -L1-CO US 9,278,987 B2 85 86 arylene-O(CH2CH), OH, and -L1-CO-arylene-O(CH CH)OCH. In some embodiments, at least one X6 is -L1 alkylene-(OCH2CH), OH. In yet a further embodiment, at N 1N1'N-1 no-1N1'n 1\on. least one X6 is selected from -L1-CO-alkylene-O(CH CH), OH and -L1-CO-alkylene-O(CHCH)OCH. In 5 some embodiments, at least one X6 is -L1-CO-alkylene-O (CHCH), OH. In some embodiments, at least one X6 is In some embodiments, at least one X6 is -L1-CO-alkylene-O(CHCH), OCH. In a further embodi ment, at least one X6 is selected from -L1-CO-arylene-O (CHCH)OHand-L1-CO-arylene-O(CHCH), OCH. In 10 some embodiments, at least one X6 is -L1-CO-arylene-O (CHCH), OH. In some embodiments, at least one X6 is -L1-CO-arylene-O(CH2CH)OCH. In a further embodi ()-r-r-s-s ment of the aforementioned embodiments, L1 is 15 In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

In another embodiment of the aforementioned embodiments, L1 is r 25 --

30 each W is O, each Y is O, at least one X is X6, and X6 is selected from -L1-CO O(CH2CH), OH and -L1-CO—O (CH2CH2)OCH. In a further embodiment, at least one X6 is -L1-CO O(CHCH), OH. In yet a further embodiment, at least one X6 is -L1-CO-O(CHCH)OCH. 35 In another embodiment is a compound of Formula (I) In yet a further embodiment of the aforementioned embodi wherein n is an integer from 1 to 35, G is ments, -alkylene- is selected from —CH2CH2—, —CH2CH2CH2—, —CH2CH2CHCH . and —CHCHCHCHCH . In some embodiments of the aforementioned embodiments, -alkylene- is —CH2CH2—. 40 In some embodiments of the aforementioned embodiments, -alkylene- is —CH2CHCH . In some embodiments of the aforementioned embodiments, -alkylene- 1S r —CHCHCHCH-. In some embodiments of the afore 45 mentioned embodiments, -alkylene- is —CH2CH2CH2CHCH-. In some embodiments of the -- aforementioned embodiments, m is 1. In some embodiments of the aforementioned embodiments, m is 2. In some embodi each W is O, each Y is O, at least one X is X6 and X6 is ments of the aforementioned embodiments, m is 3. 50 selected from N(R')-(dibenzo-18-crown-6) and an aza In another embodiment is a compound of Formula (I) crown ether. In some embodiments, at least one X6 is wherein n is an integer from 1 to 35, G is N(R)-(dibenzo-18-crown-6). In some embodiments, at least one X6 is anaza-crown ether. In further embodiments, at least one X is X6 and X6 is selected from: 55

60 - -- each Wis O, each Y is O, at least one X is X6, and at least one 65 U X6 is selected from-L1-alkylene-(OCH2CH), OH. In some embodiments, at least one X6 is US 9,278,987 B2 87 88 O -continued tioned embodiments of Formula (I) wherein at least one X is X6, n is an integer from 10-14. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least /-/ \ . one X is X6, n is an integer from 5-9. In yet further embodi ments of the aforementioned embodiments of Formula (I) N D wherein at least one X is X6, n is an integer from 1-4. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

10

15 r -- In some embodiments, at least one X6 is each Wis O, each Y is O, and at least one X is X7. In a further embodiment, at least one X7 is a heterocycle. In a further embodiment, at least one X is X7 and X7 is selected from:

- 25 N O, -- NH,

U 30 N s -- O,

In some embodiments, at least one X6 is N 35 -- O r /-/ \ . O-, O-, and -- D 40 \O / +C) In some embodiments, at least one X6 is In some embodiments, at least one X7 is 45

S \, 50 In some embodiments, at least one X7 is

* - 55 In any of the aforementioned embodiments of Formula (I) -- \ /NH wherein at least one X is X6 is another embodiment wherein n is an integer from 30-35. In further embodiments of the 60 In some embodiments, at least one X7 is aforementioned embodiments of Formula (I) wherein at least one X is X6, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X6, n is an integer from 20-24. In further embodiments of any of the aforementioned embodi 65 ments of Formula (I) wherein at least one X is X6, n is an --O) integer from 15-19. In further embodiments of the aforemen US 9,278,987 B2 89 90 In some embodiments, at least one X7 is -continued

In some embodiments, at least one X7 is 10 t; 15 In some embodiments, at least one X7 is

25 Yo 30 In some embodiments, at least one X7 is +C 35 In some embodiments, at least one X7 is 40 --> In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is In some embodiments, at least one X7 is

Z5 45 r B, kn 50 -- In some embodiments, at least one X7 is each Wis O, each Y is O, at least one X is X7, and at least one ss X7 is N(R')(R). In a further embodiment, at least one X7 is selected from:

In some embodiments, at least one X7 is 60

65 c-1 US 9,278,987 B2 91 92 In some embodiments, at least one X7 is In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is

5 Z5 V r B, N In some embodiments, at least one X7 is 10

each Wis O, each Y is O, at least one X is X7, and at least one 15 X7 is selected from: --O-( --O-O. / \, O

In some embodiments, at least one X7 is

NH2 35

40

2.

45 In another embodiment is a compound of Formula (I) O wherein n is an integer from 1 to 35, G is / CF, Z5 50 y r O N 55

each Wis O, each Y is O, at least one X is X7, and at least one X7 is N(R')(R). In a further embodiment, at least one X7 60 O is selected from -L1-CO-alkyl, -L1-CONH-alkyl, -L1-CON O. (alkyl)-alkyl, and -L1-SO-alkyl. In a further embodiment, at N -( least one X7 is -L1-CO-alkyl. In a further embodiment, at least one X7 is -L1-CONH-alkyl. In a further embodiment, at 65 N least one X7 is -L1-CONCalkyl)-alkyl. In a further embodi ment, at least one X7 is -L-SO-alkyl. US 9,278,987 B2 93 94 In some embodiments, at least one X7 is In some embodiments, at least one X7 is --O-( 5 --O-, i.

Iin Some embod1bodi menus,tS. allat least one X7 1S 10 In some embodiments, at least one X7 is --O-( 15 +C In some embodiments, at least one X7 is 2O In some embodiments at least one X7 is --O-( O. In some embodiments, at least one X7 is In some embodiments, at least one X7 is --O- 30 --O- In some embodiments, at least one X7 is In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 40 N NH Z5 y- O B,

45 N In some embodiments, at least one X7 is --

so each W is O., each Y is O, at least one X is X7, and at least one X7 is N(R')(R). In a further embodiment, at least one X7 N NH t is selected from: O 55 / V O,

In some embodiments at least one X7 is N -. HS 60 / O H O s and

O)- N N skO

HS US 9,278,987 B2 95 96 -continued each Wis O, each Y is O, and at least one X is X8. In a further embodiment, at least one X8 is -L1-CA. In a further embodi ment, at least one X8 is -L1-dCA. Inafurther embodiment, at O O least one X8 is selected from -L1-COCH(R) and -L1 Us COCH(R)NHCO,-alkyl. In some embodiments, at least one X8 is-L1-COCH2(R). In some embodiments, at least oneX8 is-L1-COCH(R)NHCO-alkyl. O' / In another embodiment is a compound of Formula (I) HS wherein n is an integer from 1 to 35, G is In some embodiments, at least one X7 is 10

-- ()-.N O. 15 r In some embodiments, at least one X7 is -- each W is O. at least one Y is NR at least one X is X8, at least oneX8 is —OR, and RandR'togetherformaring. In Us a further embodiment, the ring formed is selected from: -O-y 25 V V In some embodiments, at least one X7 is 30 , and

35 ... O HS In any of the aforementioned embodiments of Formula (I) wherein at least one X is X7 is another embodiment wherein In some embodiments, the ring formed is n is an integer from 30-35. In further embodiments of the 40 aforementioned embodiments of Formula (I) wherein at least one X is X7, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X7, n is an integer from 20-24. In further embodiments of any of the aforementioned embodi 45 ments of Formula (I) wherein at least one X is X7, n is an integer from 15-19. In further embodiments of the aforemen tioned embodiments of Formula (I) wherein at least one X is X7, n is an integer from 10-14. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least 50 one X is X7, n is an integer from 5-9. In yet further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X7, n is an integer from 1-4. In another embodiment is a compound of Formula (I) wherein n is an integer from 1 to 35, G is 55 In some embodiments, the ring formed is r 60 -- 65 US 9,278,987 B2 97 98 In any of the aforementioned embodiments of Formula (I) In another embodiment is the compound of Formula (I) wherein at least one X is X8 is another embodiment wherein wherein L1 is n is an integer from 30-35. In further embodiments of the aforementioned embodiments of Formula (I) wherein at least one X is X8, n is an integer from 25-29. In further embodi ments of the aforementioned embodiments of Formula (I) wherein at least one X is X8, n is an integer from 20-24. In further embodiments of any of the aforementioned embodi --C- ments of Formula (I) wherein at least one X is X8, n is an integer from 15-19. In further embodiments of the aforemen 10 tioned embodiments of Formula (I) wherein at least one X is wherein p is 1 and q is 2. In another embodiment is the X8, n is an integer from 10-14. In further embodiments of the compound wherein p is 2 and q is 2. In another embodiment aforementioned embodiments of Formula (I) wherein at least is the compound wherein p is 3 and q is 2. one X is X8, n is an integer from 5-9. In yet further embodi ments of the aforementioned embodiments of Formula (I) 15 In another embodiment is the compound of Formula (I) wherein at least one X is X8, n is an integer from 1-4. wherein L1 is In another embodiment is the compound of Formula (I) wherein L1 is selected from: ----O---- 3. -- In another embodiment is the compound of Formula (I) 25 wherein L1 is

t 8. d 30 ----O---- wherein Q' is N(R)-. x - 35 In another embodiment is the compound of Formula (I) wherein L1 is In another embodiment is the compound of Formula (I) wherein L1 is 40 ----O------YM -- 45 wherein Q is N(R)-. In another embodiment is the compound of Formula (I) In another embodiment is the compound of Formula (I) wherein L1 is wherein L1 is -- 3. -- 50 ----O---- 55 wherein p is 2 and q is 2. In another embodiment is the wherein E is phenyl. compound wherein p is 3 and q is 2. In another embodiment is the compound of Formula (I) In another embodiment is the compound of Formula (I) wherein L1 is wherein L1 is 60 ----O---- 65 wherein E is heteroaryl. US 9,278,987 B2 99 100 In another embodiment is the compound of Formula (I) In another embodiment is the compound of Formula (I), wherein L1 is wherein L'' is ---O--

In another embodiment is the compound of Formula (I) 10 wherein L1 is In another embodiment is the compound of Formula (I), wherein L'' is

15 ---O-- R23 P-N-R-N wherein Q' is N(R)-. In another embodiment is the compound of Formula (I) wherein L1 is and R is a C-C alkylene. In another embodiment is the compound of Formula (I), wherein L' is 25 ---O-- O RI R1 R23 R23 wherein E is pyrrolyl. In another embodiment is the com pound of Formula (I) wherein E is imidazolyl. In another embodiment is the compound of Formula (I) wherein E is 30 triazinyl. In another embodiment is the compound of Formula (I) wherein E is imidazolyl. In another embodiment is the compound of Formula (I) wherein W is O, and Y is NH. and wherein R is ethylene or propylene, and R' is hydrogen In another embodiment is the compound of Formula (I) or methyl. wherein Wis O, and Y is NR''. In another embodiment is the 35 In another embodiment is the compound of Formula (I), compound of Formula (I) wherein W is O, and Y is NR", wherein L' is wherein R'' and X8etogetherformaring. In another embodi ment is the compound of Formula (I) wherein R'' and X8e together for a structure selected from 40 R23 R23 O P-N o1 \Q s’, R 45 In another embodiment is the compound of Formula (I), wherein L' is

50 R23 R23 | O P-N o1 \Q s’, R4 H CA 55 and R is hydrogen or methyl. In another embodiment is the In another embodiment is the compound of Formula (I), compound of Formula (I), wherein L'' is wherein L'' is 60 R23 O CH3 O R23 R23 Q

-->H 65 US 9,278,987 B2 101 102 In another embodiment is the compound of Formula (I), and p, q, S and t are independently selected from 1 or 2. In wherein L' is another embodiment is the compound of Formula (I), wherein Q is X1. In another embodiment is the compound of Formula (I), wherein R’ is hydrogen, methyl or both R groups together form a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ring. In another embodiment is the compound of /8 Formula (I), wherein R is hydrogen or methyl. YM 3 In another embodiment is the compound of Formula (I), wherein Q is X2. In another embodiment is the compound of Formula (I), wherein Q is X3. In another embodiment is the In another embodiment is the compound of Formula (I), 10 compound of Formula (I), wherein Q is X4. In another wherein L' is embodiment is the compound of Formula (I), wherein Q is X5. In another embodiment is the compound of Formula (I), wherein Q is X6. In another embodiment is the compound of Formula (I), wherein Q is X7. In another embodiment is the compound of Formula (I), wherein Q is X8. ^ In another embodiment is the compound of Formula (I), Y wherein Z is C(R)-OP(=O)(OH). In another embodi ment is the compound of Formula (I), wherein R is hydro gen. and wherein p is 2, and q is 2. In another embodiment is the In another embodiment is the compound of Formula (I), compound of Formula (I), wherein L' is wherein Zis-(L')-(R') and the R' is C-C alkyl, C-C, alkylcarbonyl, C-C alkyloxycarbonyl, ora3-18 membered alkoxyalkylcarbonyl. In another embodiment is the com pound of Formula (I), wherein Zis-(L'')-(R') and the R' 25 is a C-Co alkyloxycarbonyl. In another embodiment is the 4K compound of Formula (I), wherein Z is -(L'')-(R') and the Y R" is selected from –C(=O)CCHCH-OH, -C(=O) OCHCHOCHCH-OH, O —C(=O) and wherein p is 2, and q is 3. In another embodiment is the OCHCHOCH2CHOCH2CH2OH. In another embodiment compound of Formula (I), wherein L' is 30 is the compound of Formula (I), wherein Z is -(L'')-(R') and the R' is C(=O)CCHCHOCH2CHOCH2CH2OH. In another embodiment is the compound of Formula (I), wherein Z is -(L'')-(R') and the R' is a cell-penetrating p f peptide. In another embodiment is the compound of Formula 35 (I), wherein R' is a cell-penetrating peptide and the cell -->-lg penetrating peptide is linked through a aminohexanoic acid derived linker. In another embodiment is the compound of Formula (I), wherein R' is a cell-penetrating peptide and the In another embodiment is the compound of Formula (I), wherein L' is cell-penetrating peptide is linked through a aminohexanoic 40 acid-derived linker comprising the structure:

p f R3 O

45 H (C)g . s Sr-n-r n-n- p and q are independently selected from 1 or 2, and R is CHs. In another embodiment is the compound of Formula (I), In another embodiment is the compound of Formula (I), wherein Z is selected from: wherein L' is 50

O | / \

55 At3C CH3 / / In another embodiment is the compound of Formula (I), o-1-()- O O wherein L' is % \ / Y / \ / T\ or 60 HC1 YCH, O O

--(X) - 65 US 9,278,987 B2 103 104 In another embodiment is the compound of Formula (I), In another embodiment is the compound of Formula (I), wherein Z is wherein L'' is

O R23 R23 O I w O O-P-N N O O P-N %. \ / -(/- \ / \on. O1 NQ s’, c1 YCH, R 10 and R is hydrogen or methyl. In another embodiment is the In another embodiment is the compound of Formula (I), compound of Formula (I), wherein L'' is wherein Z is -(L')-(L)-(R'). In another embodiment is the compound of Formula (I), wherein L'' is 15 R23 O CH3 O

-->H In another embodiment is the compound of Formula (I), wherein L' is

In another embodiment is the compound of Formula (I), wherein L' is 25

R23 R23 O R1 R1

30 In another embodiment is the compound of Formula (I), wherein L' is

In another embodiment is the compound of Formula (I), 35 wherein L'' is - C - and wherein p is 2, and q is 2. In another embodiment is the R23 R23 O RI R1 compound of Formula (I), wherein L' is N-R-N 40 o1 XX. Q --C – and wherein p is 2, and q is 3. In another embodiment is the and R is a C-C alkylene. In another embodiment is the 45 compound of Formula (I), wherein L' is compound of Formula (I), wherein L'' is

R23 R23 Q i --e- 50 --C- o1 In another embodiment is the compound of Formula (I), Q l– wherein L' is R6 and wherein R is ethylene or propylene, and R' is hydrogen 55 or methyl. XO-g s In another embodiment is the compound of Formula (I), p and q are independently selected from 1 or 2, and R is CHs. wherein L'' is In another embodiment is the compound of Formula (I), 60 wherein L' is R23 R23 O P-N

O1 Q S.X.4 R" 65 US 9,278,987 B2 105 106 In another embodiment is the compound of Formula (I), Formula (I), wherein Q is X3. In another embodiment is the wherein L' is compound of Formula (I), wherein Q is X4. In another embodiment is the compound of Formula (I), wherein Q is X5. In another embodiment is the compound of Formula (I), S wherein Q is X6. In another embodiment is the compound of

XOXOg N s Formula (I), wherein Q is X7. In another embodiment is the compound of Formula (I), wherein Q is X8. and p, q, S and t are independently selected from 1 or 2. In 10 In another embodiment is the compound of Formula (I), another embodiment is the compound of Formula (I), wherein wherein Z is -(L')-(L')-(R'), and L' is selected from Q is X1. In another embodiment is the compound of Formula —(C-C alkylene)-C(=O)—, or —(C-C alkoxy)-C (I), wherein R is hydrogen, methyl or both R groups (=O)—. In another embodiment is the compound of Formula together form a cyclopropyl, cyclobutyl, cyclopentyl or (I), wherein Zis-(L'')-(L)-(R'), and L' is selected from cyclohexyl ring. In another embodiment is the compound of 15 —(C-C alkoxy)-C(=O)—. Formula (I), wherein R is hydrogen or methyl. In another embodiment is the compound of Formula (I), In another embodiment is the compound of Formula (I), wherein Z is -(L')-(L)-(R') and is represented by the wherein Q is X2. In another embodiment is the compound of Structure:

NHCOEt

wherein group A is aminomethyl polystyrene resin. In another embodiment is the compound of Formula (I), wherein Zis-(L'')-(L)-(R') and R' is represented by the Structure:

NHCOEt

wherein group A is aminomethyl polystyrene resin. US 9,278,987 B2 107 108 In another embodiment is the compound of Formula (I), Table 1 provides, by way of example only, representative wherein Zis-(L'')-(L)-(R') and L' is represented by the linkages for compounds of Formula (I) Structure: TABLE 1 Representative Intersubunit Linkages (X-groups)

10 Y In another embodiment is the compound of Formula (I) O B wherein R' is an electron pair. In another embodiment is the compound of Formula (I) wherein R' is a C-C alkyl and R' is a C-C alkyl. In 15 NS another embodiment is the compound of Formula (I) wherein R" is a methyl group and R' is a methyl group. pi In another embodiment is the compound of Formula (I) wherein R' is hydrogen. In another embodiment is the compound of Formula (I) 20 No. X wherein R' is C-Co alkyl. In another embodiment is the 1 / compound of Formula (I) wherein R' is C, Co alkyl. In N another embodiment is the compound of Formula (I) wherein \ R" is C-C alkyl. In another embodimentis the compound of Formula (I) wherein R' is selected from prenyl, geranyl, 2 farnesyl or geranylgeranyl. In another embodiment is the compound of Formula (I) V M wherein R' is a cell-penetrating peptide. In another embodiment is the compound of Formula (I) so wherein R' is a guanidinyl or amidinyl group. 3 / V ( In another embodiment is the compound of Formula (I) N N wherein R' is a C-Cs cycloalkyl. \ / In another embodiment is the compound of Formula (I) wherein R' is a C-C alkylcarbonyl. In another embodi- 35 4 / \ ment is the compound of Formula (I) wherein R' is a C-Cls N N alkylcarbonyl. In another embodiment is the compound of V / Formula (I) wherein R' is a C-C alkylcarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a 5 O C-C alkylcarbonyl. In another embodiment is the com- 40 N N HN -- pound of Formula (I) wherein R' is a C-C alkylcarbonyl. In V M NH2 another embodiment is the compound of Formula (I) wherein R" is a C-C alkylcarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a C-C alkylcarbo- 6 nyl. In another embodiment is the compound of Formula (I) 45 wherein R' is a C alkylcarbonyl. In another embodiment is V the compound of Formula (I) wherein R' is CHCO . o1 'N ry In another embodiment is the compound of Formula (I) wherein R' is a C-Cls alkylcarbonyl. In another embodi- so N ment is the compound of Formula (I) wherein R' is a C-C so alkyloxycarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a C-C alkyloxycarbonyl. In 7 (;4. another embodiment is the compound of Formula (I) wherein V R" is a C-C alkyloxycarbonyl. In another embodiment is 55 Y'N the compound of Formula (I) wherein R' is a C-C alky- O rx loxycarbonyl. In another embodiment is the compound of N Formula (I) wherein R' is a C, alkyloxycarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a CHOCO . In another embodiment is the compound of 60 8 Formula (I) wherein R' is a -C(=O)CCHCH-OH, V OCHCHOCHCHOCHCH-OH.C(=O)CCHCHOCHCH-OH, O —C(=O) o PSry In another embodiment is the compound of Formula (I) Nu wherein R' is –C(=O)NHR'. In another embodiment is 65 the compound of Formula (I) wherein R' is C(=O) NHR'' and R is methyl. US 9,278,987 B2

TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups) Representative Intersubunit Linkages (X-groups)

G5 5 G5 W- – X WP-X Y O B O B r 10 r N NR 12 N YR 12 pi R13 15 R13 pi

No. X No. --O-y -- / 10 7- --O- M / 18 30

40 12

--, 21 / \ /

22 -- \ /N

60 24 US 9,278,987 B2 111 112 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups) Representative Intersubunit Linkages (X-groups)

WP-X X

w-r-Y 10 r N r NS YR 12 R12 pi R13 pi R13 15 No. No. 32 / \ , \ 25 --( N N O

25 O / 26 33 / \ \ kn-n N N O 30 27 ( )-( )—i? 35 to -o 34 40 28 --O-O) 35 29 45

50 36 30 N N C

55

C

31 60 37 O N N CF

O 65 US 9,278,987 B2 113 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups Representative Intersubunit Linkages (X-groups G5

X W -X

w-r-Y Y 10 r B N r N NR 12 YR 12

pi pi R13 R13 15 No. No. X

38

39 25

45

30

46

40 35 47

40 41

45

50

55

43 50

60 -- \ /N NO2

51

65 US 9,278,987 B2 115 116 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups Representative Intersubunit Linkages -90S H WEP-X

10 r NS R12

R13 15 No. No. X

52 61

62 53 25

30 OH

63

S4 35

55 40

64 56 45

57 65

50

58

55 66

59

60

60 67 N NH 65 US 9,278,987 B2 117 1. 18 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups) Representative Intersubunit Linkages (X-groups)

Y r 10 NN R12

R13 15

No. No. 68 79 -- \ /N-CA OH CA = cholate

69 25 -- \ /N-dCA dCA = deoxycholate 30 70

35 OH 71

w O 81 40 --\ /N NH OH

72 / \ 45 N N-NH 82 \ / O P

50

83 O

73 74 HN 55 75 OH 76 O 77

78 OH 84 O 60 \ +OR 65 OH US 9,278,987 B2 119 120 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages Representative Intersubunit Linkages (X-groups) -90S

G5 WE -x Y 10

N r NR 12

pi R13 15 No.

No. X 90

85 OH

25 91

86 O OH OH O 30 )-? 92 \ 35 87 / \ O\29 93 N N-S OH \ / 40

OH 45 O 94 88 / \ OW29 N N-S \ / 50

N 55 N \; 95 NY OH 89 O / \ We' 60 N N-S 96

OH 65 OH US 9,278,987 B2 121 122 TABLE 1-continued TABLE 1-continued

Representative Intersubunit Linkages (X-groups) Representative Intersubunit Linkages -90S G5 G5 5 H

w=1-xY w=i-xY O B r 10 r N NQ R12 NR 12 pi R13 pi R13 15 No. X No. X 104 N 97 N% YNH A w 2O I N N FN

OH

O 25

98 OH / { 105 -- N N

99 O 106

OH 35 1N-N

N NH 107

1OO OH 40 ~ 108 O 1N1,N H N N 45

109 101 / \ O N N N N / 50 OH 110 102 / \ NN s N N V / N-NH 55

111 103 O

OH 60

112

65 US 9,278,987 B2 123 124 TABLE 1-continued TABLE 1-continued Representative Intersubunit Linkages (X-groups Representative Intersubunit Linkages (X-groups) G5

w=1-xY

10

N r YR 12

R13 pi 15 No. No. X 113 123

114

124 25 115

30 116 125

117 35

126 40 118

119 45 127

120 50

128 121 55

60 129 122

65 US 9,278,987 B2

TABLE 1-continued TABLE 1-continued Representative Intersubunit Linkages (X-groups Representative Intersubunit Linkages (X-groups G5 G5 H 5 H W- – X W- -X Y Y O B O B

N NR 12 N YR 12

pi pi R13 R13 15 No. X No. X 130 /- 139 N X-NH 2O |-ON 131 ) 140 N NH N 25 1\-1

132 /- N 141 30 133 /- ~

35 142

N 134 1N1 N 40 O 143 135 1N1 ns N 45 /\,

144 O. O / M \/ 136 --- S y 145 137 55 N NH Y. Z \,

138 60 146 N N --ON

US 9,278,987 B2 129 130 TABLE 1-continued B. Oligomers with Modified Terminal Groups As noted above, the present disclosure also provides an Representative Intersubunit Linkages -90S oligomer comprising modified terminal groups. Applicants have found that modification of the 3' and/or 5' end of the oligomer with various chemical moieties provides beneficial WP-X therapeutic properties (e.g., enhanced cell delivery, potency, Y and/or tissue distribution, etc.) to the oligomers. In various embodiments, the modified terminal groups comprise a 10 hydrophobic moiety, while in other embodiments the modi fied terminal groups comprise a hydrophilic moiety. The r modified terminal groups may be present with or without the NQ R12 linkages described above. For example, in some embodi R13 ments, the oligomers comprise one or more modified terminal 15 group and modified intersubunit linkages. Combinations of No. X modified terminal groups and modified linkages also provide favorable therapeutic properties to the oligomers. In some 164 embodiments the modified terminal group is a peptide trans N NH porter as described herein. CF In another embodiment is the compound of Formula (I), wherein L'' is O

16S O 25 R23 R23 l o1 NL13

166 30

In another embodiment is the compound of Formula (I), O)- wherein L'' is 167 35

40 168

45 In another embodiment is the compound of Formula (I), 169 wherein L' is

170 50

55 and R is a C-C alkylene. In another embodiment is the compound of Formula (I), wherein L'' is

171 60

65 and wherein R is ethylene or propylene, and R' is hydrogen or methyl. US 9,278,987 B2 131 132 In another embodiment is the compound of Formula (I), In another embodiment is the compound of Formula (I), wherein L'' is wherein L' is

R23 R23 O P-N XX-XQ R" | --- 10 p and q are independently selected from 1 or 2, and R is CH. In another embodiment is the compound of Formula (I), In another embodiment is the compound of Formula (I), wherein L' is wherein L' is

R23 R23 Q H O 15 p S

o1 N Y. I g In another embodiment is the compound of Formula (I), and R is hydrogen or methyl. In another embodiment is the wherein L' is compound of Formula (I), wherein L'' is 3 O R23 R23 O CH p S 25 o1 \-sul N Q g H

In another embodiment is the compound of Formula (I), 30 and p, q, S and t are independently selected from 1 or 2. In wherein L' is another embodiment is the compound of Formula (I), wherein R" is methyl. In another embodiment is the compound of Formula (I), wherein R is hydrogen, methyl or both R' groups together form a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ring. In another embodiment is the compound 35 of Formula (I), wherein R is hydrogen or methyl. YY In another embodiment is the compound of Formula (I), ---- wherein Z is C(R)-OP(=O)(OH). In another embodi ment is the compound of Formula (I), wherein R’ is hydro In another embodiment is the compound of Formula (I), gen. wherein L' is 40 In another embodiment is the compound of Formula (I), wherein Zis-(L')-(R') and the R' is C-C alkyl, C-C, alkylcarbonyl, C-C alkyloxycarbonyl, ora3-18 membered alkoxyalkylcarbonyl. In another embodiment is the com pound of Formula (I), wherein Zis-(L')-(R')) and the R' 45 is a C-C alkyloxycarbonyl. In another embodiment is the ----YY compound of Formula (I), wherein Z is -(L'')-(R') and the R" is selected from –C(=O)CCHCH-OH, -C(=O) OCHCHOCHCH-OH, O —C(=O) and wherein p is 2, and q is 2. In another embodiment is the OCHCHOCH2CHOCH2CH2OH. In another embodiment compound of Formula (I), wherein L' is 50 is the compound of Formula (I), wherein Z is -(L'')-(R') and the R' is C(=O)CCHCHOCH2CHOCH2CH2OH. In another embodiment is the compound of Formula (I), wherein Z is selected from:

55 ----YY O | / \ and wherein p is 2, and q is 3. In another embodiment is the compound of Formula (I), wherein L' is 60 Allc1 NCH, K. / V /" O - O ! N s- O O -N- \ / Y / \ / \ . s H3C CH3 65 --- O US 9,278,987 B2 133 134 -continued alkylcarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a C-C alkylcarbonyl. In another embodiment is the compound of Formula (I) wherein R' is a (S/R \, C-Cs alkylcarbonyl. In another embodiment is the com "vuN pound of Formula (I) wherein R' is a C-C alkylcarbonyl. In %. YCH another embodiment is the compound of Formula (I) wherein R" is a C-C alkylcarbonyl. In another embodiment is the In another embodiment is the compound of Formula (I), compound of Formula (I) wherein R' is a C-C alkylcarbo wherein Z is nyl. In another embodiment is the compound of Formula (I) wherein R' is a C, alkylcarbonyl. In another embodiment is the compound of Formula (I) wherein R' is CHCO—. / \ O In another embodiment is the compound of Formula (I) O-P-N N-( O O wherein R' is a C-Cls alkylcarbonyl. In another embodi %. \ / / \ / \-on. ment is the compound of Formula (I) wherein R' is a C-C so H c1 Nchi alkyloxycarbonyl. In another embodiment is the compound 3 3 of Formula (I) wherein R' is a C-Clio alkyloxycarbonyl. In another embodiment is the compound of Formula (I) wherein In another embodiment is the compound of Formula (I) R" is a C-C alkyloxycarbonyl. In another embodiment is wherein R' is an electron pair. the compound of Formula (I) wherein R' is a C-C alky In another embodiment is the compound of Formula (I) 20 loxycarbonyl. In another embodiment is the compound of wherein R' is a C-C alkyl and R' is a C-C alkyl. In Formula (I) wherein R' is a C, alkyloxycarbonyl. In another another embodiment is the compound of Formula (I) wherein embodiment is the compound of Formula (I) wherein R' is a R" is a methyl group and R' is a methyl group. CHOCO . In another embodiment is the compound of In another embodiment is the compound of Formula (I) Formula (I) wherein R' is a -C(O)CH-CHCO.H. In wherein R' is hydrogen. ° another embodiment is the compound of Formula (I) wherein In another embodiment is the compound of Formula (I) R13 is a C(=O)CCHCH-OH, —C(=O) wherein R' is C-C alkyl. In another embodiment is the OCHCHOCH2CH2OH. O —C(=O) compound of Formula (I) wherein R' is C-Clo alkyl. In OCHCHOCHCHOCHCH-OH. another embodiment is the compound of Formula (I) wherein In another embodiment is the compound of Formula (I) R" is C-C alkyl. In anotherembodimentis the compound of wherein R' is C(=O)NHR'. In another embodiment is Formula (I) wherein R' is selected from prenyl, geranyl, the compound of Formula (I) wherein R' is C(=O) farnesyl or geranylgeranyl. NHR and R is methyl. In another embodiment is the compound of Formula (I) In some other embodiments, only the 3' terminus of the wherein R' is a cell-penetrating peptide. oligomer is conjugated to one of the groups noted above. In In another embodiment is the compound of Formula (I) some otherembodiments, only the 5' terminus of the oligomer wherein R' is a guanidinyl or amidinyl group. is conjugated to one of the groups noted above. In other In another embodiment is the compound of Formula (I) embodiments, both the 3' and 5' termini comprise one of the wherein R' is a C-Cs cycloalkyl. groups noted above. The terminal group for either the 5' In another embodiment is the compound of Formula (I) terminus, the 3' terminus, or both, may be selected from any wherein R' is a C-Co alkylcarbonyl. In another embodi- one of the groups noted above or any of the specific groups ment is the compound of Formula (I) wherein R' is a C-C, illustrated in Table 2. TABLE 2

Representative Terminal Groups

No. Name Structure

1 Trimethoxybenzoyl O H3CO

HCO OCH 2 9-fluorene-carboxyl ( ) O US 9,278,987 B2 135 136 TABLE 2-continued

Representative Terminal Groups

No. Name Structure 4-carbazolylbenzoyl

N

4-indazolylonebenzoyl O

WN NN O'A le

CH CH3

Farnesyl 21 21 rCH3 CH3 Geranyl 21 21 CH3 CH3

Prenyl HC 21 Diphenylacetyl y

Chlorodiphenylacetyl O C US 9,278,987 B2 137 138 TABLE 2-continued

Representative Terminal Groups

No. Name Structure

10 Hydroxydiphenylacetyl O OHy

11 Triphenylpropionyl O . 12 Triphenylpropyl O, 13 Triphenylacetyl ( ): O 14 Trityl (Tr)

15 Methoxytrityl (MeOTr)

N O US 9,278,987 B2 139 140 TABLE 2-continued Representative Terminal Groups

No. Name Structure

16 Methylsuccinimidyl- O cyclohexoyl O

O

17 Thioacetyl O

HS

18 COCHCHSSPy O Nn S n S ~y 2 19 Guanidinyl HN y 2 r NH

Trimethylglycine O 1. N 1.

21 Lauroyl

O

22 Triethyleneglycoloyl (EG3) HO N-1 no-1N1'N-1O no 23 Succinicacetyl "~yO O

24 Diphenylphosphoryl

25 Piperidin-4-yl US 9,278,987 B2 141 142 TABLE 2-continued

Representative Terminal Groups

No. Name Structure

26 Tritylpiperidin-4-yl

27 Boc-Piperidin-4-yl

28 Hexyn-6-yl

29 5-carboxyfluorescein

30 Benzhydryl

31 p-Chlorobenzhydryl

32 Piperazinyl (pip) US 9,278,987 B2 143 144 TABLE 2-continued Representative Terminal Groups

No. Name Structure 33 Triphenylphos O

-- P 5

34 Acetamide CHCO— 35 Methylurea CH-NHCO

1. Peptide Transporters In selected embodiments, for each X', the side chain moiety In some embodiments, the Subject oligomer is conjugated is independently selected from the group consisting of to a peptide transporter moiety, for example a cell-penetrating guanidyl (HN=C(NH)NH ), amidinyl (HN=C(NH) peptide transport moiety, which is effective to enhance trans C ), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrim port of the oligomer into cells. For example, in Some embodi idyl, 2-aminopyridinyl, and 2-aminopyrimidonyl, and it is ments the peptide transporter moiety is an arginine-rich pep 25 preferably selected from guanidyl and amidinyl. In one tide. In further embodiments, the transport moiety is attached embodiment, the side chain moiety is guanidyl, as in the to either the 5' or 3' terminus of the oligomer. When such amino acid subunit arginine (Arg). peptide is conjugated to either termini, the opposite termini is In some embodiments, the Y subunits are either contigu then available for further conjugation to a modified terminal ous, in that no X' subunits intervene between Y subunits, or group as described herein. 30 interspersed singly between X subunits. However, in some In some embodiments of the foregoing, the peptide trans embodiments the linking subunit may be betweenY' subunits. port moiety comprises 6 to 16 subunits selected from X In one embodiment, the Y subunits are at a terminus of the subunits, Y subunits, and Z subunits, where peptide transporter; in other embodiments, they are flanked (a) each X subunit independently represents lysine, argin by X' subunits. In further embodiments, each Y is —CO— ine or an arginine analog, said analog being a cationic 35 (CH), CHR NH-, where n is 2 to 7 and R is H. For C.-amino acid comprising a side chain of the structure example, when n is 5 and R is H. Y" is a 6-aminohexanoic acid RN=C(NH)R, where R is H or R; R is R, NH, subunit, abbreviated herein as Ahx. In selected embodiments NHR, or NR", where R is lower alkyl or lower alkenyland of this group, each X comprises aguanidyl side chain moiety, may further include oxygen or nitrogen; R and R* may as in an arginine Subunit. Exemplary peptides of this type togetherform a ring; and the side chain is linked to said amino 40 include those comprising arginine dimers alternating with acid via R or R: single Y subunits, where Y' is preferably Ahx. Examples (b) each Y subunit independently represents a neutral include peptides having the formula (RYR) or the formula amino acid—C(O) (CHR), NH , where n is 2 to 7 and (RRY), where Y' is preferably Ahx. In some embodiments, each R is independently H or methyl; and the nucleic acid analog is linked to a terminal Y. Subunit, 45 preferably at the C-terminus. In other embodiments, the (c) each Z subunit independently represents an O-amino linker is of the structure AhXB, where AhX is a 6-aminohex acid having a neutral aralkyl side chain; anoic acid subunit and B is a B-alanine subunit. wherein the peptide comprises a sequence represented by The peptide transport moieties as described above have one of (XY'X'), (XY), and (XZZ), where p is 2 to 5 and been shown to greatly enhance cell entry of attached oligo m is 2 to 8. 50 mers, relative to uptake of the oligomer in the absence of the In selected embodiments, for each X", the side chain moiety attached transport moiety, and relative to uptake by an is guanidyl, as in the amino acid subunit arginine (Arg). In attached transport moiety lacking the hydrophobic subunits further embodiments, each Y is —CO—(CH), CHR Y'. Such enhanced uptake may be evidenced by at least a NH , where n is 2 to 7 and R is H. For example, when n is 5 two-fold increase, or in other embodiments a four-fold and R is H. Y is a 6-aminohexanoic acid subunit, abbreviated 55 increase, in the uptake of the compound into mammaliancells hereinas Ahx; when n is 2 and R is H.Y" is a B-alanine subunit. relative to uptake of the agent by an attached transport moiety In certain embodiments, peptides of this type include those lacking the hydrophobic subunits Y". In some embodiments, comprising arginine dimers alternating with single Y. Sub uptake is enhanced at least twenty fold or at least forty fold, units, where Y is Ahx. Examples include peptides having the relative to the unconjugated compound. formula (RYR), or the formula (RRY), where Y is Ahx. In 60 A further benefit of the peptide transport moiety is its one embodiment, Y" is a 6-aminohexanoic acid subunit, R is expected ability to stabilize a duplex between an antisense arginine and p is 4. oligomer and its target nucleic acid sequence. While not In a further embodiment, each Z is phenylalanine, and mis wishing to be bound by theory, this ability to stabilize a 3 or 4. duplex may result from the electrostatic interaction between In some embodiments, the conjugated peptide is linked to 65 the positively charged transport moiety and the negatively a terminus of the oligomer via a linker Ahx-B, where Ahx is charged nucleic acid. In some embodiments, the number of a 6-aminohexanoic acid subunit and B is a B-alanine Subunit. charged subunits in the transporter is less than 14, as noted US 9,278,987 B2 145 146 above, or in other embodiments between 8 and 11, since too TABLE 3 - continued high a number of charged subunits may lead to a reduction in Sequence specificity. Arclinine-Rich Cell-Penetratinct Peptide Transporters Exemplary arginine-rich cell-penetrating peptide trans- Sequence (Amino to Carboxy SEQ porters are given below in Table 3. 5 Name Terminus, 5' to 3') ID NO RG RRRRRRG 22 TABLE 3 RG RRRRRRRG 23 Arginine-Rich Cell-Penetrating Peptide Transporters 10 R&G RRRRRRRRG 24 Sequence (Amino to Carboxy SEQ Name Terminus, 5' to 3') ID NO RSGRG RRRRRGRRRRG 25 (RFF); N-RFFRFFRFFAhxf3Ala-COOH 1. Rs. F2RG RRRRRFFRRRRG 26 CPO4. Of is Tat-G RKKRRORRRG 27 RTR N-RTRTRFLRRTAhxf3Ala-COOH 2 Tait - G RRRORRKKRG 28 RFFR N-RFFRFFRFFRAhxf3Ala-COOH 3 (RAhxRG) RAhxRRGGRAhxRGG 29 KTR N-KTRTKFLKKTAhxf3Ala-COOH 4. (RAhxRAhx) G RAhxRRRAhxRAhxRRRG 3O KFF N-KFFKFFKFFAhxf3Ala-COOH is 20 RP RRRRP 31 KFFK N-KFFKFFKFFKAhxf3Ala-COOH 6 RSP RRRRRP 32 (RFF) N-RFFRFFAhxf3Ala-COOH 7 RP RRRRRRP 33 (RFF) R N-RFFRFFRAhxf3Ala-COOH 8 25 R7P RRRRRRRP 34 RAX N-RAhxAhxRAhxAhxRAhxAhxf3Ala-COOH 9 RP RRRRRRRRP 35 (RAhxR) ; N-RAhxRRAhxRRAhxRRAhxRAhxf3Ala- O POO7 BAla = beta-alanine; 30 Ahx = 6-aminohexanoic acid rTat RRRORRKKRC 1. In some embodiments is a compound of Formula (I) RF2 RRRRRRRRRFFC 2 wherein Z is -(L')-(L')-(R') and R7 is a drug, protein or (RRAhx) B RRAhxRRAhxRRAhxRRAhxf3Ala 3 toxin. In some embodiments is a compound of Formula (I) wherein L' is a linker cleaveable under biological condi (RAhxR) AhxB; RAhxRRAhxRRAhxRRAhxRAhxf3Ala 4 3 tions. In some embodiments is a compound of Formula (I) wherein L' is selected from: (Ah:XRR)AhkRR) AhxBAhk AhxRRAhxRRAhxRRAhxRRAhxf3AlaAhkRRAhxRRAhxRRAhxRRAhxAl 5 a) —(C-C alkylene)-OC(O)C CHO : (RAhx) B RAhxRAhxRAhxRAhxRAhxRAhxf3Ala 6 b) C(O)—(C -C O alkylene)-OC(O)O CHO : c) –C(O)–(CH=CH) C(O)O CHO : (RAhx) B RAhxRAhxRAhxRAhxRAhxRAhxRAhxf3Ala 7 40 d) —(C -C O alkylene)-S S C HCHO—; O (RAhxR) AhxB RAhxRRAhxRRAhxRRAhxRRAhxRAhxf3Ala 8 e) C(O)–(C-Co alkylene)-S-S CH-CHO In some embodiments is a compound of Formula (I) (RAhxRRBR) Ah RAhxRRBAla RRAhxRRBAlaRAhxfAla 9 wherein R7 is a DNA-binding protein. In some embodiments is a compound of Formula (I) wherein R'' is a transcription RG RRRRG * * factor. In some embodiments is a compound of Formula (I) RG RRRRRG 21 wherein R'' is a zinc finger protein. In some embodiments is a compound of Formula (I) wherein R'' is a drug, protein or toxin selected from the listing provided in Table 4. TABLE 4

ANTIANDROGEN Bicalutamide, Bifluranol, Cioteronel, Cyproterone, Delmadinone Acetate, Flutamide, Nilutamide, Osaterone, Oxendolone ANTIBACTERIAL (ANTIBIOTICS) Aminoglycosides Amikacin, Arbekacin, Bambermycins, Butirosin, Dibekacin, Dihydrostreptomycin, Fortimicins, Gentarnicin, Isepamicin, Kanamycin, Micronomicin, Neomycin, Netilmicin, Paromomycin, Ribostamycin, Sisomicin, Spectinomycin, Streptomycin, Tobramycin, Trospectomycin Amphenicols AZidamfenicol, Chloramphenicol, Thiamphenicol Ansamycins Rifamide, Rifampin, Rifamycin SV. Rifapentine, Rifaximin B-Lactams US 9,278,987 B2 147 148 TABLE 4-continued Carbacephems

Loracarbef Carbapenems Biapenem, Ertapenem, Fropenem, Imipenem, Meropenem, Panipenem Cephalosporins Cefaclor, Cefadroxil, Cefamandole, Cefatrizine, Cefazedone, Cefazolin, Cefcapene Pivoxil, Cefclidin, Cefdinir, Cefditoren, Cefepime, Cefetamet, Cefixime, Cefimenoxime, Cefodizime, Cefonicid, CefoperaZone, Ceforanide, Cefoselis, Cefotaxime, Cefotiam, CefoZopran, Cefpimizole, Cefpiramide, Cefpirome, Cefbodoximc Proxetil, Cefprozil, Cefroxadine, Cefsulodin, Ceftazidime, Cefteram, Cefiezole, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefuroxime, CefuZonam, Cephacetrile Sodium, Cephalexin, Cephaloglycin, Cephaloridine, Cephalosporin C, Cephalothin, Cephapirin Sodium, Cephradine, Piveefalexin Cephamycins CefbuperaZone, Cefimetazole, Cefninox, Cefotetan, Cefoxitin Monobactams Aztreonam, Carumonam, Tigemonam Oxacephems Flomoxef, Moxalactam Penicillins Amdinocillin, Amdinocillin Pivoxil, Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, AZidocillin, AZlocillin, Bacampicillin, Carbenicillin, Carindacillin, Clometocillin, Cloxacillin, Cyclacillin, Dicloxacillin, Epicillin, Fenbenicillin, Floxacillin, Hetacillin, Lenampicillin, Metampicillin, Methicillin Sodium, Mezlocillin, Nafcillin, Oxacillin, Penamecillin, Penethamate Hydriodide, Penicillin G, Penicillin G Benzathine, Penicillin G Procaine, Penicillin N, Penicillin O, Penicillin V, Penimepicycline, Phenethicillin Potassium, Piperacillin, Pivampicillin, Propicillin, Quinacillin, Sulbenicillin, Sultamicillin, Talampicillin, Tenocillin, Ticarcillin Other Ritipenem Lincosamides Clindamycin, Lincomycin Macrollides Azithromycin, Carbomycin, Clarithromycin, Dirithromycin, Erythromycin, Erythromycin Acistrate, Erythromycin Estolate, Erythromycin Glucoheptonate, Erythromycin Lactobionate, Erythromycin Propionate, Erythromycin Stearate, Josamycin, Leucomycins, Midecamycins, Miokamycin, Oleandomycin, Primycin, Rokitamycin, Rosaramicin, Roxithromycin, Spiramycin, Telithromycin, Troleandomycin Polypeptides Amphomycin, Bacitracin, Bacitracin Zinc, Capreomycin, Colistin, Enduracidin, Enviomycin, Fusafungine, Gramicidin(s), Gramicidin S, Polymyxin, Quinupristin, Ristocetin, Teicoplanin, Thiostrepton, Tuberactinomycin, Tyrocidine, Tyrothricin, Vancomycin, Viomycin Tetracyclines Chlortetracycline, Clomocycline, Demeclocycline, Doxycycline, Guamecycline, Lymecycline, Meclocycline, Methacycline, Minocycline, Oxytetracycline, Penimepicycline, Pipacycline, Rolitetracycline, Sancycline, Tetracycline Others Cycloserine, Dalfopristin, Mupirocin, Pristinamycin, Virginiamycin ANTIBACTERIAL (SYNTHETIC) 2,4-Diaminopyrimidines Brodimoprim, Tetroxoprim, Trimethoprim, Nitrofurans Furaltadone, Furazolium Chloride, Nifuratel, Nifurfoline, Nifurpirinol, Nifurprazine, Niflurtoinol, Nitrofurantoin, Oxazolidinones Linezolid Quinolones and Analogs Balofloxacin, Cinoxacin, Ciprofloxacin, Clinafloxacin, Enoxacin, Fleroxacin, Flumequine, Gatifloxacin, Gemifloxacin, Grepafloxacin, Lomefloxacin, Miloxacin, Moxifloxacin, Nadifloxacin, Nalidixic Acid, Norfloxacin, Ofloxacin, Oxolinic Acid, PaZufloxacin, Pefloxacin, Pipemidic Acid, Piromidic Acid, Rosoxacin, Rufloxacih, Sitafloxacin, Spairfloxacin, Tosufloxacin, Trovafloxacin US 9,278,987 B2 149 150 TABLE 4-continued

Sulfonamides Acetyl Sulfamethoxypyrazine, Chloramine-B, Chloramine-T, Dichloramine T, N°-Formylsulfisomidine, N-f-D-Glucosylsulfanilamide, Mafenide, Noprylsulfamide, Phthalylsulfacetamide, Phthalylsulfathiazole, Salazosulfadimidine, Succinylsulfathiazole, Sulfabenzamide, Sulfacetamide, Sulfachlorpyridazine, Sulfachrysoidine, Sulfacytine, Sulfadiazine, Sulfadicramide, Sulfadimethoxine, Sulfadoxine, Sulfaethidole, Sulfaguanidine, Sulfaguanole, Sulfalene, Sulfaloxic Acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfamethizole, Sulfamethomidine, Sulfamethoxazole, Sulfamethoxypyridazine, Sulfametrole, Sulfamidochrysoidine, Sulfamoxole, Sulfanilamide, 4-Sulfanilamidosalicylic Acid, N-Sulfanilylsulfanilamide, Sulfanily lurea, N-Sulfani Iyl-3,4-xylamide, Sulfaperine, Sulfaphenazole, Sulfaproxyline, Sulfapyrazine, Sulfapyridine, Sulfasomizole, Sulfasymazine, Sulfathiazole, Sulfathiourea, Sulfisomidine, Sullfisoxazole Sulfones Acedapsone, Acediasulfone, Acetosulfone Sodium, Dapsone, Diathymosulfone, Glucosulfone Sodium, Solasulfone, Succisulfone, Sulfanilic Acid, p Sulfanilylbenzylamine, Sulfoxone Sodium, Thiazolsulfone Others Clofoctol, Hexedine, Methenamine, Metronidazole, Nitroxoline, Pexiganan, Taurolidine, Xibornol ANTIBACTERIAL (LEPROSTATIC) Acedapsone, Acetosulfone Sodium, Qofazimine, Dapsone, Diathymosulfone, Glucosulfone Sodium, Hydnocarpic Acid, Solasulfone, Succisulfone, Sulfoxone Sodium ANTIBACTERIAL (TUBERCULOSTATIC) p-Aminosalicylic Acid, p-Aminosalicylic Acid Hydrazide, Benzoylpas, 5-Bromosalicylhydroxamic Acid, Capreomycin, Clofazimine, Cyacetacide, Cycloserine, Dihydrostreptomycin, Enviomycin, Ethambutol, Ethionamide, Furonazide, Glyconiazide, Isoniazid, Morphazinamide, Opiniazide, Phenyl Aminosalicylate, Protionamide, Pyrazinamide, Rifabutin, Rifalazil, Rifampin, Rifapentine, Salinazid, Streptomycin, Streptonicozid, Sulfoniazide, Thiacetazone, Tiocarlide, Tuberactinomycin, Tubercidin, Viomycin, Dimethazan, Fencamine, Indalpine, indeloxazine Hydrochloride, Nefopam, Nomifensine, Oxitriptan see 4873 Oxypertine, Paroxetine, Sertraline. Thiazesim, Trazodone Hydrazides Hydrazines proclozide, Iproniazid, Isocarboxazid, Nialamide, Octamoxin, Phenelzine Phenyloxazolidinones Befloxatone, Toloxatone Pyrrollidones

Cotinine Tetracydics Maprotiline, Metralindole, Mianserin, Mirtazapine Tricyclics Adinazolam, Amineptine, Amitriptyline, Amitriptylinoxide, Amoxapine, Butriptyline, Clomipramine, Demexiptiline, Desipramine, Dibenzepin, Dimetaciine, Dothiepin, Doxepin, Fluacizine, Imipramine, Imipramine N-Oxide, Iprindole, Lofepramine, Melitracen, Metapramine, Nortriptyline, Noxiptilin, Opipramol, Pizotyline, Propizepine, Protriptyline, Quinupramine, Tianeptine, Trimipramine Others Adrafinil, Bupropion, Butacetin, Dioxadrol, Duloxetine, Etoperidone, Femoxetine, Fenpentadiol, Fluoxetine, Fluvoxamine, Hematoporphyrin, Hypericin, Levophacetoperane, Medifoxamine, Milnacipran, Minaprine, Moclobemide, Nefazodone, Oxaflozane, Piberaline, Prolintane, Pyrisuccideanol, Reboxetine, Ritanserin, Roxindole, Rubidium Chloride, Sulphide, Tandospirone, Thozalinone, Tofenacin, Tranylcypromine, Tryptophan, Venlafaxine, Viloxazine, Zimeldine ANTIDLABETIC Biguanides

Buformin, Metformin, Phenformin Hormones. Analogs Amylin, Insulin, Insulin Aspart, Insulin Glargine, Insulin Lispro, Pramlintide US 9,278,987 B2 151 152 TABLE 4-continued Sulfonylurea Derivatives Acetohexamide, Carbutamide, Chlorpropamide, Glibomuride, Gliclazide, Glimepiride, Glipizide, Gliquidone, Glisoxepid, Glyburide, Glybuthiazole), Glybuzole, Glyhexamide, Glymidine, Glypinamide, Phenbutamide, Tolazamide, Tolbutamide, Tolcyclamide Thiazolidinediones PioglitaZone, RosiglitaZone, TroglitaZone Others Acarbose, Calcium Mesoxalate, Miglitol, Nateglinide, Repaglinide, Voglibose ANTIESTROGEN Centchroman, Delmadinone Acetate, Tamoxifen, Torenifene ANTIFUNGAL (ANTIBIOTICS) Polyenes Amphotericin B, Candicidin, Dermostatin, Filipin, Fungichrornin, Hachimycin, Hamycin, Lucensomycin, Klepartricin, Natamycin, Nystatin, Pecilocin, Perimycin Others AZaserine, Caspofungin, Griseofulvin, Ohgomycins, Pyrrolnitrin, Siccanin, Tubercidin, Viridin ANTIFUNGAL (SYNTHETIC) Allylamines Butenafine, Naftifine, Terbinafine midazoles Bifonazole, Butoconazole, Chlordantoin, Chlormidazole, Coconazole, Clotrimazole, Econazole, Enilconazole, Fenticonazole, Flutrimazole, Isoconazole, Ketoconazole, Lanoconazole, Miconazole, Neticonazole, Omoconazole, Oxiconazole Nitrate, Sertaconazole, Sulconazole, Tioconazole Thiocarbamates Liranaftate, Tolciclate, Tolindate, Tolnaftate, Triazoles Fluconazole, traconazole, Posaconazole, Saperconazole, Terconazole, Voriconazole Others Acrisorcin, Amorolfine, Biphenamine, Bromosalicylchloranilide, Buclosamide, Calcium Propionate, Chlorphenesin, Ciclopirox, Cloxyquin, Coparaffinate, Diamthazole Dihydrochloride, Exalamide, Flucytosine, Hexetidine, Loflucarban, Nifuratel, Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, Sodium Propionate, Sulbentine, Tenonitrozole, Triacetin, Undecylenic Acid, Zinc Propi Onate ANTIGONADOTROPIN Danazol, Gestrinone, Paroxypropione ANTINEOPLASTIC Alkaloids 9-Aminocamptothecin, Docetaxel, Ecteinascidins, Etoposide, Irinotecan, Paclitaxel, Rubitecan, Teniposide, Topotecan, Vinblastine, Vincristine, Vindesine Alkylating Agents Alkyl Sulfonates BuSulfan, Improsulfan, Piposulfan AZiridines Carboquone, Uredepa Ethylenimines and Methylmelamines Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphoramide Nitrogen Mustards Chlorambucil, Chlornaphazine, Cyclophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan, Novembichin, Perfosfamide, Phenesterine, Prednimustine, Trichlonnethine, Trofosfamide, Uracil Mustard Nitrosoureas Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimustine US 9,278,987 B2 153 154 TABLE 4-continued Others Dacarbazine, Mannomustine, Mitobronitol, Mitolactol, Pipobroman, Temozolomide Antibiotics and Analogs Aclacinomycins, Anthramycin, AZaserine, Bleomycins, Cactinomycin, Carubicin, Chromomycins, Dactinomycin, Daunorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Idarubicin, Menogaril, Mitomycins, Mycophenolic Acid, Nogalamycin, Olivomycins, Peplomycin, Pirarubicin, Plicamycin, Porfiromycin, Puromycin, Streptonigrin, Streptozocin, TNP-470, Tubercidin, Valrubicin, Zinostatin, Zorobicin Antimetabolites Folk Acid Analogs. Antagonists Denopterin, Edatrexate, Methotrexate, Nolatrexed, Pemetrexed, Piritrexim, Pteropterin, Raltitrexed, Trimetrexate Purine Analogs Cladribine, Fludarabine, 6-Mercaptopurine, Thiamiprine, Thioguanine, Tiazofurin Pyrimidine Analogs Ancitabine, AZacitidine, 6-AZauridine, Capecitabine, Carmofur, Cytarabine, Decitabine, Doxifluridine, Emitefur, Enocitabine, Floxuridine, Fluorouracil, Gemcitabine, Tegafur Enzymes L-Asparaginase, Ranpirinase mmunomodulators Bropirimine, Interferon-C., Interferon-Y, Interleukin-2, Lentinan, Propagermanium, PSK (R), Roquinimex, Sizofiran, Ubenimex mmunotoxins

Denileukin Diftitox Monoclonal Antibodies Alemtuzumab, Edrecolomab, Gemtuzumab OZogamicin, Ibritumomab Tiuxetan, Rituximab, Tositumomab 'I, Trastuzumab Platinum Complexes Carbopiatin, Cisplatin, Lobaplatin, Milboplatin, Oxaliplatin Others Amsacrine, Arsenic Trioxide, Bisantrene, Defosfamide, Demecoline, Diaziquone, Eflornithine, Elliptinium Acetate, Etoglucid, Fenretinide, Flavopridol, Gallium Nitrate, Hydroxyurea, Imatinib, Liarozole, Lonidamine, Miltefosine, MitoguaZone, Mitoxantrone, Mopidamol, Nictracrine, Pentostatin, Phenamet, Podophyllinic Acid 2-Ethylhy-drazide, Procarbazine, Razoxane, Sobuzoxane, Spirogermanium, TenuaZonic Acid, Tirapazamine, Triaziquone, Urethan ANTINEOPLASTIC (HORMONAL) Androgens Calusterone, DromoStanolone, EpitioStanol, Mepitiostane, Testolactone Antiadrenals Aminoglutethimide, Mitotane, Trilostane Antiandrogens Bicalutamide, Flutamide, Nilutamide Antiestrogens Droloxifenc, Idoxifene, Tamoxifen, Torcmifene Antiprogestins Onapristone Aromatase Inhibitors Aminoglutethimide, Anastrozole, Exemestane, Fadrozole, Formestane, Letrozole, Vorozole Estrogens Diethylstilbestrol, Fosfestrol, Hexestrol, Polyestradiol Phosphate LH-RHAnalogs Buserelin, Cetrorelix, Goserelin, Leuprolide, Triptorelin US 9,278,987 B2 155 156 TABLE 4-continued Progestogens Chlormadinone Acetate, Medroxyprogesterone, Megestrol Acetate, Melengestrol Retinoids and Analogs Alitretinoin, Bexaroteine, Mofaroteine Somatostatin Analog Lanreotide ANTINEOPLASTIC (PHOTOSENSITIZER) Porfimer Sodium, Temopor?in, Tin Ethyl Etiopurpurin ANTINEOPLASTIC (RADIATION SOURCE) Americium, Chromic Phosphate, Radioactive, Cobalt, Gold, Radioactive, Colloidal, Ibritumomab Tiuxetan, 'I-Ethiodized Oil Iobenguane, Radium, Radon, Samarium Sm Lexidronam, Sodium Iodide, Radioactive, Sodium Phosphate, Radioactive, Strontium Chloride, Radioactive, Tositumomab 'I ANTINEOPLASTIC ADJUNCT Antimetastatic Agent Baltimastat, Prinomastat Chemomodulator

Eniluracil Chemosensitizer Biricodar, Valspodar Radioprotective

Amifostine Radiosensitzer Broxuridine, Etanidazole, RSR-13 Oroprotective

Mesna ANTINEUTROPENIC Granulocyte Colony-Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 ANTEPHEOCHROMOCYTOMA Metyrosine, Phenoxybenzamine, Phentolamine ANTIPNEUMOCYSTIC Atovaquone, Eflornithine, Pentamidine, Sulfamethoxazole ANTIPROGESTIN Onapristone ANTIPROSTATIC HYPERTROPHY Doxazosin, Dutasteride, Epristeride, Finasteride, Gestonorone Caproate, Mepartricin, OSaterone, Oxendolone, Tamsulosin, Terazosin ANTIPROTOZOAL (CRYPTOSPO-RIDIUM) Nitazoxanide ANTIPROTOZOAL (LEISHMANIA) Ethylstibamine, Hydroxystilbamidine, N-Methylglucamine, Pentamidine, Sodium Stibogluconate see 707 Stilbamidine, Urea Stibamine ANTIPROTOZOAL (TOXO-PLASMA) Pyrimethamine ANTIPROTOZOAL (TRICHO-MONAS) AcetarSone, Aminitrozole, Anisomycin, Azanidazole, Furazolidone, Hachimycin, Lauroguadine, Mepartricin, Metronidazole, Nifuratel, Nifuroxime, Nimorazole, Secnidazole, Silver Picrate, Tenonitrozole, Tinidazole ANTIPROTOZOAL (TRYPANO-SOMA) BenZnidazole, Eflomithine, Melarsoprol, Nifurtimox, Oxophenarsine Hydrochloride, Pentamidine, Propamidine, Puromycin, Quinapyramine, Stilbanidine, Suramin Sodium, Trypan Red, Tryparsamidc ANTIVIRAL Monoclonal Antibodies

PaliwiZumab US 9,278,987 B2 157 158 TABLE 4-continued Peptidomimetics

Amprenavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir Polynucleotides

Ampligen, Fomivirsen Purines? Pyrimidinones

Abacavir, Acyclovir, Adefovir, Cidofovir, Cytarabine, Didanosine, Dideoxyadenosine, Edoxudine, Emtricitabine, Famciclovir, Floxuridine, Ganciclovir, Idoxuridine, Inosine Pranobex, Lamivudine, MADU, Penciclovir, Sorivudine, Stavudine, Tenofovir, Trifluridine, Valacyclovir, Valganciclovir, Vidarabine, Zalcitabine, Zidovudine Sialic Acid Analogs

Oseltamivir, Zanamivir Others

Acemannan, Acetylleucine Monoethanolamine, Amantadine, Amidinomycin, Atevirdine, Capravirine, Delavirdine, n-Docosanol, Efavirenz, Foscamet Sodium, Interferon-W, Interferon-, Interferon-y, Kethoxal, Lysozyme, MethisaZone, Moroxydine, Nevirapine, Pentafiiside, Pleconaril, Podophyllotoxin, Ribavirin, Rimantadine, Stallimycin, Statolon, Tremacamra, Tromantadine AROMATASE INHIBITORS

Aminoglutethimide, Anastrozole, Exemestane, Fadrozole, Formestane, Letrozole, Vorozole DIAGNOSTIC AID (MRICONTRASTAGENT) Ferumoxides, Ferumoxsil, Gadobenate Dimeglumine, Gadobutrol, Gadodiamide, Gadopentetic Acid, Gadoteridol, Gadoversetamide, Gadoxetic Acid, Mangafodipir, MS-325, Perflubron DIAGNOSTIC AID (RADIOACTIVE IMAGING AGENT) Arcitumomab Complex with Tc, Butedronic Acid Complex, Capromab Pendetide, Depreotide Complex with Tc, Disofenin Complex with Tc, Exametazime Complex with Tc, Fludeoxyglucose F18, o-Iodohippurate Sodium 'I, Iofetamine 'I GLUCOCORTOCOID

21-Acetoxypregnenolone, AlclometaSone, Algestone, Amcinonide, Beclomethasone, Betamethasone, Budesonide, Chloroprednisone, Ciclesonide, Clobetasol, Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone, Cortivazol, Deflazacort, Prednisolone Sodium Phosphate, Prednisone, Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone, Triamcinolone Acetonide, Triamcinolone Benetonide, Triamcinolone Hexacetonide GROWTH HORMONE ANTAGONISTINHIBITOR

Octreotide, Pegvisomant, Somatostatin GROWTH HORMONE RELEASING FACTOR

Sermorelin GROWTH STIMULANT

Somatotropin REVERSE TRANSCRIPTASE INHIBITOR

Abacavir, Atevirdine, Capravirine, Delavirdine, Didanosine, Dideoxyadenosine, Efavirenz, Emtricitabine, Foscarnet Sodium, Lamivudine, Nevirapine, Stavudine, Suramin Sodium, Tenofovir, Zalcitabine, Zidovudine TOPOISOMERASEINEHIBITOR

9-Aminocamptothecin, Irinotecan, Topotecan TOPOISOMERASE IIINHIBITOR

Daunorubicin, Doxorubicin, Etoposide, Sobuzoxane, Teniposide US 9,278,987 B2 159 160 C. Properties of the Oligomers As noted above, the present disclosure is directed to oligo

mercomprising various modifications which impart desirable properties (e.g., increased antisense activity) to the oligo mers. In certain embodiments, the oligomer comprises a backbone comprising a sequence of morpholino ring struc tures joined by intersubunit linkages, the intersubunit link ages joining a 3'-end of one morpholino ring structure to a 5'-end of an adjacent morpholino ring structure, wherein each morpholino ring structure is bound to a base-pairing moiety, 10 Such that the oligomer can bindina sequence-specific manner to a target nucleic acid. The morpholino ring structures may have the following structure (i): 15 /or (i)

-- 25 wherein B is, at each occurrence, independently a base-pair ing moiety. Each morpholino ring structure Supports a base pairing 30 moiety (Pi), to form a sequence of base pairing moieties which is typically designed to hybridize to a selected anti sense target in a cell or in a subject being treated. The base pairing moiety may be a purine or pyrimidine found in native DNA or RNA (A, G, C, T, or U) or an analog, such as 35 hypoxanthine (the base component of the nucleoside inosine) or 5-methyl cytosine. Analog bases that confer improved binding affinity to the oligomer can also be utilized. Exem HN h HN NH plary analogs in this regard include C5-propynyl-modified 40 pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp) and NH2 the like. Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine having their respective amino groups protected by acyl pro 45 tecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromou racil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimi dine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, Xanthine, or hypoxanthine (the latter two being the natural 50 NH2 degradation products). The modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2 196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, aft 313, are also contemplated. 55 Further examples of base pairing moieties include, but are not limited to, expanded-size nucleobases in which one or more benzene rings has been added. Nucleic base replace ments described in the Glen Research catalog (www.glenre search.com); Krueger AT et al. Acc. Chem. Res., 2007, 40, 60 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943: Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543: Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622 627, are contemplated as useful for the synthesis of the oli 65 As noted above, the oligomer may be modified, in accor gomers described herein. Some examples of these expanded dance with an aspect of the invention, to include one or more size nucleobases are shown below: X2-X8 groups. In some embodiments, a compound of For US 9,278,987 B2 161 162 mula I contains at least one X3-X8 group and no X2 group. In In some embodiments is a compound of Formula I wherein Some embodiments, a compound of Formula I contains at R" is acetyl and X is not least one X2, X4, X5, X6, X7, or X8 group and no X3 group. In some embodiments, a compound of Formula I contains at least one X2, X3, X5, X6, X7, or X8 group and no X4 group. 5 In some embodiments, a compound of Formula I contains at least one X2, X3, X4, X6, X7, or X8 group and no X5 group. -- N O In some embodiments, a compound of Formula I contains at least one X2, X3, X4, X5, X7, or X8 group and no X6 group. O-ulu In some embodiments, a compound of Formula I contains at 10 least one X2, X3, X4, X5, X6, or X8 group and no X7 group. In some embodiments is a compound of Formula I wherein X In some embodiments, a compound of Formula I contains at is not least one X2-X7 group and no X8 group. In some embodi ments is a compound of Formula I wherein X is not 15

O HN -- SR N y-ll NH --O -

In some embodiments is a compound of Formula I wherein R" is acetyl and X is not 25 In some embodiments is a compound of Formula I wherein R" is acetyl and X is not

O HN -- N y-ll 30 NH SR

In some embodiments is a compound of Formula I wherein X 35 -- \ / - is not

In some embodiments is a compound of Formula I wherein X O 40 is not -- NA VN HNy-ll -- NH

45 -- N NH In some embodiments is a compound of Formula I wherein \ / NH2. R" is acetyl and X is not In some embodiments is a compound of Formula I wherein R" is acetyl and X is not O 50 -- NA VN HNy-ll -- NH / \ NH 55 --O- In some embodiments is a compound of Formula I wherein X is not In some embodiments is a compound of Formula I wherein X is not 60 -- N O -- N NH. C -u 65 \ / US 9,278,987 B2 163 164 In some embodiments is a compound of Formula I wherein In some embodiments is a compound of Formula I wherein X R" is acetyl and X is not is not

- 5 O N NH. N - NHhx ARhkARNHAc. In some embodiments is a compound of Formula I wherein X 10 is not In some embodiments is a compound of Formula I wherein R" is acetyl and X is not -- Y- 15 / \ A

NHhx ARhkARNHAc. In some embodiments is a compound of Formula I wherein R" is acetyl and X is not 2O ty In some embodiments is a compound of Formula I wherein each X group is independently selected as described above with the provision that if the X group on the phosphorous N Y- linkage proximal to the 3' terminus is 25

O N N HN -- In some embodiments is a compound of Formula I wherein X V f NH2 is not 30 NH s O O A V v M N N

-- N \-/ N -(CF. 35 V M NH2,

In some embodiments is a compound of Formula I wherein / \ O O R" is acetyl and X is not -- N O 40 \ / NH () / \ Y N N-( O s 45 N N -( N NH In some embodiments is a compound of Formula I wherein X \ / Yi, \ / . is not / \ / \ O truly- 55 -- Y-ch , or O In some embodiments is a compound of Formula I wherein N N R" is acetyl and X is not V / 60 NHhxARhkARNHAc,

-- O CF then at least one other X group is not X1. In some embodiments is a compound of Formula I wherein NH 65 each X group is independently selected as described above with the provision that if the X group on the phosphorous linkage proximal to the 3' terminus is US 9,278,987 B2 166 -continued / \ / \ , 5 --O---O-

10 , or

15 --O-(l NHhxARhkARNHAc,

then at least three other X groups are not X1. In some embodiments is a compound of Formula I wherein each X group is independently selected as described above with the provision that if the X group on the phosphorous linkage proximal to the 3' terminus is

25 --O\ / OuO HNY -- 30 / \ O s

NHhxARhx ARNHAc, 35 then at least two other X groups are not X1. In some embodiments is a compound of Formula I wherein each X group is independently selected as described above with the provision that if the X group on the phosphorous linkage proximal to the 3' terminus is

45 O HN -- -- \ /N y-ll NH s 50 O N N

--- 55

O --\ /N 60 NHhxARhkARNHAc,

then all other X groups are not X1. In some embodiments is a compound of Formula I wherein 65 each X group is independently selected as described above with the provision that if X1 is present as N(CH), and X3 is present as US 9,278,987 B2 167 168 mula I contains 5-10% X2-X8 groups. In some embodiments, NH a compound of Formula I contains less than 5% X2-X8 groups. )= NH In some embodiments is a compound of Formula I wherein HN 5 the X1 groups and the X2-X8 groups are interspersed along N the backbone. In some embodiments is a compound of For O O mula I wherein the X1 groups and the X2-X8 groups have a O strictly alternating pattern along the backbone. In some A V HN -- embodiments is a compound of Formula I wherein the X1 10 groups and the X2-X8 groups do not have a strictly alternat N N y-l. ing pattern along the backbone. In some embodiments is a NH s compound of Formula I having blocks of X1 groups and blocks of X2-X8 groups. In some embodiments is a com pound of Formula I having a central block of X1 groups then X7 is not piperidinyl. 15 flanked by blocks of X2-X8 groups. In some embodiments is In some embodiments is a compound of Formula I wherein a compound of Formula I having a central block of X2-X8 each X group is independently selected as described above groups flanked by blocks of X1 groups. with the provision that if X1 is present as N(CH), and X7 is In any of the aforementioned embodiments of Formula (I) present as piperidinyl, then X3 is not wherein at least one X is selected from X2-X8 is another embodiment wherein n is an integer from 30-35. In further embodiments of the aforementioned embodiments of For NH mula (I) wherein at least one X is selected from X2-X8, n is an )= NH integer from 25-29. In further embodiments of the aforemen HN tioned embodiments of Formula (I) wherein at least one X is 25 selected from X2-X8, n is an integer from 20-24. In further N embodiments of any of the aforementioned embodiments of O O Formula (I) wherein at least one X is selected from X2-X8, n O is an integer from 15-19. In further embodiments of the afore A w HN -- mentioned embodiments of Formula (I) wherein at least one N N y-ll 30 X is selected from X2-X8, n is an integer from 10-14. In further embodiments of the aforementioned embodiments of NH Formula (I) wherein at least one X is selected from X2-X8, in is an integer from 5-9. In yet further embodiments of the In some embodiments, a compound of Formula I contains aforementioned embodiments of Formula (I) wherein at least 35 one X is selected from X2-X8, n is an integer from 1-4. one X2-X8 group per every 2-5 X1 groups. In some embodi In any of the aforementioned embodiments of Formula (I) ments, a compound of Formula I contains 3-5 X2-X8 groups wherein n is an integer from 30-35 is another embodiment per every 10 X1 groups. In some embodiments, a compound wherein one X is X2-X8. In any of the aforementioned of Formula I contains 3-5 X2-X8 groups per every 10 X1 embodiments of Formula (I) wherein n is an integer from groups. In some embodiments, a compound of Formula I 40 30-35 is another embodiment wherein two X are indepen contains 100% X2-X8 groups. In some embodiments, a com dently selected from X2-X8. In any of the aforementioned pound of Formula I contains 95-100% X2-X8 groups. In embodiments of Formula (I) wherein n is an integer from Some embodiments, a compound of Formula I contains 30-35 is another embodiment wherein three X are indepen 90-95% X2-X8 groups. In some embodiments, a compound dently selected from X2-X8. In any of the aforementioned of Formula I contains 85-90% X2-X8 groups. In some 45 embodiments of Formula (I) wherein n is an integer from embodiments, a compound of Formula I contains 80-85% 30-35 is another embodiment wherein four X are indepen X2-X8 groups. In some embodiments, a compound of For dently selected from X2-X8. In any of the aforementioned mula I contains 75-80% X2-X8 groups. In some embodi embodiments of Formula (I) wherein n is an integer from ments, a compound of Formula I contains 70-75% X2-X8 30-35 is another embodiment wherein five X are indepen groups. In some embodiments, a compound of Formula I 50 dently selected from X2-X8. In any of the aforementioned contains 65-70% X2-X8 groups. In some embodiments, a embodiments of Formula (I) wherein n is an integer from compound of Formula I contains 60-65% X2-X8 groups. In 30-35 is another embodiment wherein six X are indepen Some embodiments, a compound of Formula I contains dently selected from X2-X8. In any of the aforementioned 55-60% X2-X8 groups. In some embodiments, a compound embodiments of Formula (I) wherein n is an integer from of Formula I contains 50-55% X2-X8 groups. In some 55 30-35 is another embodiment wherein seven X are indepen embodiments, a compound of Formula I contains 45-50% dently selected from X2-X8. In any of the aforementioned X2-X8 groups. In some embodiments, a compound of For embodiments of Formula (I) wherein n is an integer from mula I contains 40-45%. X2-X8 groups. In some embodi 30-35 is another embodiment wherein eight X are indepen ments, a compound of Formula I contains 35-40% X2-X8 dently selected from X2-X8. In any of the aforementioned groups. In some embodiments, a compound of Formula I 60 embodiments of Formula (I) wherein n is an integer from contains 30-35% X2-X8 groups. In some embodiments, a 30-35 is another embodiment wherein nine X are indepen compound of Formula I contains 25-30% X2-X8 groups. In dently selected from X2-X8. In any of the aforementioned Some embodiments, a compound of Formula I contains embodiments of Formula (I) wherein n is an integer from 20-25% X2-X8 groups. In some embodiments, a compound 30-35 is another embodiment wherein ten X are indepen of Formula I contains 15-20% X2-X8 groups. In some 65 dently selected from X2-X8. In any of the aforementioned embodiments, a compound of Formula I contains 10-15% embodiments of Formula (I) wherein n is an integer from X2-X8 groups. In some embodiments, a compound of For 30-35 is another embodiment wherein eleven X are indepen US 9,278,987 B2 169 170 dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 25-30 is another embodiment wherein fourteen X are inde 30-35 is another embodiment wherein twelve X are indepen pendently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 25-30 is another embodiment wherein fifteen X are indepen 30-35 is another embodiment wherein thirteen X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer 25-30 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein sixteen X are independently 30-35 is another embodiment wherein fourteen X are inde selected from X2-X8. In any of the aforementioned embodi pendently selected from X2-X8. In any of the aforementioned 10 ments of Formula (I) wherein n is an integer from 25-30 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein seventeen X are independently 30-35 is another embodiment wherein fifteen X are indepen selected from X2-X8. In any of the aforementioned embodi dently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 25-30 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein eighteen X are independently 30-35 is another embodiment wherein sixteen X are indepen 15 selected from X2-X8. In any of the aforementioned embodi dently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 25-30 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein nineteen X are independently 30-35 is another embodiment wherein seventeen X are inde selected from X2-X8. In any of the aforementioned embodi pendently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 25-30 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein twenty X are independently 30-35 is another embodiment wherein eighteen X are inde selected from X2-X8. pendently selected from X2-X8. In any of the aforementioned In any of the aforementioned embodiments of Formula (I) embodiments of Formula (I) wherein n is an integer from wherein n is an integer from 20-25 is another embodiment 30-35 is another embodiment wherein nineteen X are inde wherein one X is X2-X8. In any of the aforementioned pendently selected from X2-X8. In any of the aforementioned 25 embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein two X are indepen 30-35 is another embodiment wherein twenty X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. embodiments of Formula (I) wherein n is an integer from In any of the aforementioned embodiments of Formula (I) 20-25 is another embodiment wherein three X are indepen wherein n is an integer from 25-30 is another embodiment 30 dently selected from X2-X8. In any of the aforementioned wherein one X is X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein four X are indepen 25-30 is another embodiment wherein two X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 35 20-25 is another embodiment wherein five X are indepen 25-30 is another embodiment wherein three X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein six X are indepen 25-30 is another embodiment wherein four X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned 40 embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein seven X are indepen 25-30 is another embodiment wherein five X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein eight X are indepen 25-30 is another embodiment wherein six X are indepen 45 dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein nine X are indepen 25-30 is another embodiment wherein seven X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 50 20-25 is another embodiment wherein ten X are indepen 25-30 is another embodiment wherein eight X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein eleven X are indepen 25-30 is another embodiment wherein nine X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned 55 embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein twelve X are indepen 25-30 is another embodiment wherein ten X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein thirteen X are indepen 25-30 is another embodiment wherein eleven X are indepen 60 dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 20-25 is another embodiment wherein fourteen X are inde 25-30 is another embodiment wherein twelve X are indepen pendently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 65 20-25 is another embodiment wherein fifteen X are indepen 25-30 is another embodiment wherein thirteen X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer 20-25 is US 9,278,987 B2 171 172 another embodiment wherein sixteen X are independently 19-20 is another embodiment wherein nineteen X are inde selected from X2-X8. In any of the aforementioned embodi pendently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 20-25 is embodiments of Formula (I) wherein n is 20 is another another embodiment wherein seventeen X are independently embodiment wherein twenty X are independently selected selected from X2-X8. In any of the aforementioned embodi from X2-X8. ments of Formula (I) wherein n is an integer from 20-25 is In any of the aforementioned embodiments of Formula (I) another embodiment wherein eighteen X are independently wherein n is an integer from 10-15 is another embodiment selected from X2-X8. In any of the aforementioned embodi wherein one X is X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 20-25 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein nineteen X are independently 10 10-15 is another embodiment wherein two X are indepen selected from X2-X8. In any of the aforementioned embodi dently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 20-25 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein twenty X are independently 10-15 is another embodiment wherein three X are indepen selected from X2-X8. dently selected from X2-X8. In any of the aforementioned In any of the aforementioned embodiments of Formula (I) 15 embodiments of Formula (I) wherein n is an integer from wherein n is an integer from 15-20 is another embodiment 10-15 is another embodiment wherein four X are indepen wherein one X is X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein two X are indepen 10-15 is another embodiment wherein five X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein three X are indepen 10-15 is another embodiment wherein six X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein four X are indepen 25 10-15 is another embodiment wherein seven X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein five X are indepen 10-15 is another embodiment wherein eight X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from 30 embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein six X are indepen 10-15 is another embodiment wherein nine X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein seven X are indepen 10-15 is another embodiment wherein ten X are indepen dently selected from X2-X8. In any of the aforementioned 35 dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein eight X are indepen 11-15 is another embodiment wherein eleven X are indepen dently selected from X2-X8. In any of the aforementioned dently selected from X2-X8. In any of the aforementioned embodiments of Formula (I) wherein n is an integer from embodiments of Formula (I) wherein n is an integer from 15-20 is another embodiment wherein nine X are indepen 40 12-15 is another embodiment wherein twelve X are indepen dently selected 15-20 is another embodiment wherein ten X dently selected from X2-X8. In any of the aforementioned are independently selected from X2-X8. In any of the afore embodiments of Formula (I) wherein n is an integer from mentioned embodiments of Formula (I) wherein n is an inte 13-15 is another embodiment wherein thirteen X are indepen ger from 15-20 is another embodiment wherein eleven X are dently selected from X2-X8. In any of the aforementioned independently selected from X2-X8. In any of the aforemen 45 embodiments of Formula (I) wherein n is an integer from tioned embodiments of Formula (I) wherein n is an integer 14-15 is another embodiment wherein fourteen X are inde from 15-20 is another embodiment wherein twelve X are pendently selected from X2-X8. In any of the aforementioned independently selected from X2-X8. In any of the aforemen embodiments of Formula (I) wherein n is 15 is another tioned embodiments of Formula (I) wherein n is an integer embodiment wherein fifteen X are independently selected from 15-20 is another embodiment wherein thirteen X are 50 from X2-X8. independently selected from X2-X8. In any of the aforemen In any of the aforementioned embodiments of Formula (I) tioned embodiments of Formula (I) wherein n is an integer wherein n is an integer from 5-10 is another embodiment from 15-20 is another embodiment wherein fourteen X are wherein one X is X2-X8. In any of the aforementioned independently selected from X2-X8. In any of the aforemen embodiments of Formula (I) whereinn is an integer from 5-10 tioned embodiments of Formula (I) wherein n is an integer 55 is another embodiment wherein two X are independently from 15-20 is another embodiment wherein fifteen X are selected from X2-X8. In any of the aforementioned embodi independently selected from X2-X8. In any of the aforemen ments of Formula (I) wherein n is an integer from 5-10 is tioned embodiments of Formula (I) wherein n is an integer another embodiment wherein three X are independently 16-20 is another embodiment wherein sixteen X are indepen selected from X2-X8. In any of the aforementioned embodi dently selected from X2-X8. In any of the aforementioned 60 ments of Formula (I) wherein n is an integer from 5-10 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein four X are independently 17-20 is another embodiment wherein seventeen X are inde selected from X2-X8. In any of the aforementioned embodi pendently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 5-10 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein five X are independently 18-20 is another embodiment wherein eighteen X are inde 65 selected from X2-X8. In any of the aforementioned embodi pendently selected from X2-X8. In any of the aforementioned ments of Formula (I) wherein n is an integer from 6-10 is embodiments of Formula (I) wherein n is an integer from another embodiment wherein six X are independently US 9,278,987 B2 173 174 selected from X2-X8. In any of the aforementioned embodi requiring greater than 20 bases for high T, values. For some ments of Formula (I) wherein n is an integer from 7-10 is applications, longer oligomers, for example longer than 20 another embodiment wherein seven X are independently bases may have certain advantages. For example, in certain selected from X2-X8. In any of the aforementioned embodi embodiments longer oligomers may find particular utility for ments of Formula (I) wherein n is an integer from 8-10 is use in exon skippin or splice modulation. another embodiment wherein eight X are independently The targeting sequence bases may be normal DNA bases or selected from X2-X8. In any of the aforementioned embodi analogues thereof, e.g., uracil and inosine that are capable of ments of Formula (I) wherein n is an integer from 9-10 is Watson-Crick base pairing to target-sequence RNA bases. another embodiment wherein nine X are independently The oligomers may also incorporate guanine bases in place selected from X2-X8. In any of the aforementioned embodi 10 ofadenine when the target nucleotide is a uracil residue. This ments of Formula (I) wherein n is 10 is another embodiment is useful when the target sequence varies across different viral wherein ten X are independently selected from X2-X8. species and the variation at any given nucleotide residue is In any of the aforementioned embodiments of Formula (I) either cytosine or uracil. By utilizing guanine in the targeting wherein n is an integer from 1-5 is another embodiment oligomer at the position of variability, the well-known ability wherein one X is X2-X8. In any of the aforementioned 15 of guanine to base pair with uracil (termed C/U:G base pair embodiments of Formula (I) wherein n is an integer from 2-5 ing) can be exploited. By incorporatingguanine at these loca is another embodiment wherein two X are independently tions, a single oligomer can effectively target a widerrange of selected from X2-X8. In any of the aforementioned embodi RNA target variability. ments of Formula (I) wherein n is an integer from 3-5 is The compounds (e.g., oligomers, intersubunit linkages, another embodiment wherein three X are independently terminal groups) may exist in different isomeric forms, for selected from X2-X8. In any of the aforementioned embodi example structural isomers (e.g., tautomers). With regard to ments of Formula (I) wherein n is an integer from 4-5 is Stereoisomers, the compounds may have chiral centers and another embodiment wherein four X are independently may occur as racemates, enantiomerically enriched mixtures, selected from X2-X8. In any of the aforementioned embodi individual enantiomers, mixture or diastereomers or indi ments of Formula (I) wherein n is 5 is another embodiment 25 vidual diastereomers. All such isomeric forms are included wherein five X are independently selected from X2-X8. within the present invention, including mixtures thereof. The In some embodiments for antisense applications, the oli compounds may also possess axial chirality which may result gomer may be 100% complementary to the nucleic acid target in atropisomers. Furthermore, Some of the crystalline forms sequence, or it may include mismatches, e.g., to accommo of the compounds may exist as polymorphs, which are date variants, as long as a heteroduplex formed between the 30 included in the present invention. In addition, Some of the oligomer and nucleic acid target sequence is sufficiently compounds may also form Solvates with water or other stable to withstand the action of cellular nucleases and other organic solvents. Such solvates are similarly included within modes of degradation which may occur in vivo. Mismatches, the scope of this invention. if present, are less destabilizing toward the end regions of the The oligomers described herein may be used in methods of hybrid duplex than in the middle. The number of mismatches 35 inhibiting production of a protein or replication of a virus. allowed will depend on the length of the oligomer, the per Accordingly, in one embodiment a nucleic acid encoding centage of G:C base pairs in the duplex, and the position of the Such a protein is exposed to an oligomeras disclosed herein. mismatch(es) in the duplex, according to well understood In further embodiments of the foregoing, the antisense oligo principles of duplex stability. Although Such an antisense mer comprises either a 5' or 3' modified terminal group or oligomer is not necessarily 100% complementary to the 40 combinations thereof, as disclosed herein, and the base pair nucleic acid target sequence, it is effective to stably and ing moieties B form a sequence effective to hybridize to a specifically bind to the target sequence. Such that a biological portion of the nucleic acid at a location effective to inhibit activity of the nucleic acid target, e.g., expression of encoded production of the protein. In one embodiment, the location is protein(s), is modulated. an ATG start codon region of an mRNA, a splice site of a The stability of the duplex formed between an oligomer 45 pre-mRNA, or a viral target sequence as described below. and the target sequence is a function of the binding T, and the In one embodiment, the oligomer has a T, with respect to Susceptibility of the duplex to cellular enzymatic cleavage. binding to the target sequence of greater than about 50° C. The T of an antisense compound with respect to comple and it is taken up by mammalian cells or bacterial cells. In mentary-sequence RNA may be measured by conventional another embodiment, the oligomer may be conjugated to a methods, such as those described by Hames et al., Nucleic 50 transport moiety, for example an arginine-rich peptide, as Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described herein to facilitate such uptake. In another embodi described in Miyada C. G. and Wallace R. B., 1987, Oligo ment, the terminal modifications described herein can func nucleotide hybridization techniques, Methods Enzymol. Vol. tion as a transport moiety to facilitate uptake by mammalian 154 pp. 94-107. and/or bacterial cells. In some embodiments, each antisense oligomer has a bind 55 The preparation and properties of morpholino oligomers is ing T, with respect to a complementary-sequence RNA, of described in more detail below and in U.S. Pat. No. 5,185,444 greater than body temperature or in other embodiments and WO/2009/064471, each of which is hereby incorporated greater than 50° C. In other embodiments T's are in the by reference in their entirety. range 60-80° C. or greater. According to well known prin D. Formulation and Administration of the Oligomers ciples, the T of an oligomer compound, with respect to a 60 The present disclosure also provides for formulation and complementary-based RNA hybrid, can be increased by delivery of the disclosed oligomer. Accordingly, in one increasing the ratio of C:G paired bases in the duplex, and/or embodiment the present disclosure is directed to a composi by increasing the length (in base pairs) of the heteroduplex. At tion comprising an oligomer as disclosed herein and a phar the same time, for purposes of optimizing cellular uptake, it maceutically acceptable vehicle. may be advantageous to limit the size of the oligomer. For this 65 Effective delivery of the antisense oligomer to the target reason, compounds that show high T (50° C. or greater) at a nucleic acid is an important aspect of treatment. Routes of length of 20 bases or less are generally preferred over those antisense oligomer delivery include, but are not limited to, US 9,278,987 B2 175 176 various systemic routes, including oral and parenteral routes, Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers in e.g., intravenous, Subcutaneous, intraperitoneal, and intra Biology and Medicine, pp. 287-341, Academic Press, 1979). muscular, as well as inhalation, transdermal and topical deliv Hydrogels may also be used as vehicles for antisense oligo ery. The appropriate route may be determined by one of skill mer administration, for example, as described in WO in the art, as appropriate to the condition of the Subject under 5 93/01286. Alternatively, the oligonucleotides may be admin treatment. For example, an appropriate route for delivery of istered in microspheres or microparticles. (See, e.g., Wu, G.Y. an antisense oligomer in the treatment of a viral infection of and Wu, C. H., J. Biol. Chem. 262:4429-4432, 1987). Alter the skin is topical delivery, while delivery of a antisense natively, the use of gas-filled microbubbles complexed with oligomerfor the treatment of a viral respiratory infection is by the antisense oligomers can enhance delivery to targettissues, inhalation. The oligomer may also be delivered directly to the 10 as described in U.S. Pat. No. 6,245.747. Sustained release site of viral infection, or to the bloodstream. compositions may also be used. These may include semiper The antisense oligomer may be administered in any con meable polymeric matrices in the form of shaped articles Such Venient vehicle which is physiologically and/or pharmaceu as films or microcapsules. tically acceptable. Such a composition may include any of a In one embodiment, antisense inhibition is effective in variety of standard pharmaceutically acceptable carriers 15 treating infection of a host animal by a virus, by contacting a employed by those of ordinary skill in the art. Examples cell infected with the virus with an antisense agent effective to include, but are not limited to, saline, phosphate buffered inhibit the replication of the specific virus. The antisense saline (PBS), water, aqueous ethanol, emulsions, such as agent is administered to a mammalian Subject, e.g., human or oil/water emulsions or triglyceride emulsions, tablets and domestic animal, infected with a given virus, in a Suitable capsules. The choice of Suitable physiologically acceptable pharmaceutical carrier. It is contemplated that the antisense carrier will vary dependent upon the chosen mode of admin oligonucleotide arrests the growth of the RNA virus in the istration. host. The RNA virus may be decreased in number or elimi The compounds (e.g., oligomers) of the present invention nated with little or no detrimental effect on the normal growth may generally be utilized as the free acid or free base. Alter or development of the host. natively, the compounds of this invention may be used in the 25 In one aspect of the method, the Subject is a human Subject, form of acid or base addition salts. Acid addition salts of the e.g., a patient diagnosed as having a localized or systemic free amino compounds of the present invention may be pre viral infection. The condition of a patient may also dictate pared by methods well known in the art, and may be formed prophylactic administration of an antisense oligomer of the from organic and inorganic acids. Suitable organic acids invention, e.g. in the case of a patient who (1) is immunocom include maleic, fumaric, benzoic, ascorbic, Succinic, meth 30 promised; (2) is a burn victim; (3) has an indwelling catheter; anesulfonic, acetic, trifluoroacetic, oxalic, propionic, tartaric, or (4) is about to undergo or has recently undergone surgery. Salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspar In one preferred embodiment, the oligomer is a phospho tic, Stearic, palmitic, glycolic, glutamic, and benzenesulfonic rodiamidate morpholino oligomer, contained in a pharmaceu acids. Suitable inorganic acids include hydrochloric, hydro tically acceptable carrier, and is delivered orally. In another bromic, Sulfuric, phosphoric, and nitric acids. Base addition 35 preferred embodiment, the oligomer is a phosphorodiamidate salts included those salts that form with the carboxylate anion morpholino oligomer, contained in a pharmaceutically and include Salts formed with organic and inorganic cations acceptable carrier, and is delivered intravenously (i.v.). Such as those chosen from the alkali and alkaline earth metals In another application of the method, the subject is a live (for example, lithium, sodium, potassium, magnesium, Stock animal, e.g., a chicken, turkey, pig, cow or goat, etc., and barium and calcium), as well as the ammonium ion and Sub 40 the treatment is either prophylactic or therapeutic. The inven stituted derivatives thereof (for example, dibenzylammo tion also includes a livestock and poultry food composition nium, benzylammonium, 2-hydroxyethylammonium, and containing a food grain Supplemented with a subtherapeutic the like). Thus, the term “pharmaceutically acceptable salt” of amount of an antiviral antisense compound of the type structure (I) is intended to encompass any and all acceptable described above. Also contemplated is, in a method offeeding salt forms. 45 livestock and poultry with a food grain Supplemented with In addition, prodrugs are also included within the context Subtherapeutic levels of an antiviral, an improvement in of this invention. Prodrugs are any covalently bonded carriers which the food grain is Supplemented with a subtherapeutic that release a compound of structure (I) in vivo when Such amount of an antiviral oligonucleotide composition as prodrug is administered to a patient. Prodrugs are generally described above. prepared by modifying functional groups in a way Such that 50 the modification is cleaved, either by routine manipulation or In one embodiment, the antisense compound is adminis in vivo, yielding the parent compound. Prodrugs include, for tered in an amount and manner effective to result in a peak example, compounds of this invention wherein hydroxy, blood concentration of at least 200-400 nM antisense oligo amine or sulfhydryl groups are bonded to any group that, mer. Typically, one or more doses of antisense oligomer are when administered to a patient, cleaves to form the hydroxy, 55 administered, generally at regular intervals, for a period of amine or sulfhydryl groups. Thus, representative examples of about one to two weeks. Preferred doses for oral administra prodrugs include (but are not limited to) acetate, formate and tion are from about 1-1000 mg oligomer per 70 kg. In some benzoate derivatives of alcohol and amine functional groups cases, doses of greater than 1000 mg oligomer/patient may be of the compounds of structure (I). Further, in the case of a necessary. For i.v. administration, preferred doses are from carboxylic acid (-COOH), esters may be employed, such as 60 methyl esters, ethyl esters, and the like. about 0.5 mg to 1000 mg oligomer per 70 kg. The antisense In some instances, liposomes may be employed to facilitate oligomer may be administered at regular intervals for a short uptake of the antisense oligonucleotide into cells. (See, e.g., time period, e.g., daily for two weeks or less. However, in Williams, S.A., Leukemia 10(12): 1980-1989, 1996; Lappa Some cases the oligomer is administered intermittently over a lainen et al., Antiviral Res. 23:119, 1994: Uhlmann et al., 65 longer period of time. Administration may be followed by, or antisense oligonucleotides: a new therapeutic principle, concurrent with, administration of an antibiotic or other Chemical Reviews, Volume 90, No. 4, pages 544-584, 1990; therapeutic treatment. The treatment regimen may be US 9,278,987 B2 177 178 adjusted (dose, frequency, route, etc.) as indicated, based on -continued the results of immunoassays, other biochemical tests and O physiological examination of the Subject under treatment. X-P-Cl O B B An effective in vivo treatment regimen using the antisense 5 O C O X-P-O HO oligonucleotides of the invention may vary according to the 4 -a- duration, dose, frequency and route of administration, as well C as the condition of the Subject under treatment (i.e., prophy lactic administration versus administration in response to PG PG localized or systemic infection). Accordingly, Such in vivo 10 therapy will often require monitoring by tests appropriate to 5 3 the particular type of viral infection under treatment, and corresponding adjustments in the dose or treatment regimen, Referring to Reaction Scheme 1, wherein B represents a in order to achieve an optimal therapeutic outcome. Treat base pairing moiety and PG represents a protecting group, the ment may be monitored, e.g., by general indicators of disease 15 morpholino Subunits may be prepared from the correspond and/or infection, such as complete blood count (CBC), ing ribinucleoside (1) as shown. The morpholino subunit (2) may be optionally protected by reaction with a suitable pro nucleic acid detection methods, immunodiagnostic tests, tecting group precursor, for example trityl chloride. The 3' viral culture, or detection of heteroduplex. protecting group is generally removed during Solid-state oli The efficacy of an in vivo administered antiviral antisense gomer synthesis as described in more detail below. The base oligomer of the invention in inhibiting or eliminating the pairing poiety may be Suitable protected for sold phase oli growth of one or more types of RNA virus may be determined gomer synthesis. Suitable protecting groups include benzoyl from biological samples (tissue, blood, urine etc.) taken from for adenine and cytosine, phenylacetyl for guanine, and piv a subject prior to, during and Subsequent to administration of aloyloxymethyl for hypoxanthine (I). The pivaloyloxymethyl 25 group can be introduced onto the N1 position of the hypox the antisense oligomer. Assays of Such samples include (1) anthine heterocyclic base. Although an unprotected hypox monitoring the presence or absence of heteroduplex forma anthine subunit, may be employed, yields in activation reac tion with target and non-target sequences, using procedures tions are far superior when the base is protected. Other known to those skilled in the art, e.g., an electrophoretic gel Suitable protecting groups include those disclosed in mobility assay; (2) monitoring the amount of viral protein 30 co-pending U.S. application Ser. No. 12/271,040, which is production, as determined by standard techniques such as hereby incorporated by reference in its entirety. Reaction of 3 with the activated phosphorous compound 4. ELISA or Western blotting, or (3) measuring the effect on results in morpholino Subunits having the desired linkage viral titer, e.g. by the method of Spearman-Karber. (See, for moiety 5. Compounds of structure 4 can be prepared using example, Pari, G. S. et al., Antimicrob. Agents and Chemo 35 any number of methods known to those of skill in the art. For therapy 39(5):1157-1161, 1995; Anderson, K. P. et al., Anti example, Such compounds may be prepared by reaction of the microb. Agents and Chemotherapy 40(9): 2004-2011, 1996, corresponding amine and phosphorous oxychloride. In this Cottral, G. E. (ed) in: Manual of Standard Methods for Vet regard, the amine starting material can be prepared using any erinary Microbiology, pp. 60-93, 1978). method known in the art, for example those methods 40 described in the Examples and in U.S. Pat. No. 7,943,762. In some embodiments, the oligomer is actively taken up by Compounds of structure 5 can be used in Solid-phase auto mammalian cells. In further embodiments, the oligomer may mated oligomer synthesis for preparation of oligomers com be conjugated to a transport moiety (e.g., transport peptide) as prising the intersubunit linkages. Such methods are well described herein to facilitate such uptake. known in the art. Briefly, a compound of structure 5 may be E. Preparation of the Oligomers 45 modified at the 5' end to contain a linkerto a solid support. For example, compound 5 may be linked to a solid Support by a The morpholino subunits, the modified intersubunit link linker comprising L'' and L'. An exemplary method is dem ages and oligomers comprising the same can be prepared as onstrated in FIGS. 1 and 2. Once supported, the protecting described in the examples and in U.S. Pat. Nos. 5,185,444 and group (e.g., trityl) is removed and the free amine is reacted 7,943,762 which are hereby incorporated by reference in their 50 with an activated phosphorous moiety of a second compound entirety. The morpholino subunits can be prepared according of structure 5. This sequence is repeated until the desired to the following general Reaction Scheme I. length oligo is obtained. The protecting group in the termina 5' end may either be removed or left on if a 5'-modification is desired. The oligo can be removed from the solid support Reaction Scheme 1. Preparation of Morpholino Subunits 55 using any number of methods, for example treatment with DTT followed by ammonium hydroxide as depicted in FIG. B O 3. O B 1. NaIO4, MeoH (aq) HO The preparation of modified morpholino Subunits and mor 2. (NH4)2BO7 pholino oligomers are described in more detail in the HO 3. Borane- Nt 60 Examples. The morpholino oligomers containing any number triethylamine A M of modified linkages may be prepared using methods HO OH 4. Methanolic acid H. H. described herein, methods known in the art and/or described 1 (p-TsOH or HCI) 2 by reference herein. Also described in the examples are global modifications of morpholino oligomers prepared as previ 65 ously described (see e.g., PCT publication WO2008036127). The term “protecting group' refers to chemical moieties that block some or all reactive moieties of a compound and US 9,278,987 B2 179 prevent Such moieties from participating in chemical reac -continued tions until the protective group is removed, for example, those moieties listed and described in T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rded. John Wiley & Sons (1999). It may be advantageous, where different pro C l -X tecting groups are employed, that each (different) protective O group be removable by a different means. Protective groups Cbz that are cleaved under totally disparate reaction conditions allow differential removal of Such protecting groups. For H3C CH3 O example, protective groups can be removed by acid, base, and 10 Y HC O hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal HC -N-N-SO HCD1 and tert-butyldimethylsilyl are acid labile and may be used to CH, O protect carboxy and hydroxy reactive moieties in the presence TEOC BOC of amino groups protected with Cbz groups, which are 15 removable by hydrogenolysis, and Fmoc groups, which are HC Ph HC base labile. Carboxylic acid moieties may be blocked with H3C Ph base labile groups such as, without limitation, methyl, or CH Ph O ethyl, and hydroxy reactive moieties may be blocked with t-butyl trityl acetyl base labile groups such as acetyl in the presence of amines O blocked with acid labile groups such as tert-butyl carbamate or with carbamates that are both acid and base stable but hydrolytically removable. O Carboxylic acid and hydroxy reactive moieties may also be blocked with hydrolytically removable protective groups 25 Such as the benzyl group, while amine groups may be blocked with base labile groups such as Fmoc. A particulary useful amine protecting group for the synthesis of compounds of Formula (I) is the trifluoroacetamide. Carboxylic acid reac 30 tive moieties may be blocked with oxidatively-removable FMOC protective groups such as 2.4-dimethoxybenzyl, while co existing amino groups may be blocked with fluoride labile silyl carbamates. F. Antisense Activity of the Oligomers Allyl blocking groups are useful in the presence of acid RNAi and base-protecting groups since the former are stable and 35 RNA interference (RNAi) is a method of modulating gene can be subsequently removed by metal or pi-acid catalysts. expression. RNAi is a naturally occurring process. RNAi can For example, an allyl-blocked carboxylic acid can be depro also be induced by delivery of exogenous RNA sequences. tected with a palladium(0)-catalyzed reaction in the presence Naturally occurring RNAi is initiated by the recognition of of acid labile t-butyl carbamate or base-labile acetate amine 40 double-stranded RNA sequences (dsRNA) by an RNase III protecting groups. Yet another form of protecting group is a enzyme (e.g., Dicer), followed by cleavage of the dsRNA into resin to which a compound or intermediate may be attached. short fragments of ~20 nucleotides called small interfering As long as the residue is attached to the resin, that functional RNAs (siRNAs). Dicer is an RNase III enzyme with ATPase/ group is blocked and cannot react. Once released from the RNA helicase, a DUF283 (Domain of unknown function) resin, the functional group is available to react. 45 domain, a PAZ (Piwi, Argonaut and Zwille) domain which Typical blocking/protecting groups are known in the art can bind the characteristic two-base 3' overhangs of and include, but are not limited to the following moieties: microRNA (miRNA) and siRNA, two catalytic RNase III domains (RIIIa and RIIIb), and a C-terminal double-stranded RNA-binding domain (dsRBD). Following cleavage, each siRNA molecule is unwound into two single-stranded RNA sequences (SSRNAs). One strand, the passenger, is degraded. The other stand, the guide, is incorporated into the RNA -xOx induced silencing complex (RISC). RISC is a ribonucleopro Allyl Bn tein complex containing members of the Argonaute (Ago) CHO H3C CH3 55 family of proteins. Argonaute proteins have endonuclease V / activity. RNAi occurs when the guide strand base binds to a H3C Si complementary RNA sequence (for example, mRNA or pre D1 mRNA). The binding of the guide sequence induces cleavage CH3 of the guide sequence and the complementary sequence by PMB TBDMS 60 Argonaute. RNAi can also be initiated by the introduction of exog O enous RNA sequences, for example siRNA sequences, H3 C, 21\-1 miRNA sequences, and antisense oligonucleotides. Anti Me sense oligonucleotides (AONs) are single-stranded oligo O 65 nucleotides that are complementary to a target pre-mRNA or Alloc mRNA sequence. Once contacted with the complementary sequence, the AON will bind to the complementary sequence