JP2016185991A - Peptide oligonucleotide conjugates - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide peptide oligonucleotide conjugates.SOLUTION: The invention provides an oligonucleotide analog conjugated to a carrier peptide. Compounds disclosed herein are useful for treating various diseases, such as diseases where beneficial therapeutic effect is obtained by inhibiting protein expression or correcting aberrant mRNA splicing products. In one embodiment, the disclosure provides a conjugate comprising: (a) a carrier peptide comprising amino acid subunits; and (b) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence for sequence specific binding to the target nucleic acid, where two or more of the amino acid subunits are positively charged amino acids, the carrier peptide has a glycine (G) or proline (P) amino acid at the carboxy terminus of the carrier peptide, and the carrier peptide is covalently bonded to the nucleic acid analog.SELECTED DRAWING: Figure 1C

Description

Citation of Related Applications This application is a U.S. patent application to US patent application Ser. No. 13 / 101,942 filed on May 5, 2011 and US Patent Application No. 13 / 107,528 filed on May 13, 2011. Insist on benefits under Section 120 of the Patent Act. These applications are hereby incorporated by reference in their entirety.

BACKGROUND Technical Field The present invention relates generally to oligonucleotide compounds (oligomers) useful as antisense compounds, and more particularly to oligomeric compounds conjugated to cell penetrating peptides and such in antisense applications. The use of such oligomeric compounds.

Description of Related Applications The utility of many drugs with potentially useful biological activity is often hampered by the difficulty of delivering such drugs to their target. Compounds delivered into cells are generally delivered mostly from an aqueous extracellular environment and then must penetrate a lipid-soluble cell membrane to enter the cell. Many molecules, especially large molecules, are too lipophilic for actual solubilization or too hydrophilic to penetrate the membrane, unless the material is actively transported by a specific transport mechanism Either.

  A fragment of the HIV Tat protein consisting of amino acid residues 49-57 (having the sequence RKKRRQRRR, Tat49 57) has been used to deliver biologically active peptides and proteins to cells (eg, Barsom et al. 1994, International Publication No. 94/04686). Tat (49 60) has been used to improve the delivery of phosphorothioate oligonucleotides (Astraib-Fisher, Sergeev et al., 2000; Astriab-Fisher, Sergeev et al., 2002). Reverse Tat or rTat (57-49) (RRRQRRKKR) has been reported to deliver fluorescein into cells with improved efficacy compared to Tat (49 57) (Wender, Mitchell et al., 2000; Rothbard, Kreider et al., 2002). Rothbard and Wender have also disclosed other arginine-rich transport polymers (Patent Document 1 (WO 01/62297); Patent Document 2 (US Pat. No. 6,306,993); Patent Document 3 ( U.S. Patent Application Publication No. 2003/0032593)).

  Oligonucleotides are a class of potentially useful drug compounds that often interfere with delivery for therapeutic use. Phosphorodiamidate-linked morpholino oligomers (PMO; see, eg, Summerton and Weller, 1997) have been found to be more promising in this respect than charged oligonucleotide analogues, eg, phosphorothioates. I came. The PMO is an antisense molecule that is water-soluble, uncharged, or substantially uncharged, splice progression, or inhibits gene expression by interfering with the binding of translation machinery components. is there. PMO has also been shown to inhibit or prevent viral replication (Stein, Skilling et al., 2001; McCaffrey, Meuse et al., 2003). They are very resistant to enzymatic digestion (Hudziak, Barofsky et al., 1996). PMOs are in vitro in cell-free and cell culture models (Stein, Foster et al., 1997; Summerton and Weller 1997) and in vivo in zebrafish, frog and sea urchin embryos (Heasman, Kofron et al. , 2000; Nasevicius and Ekker 2000), and in adult animal models such as rats, mice, rabbits, dogs and pigs (eg, Arara and Iversen 2000; Qin, Taylor et al., 2000; Iversen 2001; Kippshidze, Keane et al., 2001; Devi 2002; Devi, Oldenkamp et al., 2002; Kipphideze, Kim et al., 2002; icker, Mata et al., see 2002), it has shown a high antisense specificity and efficacy.

  Antisense PMO oligomers have been shown to be incorporated into cells and consistently more effective in vivo and exhibit little non-specific efficacy than other widely used antisense oligonucleotides. (See, for example, P. Iversen, “Phosphoramidite Morpholino Oligomers”, in Antisense Drug Technology, ST Crooke, ed., Marque Dekker, Inc., New York, 200.) Conjugation of the PMO to arginine-rich peptides has been shown to improve their cellular uptake (see, for example, US Pat. No. 7,468,418). ). However, the toxicity of the conjugate has delayed its development as a promising drug candidate.

  Although significant progress has been made, there remains a need in the art for oligonucleotide conjugates with improved antisense or antigene performance. Such improved antisense or antigene performance includes lower toxicity, stronger affinity for DNA and RNA, without compromising sequence selectivity; improved pharmacokinetics and tissue distribution; improved Cell delivery and reliability, as well as controllability of the distribution in vivo.

International Publication No. 01/62297 US Pat. No. 6,306,993 US Patent Application Publication No. 2003/0032593 US Pat. No. 7,468,418

BRIEF SUMMARY The compounds of the present invention solve these problems and provide improvements over existing antisense molecules in the art. By conjugating a cell penetrating peptide to a substantially uncharged nucleic acid analog via a glycine or proline amino acid, we have solved the toxicity challenges associated with other peptide oligomer conjugates. did. In addition, modification of the linkage between subunits and / or conjugation of terminal moieties to the 5 ′ and / or 3 ′ ends of oligonucleotide analogues, eg, morpholino oligonucleotides, also improves the properties of the conjugate. obtain. For example, in certain embodiments, conjugates of the present disclosure reduce toxicity and / or improve cellular delivery, efficacy and / or tissue distribution and / or compared to other oligonucleotide analogs. Alternatively, it can be delivered more effectively to the target organ. These superior properties result in favorable therapeutic indicators, reduced clinical doses, as well as reduced cost of the article.

Thus, in one embodiment of the present disclosure,
(A) a carrier peptide comprising an amino acid subunit; and
(B) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence for sequence-specific binding to a target nucleic acid;
Two or more of the amino acid subunits are positively charged amino acids, the carrier peptide includes a glycine (G) or proline (P) amino acid at the carboxy terminus of the carrier peptide, and the carrier peptide is A conjugate is provided that is covalently linked to the nucleic acid analog. A composition comprising the conjugate and a pharmaceutically acceptable vehicle is also provided.

  In another embodiment, the present disclosure provides a method of inhibiting protein production comprising exposing a nucleic acid encoding the protein to a conjugate of the present disclosure.

  Another aspect of the present disclosure is a method for improving transport of a nucleic acid analog into a cell comprising conjugating the carrier peptide of claim 1 to the nucleic acid analog, wherein the cell A method wherein transport of the nucleic acid analog into the nucleic acid analog is improved as compared to the unconjugated nucleic acid analog.

を有する、少なくとも１つの中性アミノ酸を含み、式中、ｎが、２から７であり、各Ｒ^ｅが各出現箇所で独立して、水素またはメチルである、項目１記載のコンジュゲート。
（項目２５）
前記キャリアペプチドが、配列［（Ｒ^ｄＹ^ｂＲ^ｄ）_ｘ（Ｒ^ｄＲ^ｄＹ^ｂ）_ｙ］_ｚを含み、Ｒ^ｄが、各出現箇所で独立して、正電荷を有するアミノ酸であり、ｘおよびｙが、各出現箇所で独立して、０または１であり、但し、ｘ＋ｙが１または２であり、ｚが、１から６の範囲の整数である、項目２４記載のコンジュゲート。
（項目２６）
Ｒ^ｄが、各出現箇所で独立して、アルギニン、ヒスチジンまたはリジンである、項目２５記載のコンジュゲート。
（項目２７）
各Ｒ^ｄが、アルギニンである、項目２５記載のコンジュゲート。
（項目２８）
少なくとも１つのＹ^ｂが、６−アミノヘキサン酸またはβ−アラニンである、項目２５記載のコンジュゲート。
（項目２９）
前記キャリアペプチドが、配列ＩＬＦＱＹを含む、項目１記載のコンジュゲート。
（項目３０）
前記キャリアペプチドが、配列ＩＬＦＱ、ＩＷＦＱまたはＩＬＩＱを含む、項目１記載のコンジュゲート。
（項目３１）
前記キャリアペプチドが、配列ＰＰＭＷＳ、ＰＰＭＷＴ、ＰＰＭＦＳまたはＰＰＭＹＳを含む、項目１記載のコンジュゲート。
（項目３２）
前記キャリアペプチドが、アラニン、アスパラギン、システイン、グルタミン、グリシン、ヒスチジン、リジン、メチオニン、セリンまたはスレオニンのうちの少なくとも１つを含む、項目１記載のコンジュゲート。
（項目３３）
前記キャリアペプチドが、前記キャリアペプチドのアミノ末端に、アセチル、ベンゾイルまたはステアロイル部分を含む、項目１記載のコンジュゲート。
（項目３４）
前記アミノ酸サブユニットの各々が、天然アミノ酸である、項目１記載のコンジュゲート。
（項目３５）
前記荷電していない骨格が、サブユニット間リンケージにより結合されたモルホリノ環構造の配列を含み、前記サブユニット間リンケージが、１つのモルホリノ環構造の３’端を、隣接するモルホリノ環構造の５’端に結合し、前記オリゴマーが前記標的核酸に、配列特異的な方法で結合し得るように、各モルホリノ環構造が、塩基対合部分に結合され、前記サブユニット間リンケージが、下記一般構造（Ｉ）：

または、その塩もしくはその異性体を有し、該サブユニット間リンケージ（Ｉ）の各々が、独立して、リンケージ（Ａ）またはリンケージ（Ｂ）であり：
リンケージ（Ａ）に関して：
Ｗが各出現箇所で独立して、ＳまたはＯであり；
Ｘが各出現箇所で独立して、−Ｎ（ＣＨ_３）_２、−ＮＲ^１Ｒ^２、−ＯＲ^３または；

であり；
Ｙが各出現箇所で独立して、Ｏまたは−ＮＲ^２であり；
Ｒ^１が各出現箇所で独立して、水素またはメチルであり；
Ｒ^２が各出現箇所で独立して、水素または−ＬＮＲ^４Ｒ^５Ｒ^７であり；
Ｒ^３が各出現箇所で独立して、水素またはＣ_１−Ｃ_６アルキルであり；
Ｒ^４が各出現箇所で独立して、水素、メチル、−Ｃ（＝ＮＨ）ＮＨ_２、−Ｚ−Ｌ−ＮＨＣ（＝ＮＨ）ＮＨ_２または−［Ｃ（Ｏ）ＣＨＲ’ＮＨ］_ｍＨであり、Ｚが、カルボニル（Ｃ（Ｏ））または直接結合であり、Ｒ’が、天然に存在するアミノ酸の側鎖またはその１つもしくは２つの炭素同族体であり、ｍが、１から６であり；
Ｒ^５が各出現箇所で独立して、水素、メチルまたは電子対であり；
Ｒ^６が各出現箇所で独立して、水素またはメチルであり；
Ｒ^７が各出現箇所で独立して、水素、Ｃ_１−Ｃ_６アルキルまたはＣ_１−Ｃ_６アルコキシアルキルであり；
Ｌが、アルキル、アルコキシもしくはアルキルアミノの基またはそれらの組み合わせを含む、長さ１８原子以下の必要に応じたリンカーであり、ならびに、
リンケージ（Ｂ）に関して：
Ｗが各出現箇所で独立して、ＳまたはＯであり；
Ｘが各出現箇所で独立して、−ＮＲ^８Ｒ^９または−ＯＲ^３であり；および、
Ｙが各出現箇所で独立して、Ｏまたは−ＮＲ^１０であり、
Ｒ^８が各出現箇所で独立して、水素またはＣ_２−Ｃ_１２アルキルであり；
Ｒ^９が各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アラルキルまたはアリールであり；
Ｒ^１０が各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキルまたは−ＬＮＲ^４Ｒ^５Ｒ^７であり；
Ｒ^８およびＲ^９が結合して、５〜１８員の単環複素環もしくは二環複素環を形成してもよく、または、Ｒ^８、Ｒ^９またはＲ^３がＲ^１０と結合して、５〜７員の複素環を形成し、Ｘが４−ピペラジノである場合、Ｘが下記構造（ＩＩＩ）：

を有し、式中：
Ｒ^１１が各出現箇所で独立して、Ｃ_２−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アミノアルキル、Ｃ_１−Ｃ_１２アルキルカルボニル、アリール、ヘテロアリールまたはヘテロシクリルであり；および、
Ｒが各出現箇所で独立して、電子対、水素またはＣ_１−Ｃ_１２アルキルであり；および、
Ｒ^１２が各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アミノアルキル、−ＮＨ_２、−ＣＯＮＨ_２、−ＮＲ^１３Ｒ^１４、−ＮＲ^１３Ｒ^１４Ｒ^１５、Ｃ_１−Ｃ_１２アルキルカルボニル、オキソ、−ＣＮ、トリフルオロメチル、アミジル、アミジニル、アミジニルアルキル、アミジニルアルキルカルボニル グアニジニル、グアニジニルアルキル、グアニジニルアルキルカルボニル、コラート、デオキシコラート、アリール、ヘテロアリール、複素環、−ＳＲ^１３またはＣ_１−Ｃ_１２アルコキシであり、Ｒ^１３、Ｒ^１４およびＲ^１５が、各出現箇所で独立して、Ｃ_１−Ｃ_１２アルキルである、項目１記載のコンジュゲート。
（項目３６）
前記モルホリノ環構造のうちの少なくとも１つが、下記構造（ｉ）：

を有し、式中、Ｐｉが、各出現箇所で独立して、塩基対合部分である、項目３５記載のコンジュゲート。
（項目３７）
前記サブユニット間リンケージのうちの少なくとも１つが、リンケージ（Ａ）である、項目３５記載のコンジュゲート。
（項目３８）
前記サブユニット間リンケージの各々が、リンケージ（Ａ）である、項目３５記載のコンジュゲート。
（項目３９）
各リンケージ（Ａ）が、各出現箇所で同じ構造を有する、項目３５記載のコンジュゲート。
（項目４０）
リンケージ（Ａ）の少なくとも１つ出現箇所に関して、Ｘが、−Ｎ（ＣＨ_３）_２である、項目３５記載のコンジュゲート。
（項目４１）
リンケージ（Ａ）の各出現箇所で、Ｘが、−Ｎ（ＣＨ_３）_２である、項目３５記載のコンジュゲート。
（項目４２）
各リンケージが、リンケージ（Ａ）であり、Ｘが、−Ｎ（ＣＨ_３）_２である、項目３５記載のコンジュゲート。
（項目４３）
リンケージ（Ａ）の少なくとも１つの出現箇所に関して、Ｘが、下記構造：

有する、項目３５記載のコンジュゲート。
（項目４４）
リンケージ（Ｂ）の少なくとも１つの出現箇所に関して、Ｘが、下記構造：

有する、項目３５記載のコンジュゲート。
（項目４５）
リンケージ（Ｂ）の少なくとも１つの出現箇所に関して、Ｘが、下記構造：

有する、項目３５記載のコンジュゲート。
（項目４６）
ＷおよびＹが、各出現箇所でＯである、項目３５記載のコンジュゲート。
（項目４７）
前記サブユニット間リンケージのうちの少なくとも５％が、リンケージ（Ｂ）である、項目３５記載のコンジュゲート。
（項目４８）
各リンケージ（Ｂ）が、各出現箇所で同じ構造を有する、項目３５記載のコンジュゲート。
（項目４９）
前記核酸類似体が、下記構造（ＸＶＩＩ）：

または、その塩もしくは異性体を有し、式中：
Ｒ^１７が各出現箇所で独立して、存在しないか、水素またはＣ_１−Ｃ_６アルキルであり；
Ｒ^１８およびＲ^１９が各出現箇所で独立して、存在しないか、水素、前記キャリアペプチド、Ｃ_２−Ｃ_３０アルキルカルボニル、−Ｃ（＝Ｏ）ＯＲ^２１またはＲ^２０であり；
Ｒ^２０が各出現箇所で独立して、グアニジニル、ヘテロシクリル、Ｃ_１−Ｃ_３０アルキル、Ｃ_３−Ｃ_８シクロアルキル；Ｃ_６−Ｃ_３０アリール、Ｃ_７−Ｃ_３０アラルキル、Ｃ_３−Ｃ_３０アルキルカルボニル、Ｃ_３−Ｃ_８シクロアルキルカルボニル、Ｃ_３−Ｃ_８シクロアルキルアルキルカルボニル、Ｃ_７−Ｃ_３０アリールカルボニル、Ｃ_７−Ｃ_３０アラルキルカルボニル、Ｃ_２−Ｃ_３０アルキルオキシカルボニル、Ｃ_３−Ｃ_８シクロアルキルオキシカルボニル、Ｃ_７−Ｃ_３０アリールオキシカルボニル、Ｃ_８−Ｃ_３０アラルキルオキシカルボニルまたは−Ｐ（＝Ｏ）（Ｒ^２２）_２であり；
Ｒ^２１が、１つ以上のエーテル結合、ヒドロキシル部分またはそれらの組み合わせを含むＣ_１−Ｃ_３０アルキルであり；
各Ｒ^２２が独立して、Ｃ_６−Ｃ_１２アリールオキシであり；
Ｐｉが、塩基対合部分であり；
Ｌ^１およびＬ^２がそれぞれ、直接結合または、アルキル、ヒドロキシル、アルコキシ、アルキルアミノ、アミド、エステル、カルボニル、カルバマート、ホスホロジアミダート、ホスホロアミダート、ホスホロチオアートおよびホスホジエステルから選択される結合を含む、長さ１８原子以下のリンカーであり；
ｘが、０またはそれより大きい整数であり；および、
Ｒ^１７およびＲ^１８が両方とも存在し、Ｒ^１８またはＲ^１９のうちの少なくとも１つが、前記キャリアペプチドである、項目３５記載のコンジュゲート。
（項目５０）
Ｒ^１８が、前記キャリアペプチドである、項目４９記載のコンジュゲート。
（項目５１）
Ｒ^１９が、前記キャリアペプチドである、項目４９記載のコンジュゲート。
（項目５２）
Ｒ^１８またはＲ^１９のうちの少なくとも１つが、Ｒ^２０である、項目４９記載のコンジュゲート。
（項目５３）
Ｒ^１９が、−Ｃ（＝Ｏ）ＯＲ^２１である、項目４９記載のコンジュゲート。
（項目５４）
Ｒ^１９が、

である、項目５３記載のコンジュゲート。
（項目５５）
Ｌ^１が、ホスホロジアミダートおよびピペラジン結合を含む、項目４９記載のコンジュゲート。
（項目５６）
Ｌ^１が、下記構造（ＸＸＩＸ）：

を有し、式中、Ｒ^２４が存在しないか、ＨまたはＣ_１−Ｃ_６アルキルである、項目５５記載のコンジュゲート。
（項目５７）
Ｒ^２４が、存在しない、項目５６記載のコンジュゲート。
（項目５８）
Ｒ^１８が、前記キャリアペプチドであり、Ｌ^２が、直接結合であり、前記キャリアペプチドが、前記キャリアペプチドのカルボキシ末端と前記核酸類似体の３’末端窒素との間の結合を形成する、項目４９記載のコンジュゲート。
（項目５９）
項目１記載のコンジュゲートと、薬学的に許容され得る媒体とを含む、組成物。
（項目６０）
被験体における疾患を処置する方法であって、治療的に有効量の項目１記載のコンジュゲートを、前記被験体に投与する工程を含む、方法。
（項目６１）
前記疾患が、ウイルス感染、神経筋疾患、細菌感染、炎症または多発性嚢胞腎である、請求項６０記載の方法。
（項目６２）
前記ウイルス感染が、インフルエンザである、項目６１記載の方法。
（項目６３）
前記神経筋疾患が、デュシェンヌ型筋ジストロフィーである、項目６１記載の方法。
（項目６４）
細胞内への核酸類似体の輸送を向上するための方法であって、項目１記載のキャリアペプチドを、核酸類似体にコンジュゲートする工程を含み、該細胞内への該核酸類似体の輸送が、非コンジュゲート型の該核酸類似体と比較して向上される、方法。
（項目６５）
前記核酸類似体が、モルホリノオリゴマーである、項目６４記載の方法。 In another embodiment, the present disclosure relates to a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed conjugate. Also provided are methods of making the conjugates, methods for using the same, and carrier peptides useful for conjugation to nucleic acid analogs.
In certain embodiments, for example, the following are provided:
(Item 1)
(A) a carrier peptide comprising an amino acid subunit; and
(B) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence for sequence-specific binding to a target nucleic acid,
Here, two or more of the amino acid subunits are positively charged amino acids, and the carrier peptide contains a glycine (G) or proline (P) amino acid subunit at the carboxy terminus of the carrier peptide. , 7 or fewer consecutive amino acid subunits are arginine, and the carrier peptide is covalently bound to the nucleic acid analog;
Conjugate.
(Item 2)
The conjugate according to item 1, wherein the carrier peptide contains a glycine amino acid at the carboxy terminus.
(Item 3)
Item 2. The conjugate according to Item 1, wherein the carrier peptide contains a proline amino acid at the carboxy terminus.
(Item 4)
The conjugate of item 1, wherein the carrier peptide comprises 4 to 40 amino acid subunits.
(Item 5)
The conjugate of item 1, wherein the carrier peptide comprises 6 to 20 amino acid subunits.
(Item 6)
Item 2. The conjugate according to Item 1, wherein the positively charged amino acid is histidine (H), lysine (K), arginine (R), or a combination thereof.
(Item 7)
The conjugate according to item 1, wherein at least one of the positively charged amino acids is arginine.
(Item 8)
Item 2. The conjugate according to Item 1, wherein each of the amino acid subunits other than the carboxy-terminal glycine or proline is a positively charged amino acid.
(Item 9)
Item 2. The conjugate according to Item 1, wherein each of the positively charged amino acids is arginine.
(Item 10)
At least one of the positively charged amino acids is an arginine analog, and the arginine analog is a cationic α-amino acid comprising a side chain of the structure R ^a N═C (NH ₂ ) R ^b Where R ^a is H or R ^c ; R ^b is R ^c , NH ₂ , NHR or N (R ^c ) ₂ , R ^c is lower alkyl or lower alkenyl, and optionally The conjugate according to item 1, comprising oxygen or nitrogen, or R ^a and R ^b may form a ring together; the side chain is linked to the amino acid via R ^a or R ^b. Gate.
(Item 11)
The conjugate according to item 1, wherein at least 20% of the amino acid subunits are amino acids having a positive charge.
(Item 12)
The conjugate according to item 1, wherein at least 50% of the amino acid subunits are amino acids having a positive charge.
(Item 13)
The conjugate according to item 1, wherein at least 80% of said amino acid subunits are positively charged amino acids.
(Item 14)
The conjugate according to item 1, wherein all the amino acid subunits other than the carboxy-terminal glycine or proline are positively charged amino acids.
(Item 15)
Item 1. The carrier peptide comprises the sequence (R ^d ) _m , R ^d is an amino acid having a positive charge independently at each occurrence, and m is an integer ranging from 2 to 12. Conjugate of.
(Item 16)
R ^d is, independently at each occurrence, arginine, histidine or lysine, item 15 conjugate according.
(Item 17)
16. A conjugate according to item 15, wherein each R ^d is arginine.
(Item 18)
Item 2. The conjugate according to Item 1, wherein each amino acid subunit other than the carboxy-terminal glycine or proline is arginine.
(Item 19)
The carrier peptide comprises at least one hydrophobic amino acid, the hydrophobic amino acid comprising a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain, wherein the alkyl, alkenyl and alkynyl side chains are The conjugate according to item 1, comprising at most one heteroatom for every 6 carbon atoms.
(Item 20)
20. A conjugate according to item 19, wherein the carrier peptide comprises two or more hydrophobic amino acids.
(Item 21)
20. A conjugate according to item 19, wherein the carrier peptide comprises two or more consecutive hydrophobic amino acids.
(Item 22)
20. A conjugate according to item 19, wherein at least one of the hydrophobic amino acids is phenylalanine.
(Item 23)
20. A conjugate according to item 19, wherein each of the hydrophobic amino acids is phenylalanine.
(Item 24)
The carrier peptide has the following structure (Y ^b ):

The conjugate according to item 1, comprising at least one neutral amino acid having the formula: wherein n is 2 to 7 and each R ^e is independently hydrogen or methyl at each occurrence.
(Item 25)
The carrier peptide comprises the sequence [(R ^d Y ^b R ^d ) _x (R ^d R ^d Y ^b ) _y ] _z , wherein R ^d is an amino acid having a positive charge independently at each occurrence; 25. A conjugate according to item 24, wherein x and y are independently 0 or 1 at each occurrence, provided that x + y is 1 or 2 and z is an integer in the range of 1-6.
(Item 26)
26. A conjugate according to item 25, wherein R ^d is arginine, histidine or lysine independently at each occurrence.
(Item 27)
26. A conjugate according to item 25, wherein each R ^d is arginine.
(Item 28)
26. A conjugate according to item 25, wherein at least one Y ^b is 6-aminohexanoic acid or β-alanine.
(Item 29)
The conjugate of item 1, wherein the carrier peptide comprises the sequence ILFQY.
(Item 30)
The conjugate according to item 1, wherein said carrier peptide comprises the sequence ILFQ, IWFQ or ILIQ.
(Item 31)
The conjugate according to item 1, wherein the carrier peptide comprises the sequence PPMWS, PPMWT, PPMFS or PPMYS.
(Item 32)
The conjugate according to item 1, wherein the carrier peptide comprises at least one of alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.
(Item 33)
The conjugate according to item 1, wherein the carrier peptide comprises an acetyl, benzoyl or stearoyl moiety at the amino terminus of the carrier peptide.
(Item 34)
The conjugate of item 1, wherein each of said amino acid subunits is a natural amino acid.
(Item 35)
The uncharged skeleton comprises a sequence of morpholino ring structures joined by an inter-subunit linkage, and the inter-subunit linkage connects the 3 ′ end of one morpholino ring structure to the 5 ′ of an adjacent morpholino ring structure. Each morpholino ring structure is bound to a base-pairing moiety so that the oligomer can bind to the target nucleic acid in a sequence specific manner, and the intersubunit linkage has the following general structure ( I):

Or a salt thereof or an isomer thereof, and each of the intersubunit linkages (I) is independently linkage (A) or linkage (B):
Regarding linkage (A):
W is S or O independently at each occurrence;
X is independently at each occurrence, —N (CH ₃ ) ₂ , —NR ¹ R ² , —OR ³ or;

Is;
Y is independently O or —NR ² at each occurrence;
R ¹ is independently hydrogen or methyl at each occurrence;
R ² is independently hydrogen or —LNR ⁴ R ⁵ R ⁷ at each occurrence;
R ³ is independently at each occurrence hydrogen or C ₁ -C ₆ alkyl;
R ⁴ is independently at each occurrence hydrogen, methyl, —C (═NH) NH ₂ , —ZL—NHC (═NH) NH ₂ or — [C (O) CHR′NH] _m H And Z is carbonyl (C (O)) or a direct bond, R ′ is a side chain of a naturally occurring amino acid or one or two carbon analogues thereof, and m is 1 to 6 Yes;
R ⁵ is independently hydrogen, methyl or an electron pair at each occurrence;
R ⁶ is independently hydrogen or methyl at each occurrence;
R ⁷ is independently hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxyalkyl at each occurrence;
L is an optional linker no longer than 18 atoms in length, comprising an alkyl, alkoxy or alkylamino group or combinations thereof; and
Regarding linkage (B):
W is S or O independently at each occurrence;
X is independently —NR ⁸ R ⁹ or —OR ³ at each occurrence; and
Y is independently O or —NR ¹⁰ at each occurrence;
R ⁸ is independently hydrogen or C ₂ -C ₁₂ alkyl at each occurrence;
R ⁹ is independently at each occurrence, hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aralkyl or aryl;
R ¹⁰ is independently at each occurrence, hydrogen, C ₁ -C ₁₂ alkyl, or —LNR ⁴ R ⁵ R ⁷ ;
R ⁸ and R ⁹ may combine to form a 5-18 membered monocyclic or bicyclic heterocycle, or R ⁸ , R ⁹ or R ³ may combine with R ¹⁰ to form 5 When a -7 membered heterocyclic ring is formed and X is 4-piperazino, X is the following structure (III):

In the formula:
R ¹¹ is independently at each occurrence, C ₂ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, C ₁ -C ₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl; and
R is independently an electron pair, hydrogen or C ₁ -C ₁₂ alkyl at each occurrence; and
R ¹² is independently at each occurrence, hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, —NH ₂ , —CONH ₂ , —NR ¹³ R ¹⁴ , —NR ¹³ R ¹⁴ R ¹⁵ , C ₁ -C ₁₂ alkylcarbonyl, oxo, -CN, trifluoromethyl, amidyl, amidinyl, amino isoxazolidinyl alkyl, amino isoxazolidinyl alkylcarbonyl guanidinyl, guar Nizhny Le alkyl, guar Nizhny Le alkylcarbonyl, alcoholates, deoxycholate, aryl 1, heteroaryl, heterocycle, —SR ¹³ or C ₁ -C ₁₂ alkoxy, wherein R ¹³ , R ¹⁴ and R ¹⁵ are each independently C ₁ -C ₁₂ alkyl Conjugate of.
(Item 36)
At least one of the morpholino ring structures has the following structure (i):

36. A conjugate according to item 35, wherein Pi is a base-pairing moiety independently at each occurrence.
(Item 37)
36. The conjugate of item 35, wherein at least one of the intersubunit linkage is linkage (A).
(Item 38)
36. The conjugate of item 35, wherein each of the intersubunit linkages is a linkage (A).
(Item 39)
36. The conjugate of item 35, wherein each linkage (A) has the same structure at each occurrence.
(Item 40)
For at least one occurrence of the linkage (A), X is a -N _{(CH 3) 2,} a conjugate of item 35, wherein.
(Item 41)
36. The conjugate according to item 35, wherein X is —N (CH ₃ ) ₂ at each occurrence of linkage (A).
(Item 42)
Each linkage is a linkage (A), X is a -N _{(CH 3) 2,} a conjugate of item 35, wherein.
(Item 43)
For at least one occurrence of linkage (A), X is the following structure:

36. The conjugate according to item 35.
(Item 44)
For at least one occurrence of linkage (B), X is the following structure:

36. The conjugate according to item 35.
(Item 45)
For at least one occurrence of linkage (B), X is the following structure:

36. The conjugate according to item 35.
(Item 46)
36. The conjugate of item 35, wherein W and Y are O at each occurrence.
(Item 47)
36. The conjugate of item 35, wherein at least 5% of the intersubunit linkage is linkage (B).
(Item 48)
36. The conjugate of item 35, wherein each linkage (B) has the same structure at each occurrence.
(Item 49)
The nucleic acid analog has the following structure (XVII):

Or a salt or isomer thereof, wherein:
R ¹⁷ is independently absent at each occurrence or is hydrogen or C ₁ -C ₆ alkyl;
R ¹⁸ and R ¹⁹ are independently absent at each occurrence or are hydrogen, said carrier peptide, C ₂ -C ₃₀ alkylcarbonyl, —C (═O) OR ²¹ or R ²⁰ ;
R ²⁰ is independently at each occurrence, guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C ₆ -C ₃₀ aryl, C ₇ -C ₃₀ aralkyl, C ₃ -C ₃₀ alkyl _carbonyl, C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 7 _{-C 30 arylcarbonyl,} C 7 _{-C 30 aralkylcarbonyl,} C 2 _{-C 30 alkyloxycarbonyl,} C 3 -C ₈ cycloalkyloxycarbonyl, C ₇ -C ₃₀ aryloxycarbonyl, C ₈ -C ₃₀ aralkyloxycarbonyl or —P (═O) (R ²² ) ₂ ;
R ²¹ is C ₁ -C ₃₀ alkyl containing one or more ether linkages, hydroxyl moieties, or combinations thereof;
Each R ²² is independently C ₆ -C ₁₂ aryloxy;
Pi is a base-pairing moiety;
L ¹ and L ² are each selected from a direct bond or alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, carbonyl, carbamate, phosphorodiamidate, phosphoramidate, phosphorothioate and phosphodiester A linker that is 18 atoms or less in length, including a bond;
x is an integer of 0 or greater; and
36. The conjugate of item 35, wherein R ¹⁷ and R ¹⁸ are both present and at least one of R ¹⁸ or R ¹⁹ is the carrier peptide.
(Item 50)
R ¹⁸ is a said carrier peptide conjugate of item 49, wherein.
(Item 51)
50. A conjugate according to item 49, wherein R ¹⁹ is the carrier peptide.
(Item 52)
50. The conjugate of item 49, wherein at least one of R ¹⁸ or R ¹⁹ is R ²⁰ .
(Item 53)
R ¹⁹ is, -C (= O) is ^{OR 21,} the conjugate of item 49, wherein.
(Item 54)
R ¹⁹ is

54. The conjugate according to item 53.
(Item 55)
L ¹ is, phosphorodiamidate and piperazine binding conjugate of item 49, wherein.
(Item 56)
L ¹ is the following structure (XXIX):

It has, in the ^formula, does not exist or ^{R 24,} is H or _C 1 -C ₆ alkyl, a conjugate of item 55, wherein.
(Item 57)
57. The conjugate according to item 56, wherein R ²⁴ is absent.
(Item 58)
R ¹⁸ is the carrier peptide, L ² is a direct bond, and the carrier peptide forms a bond between the carboxy terminus of the carrier peptide and the 3 ′ terminal nitrogen of the nucleic acid analog. 49. The conjugate according to 49.
(Item 59)
A composition comprising the conjugate of item 1 and a pharmaceutically acceptable medium.
(Item 60)
A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate of item 1.
(Item 61)
61. The method of claim 60, wherein the disease is viral infection, neuromuscular disease, bacterial infection, inflammation or polycystic kidney disease.
(Item 62)
62. The method of item 61, wherein the viral infection is influenza.
(Item 63)
62. The method of item 61, wherein the neuromuscular disease is Duchenne muscular dystrophy.
(Item 64)
A method for improving transport of a nucleic acid analog into a cell comprising conjugating the carrier peptide according to item 1 to the nucleic acid analog, wherein transport of the nucleic acid analog into the cell comprises Improved as compared to the non-conjugated nucleic acid analogue.
(Item 65)
65. A method according to item 64, wherein the nucleic acid analog is a morpholino oligomer.

  These and other aspects of the invention will be apparent upon reference to the following detailed description. To accomplish this goal, various references are described herein that describe specific background information, procedures, compounds and / or compositions in more detail, each of which is incorporated by reference in its entirety. This is incorporated herein.

FIG. 1A shows a typical morpholino oligomeric structure containing a phosphorodiamidate linkage. FIG. 1B shows a morpholino oligomer conjugated to a carrier peptide at the 5 'end. FIG. 1C shows a morpholino oligomer conjugated to the carrier peptide at the 3 'end. 1D-G show designated 1D to 1G repeating subunit segments, which are typical morpholino oligonucleotides. 1D-G show designated 1D to 1G repeating subunit segments, which are typical morpholino oligonucleotides. 1D-G show designated 1D to 1G repeating subunit segments, which are typical morpholino oligonucleotides. 1D-G show designated 1D to 1G repeating subunit segments, which are typical morpholino oligonucleotides. FIG. 2 shows a typical intersubunit linkage attached to the morpholino-T moiety. FIG. 3 is a reaction scheme showing the preparation of a linker for solid phase synthesis. FIG. 4 shows the preparation of a solid support for oligomer synthesis. FIGS. 5A, 5B and 5C show exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. FIGS. 5A, 5B and 5C show exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. FIGS. 5A, 5B and 5C show exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. FIGS. 6A, 6B and 6C are alternative representations of exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. FIGS. 6A, 6B and 6C are alternative representations of exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. FIGS. 6A, 6B and 6C are alternative representations of exon skipping data for a typical conjugate compared to a known conjugate in each of the mouse quadriceps, diaphragm and heart. 7A and 7B are graphs showing blood urea nitrogen (BUN) levels and viability in mice treated with various peptide-oligomer conjugates, respectively. 7A and 7B are graphs showing blood urea nitrogen (BUN) levels and viability in mice treated with various peptide-oligomer conjugates, respectively. Figures 8A and 8B show renal injury marker (KIM) and clusterin (Clu) data for mice treated with various peptide-oligomer conjugates, respectively. Figures 8A and 8B show renal injury marker (KIM) and clusterin (Clu) data for mice treated with various peptide-oligomer conjugates, respectively. Figures 9A, 9B, 9C and 9D are graphs comparing exon skipping, BUN levels, percent survival and KIM levels, respectively, in mice treated with a typical conjugate compared to known conjugates. Figures 9A, 9B, 9C and 9D are graphs comparing exon skipping, BUN levels, percent survival and KIM levels, respectively, in mice treated with a typical conjugate compared to known conjugates. Figures 9A, 9B, 9C and 9D are graphs comparing exon skipping, BUN levels, percent survival and KIM levels, respectively, in mice treated with a typical conjugate compared to known conjugates. Figures 9A, 9B, 9C and 9D are graphs comparing exon skipping, BUN levels, percent survival and KIM levels, respectively, in mice treated with a typical conjugate compared to known conjugates. FIG. 10 shows KIM data for mice treated with various conjugates. FIG. 11 shows the results of BUN analysis of mice treated with various conjugates. FIG. 12 is a graph showing the concentration of various oligomers in mouse kidney tissue.

Detailed description Definitions In the following description, certain specific details are set forth to provide through an understanding of various embodiments. However, one skilled in the art will understand that the invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless stated otherwise, throughout the following specification and claims, the word “comprise” and variations thereof, eg, “comprises” and “comprising”, are openly inclusive meanings, ie Interpreted as “including, but not limited to”. Furthermore, the headings provided herein are for convenience only and do not explain the scope or meaning of the claimed invention.

  Throughout this specification, “one embodiment” or “an embodiment” has at least one specific property, structure, or characteristic described in connection with that embodiment. It is meant to be included in one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification need not refer to all of the same embodiments. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Further, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include a plurality of instructions unless otherwise specified. It should be noted that the term “or” is generally used in its sense including “and / or” unless stated otherwise.

  As used herein, the following terms have the following meanings unless otherwise indicated.

“Amino” refers to the group —NH ₂ .

  “Cyano” or “nitrile” refers to the group —CN.

  “Hydroxy” or “hydroxyl” means an —OH group.

  “Imino” refers to the ═NH substituent.

“Guanidinyl” refers to the —NHC (═NH) NH ₂ substituent.

“Amidinyl” means a —C (═NH) NH ₂ substituent.

“Nitro” refers to the —NO ₂ radical.

  “Oxo” means the ═O substituent.

  “Thioxo” refers to the ═S substituent.

を意味する。 “Colato” has the following structure:

Means.

を意味する。 “Deoxycholate” has the following structure:

Means.

“Alkyl” is saturated or unsaturated (ie, contains one or more double and / or triple bonds) having 1 to 30 carbon atoms and attached to the rest of the molecule by a single bond. Means a linear or branched hydrocarbon chain group. Alkyl containing any number of 1 to 30 carbon atoms is included. It is alkyl containing 30 or fewer carbon _atoms, C 1 _{-C 30} alkyl, likewise e.g., alkyl containing up to 12 carbon _atoms, a C 1 _{-C 12} alkyl. Alkyl containing other numbers of carbon atoms (and other moieties as defined herein) are similarly represented. The alkyl group is not limited and is C ₁ -C ₃₀ alkyl, C ₁ -C ₂₀ alkyl, C ₁ -C ₁₅ alkyl, C ₁ -C ₁₀ alkyl, C ₁ -C ₈ alkyl, C ₁ -C ₆ alkyl. C ₁ -C ₄ alkyl, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkyl, C ₂ -C ₈ alkyl, C ₃ -C ₈ alkyl and C ₄ -C ₈ alkyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1- Dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, Examples include but-2-ynyl, but-3-ynyl, pentynyl, hexynyl and the like. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below.

“Alkylene” or “alkylene chain” means a linear or branched divalent hydrocarbon chain that connects the remainder of the molecule to a radical group. Alkylene can be saturated or unsaturated (ie, contain one or more double and / or triple bonds). Typical alkylenes are not limited and are C ₁ -C ₁₂ alkylene, C ₁ -C ₈ alkylene, C ₁ -C ₆ alkylene, C ₁ -C ₄ alkylene, C ₁ -C ₃ alkylene, C ₁ -C ₂ alkylene, _{C 1} alkylene, and the like. Typical alkylene groups are not limited and include methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene and the like. The alkylene chain is attached to the rest of the molecule by a single bond or a double bond, and is attached to the radical group by a single bond or a double bond. The point of attachment of the alkylene chain to the rest of the molecule and the radical group can be through one carbon or any two carbons in the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted as described below.

“Alkoxy” means _a group of the formula —OR _a , where R _a is an alkyl group, as defined. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted as described below.

“Alkoxyalkyl” means _a group of the formula —R _b OR _a where R _a is an alkyl group, as defined, and R _b is an alkylene group, as defined. Unless stated otherwise specifically in the specification, an alkoxyalkyl group may be optionally substituted as described below.

“Alkylcarbonyl” means a radical of the formula —C (═O) R _a , where R _a is an alkyl group, as defined. Unless stated otherwise specifically in the specification, an alkylcarbonyl group may be optionally substituted as described below.

“Alkyloxycarbonyl” means a radical of the formula —C (═O) OR _a , where R _a is an alkyl group, as defined. Unless stated otherwise specifically in the specification, an alkyloxycarbonyl group may be optionally substituted as described below.

“Alkylamino” means _a group of the formula —NHR _a or —NR _a R _a , where each R _a is independently an alkyl group, as defined above. Unless stated otherwise specifically in the specification, an alkylamino group may be optionally substituted as described below.

“Amidyl” means _a group of the formula —N (H) C (═O) R _a , where R _a is an alkyl group or an aryl group, as defined herein. Unless stated otherwise specifically in the specification, an amidyl group may be optionally substituted as described below.

“Amidinylalkyl” means a group of the formula —R _b —C (═NH) NH ₂ , wherein R _b is an alkylene group, as defined above. Unless stated otherwise specifically in the specification, an amidinylalkyl group may be optionally substituted as described below.

“Amidinylalkylcarbonyl” means a group of the formula —C (═O) R _b —C (═NH) NH ₂ , wherein R _b is an alkylene group, as defined above. Unless stated otherwise specifically in the specification, an amidinylalkylcarbonyl group may be optionally substituted as described below.

“Aminoalkyl” means _a group of formula —R _b —NR _a R _a , where R _b is an alkylene group, as defined above, and each R _a is independently hydrogen or It is an alkyl group.

“Thioalkyl” means _a radical of the formula —SR _a where R _a is an alkyl group as defined above. Unless stated otherwise specifically in the specification, a thioalkyl group may be optionally substituted.

  “Aryl” means a group derived from a hydrocarbon ring system containing hydrogen, 6 to 30 carbon atoms, and at least one aromatic ring. The aryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic system and may include fused ring systems or bridged ring systems. The aryl group is not limited, but is asanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, And aryl groups derived from the hydrocarbon ring systems of preaden, pyrene and triphenylene. Unless stated otherwise specifically in the specification, the term “aryl” or the prefix “ar-” (eg, “aralkyl”) is meant to include optionally substituted aryl groups.

“Aralkyl” means a group of the formula —R _b —R _c , where R _b is an alkylene chain, as defined above, and R _c is one or more aryl groups, as defined above, For example, benzyl, diphenylmethyl, trityl and the like. Unless stated otherwise specifically in the specification, an aralkyl group may be optionally substituted.

“Arylcarbonyl” means a group of the formula —C (═O) R _c , where R _c is one or more aryl groups, eg, phenyl, as defined above. Unless stated otherwise specifically in the specification, an arylcarbonyl group may be optionally substituted.

“Aryloxycarbonyl” means a group of the formula —C (═O) OR _c , where R _c is one or more aryl groups, eg, phenyl, as defined above. Unless stated otherwise specifically in the specification, an aryloxycarbonyl group may be optionally substituted.

“Aralkylcarbonyl” means a group of the formula —C (═O) R _b —R _c where R _b is an alkylene chain as defined above and R _c is 1 as defined above. One or more aryl groups, for example phenyl. Unless stated otherwise specifically in the specification, an aralkylcarbonyl group may be optionally substituted.

“Aralkyloxycarbonyl” means a group of the formula —C (═O) OR _b —R _c , where R _b is an alkylene chain, as defined above, and R _c is as defined above. One or more aryl groups, for example phenyl. Unless stated otherwise specifically in the specification, an aralkyloxycarbonyl group may be optionally substituted.

“Aryloxy” means a group of the formula —OR _c where R _c is one or more aryl groups, eg, phenyl, as defined above. Unless stated otherwise specifically in the specification, an arylcarbonyl group may be optionally substituted.

  “Cycloalkyl” means a stable, non-aromatic, mono- or polycyclic carbocyclic ring, which may include fused or bridged ring systems, may be saturated, or unsaturated. It may be attached to the rest of the molecule by a single bond. Exemplary cycloalkyls include, but are not limited to, cycloalkyls having 3 to 15 carbon atoms and 3 to 8 carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of the polycyclic group include adamantyl, norbornyl, decalinyl, and 7,7-dimethyl-bicyclo [2.2.1] heptanyl. Unless stated otherwise specifically in the specification, a cycloalkyl group may be optionally substituted.

“Cycloalkylalkyl” means a group of the formula —R _b R _d , where R _b is an alkylene chain, as defined above, and R _d is a cycloalkyl group, as defined above. Unless stated otherwise specifically in the specification, a cycloalkylalkyl group may be optionally substituted.

“Cycloalkylcarbonyl” means a radical of the formula —C (═O) R _d , where R _d is a cycloalkyl group, as defined above. Unless stated otherwise specifically in the specification, a cycloalkylcarbonyl group may be optionally substituted.

“Cycloalkyloxycarbonyl” means a group of the formula —C (═O) OR _d , where R _d is a cycloalkyl group, as defined above. Unless stated otherwise specifically in the specification, a cycloalkyloxycarbonyl group may be optionally substituted.

  A “fused” is any ring structure described herein that is fused to an existing ring structure. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that becomes a part of the fused heterocyclyl ring or the fused heteroaryl ring may be substituted with a nitrogen atom.

“Guanidinylalkyl” means a group of the formula —R _b —NHC (═NH) NH ₂ , where R _b is an alkylene group, as defined above. Unless stated otherwise specifically in the specification, a guanidinylalkyl group may be optionally substituted as described below.

“Guanidinylalkylcarbonyl” means a radical of the formula —C (═O) R _b —NHC (═NH) NH ₂ , where R _b is an alkylene group, as defined above. Unless stated otherwise specifically in the specification, a guanidinylalkylcarbonyl group may be optionally substituted as described below.

  “Halo” or “halogen” means bromo, chloro, fluoro or iodo.

  “Haloalkyl” is one or more halo groups, as defined above, eg, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3 -Means an alkyl group as defined above, substituted by bromo-2-fluoropropyl, 1,2-dibromoethyl or the like; Unless stated otherwise specifically in the specification, a haloalkyl group may be optionally substituted.

  “Perhalo” or “perfluoro” means a moiety in which each hydrogen atom is replaced by a halo atom or a fluorine atom.

  “Heterocyclyl”, “heterocycle” or “heterocycle” is a stable containing 2 to 23 carbon atoms and 1 to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur A non-aromatic ring group of 3 to 24 members. Unless stated otherwise specifically in the specification, the heterocyclyl group may be monocyclic, bicyclic, tricyclic or tetracyclic, and may include fused or bridged ring systems. The nitrogen, carbon or sulfur atom in the heterocyclyl group may be optionally oxidized and the nitrogen atom may optionally be quaternized. The heterocyclyl group may be partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroin Drill, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, Trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-c Un -6,21- crown -7, aza-18-crown-6, diaza-18-crown-6, aza-21-crown -7 and diaza-21-crown-7 and the like. Unless stated otherwise specifically in the specification, a heterocyclyl group may be optionally substituted.

  “Heteroaryl” includes a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur, and at least one aromatic ring Means a 5- to 14-membered ring system group. For the purposes of the present invention, the heteroaryl group may be monocyclic, bicyclic, tricyclic or tetracyclic and may include fused ring systems or bridged ring systems. The nitrogen, carbon or sulfur atom in the heteroaryl group may be optionally oxidized and the nitrogen atom may optionally be quaternized. Examples include non-limiting, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo [b] [1,4] dioxepinyl 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranyl, benzothienyl (benzothiophenyl), benzotriazolyl, Benzo [4,6] imidazo [1,2-a] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indoli , Indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyrazinyl, 1-oxidepyri Dazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, isoquinolinyl Nyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thiof Alkenyl (i.e., thienyl) and the like. Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

All the above groups may be substituted or unsubstituted. As used herein, the term “substituted” refers to any of the above groups that can be further functionalized (ie, alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl, alkylamino, Amidyl, amidinylalkyl, amidinylalkylcarbonyl, aminoalkyl, aryl, aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl, cycloalkyl Alkylcarbonyl, cycloalkyloxycarbonyl, guanidinylalkyl, guanidinylalkylcarbonyl, haloalkyl, heterocyclyl and / or heteroary ) Means. At least one hydrogen atom is replaced by a bond to a non-hydrogen atom substituent. Unless otherwise specified herein, substituents are oxo, —CO ₂ H, nitrile, nitro, —CONH ₂ , hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl, aryl, Aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl, heteroaryl, dialkylamine, arylamine Alkylarylamines, diarylamines, N-oxides, imides and enamines; Including one or more substituents selected from silicon atoms, perfluoroalkyl or perfluoroalkoxy such as trifluoromethyl or trifluoromethoxy in the group of alkylarylsilyl, alkyldiarylsilyl, triarylsilyl But you can. “Substituted” means that one or more hydrogen atoms are replaced by a higher order bond (eg, a double or triple bond) oxygen in a heteroatom, eg, an oxo, carbonyl, carboxyl, and ester group; Means, for example, any of the above groups that are substituted by nitrogen in the groups of imine, oxime, hydrazone and nitrile. For example, “substituted” means that one or more hydrogen atoms are —NR _g C (═O) NR _g R _h , —NR _g C (═O) OR _h , —NR _g SO ₂ R _h , — _{OC (= O) NR g R} h, -OR g, -SR g, -SOR g, -SO 2 R g, -OSO 2 R g, -SO 2 oR g, = NSO 2 R g and _-SO 2 NR is substituted with _g R _h, it includes any of the above groups. “Substituted” means that one or more hydrogen atoms are —C (═O) R _g , —C (═O) OR _g , —CH ₂ SO ₂ R _g , —CH ₂ SO ₂ NR _g R _h , -SH, -SR _g, or -SSR _g means any of the above groups. In the foregoing, R _g and R _h may be the same or different and are independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl. , Heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. Furthermore, each of the aforementioned substituents may optionally be substituted with one or more of the above substituents. Furthermore, any of the above groups may be substituted to contain one or more internal oxygen or sulfur atoms. For example, an alkyl group may be substituted with one or more internal oxygen atoms to form an ether or polyether group. Similarly, alkyl groups may be substituted with one or more internal sulfur atoms to form thioethers, disulfides, and the like. The amidyl moiety may be substituted with no more than two halo atoms, while the other groups may be substituted with one or more halo atoms. Any of the above groups may be substituted with amino, monoalkylamino, guanidinyl or amidinyl. Optional substituents for any of the above groups also include arylphosphoryl, eg, —R _a P (Ar) ₃ , where R _a is alkylene and Ar is an aryl moiety, eg, phenyl.

  The terms “antisense oligomer” or “antisense compound” are used interchangeably and are sequences of subunits each having a base carried in a backbone subunit composed of ribose or other pentose sugars or morpholino groups. Means. The backbone group hybridizes the base in the compound to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid: oligomer heteroduplex within the target sequence. Are linked by intersubunit linkage. The oligomer may have exact sequence complementarity or near complementarity to the target sequence. Such an antisense oligomer can be said to “direct” to the sequence to which it interferes with or inhibits translation of mRNA containing the target sequence and to which it hybridizes.

  By “morpholino oligomer” or “PMO” is meant a polymer molecule having a backbone that supports a base capable of hydrogen bonding to a typical polynucleotide. The polymer lacks a pentose sugar backbone moiety, more specifically a ribose backbone linked by a phosphodiester bond, characteristic of nucleotides and nucleosides, but instead contains a ring nitrogen linked by a ring nitrogen. Typical “morpholino” oligomers have (thio) phosphoramidate or (thio) phosphorodiamidate linkages that link the morpholino nitrogen of one subunit to the 5 ′ exocyclic carbon of an adjacent subunit. Have morpholino subunit structures that are linked together. Each subunit contains a purine or pyrimidine base-pairing moiety that is effective to bind to a base in a polynucleotide by base-specific hydrogen bonding. Morpholino oligomers (including antisense oligomers) are described, for example, in US Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 444; 5,521,063; 5,506,337 and pending US patent applications 12 / 271,036; 12 / 271,040; and WO 2009/064471. They are described in detail, all of which are incorporated herein by reference in their entirety. A typical PMO is a PMO in which the inter-subunit linkage is linkage (A1).

  “PMO +” may be any number of (1-piperazino) phosphini described previously (eg, see WO 2008/036127, which is incorporated herein by reference in its entirety). It means a phosphorodiamidate morpholino oligomer comprising the linkage (A2 and A3) of lideneoxy, (1- (4- (ω-guanidino-alkanoyl))-piperazino) phosphinylideneoxy.

  “PMO-X” means a phosphorodiamidate morpholino oligomer disclosed herein comprising at least one linkage (B) or at least one disclosed terminal modification.

  A “phosphoramidate” group includes a phosphorus atom having three attached oxygen atoms and one attached nitrogen atom. On the other hand, a “phosphorodiamidate” group (see, eg, FIGS. 1D-E) contains phosphorus with two attached oxygen atoms and two attached nitrogen atoms. The uncharged inter-subunit linkage or modified of the oligomer described in this application and in co-pending US patent applications 61 / 349,783 and 11 / 801,885 In the intersubunit linkage, one nitrogen is always hanging on the skeleton chain. The second nitrogen in the phosphorodiamidate linkage is typically the ring nitrogen in the morpholino ring structure.

  The linkage of “thiophosphoramidate” or “thiophosphorodiamidate” is a phosphoramidate or phosphorylation wherein one oxygen atom, typically the oxygen hanging from the backbone, is replaced by sulfur, respectively. This is a linkage of rosamidate.

  “Intersubunit linkage” means a linkage, eg, structure (I), that connects two morpholino subunits.

  As used herein, “charged”, “uncharged”, “cationic” and “anionic” are the main states of a chemical moiety near neutral pH, eg, about 6-8. Means. For example, the term may refer to the main state of the chemical moiety at physiological pH, ie about 7.4.

  “Lower alkyl” means an alkyl group of 1 to 6 carbon atoms, for example methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-pentyl and isopentyl. In certain embodiments, “lower alkyl” groups have 1 to 4 carbon atoms. In other embodiments, a “lower alkyl” group has 1 to 2 carbon atoms, ie, methyl or ethyl. Similarly, “lower alkenyl” refers to 2 to 6 alkenyl groups, preferably 3 or 4 carbon atoms, such as allyl and butenyl.

  “Non-interfering” substituents are those that do not adversely affect the ability of the antisense oligomers described herein to bind to the target of interest. Such substituents include small and / or relatively non-polar groups such as methyl, ethyl, methoxy, ethoxy or fluoro.

Oligonucleotides or antisense oligomers have a T _m of greater than 37 ° C., greater than 45 ° C., preferably at least 50 ° C., and typically from 60 ° C. to 80 ° C. When hybridized to a target, “specifically hybridizes” to the target polynucleotide. “T _m ” in the oligomer is a temperature at which 50% hybridizes to a complementary polynucleotide. _Tm is described, for example, in Miyada et al., Methods Enzymol. 154: 94-107 (1987), determined under standard conditions in saline. Such hybridization can occur by “approximate” or “substantial” complementation of the antisense oligomer to the target sequence, as well as exact complementation.

  A polynucleotide is described as being “complementary” to each other when hybridization occurs in an anti-equilibrium configuration between two single stranded polynucleotides. Complementarity (the degree to which one polynucleotide is complementary to another) is the bases in opposite strands that are expected to form hydrogen bonds with each other based on commonly accepted base-pairing rules. It can be quantified in terms of percentage.

  If the first sequence is a sequence of a polynucleotide that specifically binds to or specifically hybridizes to the second polynucleotide sequence under physiological conditions, the first sequence is the second sequence Is an “antisense sequence”.

  The term “targeting sequence” is a sequence in the oligonucleotide analog that is complementary (meaning that it is substantially complementary) to the target sequence in the RNA genome. The entire sequence or only part of the analog compound may be complementary to the target sequence. For example, in an analog having 20 bases, only 12-14 may be targeting sequences. Typically, the targeting sequence is formed with contiguous bases in the analog, or is discontinuous if the constituent sequence spanning the target sequence is located, for example, from the opposite side of the analog. It may be formed in any arrangement.

  The “backbone” of an oligonucleotide analog (eg, an uncharged oligonucleotide analog) refers to a structure that supports the base pairing moiety; eg, a morpholino oligomer, as described herein. The “skeleton” includes a morpholino ring structure connected by an intersubunit linkage (eg, a phosphorus-containing linkage). By “substantially uncharged backbone” is meant an oligonucleotide analog backbone in which less than 50% of the intersubunit linkages are charged at near neutral pH. For example, a substantially uncharged skeleton is charged at a near neutral pH of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or even 0%. Intersubunit linkages may be included. In some embodiments, the substantially uncharged scaffold is between at most one (at physiological pH) charged subunit for every 4 (at physiological pH) uncharged linkages. Linkage, including at most 1 for every 8 uncharged linkages, or at most 1 for every 16 uncharged linkages. In some embodiments, the nucleic acid analogs described herein are not fully charged.

  Target and targeting sequences are described as being “complementary” to each other when hybridization occurs in an antiparallel configuration. The targeting sequence may have “approximate” or “substantial” complementarity to the target sequence and the intended function of the presently described method, ie, “complementary”. Preferably, the oligonucleotide analogue compound used in the presently described method has at most one mismatch to the target sequence every 10 nucleotides, preferably from 20 to at most 1 Have a mismatch. Alternatively, the antisense oligomer used is at least 80%, at least 90% sequence homology, or at least 95% sequence homology for a typical targeting sequence, as specified herein. Have For purposes of complementary binding to an RNA target, the guanine base may be complementary to either cytosine or uracil RNA base, as described below.

  “Heteroduplex” means a duplex between an oligonucleotide analog and a complementary portion of a target RNA. “Nuclease-resistant heteroduplex” refers to in vivo, by intracellular and extracellular nucleases, eg RNAseH, wherein the heteroduplex is capable of cleaving double-stranded RNA / RNA or RNA / DNA complexes. Means a heteroduplex formed by binding an antisense oligomer to its complementary target so that it is substantially resistant to degradation.

  “Actively taken up by mammalian cells” when the drug can enter the cell by a mechanism other than passive diffusion through the cell membrane. The drug requires “active transport”, which means transporting the drug across mammalian cell membranes, eg, by an ATP-dependent transport mechanism, or binding of the drug to transport proteins and then bound through the membrane The drug may be transported by “facilitated transport” which means transporting the antisense agent through the cell membrane by a transport mechanism that facilitates passage of the bound drug.

  The terms “modulate expression” and / or “antisense activity” either increase or more typically reduce the expression of a given protein by interfering with RNA expression or translation. Means the ability of antisense oligomers. In the case of reducing protein expression, the antisense oligomer may directly interfere with the expression of a given gene or may contribute to accelerated degradation of RNA transcribed from that gene. As described herein, morpholino oligomers are believed to function via the former (steric hindrance) mechanism. Preferred antisense targets sterically hindering oligomers, including the ATG initiation codon region, splice site, regions in close proximity to the splice site, and the 5'-untranslated region of mRNA, while other regions target morpholino oligomers. Use and be well targeted.

“Amino acid subunits” are generally α-amino acid residues (—CO—CHR—NH—), but β- or other amino acid residues (eg, —CO—CH ₂ CHR—NH—). However, R is an amino acid side chain.

  The term “naturally occurring amino acid” means an amino acid present in a protein found in nature. The term “unnatural amino acid” refers to those amino acids that are not present in proteins found in nature, including, for example, beta-alanine (β-Ala) and 6-aminohexanoic acid (Ahx).

  An “effective amount” or “therapeutically effective amount” is administered to a mammalian subject, either as a single dose or as part of a series of doses, typically selected target nucleic acid sequences. Means the amount of antisense oligomer effective to produce the desired therapeutic effect by inhibiting the translation of.

  “Treatment” of an individual (eg, a mammal such as a human) or cell is any type of therapeutic intervention used in an attempt to change the natural history of said individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition and may be performed either prophylactically or at the beginning of a pathological event or after contact with a pathogen.

II. Carrier peptide A. Properties of the Carrier Peptide As noted above, the present disclosure relates to conjugates of carrier peptides and nucleic acid analogs. The carrier peptide is generally effective in improving cell penetration of the nucleic acid analog. In addition, Applicants have surprisingly found that glycine (G) or proline (P) between the nucleic acid analog and the remainder of the carrier peptide (eg, at the carboxy terminus or amino terminus of the carrier peptide). Containing an amino acid subunit reduces the toxicity of the conjugate while the efficacy is comparable or improved compared to a conjugate having a different linkage between the carrier peptide and the nucleic acid analog I found out that For this reason, the conjugates of the present disclosure have better therapeutic means than other peptide-oligomer conjugates and are promising drug candidates.

  In addition to toxicity reduction, the presence of a glycine or proline amino acid subunit between the nucleic acid analog and the carrier peptide is believed to provide further advantages. For example, glycine is inexpensive and easily linked to the nucleic acid analog (or optional linker) without the possibility of racemization. Similarly, proline also provides a carrier peptide that is easily linked without racemization and is not a helix-forming agent. The hydrophobicity of proline may have certain advantages for the interaction between the carrier peptide and the lipid bilayer of the cell, for example (in one embodiment) a carrier peptide comprising multiple prolines is You may resist formation of G quadruple. Finally, in certain embodiments, when the proline moiety is adjacent to an arginine amino acid subunit, the proline moiety is metabolized to the conjugate because the arginine-proline amide bond cannot be cleaved by a common endopeptidase. Gives sex.

  As described above, conjugates comprising a carrier peptide linked to a nucleic acid analog via a glycine or proline amino acid subunit have comparable efficacy with lower toxicity compared to other known conjugates. . Experiments conducted in support of this application show that the conjugates of the present disclosure have very low nephrotoxicity markers compared to other conjugates (eg, renal injury as described in Example 30). (See marker (KIM) and blood urea nitrogen (BUN) data). Without being bound by theory, the inventor has shown that the reduced toxicity of the disclosed conjugates is due to the non-natural amino acids at the portion of the peptide attached to the nucleic acid analog (eg, the carboxy terminus). For example, it is believed to be related to the absence of aminohexanoic acid or β-alanine. Since these unnatural amino acids are not cleaved in vivo, it is believed that toxic concentrations of uncleaved peptides accumulate and can cause toxicity.

  The glycine or proline moiety can be either the amino terminus or the carboxy terminus of the carrier peptide, and in some examples, the carrier peptide is directly linked to the nucleic acid analog via the glycine or proline subunit. Alternatively, the carrier peptide may be bound to the nucleic acid analog via an optional linker.

In one embodiment, the present disclosure provides:
(A) a carrier peptide comprising an amino acid subunit; and
(B) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence for sequence-specific binding to a target nucleic acid;
Two or more of the amino acid subunits are positively charged amino acids, the carrier peptide comprises a glycine (G) or proline (P) amino acid subunit at the carboxy terminus of the carrier peptide, the carrier peptide comprising: And conjugates covalently linked to the nucleic acid analog. In some embodiments, no more than 7 consecutive amino acid subunits are arginine, for example, no more than 6 consecutive amino acid subunits are arginine. In some embodiments, the carrier peptide comprises a glycine amino acid subunit at the carboxy terminus. In another embodiment, the carrier peptide comprises a proline amino acid subunit at the carboxy terminus. In yet another embodiment, the carrier peptide comprises a single glycine or proline at the carboxy terminus (ie, no glycine or proline dimer or trimer etc. at the carboxy terminus).

In certain embodiments, (i) provided by an unconjugated oligomer when binding of the antisense oligomer to its target sequence is effective to interfere with a translation initiation codon for the encoded protein. A decrease in the expression of the encoded protein compared to that, or
(Ii) unconjugated if the binding of the antisense oligomer to its target sequence is effective to block an aberrantly spliced site in the pre-mRNA encoding the protein that is correctly spliced The carrier peptide, when conjugated to an antisense oligomer having a substantially uncharged backbone, as evidenced by an increase in expression of the encoded protein compared to that provided by the oligomer, It is effective to improve the binding of the antisense oligomer to its target sequence as compared to the antisense oligomer in an unconjugated state. Suitable assays for measuring these effects are further described below. In one embodiment, conjugation of the peptide provides this activity in a cell-free translation assay as described herein. In some embodiments, the activity is enhanced by at least 2 factors, at least 5 factors, or at least 10 factors.

  Alternatively, or additionally, the carrier peptide is effective to improve transport of the nucleic acid analog into the cell as compared to an unconjugated analog. In certain embodiments, transport is enhanced by at least two factors, at least two factors, at least five factors, or at least ten factors.

  In other embodiments, the carrier peptide reduces the toxicity of the conjugate (ie, increases the maximum tolerated dose) compared to a conjugate comprising a carrier peptide that lacks a terminal glycine or proline amino subunit. It is effective. In certain embodiments, toxicity is reduced by at least 2 factors, at least 2 factors, at least 5 factors or at least 10 factors.

  A further advantage of the peptide transport moiety is its expected ability to stabilize the duplex between the antisense oligomer and its target nucleic acid sequence. Without being bound by theory, this ability to stabilize the duplex can be attributed to electrostatic interactions between a positively charged transport moiety and a negatively charged nucleic acid.

  The length of the carrier peptide is not particularly limited and varies in various embodiments. In some embodiments, the carrier peptide comprises 4 to 40 amino acid subunits. In other embodiments, the carrier peptide comprises 6 to 30, 6 to 20, 8 to 25, or 10 to 20 amino acid subunits. In some embodiments, the carrier peptide is linear, while in other embodiments, the carrier peptide is branched.

  In some embodiments, the carrier peptide is rich in positively charged amino acid subunits, eg, arginine amino acid subunits. A carrier peptide is “rich” with a positive charge if at least 10% of the amino acid subunits have a positive charge. For example, in some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid subunits have a positive charge. Have. In yet other embodiments, all amino acid subunits, except glycine or proline amino acid subunits, have a positive charge. In yet another embodiment, all of the positively charged amino acid subunits are arginine.

  In another embodiment, the number of positively charged amino acid subunits in the carrier peptide ranges from 1 to 20, for example 1 to 10 or 1 to 6. In one embodiment, the number of positively charged amino acid subunits in the carrier peptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

  The positively charged amino acid can be a naturally occurring amino acid, a non-naturally occurring amino acid, a synthetic amino acid, a modified amino acid, or an analog of a natural amino acid. For example, modified amino acids having a net positive charge may be specifically designed for use in the present invention, as described in more detail below. Many different types of modifications to amino acids are well known in the art. In one embodiment, the positively charged amino acid is histidine (H), lysine (K) or arginine (R). In another embodiment, the carrier peptide includes only natural amino acid subunits (ie, does not include unnatural amino acids). In other embodiments, the terminal amino acid may be capped, eg, at the N-terminus, eg, with an acetyl, benzoyl, stearyl moiety.

  Any number, combination and / or sequence of H, K and / or R may be present in the carrier peptide. In some embodiments, all amino acid subunits other than the carboxy-terminal glycine or proline are positively charged amino acids. In another embodiment, at least one of the positively charged amino acids is arginine. For example, in some embodiments, all of the positively charged amino acids are arginine, and in yet other embodiments, the carrier peptide consists of arginine and the carboxy-terminal glycine or proline. In yet another embodiment, the carrier peptide comprises no more than 7 consecutive arginines, eg, no more than 6 consecutive arginines.

Other types of positively charged amino acids are also envisioned. For example, in one embodiment, at least one of the positively charged amino acids is an arginine analog. For example, the arginine analog has the structure R ^a N═C (NH ₂ ) R ^b, where R ^a is H or R ^c ; R ^b is R ^c , NH ₂ , NHR or N (R ^c ) ₂ and R ^c is lower alkyl or lower alkenyl and optionally contains oxygen or nitrogen, or R ^a and R ^b together form a ring) It may be an amino acid and the side chain is linked to the amino acid via R ^a or R ^b . The carrier peptide may comprise any number of these arginine analogs.

The positively charged amino acid may occur in any sequence within the carrier peptide. For example, in some embodiments, the positively charged amino acids may be alternating or consecutive. For example, the carrier peptide may include the sequence (R ^d ) _m , where R ^d is an amino acid having a positive charge independently at each occurrence, and m is 2 to 12, 2 to 10, 2 To an integer in the range of 8 or 2 to 6. For example, in certain embodiments, R ^d is arginine and the carrier peptide is selected from (R) ₄ , (R) ₅ , (R) ₆ , (R) ₇ and (R) ₈ , or A sequence selected from (R) ₄ , (R) ₅ , (R) ₆ and (R) ₇ . For example, in certain embodiments, the carrier peptide comprises the sequence (R) ₆ , eg, (R) ₆ G or (R) ₆ P.

In another embodiment, the carrier peptide consists of the sequence (R ^d ) _m and the carboxy-terminal glycine or proline, wherein R ^d is an amino acid having a positive charge, independently at each occurrence; m is an integer ranging from 2 to 12, 2 to 10, 2 to 8 or 2 to 6. In certain embodiments, R ^d is arginine, histidine, or lysine, independently at each occurrence. For example, in certain embodiments, R ^d is arginine and the carrier peptide is a sequence selected from (R) ₄ , (R) ₅ , (R) ₆ , (R) ₇ and (R) _8. And carboxy-terminal glycine or proline. For example, in certain embodiments, the carrier peptide consists of the sequence (R) ₆ G or (R) ₆ P.

  In some other embodiments, the carrier peptide may comprise one or more hydrophobic amino acid subunits. The hydrophobic amino acid subunit comprises a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain, wherein the alkyl, alkenyl and alkynyl side chains are at most every six carbon atoms. Contains one heteroatom. In some embodiments, the hydrophobic amino acid is phenylalanine (F). For example, the carrier peptide may comprise two or more consecutive hydrophobic amino acids, eg, phenylalanine (F), eg, two consecutive phenylalanine moieties. The hydrophobic amino acid can be present at any location in the carrier peptide sequence.

であり、
式中、ｎは、２から７であり、各Ｒ^ｅは、各出現箇所で独立して、水素またはメチルである。一部のこれらの実施形態では、Ｒ^ｄは、各出現箇所で独立して、アルギニン、ヒスチジンまたはリジンである。他の実施形態では、各Ｒ^ｄは、アルギニンである。他の実施形態では、ｎは５であり、Ｙ^ｂは、アミノヘキサン酸部分である。他の実施形態では、ｎは２であり、Ｙ^ｂは、β−アラニン部分である。さらに他の実施形態では、Ｒ^ｅは、水素である。 In another embodiment, the carrier peptide has the sequence [(R ^d Y ^b R ^d ) _x (R ^d R ^d Y ^b ) _y ] _z or [(R ^d R ^d Y ^b ) _y (R ^d Y ^b R ^d ) _x ] _z , wherein R ^d is a positively charged amino acid independently at each occurrence, x and y are independently 0 or 1 at each occurrence, and x + y is 1 or 2 is provided, z is 1, 2, 3, 4, 5 or 6, and Y ^b is

And
Where n is 2 to 7, and each R ^e is independently hydrogen or methyl at each occurrence. In some of these embodiments, R ^d is independently arginine, histidine, or lysine at each occurrence. In other embodiments, each R ^d is arginine. In other embodiments, n is 5 and Y ^b is an aminohexanoic acid moiety. In other embodiments, n is 2 and Y ^b is a β-alanine moiety. In still other embodiments, R ^e is hydrogen.

In certain embodiments described above, x is 1 and y is 0, and the carrier peptide comprises the sequence (R ^d Y ^b R ^d ) _z . In other embodiments, n is 5 and Y ^b is an aminohexanoic acid moiety. In other embodiments, n is 2 and Y ^b is a β-alanine moiety. In still other embodiments, R ^e is hydrogen.

In still other embodiments described above, x is 0, y is 1, and the carrier peptide comprises the sequence (R ^d R ^d Y ^b ) _z . In other embodiments, n is 5 and Y ^b is an aminohexanoic acid moiety. In other embodiments, n is 2 and Y ^b is a β-alanine moiety. In still other embodiments, R ^e is hydrogen.

In another embodiment, the carrier peptide comprises the sequence (R ^d Y ^b ) _p . R ^d and Y ^b are as defined above, and p is an integer ranging from 2 to 8. In other embodiments, each R ^d is arginine. In other embodiments, n is 5 and Y ^b is an aminohexanoic acid moiety. In other embodiments, n is 2 and Y ^b is a β-alanine moiety. In still other embodiments, R ^e is hydrogen.

In another embodiment, the carrier peptide comprises the sequence ILFQY. In addition to the ILFQY sequence, the peptide may include any other sequence disclosed herein. For example, the carrier peptide may be ILFQY and [(R ^d Y ^b R ^d ) _x (R ^d R ^d Y ^b ) _y ] _z , [(R ^d R ^d Y ^b ) _y (R ^d Y ^b R ^d ). _x ] _z , (R ^d Y ^b ) _p, or a combination thereof, where R ^d , x, y and Y ^b are as defined above. [(R ^d Y ^b R ^d ) _x (R ^d R ^d Y ^b ) _y ] _z , [(R ^d R ^d Y ^b ) _y (R ^d Y ^b R ^d ) _x ] _z or (R ^d Y ^b The sequence of _p may be present at the amino terminus, the carboxy terminus or both of the ILFQY sequence. In certain embodiments, x is 1 and y is 0, and the carrier peptide comprises (R ^d Y ^b R ^d ) _z attached to the ILFQY sequence via an optional Z linker.

In another related embodiment, the carrier peptide comprises the sequence ILFQ, IWFQ or ILIQ. Other embodiments comprise a carrier peptide comprising the sequence PPMWS, PPMWT, PPMFS or PPMYS. In addition to these sequences, the carrier peptide may include any other sequences described herein, for example, the sequence [(R ^d Y ^b R ^d ) _x (R ^d R ^d Y ^b ) _y ] _z , [(R ^d R ^d Y ^b ) _y (R ^d Y ^b R ^d ) _x ] _z , or (R ^d Y ^b ) _p , wherein R ^d , x, y and Y ^b are as defined above It is as follows.

  Some embodiments of the carrier peptide have modifications to naturally occurring amino acid subunits, for example, the amino-terminal or carboxy-terminal amino acid subunits can be modified. Such modifications include capping unbound amino or unbound carboxy with a hydrophobic group. For example, the amino terminus may be capped with an acetyl, benzoyl or stearoyl moiety. For example, any peptide sequence in Table 1 may have such modifications even if not specifically indicated in the table. In these embodiments, the amino terminus of the carrier peptide may be indicated as follows:

  In yet another embodiment, the carrier peptide comprises at least one of alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.

  In some embodiments disclosed herein, the carrier peptide consists of the above sequence and the carboxy-terminal glycine or proline amino acid subunit.

であり、式中、ｎは、２から７であり、各Ｒ^ｅは、各出現箇所で独立して、水素またはメチルであり、
（ｃ）Ｚは、アラニン、アスパラギン、システイン、グルタミン、グリシン、ヒスチジン、リジン、メチオニン、セリン、スレオニンおよび、生理学的ｐＨで負に荷電している側鎖（例えば、カルボキシレート側鎖）を除く、天然に存在する側鎖の１つまたは２つの炭素同族体である側鎖を有するアミノ酸から選択されるアミノ酸サブユニットを表わす、ペプチド−核酸類似体コンジュゲートを提供する。一部の実施形態では、前記側鎖は、中性である。他の実施形態では、前記Ｚ側鎖は、天然に存在するアミノ酸の側鎖である。一部の実施形態では、前記任意のＺサブユニットは、アラニン、グリシン、メチオニン、セリンおよびスレオニンから選択される。前記キャリアペプチドは、０、１、２または３つのＺサブユニットを含んでもよく、一部の実施形態では、多くても２つのＺサブユニットを含む。 In some embodiments, the carrier peptide has the following sequence (amino terminus to carboxy terminus): R ₆ G, R ₇ G, R ₈ G, R ₅ GR ₄ G, R ₅ F ₂ R ₄ G, Tat _{-G, rTat-G, (RXR} 2 G 2) 2 or _(RXR 3 _X) does not constitute a ₂ G. In yet other embodiments, the carrier peptide does not constitute R ₈ G, R ₉ G, or R ₉ F ₂ G. In yet another embodiment, the carrier peptide is the following _{sequence: Tat-G, rTat-G} , R 9 F 2 G, R 5 F 2 R 4, R 4 G, R 5 G, R 6 G, R ₇ G, R ₈ G, R ₉ G, (RXR) ₄ G, (RXR) ₅ G, (RXRRBR) ₂ G, (RAR) ₄ F ₂ or (RGR) ₄ F ₂ are not configured. In another embodiment, the carrier peptide does not constitute “Penetratin” or “R ₆ Pen”.
In another aspect, the present disclosure provides:
A nucleic acid analog having a substantially uncharged backbone and a targeting base sequence, and
8 to 16 additional others covalently linked to the nucleic acid analog, comprising the carboxy-terminal glycine or proline amino acid subunit, selected from an R ^d subunit, a Y subunit and an optional Z subunit A peptide comprising at least 8 R ^d subunits, at least 2 Y subunits and at most 3 Z subunits, wherein> 50% of said subunits are R ^d subunits Including
(A) Each R ^d subunit is independently represented by arginine or an arginine analog, wherein the arginine analog has the structure R ^a N═C (NH ₂ ) R ^b (where R ^a is H Or R ^c ; R ^b is R ^c , NH ₂ , NHR or N (R ^c ) ₂ , R ^c is lower alkyl or lower alkenyl, and optionally contains oxygen or nitrogen, or R ^a and R ^b together form a ring) a cationic α-amino acid, said side chain being linked to an amino acid via R ^a or R ^b ,
(B) the at least two Y subunits are Y ^a or Y ^b ;
(I) each Y ^a is independently a neutral α-amino acid subunit having a side chain independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl and aralkyl. When selected from alkyls, alkenyls and alkynyls, the side chain contains at most one heteroatom for every two, preferably every four, and more preferably every six carbon atoms, The subunits are contiguous or adjacent to the linker moiety; and
(Ii) Y ^b is

Where n is 2 to 7, and each R ^e is independently hydrogen or methyl at each occurrence,
(C) Z excludes alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, threonine and side chains that are negatively charged at physiological pH (eg, carboxylate side chains) Peptide-nucleic acid analog conjugates are provided that represent amino acid subunits selected from amino acids having side chains that are one or two carbon homologues of a naturally occurring side chain. In some embodiments, the side chain is neutral. In another embodiment, the Z side chain is a side chain of a naturally occurring amino acid. In some embodiments, the optional Z subunit is selected from alanine, glycine, methionine, serine and threonine. The carrier peptide may include 0, 1, 2, or 3 Z subunits, and in some embodiments, includes at most 2 Z subunits.

In selected embodiments, the carrier peptide is a Y ^a type Y subunit has two accurately. The Y subunit is contiguous or adjacent to the cysteine subunit. In some embodiments, the two Y ^a subunit is continuous. In other embodiments, the side chain of Y ^a subunit, excluding side chains that are charged at physiological pH, the side chain of a naturally occurring amino acid, and, one of them or two carbon homolog including. Another possible side chain is the side chain of a naturally occurring amino acid. In a further embodiment, the side chain is an aryl or aralkyl side chain, for example, each Y ^a may be independently selected from phenylalanine, tyrosine, tryptophan, leucine, isoleucine and valine.

In selected embodiments, each Y ^a is independently selected from phenylalanine and tyrosine. In a further embodiment, each Y ^a is phenylalanine. This includes, for example, conjugates consisting of an arginine subunit, a phenylalanine subunit, the glycine or proline amino acid subunit, an optional linker moiety and a nucleic acid analog. One such conjugate comprises a peptide having the formula Arg ₉ Phe ₂ aa, where aa is glycine or proline.

  The aforementioned carrier peptide may comprise ILFQY, ILFQ, IWFQ or ILIQ. Other embodiments comprise the aforementioned carrier peptides comprising the sequence PPMWS, PPMWT, PPMFS or PPMYS

  The peptide-oligomer conjugate of the present invention is more effective than the unconjugated oligomer in inhibiting expression of target mRNA in protein expression systems including cell-free translation systems; inhibiting splicing of target pre-mRNA; and nucleic acid replication of viruses. Alternatively, it is effective in a variety of functions, including inhibiting viral replication by targeting cis-acting elements that control mRNA transcription.

Conjugates of other drugs (ie not nucleic acid analogs) with the carrier peptide are also included within the scope of the present invention. Specifically, some embodiments:
(A) a carrier peptide comprising an amino acid subunit; and (b) a drug;
Two or more of the amino acid subunits are positively charged amino acids, the carrier peptide comprises a glycine (G) or proline (P) amino acid subunit at the carboxy terminus of the carrier peptide, the carrier peptide comprising: Providing a conjugate covalently bound to the drug. The carrier peptide in these embodiments may be any carrier peptide described herein. Also provided is a method for delivering the drug by conjugating the drug to the carrier peptide.

  The drug to be delivered may be a biologically active agent, such as a therapeutic or diagnostic agent, and the drug may be a compound used for detection, such as a fluorescent compound. Biologically active agents include biomolecules such as peptides, proteins, sugars or nucleic acids, especially antisense oligonucleotides, or drug substances selected from “small molecules” organic or inorganic compounds. . “Small molecule” compounds may be broadly defined as organic, inorganic or organometallic compounds that are not the aforementioned biomolecules. Typically, such compounds have a molecular weight of less than 1000, and in one embodiment less than 500.

  In one embodiment, the drug to be delivered will not contain a single amino acid, dipeptide or tripeptide. In another embodiment, the drug to be delivered does not comprise a short oligopeptide; ie, an oligopeptide having 6 or fewer amino acid subunits. In a further embodiment, the drug to be delivered does not include longer oligopeptides; that is, oligopeptides having between 7 and 20 amino acid subunits. In a further embodiment, the drug to be delivered does not comprise a polypeptide or protein having more than 20 amino acid subunits.

  The carrier peptide is effective to improve transport of the drug into cells and / or lacks a glycine or proline subunit compared to the drug in an unconjugated state. It has lower toxicity compared to the drug conjugated to the corresponding peptide. In some embodiments, transport is enhanced by at least 2, at least 5 or at least 10 factors. In other embodiments, toxicity is reduced by at least 2, at least 5 or at least 10 factors (ie, the maximum tolerated dose is increased).

B. Peptide Linker The carrier peptide can be coupled to the drug (eg, nucleic acid analog, drug, etc.) to be delivered by various methods available to those skilled in the art. In some embodiments, the carrier peptide is directly linked to the nucleic acid analog without an intervening linker. In this regard, the formation of an amide bond between the terminal amino acid and an unbound carboxyl-unbound amine on the nucleic acid analog may be useful for the formation of the conjugate. In certain embodiments, the carboxy-terminal glycine or proline subunit is directly linked to the 3 ′ end of the nucleic acid analog. For example, the carrier peptide may be linked by forming an amide bond between the carboxy-terminal glycine or proline moiety and the 3 ′ morpholino ring nitrogen (see, eg, FIG. 1C).

In some embodiments, the nucleic acid analog, Y ^a or Y ^b subunit, cysteine subunits and, via a linker moiety selected from non-amino acid linker moiety uncharged, conjugated to the carrier peptide Is done. In another embodiment, the nucleic acid analog is directly linked to the carrier peptide via the glycine or proline moiety, either to the 5 ′ end or the 3 ′ end of the nucleic acid analog. In some embodiments, the carrier peptide is directly linked to the 3 ′ of the nucleic acid analog via the glycine or proline amino acid subunit, eg, directly to the 3 ′ morpholino nitrogen via an amide bond. Combined.

を有し、ここで、Ｒ^２４は存在しないか、ＨまたはＣ_１−Ｃ_６アルキルである。ある実施形態では、Ｒ^２４は存在せず、他の実施形態では、構造（ＸＸＩＸ）は、核酸類似体（例えば、モルホリノオリゴマー）の５’端を、前記キャリアペプチドに結合する（例えば、図１Ｂを参照のこと）。 In another embodiment, the conjugate includes a binding moiety between the terminal glycine or proline amino acid subunits. In some embodiments, the linker is a bond selected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, carbonyl, carbamate, phosphorodiamidate, phosphoramidate, phosphorothioate and phosphodiester. And 18 atoms or less in length. In certain embodiments, the linker comprises a phosphorodiamidate and a piperazine bond. For example, in some embodiments, the linker has the following structure (XXIX):

Where R ²⁴ is absent or is H or C ₁ -C ₆ alkyl. In some embodiments, R ²⁴ is absent, and in other embodiments, the structure (XXIX) attaches the 5 ′ end of a nucleic acid analog (eg, morpholino oligomer) to the carrier peptide (eg, FIG. 1B). checking).

In some embodiments, the side chain portion of the R ^d subunit is independently guanidyl (HN═C (NH ₂ ) NH—), amidinyl (HN═C (NH ₂ ) C <), 2- Selected from aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridyl and 2-aminopyrimidyl.

  Multiple carrier peptides can be attached to a single compound, if desired. Alternatively, multiple compounds can be conjugated to a single transporter. The linker between the carrier peptide and the nucleic acid analog may be composed of a natural amino acid or a non-natural amino acid (eg, 6-aminohexanoic acid or β-alanine). The linker is formed, for example, by carbodiimide promoted condensation between the carboxy terminus of the transporter peptide and an amine or hydroxy group (eg, 3 ′ morpholino nitrogen or 5 ′ OH) of the nucleic acid analog. Direct bonds may also be included.

  In general, the linker may include any non-reactive moiety that does not interfere with the transport or function of the conjugate. The linker may be selected from those that are non-cleavable under normal use conditions, including, for example, ether, thioether, amide or carbamate linkages. In other embodiments, it is desirable that the linker comprises a linkage between the carrier peptide and a compound (eg, an oligonucleotide analog, drug, etc.) that is cleavable in vivo. In vivo cleavable bonds are known in the art and include, for example, enzymatically hydrolyzed carboxylic esters and disulfides that are cleaved in the presence of glutathione. By application of radiation of the appropriate wavelength, a photolytically cleavable linkage, such as ortho-nitrophenyl ether, may be suitable for cleavage in vivo. Exemplary heterobifunctional binders further comprising a cleavable disulfide group include N-hydroxysuccinimidyl 3-[(4-azidophenyl) dithio] propionate and Vanin, E .; F. and Ji, T .; H. Biochemistry 20: 6754-6760 (1981).

C. Exemplary Carrier Peptides A table of exemplary carrier peptide sequences and oligonucleotide sequences is provided in Table 1 below. In some embodiments, the disclosure provides a peptide oligomer conjugate, wherein the peptide comprises or consists of any one of the peptide sequences in Table 1. In another embodiment, the nucleic acid analog comprises or consists of any of the oligonucleotide sequences in Table 1. In yet another embodiment, the present disclosure provides a peptide oligomer conjugate, wherein the peptide comprises or consists of any one of the peptide sequences in Table 1, and the nucleic acid The analog comprises or consists of any of the oligonucleotide sequences in Table 1. In another embodiment, the disclosure provides a peptide comprising or consisting of any one of the sequences in Table 1.

aa = glycine or proline; B = β-alanine; X = 6-aminohexanoic acid; tg = unmodified amino terminus or amino terminus capped by an acetyl, benzoyl or stearoyl group (ie acetylamide, benzoylamide or Stearoylamide), and Y ^b is —C (O) — (CHR ^e ) _n —NH—, n is 2 to 7, and each R ^e is independently hydrogen at each occurrence. Or methyl. For the sake of clarity, not all sequences are mentioned to have a terminal tg group. However, each of the above sequences may comprise an unmodified amino terminus or an amino terminus capped with an acetyl, benzoyl or stearoyl group.

III. Antisense Oligomers Nucleic acid analogs included in the conjugates of the invention are synthetic oligomers that are substantially uncharged, such as antisense oligonucleotide analogs, that are capable of specifically binding to a polynucleotide target sequence. is there. Such analogs include, for example, methylphosphonates, peptide nucleic acids, substantially uncharged N3 ′ → P5 ′ phosphoramidates and morpholino oligomers.

  The base sequence of the nucleic acid analog provided by a base pairing group supported by an analog backbone can be any sequence, and the supported base pairing group can be standard or modified A, T, C, G and U bases, or non-standard inosine (I) and 7-deaza-G bases.

  In some embodiments, the nucleic acid analog is a morpholino oligomer, ie, an oligonucleotide analog composed of a morpholino subunit structure of the type shown in FIG. (I) The structure is such that a phosphorus-containing linkage of 1 to 3 atoms in length, preferably 2 atoms in length, bonds the morpholino nitrogen of one subunit to the 5 ′ exocyclic carbon of an adjacent subunit. Are coupled to each other. (Ii) Pi and Pj are purine or pyrimidine base-pairing moieties effective to bind to bases in polynucleotides by base-specific hydrogen bonding. The base pairing moiety of the purine or pyrimidine is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structure and binding properties of the morpholino oligomers are further described below and are described in US Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315. 5, 521,063 and 5,506,337, all of which are incorporated herein by reference.

Desirable chemical properties of the morpholino-based oligomers include the ability to selectively hybridize with complementary base target nucleic acids, including target RNAs with high T _m , even oligomers as short as 8-14 bases, mammals The ability to be actively transported into cells and the ability of oligomer: RNA heteroduplexes resistant to RNAse degradation.

  In a preferred embodiment, the morpholino oligomer is about 8-40 subunits in length. More typically, the oligomer is about 8-20, about 8-16, about 10-30, or about 12-25 subunits in length. For some applications, such as antibacterials, it is particularly advantageous, for example, when short oligomers of about 8-12 subunits in length are attached to peptide transporters as disclosed herein. obtain.

A. Oligomers with modified intersubunit linkages One embodiment of the present disclosure relates to peptide-oligomer conjugates comprising nucleic acid analogs (eg, morpholino oligomers) comprising modified intersubunit linkages. In some embodiments, the conjugate has an improved cell compared to an oligomer having a higher affinity for DNA and RNA and having other inter-subunit linkages than the corresponding unmodified oligomer has. Shows delivery, efficacy and / or tissue distribution characteristics. In one embodiment, the conjugate comprises one or more (A) type intersubunit linkages, as defined below. In another embodiment, the conjugate comprises at least one type (B) intersubunit linkage, as defined below. In yet another embodiment, the conjugate comprises (A) and (B) type intersubunit linkages. In yet other embodiments, the conjugate comprises a morpholino oligomer, as described in more detail below. The structural characteristics and properties of various linkage types and oligomers are described in more detail in the description below.

または、その塩もしくはその異性体を有し、式中、
Ｗは、各出現箇所で独立して、ＳまたはＯであり；
Ｘは、各出現箇所で独立して、−Ｎ（ＣＨ_３）_２、−ＮＲ^１Ｒ^２、−ＯＲ^３または

であり；
Ｙは、各出現箇所で独立して、Ｏまたは−ＮＲ^２であり；
Ｒ^１は、各出現箇所で独立して、水素またはメチルであり；
Ｒ^２は、各出現箇所で独立して、水素または−ＬＮＲ^４Ｒ^５Ｒ^７であり；
Ｒ^３は、各出現箇所で独立して、水素またはＣ_１−Ｃ_６アルキルであり；
Ｒ^４は、各出現箇所で独立して、水素、メチル、−Ｃ（＝ＮＨ）ＮＨ_２、−Ｚ−Ｌ−ＮＨＣ（＝ＮＨ）ＮＨ_２または−［Ｃ（Ｏ）ＣＨＲ’ＮＨ］_ｍＨであり、Ｚは、−Ｃ（＝Ｏ）−または直接結合であり、Ｒ’は、天然に存在するアミノ酸または１つもしくは２つのその炭素同族体の側鎖であり、ｍは、１から６であり；
Ｒ^５は、各出現箇所で独立して、水素、メチルまたは電子対であり；
Ｒ^６は、各出現箇所で独立して、水素またはメチルであり；
Ｒ^７は、各出現箇所で独立して、水素、Ｃ_１−Ｃ_６アルキルまたはＣ_１−Ｃ_６アルコキシアルキルであり；
Ｌは、アルキル、アルコキシもしくはアルキルアミノの基またはそれらの組み合わせを含む、長さ１８個原子以下の必要に応じたリンカーである。 1. Linkage (A)
Applicants have found that improvements in antisense activity, biodistribution, and / or other desired properties can be optimized by preparing oligomers with various intersubunit linkages. For example, the oligomer may optionally include one or more (A) type intersubunit linkages. In one embodiment, the oligomer comprises at least one (A) type linkage, for example, each linkage may be (A) type. In some other embodiments, each (A) type linkage has the same structure. Type (A) linkages may include those disclosed in co-owned US Pat. No. 7,943,762, which is incorporated herein by reference in its entirety. Linkage (A) has the following structure (I) wherein 3 ′ and 5 ′ indicate attachment points to the 3 ′ end and 5 ′ end of the morpholino ring (ie, structure (i) described below), respectively.

Or a salt or isomer thereof, wherein
W is S or O independently at each occurrence;
X is independently —N (CH ₃ ) ₂ , —NR ¹ R ² , —OR ³ or

Is;
Y is independently O or —NR ² at each occurrence;
R ¹ is independently hydrogen or methyl at each occurrence;
R ² is independently hydrogen or —LNR ⁴ R ⁵ R ⁷ at each occurrence;
R ³ is independently hydrogen or C ₁ -C ₆ alkyl at each occurrence;
R ⁴ is independently hydrogen, methyl, —C (═NH) NH ₂ , —ZL—NHC (═NH) NH ₂ or — [C (O) CHR′NH] _m H independently at each occurrence. Z is —C (═O) — or a direct bond, R ′ is the side chain of a naturally occurring amino acid or one or two of its carbon analogs, and m is 1 to 6 Is;
R ⁵ is independently hydrogen, methyl, or an electron pair at each occurrence;
R ⁶ is independently hydrogen or methyl at each occurrence;
R ⁷ is independently at each occurrence, hydrogen, C ₁ -C ₆ alkyl, or C ₁ -C ₆ alkoxyalkyl;
L is an optional linker containing a group of alkyl, alkoxy or alkylamino or a combination thereof and having a length of 18 atoms or less.

In some examples, the oligomer comprises at least one (A) type linkage. In some other embodiments, the oligomer comprises at least two consecutive (A) type linkages. In a further embodiment, at least 5% of the linkages in the oligomer are of the (A) type. For example, in some embodiments, 5% -95%, 10% to 90%, 10% to 50%, or 10% to 35% of the linkage may be (A) type linkage. In some specific embodiments, at least one (A) type linkage is —N (CH ₃ ) ₂ . In other embodiments, the (A) type linkage is a -N _{(CH 3) 2.} In yet another embodiment, each linkage in the oligomer is —N (CH ₃ ) ₂ . In other embodiments, at least one (A) type linkage is piperidin-1-yl, eg, unsubstituted piperazin-1-yl (eg, A2 or A3). In other embodiments, each (A) type linkage is piperidin-1-yl, eg, unsubstituted piperazin-1-yl.

  In some embodiments, W is S or O, independently at each occurrence, and in certain embodiments, W is O.

In some embodiments, X is independently —N (CH ₃ ) ₂ , —NR ¹ R ² , —OR ³ at each occurrence. In some embodiments, X is —N (CH ₃ ) ₂ . In other embodiments, X is —NR ¹ R ² , and in other examples, X is —OR ³ .

In some embodiments, R ¹ is independently hydrogen or methyl at each occurrence. In some embodiments, R ¹ is hydrogen. In other embodiments, X is methyl.

In some embodiments, R ² is hydrogen at each occurrence. In other embodiments, R ² is —LNR ⁴ R ⁵ R ⁷ at each occurrence. In some embodiments, R ³ is independently hydrogen or C ₁ -C ₆ alkyl at each occurrence. In other embodiments, R ³ is methyl. In yet other embodiments, R ³ is ethyl. In some other embodiments, R ³ is n-propyl or isopropyl. In some other embodiments, R ³ is C ₄ alkyl. In other embodiments, R ³ is C ₅ alkyl. In some other embodiments, R ³ is C ₆ alkyl.

In certain embodiments, R ⁴ is independently at each occurrence. In other embodiments, R ⁴ is methyl. In still other embodiments, R ⁴ is —C (═NH) NH ₂ . In other embodiments, R ⁴ is —ZL—NHC (═NH) NH ₂ . In still other embodiments, R ⁴ is — [C (═O) CHR′NH] _m H. In one embodiment, Z is —C (═O) —, and in another embodiment, Z is a direct bond. R ′ is the side chain of a naturally occurring amino acid. In some embodiments, R ′ is a carbon homologue of one or two naturally occurring amino acid side chains.

  m is an integer of 1 to 6. m may be 1. m may be 2. m may be 3. m may be four. m may be 5. m may be 6.

In some embodiments, R ⁵ is independently at each occurrence, hydrogen, methyl, or an electron pair. In some embodiments, R ⁵ is hydrogen. In other embodiments, R ⁵ is methyl. In yet other embodiments, R ⁵ is an electron pair.

In some embodiments, R ⁶ is independently hydrogen or methyl at each occurrence. In some embodiments, R ⁶ is hydrogen. In other embodiments, R ⁶ is methyl.

In other embodiments, R ⁷ is independently at each occurrence, hydrogen, C ₁ -C ₆ alkyl, or C ₂ -C ₆ alkoxyalkyl. In some embodiments, R ⁷ is hydrogen. In other embodiments, R ⁷ is C ₁ -C ₆ alkyl. In still other embodiments, R ⁷ is C ₂ -C ₆ alkoxyalkyl. In some embodiments, R ⁷ is methyl. In other embodiments, R ⁷ is ethyl. In yet other embodiments, R ⁷ is n-propyl or isopropyl. In some other embodiments, R ⁷ is C ₄ alkyl. In some embodiments, R ⁷ is C ₅ alkyl. In some embodiments, R ⁷ is C ₆ alkyl. In still other embodiments, R ⁷ is C ₂ alkoxyalkyl. In some other embodiments, R ⁷ is C ₃ alkoxyalkyl. In still other embodiments, R ⁷ is C ₄ alkoxyalkyl. In some embodiments, R ⁷ is C ₅ alkoxyalkyl. In other embodiments, R ⁷ is C ₆ alkoxyalkyl.

As described above, the linker group L is alkyl (eg, —CH ₂ —CH ₂ —), alkoxy (eg, , including binding in its backbone selected from -C-O-C-) and alkylamino _(e.g., -CH 2 -NH-). Although a branched linkage (eg, —CH ₂ —CHCH ₃ —) is possible, the linker is generally unbranched. In one embodiment, the linker is a hydrocarbon linker. Such linkers structure _{(CH 2) n} - may have, n is 1-12, preferably 2-8, and more preferably 2-6.

  Oligomers having any number of (A) type linkages are provided. In some embodiments, the oligomer does not comprise (A) type linkage. In certain embodiments, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent of the linkage is (A) type linkage. In selected embodiments, 10 to 80, 20 to 80, 20 to 60, 20 to 50, 20 to 40 or 20 to 35 percent of the linkage is (A) type linkage. In yet another embodiment, each linkage is of type (A).

または、その塩もしくはその異性体を有する。式中、
Ｗは、各出現箇所で独立して、ＳまたはＯであり；
Ｘは、各出現箇所で独立して、−ＮＲ^８Ｒ^９または−ＯＲ^３であり；および、
Ｙは、各出現箇所で独立して、Ｏまたは−ＮＲ^１０であり、
Ｒ^３は、各出現箇所で独立して、水素またはＣ_１−Ｃ_６アルキルであり；
Ｒ^８は、各出現箇所で独立して、水素またはＣ_２−Ｃ_１２アルキルであり；
Ｒ^９は、各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アラルキルまたはアリールであり；
Ｒ^１０は、各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキルまたは−ＬＮＲ^４Ｒ^５Ｒ^７であり；
Ｒ^８およびＲ^９が結合して、５〜１８員の単環もしくは二環複素環、または、Ｒ^８、Ｒ^９またはＲ^３がＲ^１０と結合して、５〜７員の複素環を形成する。Ｘが４−ピペラジノである場合、Ｘは、下記構造（ＩＩＩ）：

を有する。式中、
Ｒ^１１は、各出現箇所で独立して、Ｃ_２−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アミノアルキル、Ｃ_１−Ｃ_１２アルキルカルボニル、アリール、ヘテロアリールまたはヘテロシクリルであり；
Ｒは、各出現箇所で独立して、電子対、水素またはＣ_１−Ｃ_１２アルキルであり；および、
Ｒ^１２は、各出現箇所で独立して、水素、Ｃ_１−Ｃ_１２アルキル、Ｃ_１−Ｃ_１２アミノアルキル、−ＮＨ_２、−ＣＯＮＨ_２、−ＮＲ^１３Ｒ^１４、−ＮＲ^１３Ｒ^１４Ｒ^１５、Ｃ_１−Ｃ_１２アルキルカルボニル、オキソ、−ＣＮ、トリフルオロメチル、アミジル、アミジニル、アミジニルアルキル、アミジニルアルキルカルボニル、グアニジニル、グアニジニルアルキル、グアニジニルアルキルカルボニル、コラート、デオキシコラート、アリール、ヘテロアリール、複素環、−ＳＲ^１３またはＣ_１−Ｃ_１２アルコキシであり、Ｒ^１３、Ｒ^１４およびＲ^１５は、各出現箇所で独立して、Ｃ_１−Ｃ_１２アルキルである。 2. Linkage (B)
In some embodiments, the oligomer comprises at least one (B) type linkage. For example, the oligomer may comprise 1, 2, 3, 4, 5, 6 or more (B) type linkages. The (B) type linkage may be adjacent or may be dispersed throughout the oligomer. (B) type linkage has the following compound structure (I):

Or it has the salt or its isomer. Where
W is S or O independently at each occurrence;
X is independently —NR ⁸ R ⁹ or —OR ³ at each occurrence; and
Y is independently O or —NR ¹⁰ at each occurrence;
R ³ is independently hydrogen or C ₁ -C ₆ alkyl at each occurrence;
R ⁸ is independently hydrogen or C ₂ -C ₁₂ alkyl at each occurrence;
R ⁹ is independently at each occurrence, hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aralkyl or aryl;
R ¹⁰ is independently hydrogen, C ₁ -C ₁₂ alkyl or —LNR ⁴ R ⁵ R ⁷ at each occurrence;
R ⁸ and R ⁹ combine to form a 5- to 18-membered monocyclic or bicyclic heterocyclic ring, or R ⁸ , R ⁹ or R ³ combine with R ¹⁰ to form a 5- to 7-membered heterocyclic ring To do. When X is 4-piperazino, X is the following structure (III):

Have Where
R ¹¹ is independently C ₂ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, C ₁ -C ₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl at each occurrence;
R is, independently at each occurrence, an electron pair, hydrogen or C ₁ -C ₁₂ alkyl; and
R ¹² is independently hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, —NH ₂ , —CONH ₂ , —NR ¹³ R ¹⁴ , —NR ¹³ R ¹⁴ R ¹⁵ at each occurrence. , _C 1 _{-C 12} alkylcarbonyl, oxo, -CN, trifluoromethyl, amidyl, amidinyl, amino isoxazolidinyl alkyl, amino isoxazolidinyl alkylcarbonyl, guanidinyl, guar Nizhny Le alkyl, guar Nizhny Le alkylcarbonyl, alcoholates, deoxycholate , Aryl, heteroaryl, heterocycle, —SR ¹³ or C ₁ -C ₁₂ alkoxy, wherein R ¹³ , R ¹⁴ and R ¹⁵ are independently C ₁ -C ₁₂ alkyl at each occurrence.

  In some embodiments, the oligomer comprises one (B) type linkage. In some other embodiments, the oligomer comprises two (B) type linkages. In some other embodiments, the oligomer comprises three (B) type linkages. In some other embodiments, the oligomer comprises four (B) type linkages. In yet another embodiment, the (B) type linkage is continuous (ie, the (B) type linkages are adjacent to each other). In a further embodiment, at least 5% of the linkage in the oligomer is of type (B). For example, in some embodiments, 5% -95%, 10% to 90%, 10% to 50%, or 10% to 35% of the linkage may be (B) type linkage.

In other embodiments, R ³ is independently hydrogen or C ₁ -C ₆ alkyl at each occurrence. In still other embodiments, R ³ may be methyl. In some embodiments, R ³ can be ethyl. In some other embodiments, R ³ can be n-propyl or isopropyl. In still other embodiments, R ³ may be C ₄ alkyl. In some embodiments, R ³ can be C ₅ alkyl. In some embodiments, R ³ can be C ₆ alkyl.

In some embodiments, R ⁸ is independently hydrogen or C ₂ -C ₁₂ alkyl at each occurrence. In some embodiments, R ⁸ is hydrogen. In yet other embodiments, R ⁸ is ethyl. In some other embodiments, R ⁸ is n-propyl or isopropyl. In some embodiments, R ⁸ is C ₄ alkyl. In yet other embodiments, R ⁸ is C ₅ alkyl. In other embodiments, R ⁸ is C ₆ alkyl. In some embodiments, R ⁸ is C ₇ alkyl. In yet other embodiments, R ⁸ is C ₈ alkyl. In other embodiments, R ⁸ is C ₉ alkyl. In still other embodiments, R ⁸ is C ₁₀ alkyl. In some other embodiments, R ⁸ is C ₁₁ alkyl. In yet other embodiments, R ⁸ is C ₁₂ alkyl. In some other embodiments, R ⁸ is C ₂ -C ₁₂ alkyl, wherein the C ₂ -C ₁₂ alkyl is one or more double bonds (eg, alkenes), triple bonds (eg, alkynes). ) Or both. In some embodiments, R ⁸ is unsubstituted C ₂ -C ₁₂ alkyl.

In some embodiments, R ⁹ is independently hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aralkyl, or aryl at each occurrence. In some embodiments, R ⁹ is hydrogen. In yet other embodiments, R ⁹ is C ₁ -C ₁₂ alkyl. In other embodiments, R ⁹ is methyl. In yet other embodiments, R ⁹ is ethyl. In some other embodiments, R ⁹ is n-propyl or isopropyl. In some embodiments, R ⁹ is C ₄ alkyl. In some embodiments, R ⁹ is C ₅ alkyl. In still other embodiments, R ⁹ is C ₆ alkyl. In some other embodiments, R ⁹ is C ₇ alkyl. In some embodiments, R ⁹ is C ₈ alkyl. In some embodiments, R ⁹ is C ₉ alkyl. In some other embodiments, R ⁹ is C ₁₀ alkyl. In some other embodiments, R ⁹ is C ₁₁ alkyl. In still other embodiments, R ⁹ is C ₁₂ alkyl.

を有する。 In some other embodiments, R ⁹ is C ₁ -C ₁₂ aralkyl. For example, in some embodiments, R ⁹ is benzyl, which can be optionally substituted on either the phenyl ring or the benzyl carbon. In this regard, substituents include alkyl and alkoxy groups such as methyl or methoxy. In some embodiments, the benzyl group is substituted with methyl at the benzyl carbon. For example, in some embodiments, R ⁹ has the following structure (XIV):

Have

の１つを有する。 In other embodiments, R ⁹ is aryl. For example, in some embodiments, R ⁹ is phenyl, which may be optionally substituted. In this regard, substituents include alkyl and alkoxy groups such as methyl or methoxy. In other embodiments, R ⁹ is phenyl, wherein the phenyl comprises a crown ether moiety, eg, a 12-18 membered crown ether. In one embodiment, the crown ether is an 18-membered ring and may contain a still further phenyl moiety. For example, in one embodiment, R ⁹ has the following structure (XV) or (XVI):

One of them.

In some embodiments, R ¹⁰ is independently hydrogen, C ₁ -C ₁₂ alkyl, or —LNR ⁴ R ⁵ R ⁷ , and R ⁴ , R ^5, and R ⁷ are linked ( As defined above for A). In other embodiments, R ¹⁰ is hydrogen. In other embodiments, R ¹⁰ is C ₁ -C ₁₂ alkyl. In other embodiments, R ¹⁰ is —LNR ⁴ R ⁵ R ⁷ . In some embodiments, R ¹⁰ is methyl. In yet other embodiments, R ¹⁰ is ethyl. In some embodiments, R ¹⁰ is C ₃ alkyl. In some embodiments, R ¹⁰ is C ₄ alkyl. In yet other embodiments, R ¹⁰ is C ₅ alkyl. In some other embodiments, R ¹⁰ is C ₆ alkyl. In other embodiments, R ¹⁰ is C ₇ alkyl. In yet other embodiments, R ¹⁰ is C ₈ alkyl. In some embodiments, R ¹⁰ is C ₉ alkyl. In other embodiments, R ¹⁰ is C ₁₀ alkyl. In still other embodiments, R ¹⁰ is C ₁₁ alkyl. In some other embodiments, R ¹⁰ is C ₁₂ alkyl.

を有する。式中、Ｚは、５または６員の単環複素環を表わす。 In some embodiments, R ⁸ and R ⁹ are joined to form a 5-18 membered mono- or bicyclic heterocycle. In some embodiments, the heterocycle is a 5- or 6-membered monocyclic heterocycle. For example, in some embodiments, the linkage (B) has the following structure (IV):

Have In the formula, Z represents a 5- or 6-membered monocyclic heterocyclic ring.

が挙げられる。 In other embodiments, the heterocycle is a bicycle, eg, a 12 membered bicyclic heterocycle. The heterocycle may be piperidine. The heterocycle may be morpholino. The heterocycle may be piperidinyl. The heterocycle may be decahydroisoquinoline. Typical heterocycles include the following:

Is mentioned.

In some embodiments, R ¹¹ is C ₂ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, aryl, heteroaryl or heterocyclyl independently at each occurrence.

In some embodiments, R ¹¹ is C ₂ -C ₁₂ alkyl. In some embodiments, R ¹¹ is ethyl. In other embodiments, R ¹¹ is C ₃ alkyl. In yet other embodiments, R ¹¹ is isopropyl. In some other embodiments, R ¹¹ is C ₄ alkyl. In other embodiments, R ¹¹ is C ₅ alkyl. In some embodiments, R ¹¹ is C ₆ alkyl. In other embodiments, R ¹¹ is C ₇ alkyl. In some embodiments, R ¹¹ is C ₈ alkyl. In other embodiments, R ¹¹ is C ₉ alkyl. In still other embodiments, R ¹¹ is C ₁₀ alkyl. In some other embodiments, R ¹¹ is C ₁₁ alkyl. In some embodiments, R ¹¹ is C ₁₂ alkyl.

In other embodiments, R ¹¹ is C ₁ -C ₁₂ aminoalkyl. In some embodiments, R ¹¹ is methylamino. In some embodiments, R ¹¹ is ethylamino. In other embodiments, R ¹¹ is C ₃ aminoalkyl. In yet other embodiments, R ¹¹ is C ₄ aminoalkyl. In some other embodiments, R ¹¹ is C ₅ aminoalkyl. In other embodiments, R ¹¹ is C ₆ aminoalkyl. In still other embodiments, R ¹¹ is C ₇ aminoalkyl. In some embodiments, R ¹¹ is C ₈ aminoalkyl. In other embodiments, R ¹¹ is C ₉ aminoalkyl. In still other embodiments, R ¹¹ is C ₁₀ aminoalkyl. In some other embodiments, R ¹¹ is C ₁₁ aminoalkyl. In other embodiments, R ¹¹ is C ₁₂ aminoalkyl.

In another embodiment, R ¹¹ is C ₁ -C ₁₂ alkylcarbonyl. In yet other embodiments, R ¹¹ is C ₁ alkylcarbonyl. In other embodiments, R ¹¹ is C ₂ alkylcarbonyl. In some embodiments, R ¹¹ is C ₃ alkylcarbonyl. In yet other embodiments, R ¹¹ is C ₄ alkylcarbonyl. In some embodiments, R ¹¹ is C ₅ alkylcarbonyl. In some other embodiments, R ¹¹ is C ₆ alkylcarbonyl. In other embodiments, R ¹¹ is C ₇ alkylcarbonyl. In still other embodiments, R ¹¹ is C ₈ alkylcarbonyl. In some embodiments, R ¹¹ is C ₉ alkylcarbonyl. In still other embodiments, R ¹¹ is C ₁₀ alkylcarbonyl. In some other embodiments, R ¹¹ is C ₁₁ alkylcarbonyl. In some embodiments, R ¹¹ is C ₁₂ alkylcarbonyl. In still other embodiments, R ¹¹ is —C (═O) (CH ₂ ) _n CO ₂ H, wherein n is 1-6. For example, in some embodiments, n is 1. In another embodiment, n is 2. In still other embodiments, n is 3. In some other embodiments, n is 4. In still other embodiments, n is 5. In another embodiment, n is 6.

In other embodiments, R ¹¹ is aryl. For example, in some embodiments, R ¹¹ is phenyl. In some embodiments, the phenyl is substituted with, for example, a nitro group.

In other embodiments, R ¹¹ is heteroaryl. For example, in some embodiments, R ¹¹ is pyridinyl. In other embodiments, R ¹¹ is pyrimidinyl.

In other embodiments, R ¹¹ is heterocyclyl. For example, in some embodiments, R ¹¹ is piperidinyl, such as piperidin-4-yl.

In some embodiments, R ¹¹ is ethyl, isopropyl, piperidinyl, pyrimidinyl, cholate, deoxycholate, or —C (═O) (CH ₂ ) _n CO ₂ H, and n is 1-6.

In some embodiments, R is an electron pair. In other embodiments, R is hydrogen. In other embodiments, R is C ₁ -C ₁₂ alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In other embodiments, R is C ₃ alkyl. In yet other embodiments, R is isopropyl. In some other embodiments, R is C ₄ alkyl. In yet other embodiments, R is C ₅ alkyl. In some embodiments, R is C ₆ alkyl. In other embodiments, R is C ₇ alkyl. In yet other embodiments, R is C ₈ alkyl. In other embodiments, R is C ₉ alkyl. In some embodiments, R is C ₁₀ alkyl. In still other embodiments, R is C ₁₁ alkyl. In some embodiments, R is C ₁₂ alkyl.

In some embodiments, R ¹² is independently at each occurrence, hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ aminoalkyl, —NH ₂ , —CONH ₂ , —NR ¹³ R ¹⁴ , -NR ¹³ R ¹⁴ R ^15, oxo, -CN, trifluoromethyl, amidyl, amidinyl, amino isoxazolidinyl alkyl, amino isoxazolidinyl alkylcarbonyl guanidinyl, guar Nizhny Le alkyl, guar Nizhny Le alkylcarbonyl, alcoholates, deoxycholate, aryl , Heteroaryl, heterocycle, —SR ¹³ or C ₁ -C ₁₂ alkoxy, wherein R ¹³ , R ¹⁴ and R ¹⁵ are independently C ₁ -C ₁₂ alkyl at each occurrence.

In some embodiments, R ¹² is hydrogen. In some embodiments, R ¹² is C ₁ -C ₁₂ alkyl. In some embodiments, R ¹² is C ₁ -C ₁₂ aminoalkyl. In some embodiments, R ¹² is —NH ₂ . In some embodiments, R ¹² is —CONH ₂ . In some embodiments, R ¹² is —NR ¹³ R ¹⁴ . In some embodiments, R ¹² is —NR ¹³ R ¹⁴ R ¹⁵ . In some embodiments, R ¹² is C ₁ -C ₁₂ alkylcarbonyl. In some embodiments, R ¹² is oxo. In some embodiments, R ¹² is —CN. In some embodiments, R ¹² is trifluoromethyl. In some embodiments, R ¹² is amidyl. In some embodiments, R ¹² is amidinyl. In some embodiments, R ¹² is amidinylalkyl. In some embodiments, R ¹² is amidinylalkylcarbonyl. In some embodiments, R ¹² is guanidinyl, such as monomethylguanidinyl or dimethylguanidinyl. In some embodiments, R ¹² is guanidinylalkyl. In some embodiments, R ¹² is amidinylalkylcarbonyl. In some embodiments, R ¹² is cholate. In some embodiments, R ¹² is deoxycholate. In some embodiments, R ¹² is aryl. In some embodiments, R ¹² is heteroaryl. In some embodiments, R ¹² is a heterocycle. In some embodiments, R ¹² is -SR ¹³ . In some embodiments, R ¹² is C ₁ -C ₁₂ alkoxy. In some embodiments, R ¹² is dimethylamine.

In other embodiments, R ¹² is methyl. In yet other embodiments, R ¹² is ethyl. In some embodiments, R ¹² is C ₃ alkyl. In some embodiments, R ¹² is isopropyl. In some embodiments, R ¹² is C ₄ alkyl. In other embodiments, R ¹² is C ₅ alkyl. In still other embodiments, R ¹² is C ₆ alkyl. In some other embodiments, R ¹² is C ₇ alkyl. In some embodiments, R ¹² is C ₈ alkyl. In yet other embodiments, R ¹² is C ₉ alkyl. In some embodiments, R ¹² is C ₁₀ alkyl. In yet other embodiments, R ¹² is C ₁₁ alkyl. In other embodiments, R ¹² is C ₁₂ alkyl. In yet other embodiments, the alkyl moiety is substituted with one or more oxygen atoms to form an ether moiety, eg, a methoxymethyl moiety.

In some embodiments, R ¹² is methylamino. In other embodiments, R ¹² is ethylamino. In still other embodiments, R ¹² is C ₃ aminoalkyl. In some embodiments, R ¹² is C ₄ aminoalkyl. In still other embodiments, R ¹² is C ₅ aminoalkyl. In some other embodiments, R ¹² is C ₆ aminoalkyl. In some embodiments, R ¹² is C ₇ aminoalkyl. In some embodiments, R ¹² is C ₈ aminoalkyl. In still other embodiments, R ¹² is C ₉ aminoalkyl. In some other embodiments, R ¹² is C ₁₀ aminoalkyl. In still other embodiments, R ¹² is C ₁₁ aminoalkyl. In other embodiments, R ¹² is C ₁₂ aminoalkyl. In some embodiments, the aminoalkyl is dimethylalkylaminoalkyl.

In yet other embodiments, R ¹² is acetyl. In some other embodiments, R ¹² is C ₂ alkylcarbonyl. In some embodiments, R ¹² is C ₃ alkylcarbonyl. In still other embodiments, R ¹² is C ₄ alkylcarbonyl. In some embodiments, R ¹² is C ₅ alkylcarbonyl. In still other embodiments, R ¹² is C ₆ alkylcarbonyl. In some other embodiments, R ¹² is C ₇ alkylcarbonyl. In some embodiments, R ¹² is C ₈ alkylcarbonyl. In still other embodiments, R ¹² is C ₉ alkylcarbonyl. In some other embodiments, R ¹² is C ₁₀ alkylcarbonyl. In some embodiments, R ¹² is C ₁₁ alkylcarbonyl. In another embodiment, R ¹² is C ₁₂ alkylcarbonyl. The alkylcarbonyl is substituted with a carboxy moiety. For example, the alkylcarbonyl is substituted to form a succinic acid moiety (ie, 3-carboxyalkylcarbonyl). In another embodiment, the alkylcarbonyl is substituted with a terminal -SH group.

を有してもよい。 In some embodiments, R ¹² is amidyl. In some embodiments, the amidyl includes an alkyl moiety that is further substituted, eg, with —SH, carbamate, or a combination thereof. In another embodiment, the amidyl is substituted with an aryl moiety, such as phenyl. In certain embodiments, R ¹² has the following structure (IX):

You may have.

Wherein R ¹⁶ is independently hydrogen, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, —CN, aryl, or heteroaryl at each occurrence.

In some embodiments, R ¹² is methoxy. In other embodiments, R ¹² is ethoxy. In still other embodiments, R ¹² is C ₃ alkoxy. In some embodiments, R ¹² is C ₄ alkoxy. In some embodiments, R ¹² is C ₅ alkoxy. In some other embodiments, R ¹² is C ₆ alkoxy. In other embodiments, R ¹² is C ₇ alkoxy. In some other embodiments, R ¹² is C ₈ alkoxy. In some embodiments, R ¹² is C ₉ alkoxy. In other embodiments, R ¹² is C ₁₀ alkoxy. In some embodiments, R ¹² is C ₁₁ alkoxy. In yet other embodiments, R ¹² is C ₁₂ alkoxy.

In certain embodiments, R ¹² is pyrrolidinyl, eg, pyrrolidin-1-yl. In other embodiments, R ¹² is piperidinyl, such as piperidin-1-yl or piperidin-4-yl. In other embodiments, R ¹² is morpholino, eg, morpholin-4-yl. In other embodiments, R ¹² is phenyl, and in yet further embodiments, the phenyl is substituted with, for example, a nitro group. In yet other embodiments, R ¹² is pyrimidinyl, eg, pyrimidin-2-yl.

In other embodiments, R ¹³ , R ^14, and R ¹⁵ are independently C ₁ -C ₁₂ alkyl at each occurrence. In some embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is methyl. In yet other embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is ethyl. In other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₃ alkyl. In yet other embodiments, R ¹³ , R ^14, or R ¹⁵ is isopropyl. In other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₄ alkyl. In some embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is C ₅ alkyl. In some other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₆ alkyl. In other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₇ alkyl. In still other embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is C ₈ alkyl. In other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₉ alkyl. In some embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is C ₁₀ alkyl. In some embodiments, R ¹³ , R ¹⁴ or R ¹⁵ is C ₁₁ alkyl. In still other embodiments, R ¹³ , R ^14, or R ¹⁵ is C ₁₂ alkyl.

As noted above, in some embodiments, R ¹² is amidyl substituted with an aryl moiety. In this regard, each R ¹⁶ may be the same or different. In certain of these embodiments, R ¹⁶ is hydrogen. In other embodiments, R ¹⁶ is —CN. In other embodiments, R ¹⁶ is heteroaryl, eg, tetrazolyl. In certain other embodiments, R ¹⁶ is methoxy. In other embodiments, R ¹⁶ is aryl, wherein said aryl is optionally substituted. In this regard, optional substituents include C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, such as methoxy; trifluoromethoxy; halo, such as chloro; and trifluoromethyl.

In other embodiments, R ¹⁶ is methyl. In yet other embodiments, R ¹⁶ is ethyl. In some embodiments, R ¹⁶ is C ₃ alkyl. In some other embodiments, R ¹⁶ is isopropyl. In still other embodiments, R ¹⁶ is C ₄ alkyl. In other embodiments, R ¹⁶ is C ₅ alkyl. In still other embodiments, R ¹⁶ is C ₆ alkyl. In some other embodiments, R ¹⁶ is C ₇ alkyl. In some embodiments, R ¹⁶ is C ₈ alkyl. In still other embodiments, R ¹⁶ is C ₉ alkyl. In some other embodiments, R ¹⁶ is C ₁₀ alkyl. In other embodiments, R ¹⁶ is C ₁₁ alkyl. In some other embodiments, R ¹⁶ is C ₁₂ alkyl.

In some embodiments, R ¹⁶ is methoxy. In some embodiments, R ¹⁶ is ethoxy. In still other embodiments, R ¹⁶ is C ₃ alkoxy. In some other embodiments, R ¹⁶ is C ₄ alkoxy. In other embodiments, R ¹⁶ is C ₅ alkoxy. In some other embodiments, R ¹⁶ is C ₆ alkoxy. In still other embodiments, R ¹⁶ is C ₇ alkoxy. In some other embodiments, R ¹⁶ is C ₈ alkoxy. In still other embodiments, R ¹⁶ is C ₉ alkoxy. In some other embodiments, R ¹⁶ is C ₁₀ alkoxy. In some embodiments, R ¹⁶ is C ₁₁ alkoxy. In some other embodiments, R ¹⁶ is C ₁₂ alkoxy.

の１つを有する複素環を形成する。 In some other embodiments, R ⁸ and R ⁹ are joined to form a 12-18 membered crown ether. For example, in some embodiments, the crown ether is 18 members, and in other embodiments, the crown ether is 15 members. In certain embodiments, R ⁸ and R ⁹ are combined to form the following structure (X) or (XI):

To form a heterocycle having one of

で表わされる。式中、Ｚ’は、５〜７員の複素環を表わす。構造（ＸＩ）のある実施形態では、Ｒ^１２は、各出現箇所で水素である。例えば、リンケージ（Ｂ）が、下記構造（Ｂ１）、（Ｂ２）または（Ｂ３）：

の１つを有してもよい。 In some embodiments, R ⁸ , R ⁹ or R ³ is joined with R ¹⁰ to form a 5-7 membered heterocycle. For example, in some embodiments, R ³ is combined with R ¹⁰ to form a 5-7 membered heterocycle. In some embodiments, the heterocycle is 5-membered. In another embodiment, the heterocycle is 6-membered. In another embodiment, the heterocycle is 7-membered. In some embodiments, the heterocyclic ring has the following structure (XII):

It is represented by In the formula, Z ′ represents a 5- to 7-membered heterocyclic ring. In certain embodiments of structure (XI), R ¹² is hydrogen at each occurrence. For example, the linkage (B) has the following structure (B1), (B2) or (B3):

You may have one of.

In certain other embodiments, R ¹² is C ₁ -C ₁₂ alkylcarbonyl or amidyl further substituted with an arylphosphoryl moiety, eg, a triphenylphosphoryl moiety. Examples of linkages having this structure are B56 and B55.

In certain embodiments, linkage (B) does not have any of structures A1-A5.
Table 2 shows typical linkages of types (A) and (B).

In the following sequences and descriptions, the above names for the linkages are often used. For example, the base containing the PMO ^apn linkage is shown as ^apn B. B is a base. Other linkages are nominated as well. Furthermore, abbreviations may be used, for example, abbreviations in the above parentheses may be used (for example, ^a B means ^apn B). Other readily identifiable abbreviations can also be used.

B. Oligomers with modified end groups In addition to the carrier peptide, the conjugate may comprise an oligomer with modified end groups. Applicants have noted that modification of the 3 'and / or 5' ends of the oligomer with various chemical moieties may be beneficial to the conjugate for therapeutic properties (eg, improved cellular delivery, efficacy and / or tissue). Distribution etc.). In various embodiments, the modified end group includes a hydrophobic moiety. On the other hand, in another embodiment, the modified end group includes a hydrophilic moiety. The modified end group may be present with or without the aforementioned linkage. For example, in some embodiments, the oligomer conjugated to the carrier peptide comprises one or more modified end groups and a linkage of type (A), eg, X is —N (CH ₃ ) _2. Including. In other embodiments, the oligomer comprises one or more modified end groups and a linkage of type (B), eg, X is 4-aminopiperidin-1-yl (ie, APN). In yet another embodiment, the oligomer comprises one or more modified end groups and a mixture of linkages (A) and (B). For example, the oligomer may comprise one or more modified end groups (eg, trityl or triphenylacetyl), a linkage where X is —N (CH ₃ ) ₂ , and X is 4-aminopiperidin-1-yl. Some linkages may be included. Other combinations of modified end groups and modified linkages also provide favorable therapeutic properties to the oligomer.

または、その塩もしくはその異性体を有し、式中、Ｘ、ＷおよびＹは、リンケージ（Ａ）および（Ｂ）のいずれかについての上記定義の通りであり、
Ｒ^１７は、各出現箇所で独立して、存在しないか、水素またはＣ_１−Ｃ_６アルキルであり；
Ｒ^１８およびＲ^１９は、各出現箇所で独立して、存在しないか、水素、前記キャリアペプチド、天然もしくは非天然のアミノ酸、Ｃ_２−Ｃ_３０アルキルカルボニル、−Ｃ（＝Ｏ）ＯＲ^２１またはＲ^２０であり；
Ｒ^２０は、各出現箇所で独立して、グアニジニル、ヘテロシクリル、Ｃ_１−Ｃ_３０アルキル、Ｃ_３−Ｃ_８シクロアルキル；Ｃ_６−Ｃ_３０アリール、Ｃ_７−Ｃ_３０アラルキル、Ｃ_３−Ｃ_３０アルキルカルボニル、Ｃ_３−Ｃ_８シクロアルキルカルボニル、Ｃ_３−Ｃ_８シクロアルキルアルキルカルボニル、Ｃ_７−Ｃ_３０アリールカルボニル、Ｃ_７−Ｃ_３０アラルキルカルボニル、Ｃ_２−Ｃ_３０アルキルオキシカルボニル、Ｃ_３−Ｃ_８シクロアルキルオキシカルボニル、Ｃ_７−Ｃ_３０アリールオキシカルボニル、Ｃ_８−Ｃ_３０アラルキルオキシカルボニルまたは−Ｐ（＝Ｏ）（Ｒ^２２）_２であり；
Ｐｉは、各出現箇所で独立して、塩基対合部分であり；
Ｌ^１は、アルキル、ヒドロキシル、アルコキシ、アルキルアミノ、アミド、エステル、ジスルフィド、カルボニル、カルバマート、ホスホロジアミダート、ホスホロアミダート、ホスホロチオアート、ピペラジンおよびホスホジエステルから選択される結合を含む、長さ１８個原子以下の必要に応じたリンカーであり；および、
ｘは、０またはそれより大きい整数であり；および、
Ｒ^１８またはＲ^１９の少なくとも１つが、Ｒ^２０であり；および、
Ｒ^１８またはＲ^１９の少なくとも１つが、Ｒ^２０であり、但し、Ｒ^１７およびＲ^１８は両方とも存在する。 In one embodiment, the oligomer comprising a modified end has the following structure (XVII):

Or a salt or isomer thereof, wherein X, W and Y are as defined above for any of linkages (A) and (B);
R ¹⁷ is independently absent at each occurrence or is hydrogen or C ₁ -C ₆ alkyl;
R ¹⁸ and R ¹⁹ are independently absent at each occurrence, hydrogen, the carrier peptide, a natural or unnatural amino acid, a C ₂ -C ₃₀ alkylcarbonyl, —C (═O) OR ²¹ or R ²⁰ ;
R ²⁰ is independently guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C ₆ -C ₃₀ aryl, C ₇ -C ₃₀ aralkyl, C ₃ -C ₃₀ independently at each occurrence. _{alkylcarbonyl,} C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 7 _{-C 30 arylcarbonyl,} C 7 _{-C 30 aralkylcarbonyl,} C 2 _{-C 30} alkyloxycarbonyl, _{C 3} - C ₈ cycloalkyloxycarbonyl, C ₇ -C ₃₀ aryloxycarbonyl, C ₈ -C ₃₀ aralkyloxycarbonyl or —P (═O) (R ²² ) ₂ ;
Pi is a base-pairing moiety independently at each occurrence;
L ¹ includes a bond selected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, disulfide, carbonyl, carbamate, phosphorodiamidate, phosphoramidate, phosphorothioate, piperazine and phosphodiester, An optional linker no longer than 18 atoms in length; and
x is an integer of 0 or greater; and
At least one of R ¹⁸ or R ¹⁹ is R ²⁰ ; and
At least one of R ¹⁸ or R ¹⁹ is R ²⁰ , provided that both R ¹⁷ and R ¹⁸ are present.

Said oligomer having modified end groups may comprise any number of type (A) and (B) linkages. For example, the oligomer may include only the (A) type linkage. For example, X in each linkage, -N _{(CH 3)} may be _2. Alternatively, the oligomer may include only the linkage (B). In one embodiment, the oligomer comprises a mixture of linkages (A) and (B), for example, 1 to 4 linkages are in the (B) form and the remaining linkages are in the (A) form. In this regard, the linkage is not limited and includes linkages in which X is aminopiperidinyl for type (B) and dimethylamino for type (A).

In some embodiments, R ¹⁷ is absent. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ¹⁷ is methyl. In still other embodiments, R ¹⁷ is ethyl. In some embodiments, R ¹⁷ is C ₃ alkyl. In some other embodiments, R ¹⁷ is isopropyl. In other embodiments, R ¹⁷ is C ₄ alkyl. In still other embodiments, R ¹⁷ is C ₅ alkyl. In some other embodiments, R ¹⁷ is C ₆ alkyl.

In other embodiments, R ¹⁸ is absent. In some embodiments, R ¹⁸ is hydrogen. In some embodiments, R ¹⁸ is the carrier peptide. In some embodiments, R ¹⁸ is a natural or unnatural amino acid, eg, trimethylglycine. In some embodiments, R ¹⁸ is R ²⁰ .

を有してもよい。 In other embodiments, R ¹⁹ is absent. In some embodiments, R ¹⁹ is hydrogen. In some embodiments, R ¹⁹ is the carrier peptide. In some embodiments, R ¹⁹ is a natural or unnatural amino acid, eg, trimethylglycine. In some embodiments, R ¹⁹ is —C (═O) OR ¹⁷ , for example, R ¹⁹ has the structure:

You may have.

In other embodiments, R ¹⁸ or R ¹⁹ is C ₂ -C ₃₀ alkylcarbonyl, eg, —C (═O) (CH ₂ ) _n CO ₂ H, where n is 1 to 6, eg, 2 It is. In other examples, R ¹⁸ or R ¹⁹ is acetyl.

In some embodiments, R ²⁰ is independently at each occurrence, guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C ₆ -C ₃₀ aryl, C ₇ -C _{30. aralkyl,} C 3 _{-C 30 alkylcarbonyl,} C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 6 _{-C 30 arylcarbonyl,} C 7 _{-C 30 aralkylcarbonyl,} C 2 _{-C 30 alkyloxycarbonyl,} C 3 -C ₈ cycloalkyl _{alkyloxycarbonyl,} C 7 _{-C 30 aryloxycarbonyl,} C 8 _{-C 30} aralkyloxycarbonyl, -C (= O) ^{oR 21} or -P ⁽⁼ O) ^{(R 22} ₂ ). R ²¹ is C ₁ -C ₃₀ alkyl containing one or more oxygen or hydroxyl moieties or combinations thereof. Each R ²² is C ₆ -C ₁₂ aryloxy.

In certain other embodiments, R ¹⁹ is —C (═O) OR ²¹ , and R ¹⁸ is hydrogen, guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C _6- C _{30 aryl,} C 3 _{-C 30 alkylcarbonyl,} C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 7 _{-C 30 arylcarbonyl,} C 7 _{-C 30} aralkylcarbonyl, _{C 2} - C ₃₀ alkyloxy _carbonyl, C 3 -C ₈ cycloalkyl _{alkyloxycarbonyl,} C 7 _{-C 30 aryloxycarbonyl,} C 8 _{-C 30} aralkyloxycarbonyl or ^{-P (= O) (R 22} ) 2. Each R ²² is C ₆ -C ₁₂ aryloxy.

In other embodiments, R ²⁰ is independently at each occurrence, guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C ₆ -C ₃₀ aryl, C ₃ -C ₃₀ alkyl. _carbonyl, C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 7 _{-C 30 arylcarbonyl,} C 7 _{-C 30 aralkylcarbonyl,} C 2 _{-C 30 alkyloxycarbonyl,} C 3 -C ₈ cycloalkyloxycarbonyl, C ₇ -C ₃₀ aryloxycarbonyl, C ₈ -C ₃₀ aralkyloxycarbonyl or —P (═O) (R ²² ) ₂ . On the other hand, in other examples, R ²⁰ is independently guanidinyl, heterocyclyl, C ₁ -C ₃₀ alkyl, C ₃ -C ₈ cycloalkyl; C ₆ -C ₃₀ aryl, C ₇ -C ₃₀ independently at each occurrence. _aralkyl, C 3 -C ₈ cycloalkyl _carbonyl, C 3 -C ₈ cycloalkyl _{alkylcarbonyl,} C 7 _{-C 30 arylcarbonyl,} C 7 _{-C 30 aralkylcarbonyl,} C 2 _{-C 30 alkyloxycarbonyl,} C 3 -C ₈ cycloalkyloxycarbonyl, C ₇ -C ₃₀ aryloxycarbonyl, C ₈ -C ₃₀ aralkyloxycarbonyl or —P (═O) (R ²² ) ₂ .

In some embodiments, R ²⁰ is guanidinyl, such as monomethylguanidinyl or dimethylguanidinyl. In other embodiments, R ²⁰ is heterocyclyl. For example, in some embodiments, R ²⁰ is piperidin-4-yl. In some embodiments, piperidin-4-yl is substituted with a trityl or Boc group. In other embodiments, R ²⁰ is C ₃ -C ₈ cycloalkyl. In other embodiments, R ²⁰ is C ₆ -C ₃₀ aryl.

を有する。式中、Ｒ^２３は、各出現箇所で独立して、水素、ハロ、Ｃ_１−Ｃ_３０アルキル、Ｃ_１−Ｃ_３０アルコキシ、Ｃ_１−Ｃ_３０アルキルオキシカルボニル、Ｃ_７−Ｃ_３０アラルキル、アリール、ヘテロアリール、ヘテロシクリルまたはヘテロシクロアルキル（ｈｅｔｅｒｏｃｙｃｌａｌｋｙｌ）である。１つのＲ^２３は、もう１つのＲ^２３と結合して、ヘテロシクリル環を形成してもよい。一部の実施形態では、少なくとも１つのＲ^２３は、水素であり、例えば、一部の実施形態では、各Ｒ^２３は、水素である。他の実施形態では、少なくとも１つのＲ^２３は、Ｃ_１−Ｃ_３０アルコキシであり、例えば、一部の実施形態では、各Ｒ^２３は、メトキシである。他の実施形態では、少なくとも１つのＲ^２３は、ヘテロアリールであり、例えば、一部の実施形態では、少なくとも１つのＲ^２３は、下記構造（ＸＶＩＩＩａ）または（ｏｆ）（ＸＶＩＩＩｂ）：

の１つを有する。 In some embodiments, R ²⁰ is C ₇ -C ₃₀ arylcarbonyl. For example, in some embodiments, R ²⁰ has the following structure (XVIII):

Have In the formula, R ²³ is independently hydrogen, halo, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ alkoxy, C ₁ -C ₃₀ alkyloxycarbonyl, C ₇ -C ₃₀ aralkyl, aryl independently at each occurrence site. , Heteroaryl, heterocyclyl or heterocycloalkyl. One R ²³ may combine with another R ²³ to form a heterocyclyl ring. In some embodiments, at least one R ²³ is hydrogen, for example, in some embodiments, each R ²³ is hydrogen. In other embodiments, at least one R ²³ is C ₁ -C ₃₀ alkoxy, for example, in some embodiments, each R ²³ is methoxy. In other embodiments, at least one R ²³ is heteroaryl, for example, in some embodiments, at least one R ²³ has the following structure (XVIIIa) or (of) (XVIIIb):

One of them.

In still other embodiments, one R ^23, combine with another R ^23, form a heterocyclyl ring. For example, in one embodiment, R ²⁰ is 5-carboxyfluorescein.

の１つを有する。 In other embodiments, R ²⁰ is C ₇ -C ₃₀ aralkylcarbonyl. For example, in various embodiments, R ²⁰ has the following structure (XIX), (XX) or (XXI):

One of them.

In the formula, R ²³ is independently hydrogen, halo, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ alkoxy, C ₁ -C ₃₀ alkyloxycarbonyl, C ₇ -C ₃₀ aralkyl, aryl independently at each occurrence site. , Heteroaryl, heterocyclyl or heterocycloalkyl. One R ²³ may combine with another R ²³ to form a heterocyclyl ring. X is —OH or halo. m is an integer of 0 to 6. In some specific embodiments, m is 0. In another embodiment, m is 1. On the other hand, in other embodiments, m is 2. In other embodiments, at least one R ²³ is hydrogen, for example, in some embodiments, each R ²³ is hydrogen. In some embodiments, X is hydrogen. In other embodiments, X is —OH. In other embodiments, X is Cl. In other embodiments, at least one R ²³ is C ₁ -C ₃₀ alkoxy, such as methoxy.

を有する。ここで、Ｒ^２３は、各出現箇所で独立して、水素、ハロ、Ｃ_１−Ｃ_３０アルキル、Ｃ_１−Ｃ_３０アルコキシ、Ｃ_１−Ｃ_３０アルキルオキシカルボニル、Ｃ_７−Ｃ_３０アラルキル、アリール、ヘテロアリール、ヘテロシクリルまたはヘテロシクロアルキル（ｈｅｔｅｒｏｃｙｃｌａｌｋｙｌ）である。１つのＲ^２３は、もう１つのＲ^２３と結合して、ヘテロシクリル環を形成してもよい。例えば、一部の実施形態では、各Ｒ^２３は、水素である。他の実施形態では、少なくとも１つのＲ^２３は、Ｃ_１−Ｃ_３０アルコキシであり、例えば、メトキシである。 In still other embodiments, R ²⁰ is C ₇ -C ₃₀ aralkyl, eg, trityl. In other embodiments, R ²⁰ is methoxytrityl. In some embodiments, R ²⁰ has the following structure (XXII):

Have Here, R ²³ is independently hydrogen, halo, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ alkoxy, C ₁ -C ₃₀ alkyloxycarbonyl, C ₇ -C ₃₀ aralkyl, aryl independently at each occurrence location. , Heteroaryl, heterocyclyl or heterocycloalkyl. One R ²³ may combine with another R ²³ to form a heterocyclyl ring. For example, in some embodiments, each R ²³ is hydrogen. In other embodiments, at least one R ²³ is C ₁ -C ₃₀ alkoxy, such as methoxy.

を有する。 In yet other embodiments, R ²⁰ is a C ₇ -C ₃₀ aralkyl, and R ²⁰ is the following structure (XXIII):

Have

In some embodiments, at least one R ²³ is halo, eg, chloro. In some other embodiments, one R ²³ is chloro in the para position.

の１つを有する。 In other embodiments, R ²⁰ is C ₁ -C ₃₀ alkyl. For example, in some embodiments, R ²⁰ is C ₄ -C ₂₀ alkyl, optionally including one or more double bonds. For example, in some embodiments, R ²⁰ is a triple bond, eg, C ₄ -C ₁₀ alkyl, including a terminal triple bond. In some embodiments, R ²⁰ is hexyn-6-yl. In some embodiments, R ²⁰ has the following structure (XXIV), (XXV), (XXVI), or (XXVII):

One of them.

In yet other embodiments, R ²⁰ is C ₃ -C ₃₀ alkylcarbonyl, eg, C ₃ -C ₁₀ alkylcarbonyl. In some embodiments, R ²⁰ is —C (═O) (CH ₂ ) _p SH or —C (═O) (CH ₂ ) _p SSHet, p is an integer from 1 to 6, Het is heteroaryl. For example, p may be 1 or p may be 2. In other examples, Het is pyridinyl, such as pyridin-2-yl. In other _embodiments, C 3 _{-C 30} alkylcarbonyl is substituted by a further oligomer. For example, in some embodiments, the oligomer comprises a C ₃ -C ₃₀ alkyl carbonyl at the 3 ′ position that connects the oligomer to the 3 ′ position of another oligomer. Such end modifications are included within the scope of the present disclosure.

In another embodiment, R ²⁰ is C ₃ -C ₃₀ alkylcarbonyl further substituted with an aryl phosphoryl moiety, eg, triphenylphosphoryl. Examples of such R ²⁰ groups include structure 33 in Table 3.

を有する。式中、Ｒ^２３は、各出現箇所で独立して、水素、ハロ、Ｃ_１−Ｃ_３０アルキル、Ｃ_１−Ｃ_３０アルコキシ、Ｃ_１−Ｃ_３０アルキルオキシカルボニル、Ｃ_７−Ｃ_３０アラルキル、アリール、ヘテロアリール、ヘテロシクリルまたはヘテロシクロアルキル（ｈｅｔｅｒｏｃｙｃｌａｌｋｙｌ）である。１つのＲ^２３は、もう１つのＲ^２３と結合して、ヘテロシクリル環を形成してもよい。一部の実施形態では、Ｒ^２３は、ヘテロシクロアルキル（ｈｅｔｅｒｏｃｙｃｌａｌｋｙｌ）であり、例えば、一部の実施形態では、Ｒ^２３は、下記構造：

を有する。 In other examples, R ²⁰ is C ₃ -C ₈ cycloalkylcarbonyl, eg, C ₅ -C ₇ alkylcarbonyl. In these embodiments, R ²⁰ has the following structure (XXVIII):

Have In the formula, R ²³ is independently hydrogen, halo, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ alkoxy, C ₁ -C ₃₀ alkyloxycarbonyl, C ₇ -C ₃₀ aralkyl, aryl independently at each occurrence site. , Heteroaryl, heterocyclyl or heterocycloalkyl. One R ²³ may combine with another R ²³ to form a heterocyclyl ring. In some embodiments, R ²³ is heterocycloalkyl, for example, in some embodiments, R ²³ has the structure:

Have

を有する。 In some other embodiments, R ²⁰ is C ₃ -C ₈ cycloalkylalkylcarbonyl. In other embodiments, R ²⁰ is C ₂ -C ₃₀ alkyloxycarbonyl. In other embodiments, R ²⁰ is C ₃ -C ₈ cycloalkyloxycarbonyl. In other embodiments, R ²⁰ is C ₇ -C ₃₀ aryloxycarbonyl. In other embodiments, R ²⁰ is C ₈ -C ₃₀ aralkyloxycarbonyl. In other embodiments, R ²⁰ is —P (═O) (R ²² ) ₂ , and each R ²² is C ₆ -C ₁₂ aryloxy. For example, in some embodiments, R ²⁰ has the following structure (C24):

Have

In other embodiments, R ²⁰ contains one or more halo atoms. For example, in some embodiments, R ²⁰ comprises a perfluoro analog of any of the above R ²⁰ moieties. In other embodiments, R ²⁰ comprises p-trifluoromethylphenyl, p-trifluoromethyltrityl, perfluoropentyl, or pentafluorophenyl.

In some embodiments, the 3 ′ end includes a modification, and in other embodiments the 5 ′ end includes a modification. In other embodiments, both the 3 ′ end and the 5 ′ end include modifications. Thus, in some embodiments, R ¹⁸ is absent and R ¹⁹ is R ²⁰ . In other embodiments, R ¹⁹ is absent and R ¹⁸ is R ²⁰ . In still other embodiments, R ¹⁸ and R ¹⁹ are each R ²⁰ .

In some embodiments, the oligomer comprises a cell penetrating peptide in addition to a 3 ′ or 5 ′ modification. Thus, in some embodiments, R ¹⁹ is a cell penetrating peptide and R ¹⁸ is R ²⁰ . In other embodiments, R ¹⁸ is a cell penetrating peptide and R ¹⁹ is R ²⁰ . In further embodiments of the foregoing, the cell penetrating peptide is an arginine rich peptide.

を有する。 In some embodiments, the linker L ¹ that connects the 5 ′ end group (ie, R ¹⁹ ) to the oligomer may or may not be present. The linker is provided with any number of functional groups and the linker retains its ability to attach the 5 ′ end group to the oligomer, wherein the linker is sequence-specifically attached to the target sequence. Length, provided that it does not interfere with the ability of the oligomer to bind. In one embodiment, L comprises a phosphorodiamidate and a piperazine linkage. For example, in some embodiments, L is the following structure (XXIX):

Have

Wherein R ²⁴ is absent or is hydrogen or C ₁ -C ₆ alkyl. In some embodiments, R ²⁴ is absent. In some embodiments, R ²⁴ is hydrogen. In some embodiments, R ²⁴ is C ₁ -C ₆ alkyl. In some embodiments, R ²⁴ is methyl. In other embodiments, R ²⁴ is ethyl. In still other embodiments, R ²⁴ is C ₃ alkyl. In some other embodiments, R ²⁴ is isopropyl. In still other embodiments, R ²⁴ is C ₄ alkyl. In some embodiments, R ²⁴ is C ₅ alkyl. In still other embodiments, R ²⁴ is C ₆ alkyl.

を有する。式中、Ｒ^２５は、水素または−ＳＲ^２６であり、Ｒ^２６は、水素、Ｃ_１−Ｃ_３０アルキル、ヘテロシクリル、アリールまたはヘテロアリールである。ｑは、０から６の整数である。 In yet other embodiments, R ²⁰ is C ₃ -C ₃₀ alkylcarbonyl. R ²⁰ has the following structure (XXX):

Have Wherein R ²⁵ is hydrogen or —SR ²⁶ and R ²⁶ is hydrogen, C ₁ -C ₃₀ alkyl, heterocyclyl, aryl or heteroaryl. q is an integer of 0 to 6.

In any further embodiments of any of the above, R ²³ is independently at each occurrence hydrogen, halo, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ alkoxy, aryl, heteroaryl, heterocyclyl, or heterocyclo It is alkyl (heterocyclyl).

  In some other embodiments, only the 3 'end of the oligomer is conjugated to one of the groups. In some other embodiments, only the 5 'end of the oligomer is conjugated to one of the groups. In another embodiment, both the 3 'and 5' ends contain one of the above groups. The terminal group may be selected from any one of the above groups or any of the specific groups shown in Table 3.

を有してもよい。
式中、Ｐｉは、各出現箇所で独立して、塩基対合部分である。 C. Conjugate Properties As noted above, the present disclosure relates to conjugates of carrier peptides and oligonucleotide analogs (ie, oligomers). The oligomer may include various modifications that impart desired properties (eg, enhanced antisense activity) to the oligomer. In one embodiment, the oligomer comprises a backbone comprising a sequence of morpholino ring structures joined by an intersubunit linkage. The inter-subunit linkage connects the 3 ′ end of one morpholino ring structure to the 5 ′ end of an adjacent morpholino ring structure. Each morpholino ring structure is bound to a base pairing moiety so that the oligomer can bind to the target nucleic acid in a sequence specific manner. The morpholino ring structure has the following structure (i):

You may have.
In the formula, Pi is a base-pairing moiety independently at each occurrence location.

  Each morpholino ring structure supports a base-pairing moiety (Pi) and comprises a sequence of base-pairing moieties that are typically designed to hybridize to a selected antisense target in a cell or treated subject. Form. Said base-pairing moiety may be a purine or pyrimidine (A, G, C, T or U) or analog such as hypoxanthine (the base component of nucleoside inosine) or 5-methyl found in natural DNA or RNA. Can be cytosine. Analog bases that confer improved binding affinity to the oligomer may also be used. In this regard, typical analogs include C5-propynyl-modified pyrimidine, 9- (aminoethoxy) phenoxazine (G-clamp), and the like.

  As described above, the oligomer may be modified according to aspects of the present invention to produce one or more linkages (B), for example, no more than about 1 for every 2-5 uncharged linkages, typically Specifically, 3 to 5 may be included for every 10 uncharged linkages. Some embodiments also include one or more (B) type linkages. In some embodiments, the optimal improvement in antisense activity is seen when less than about half of the skeletal linkage is of type (B). Roughly, the maximum improvement is typically also seen with a small amount of linkage (B), eg, 10-20%.

  In one embodiment, type (A) and type (B) linkages are interspersed along the skeleton. In some embodiments, the oligomer does not have an exact pattern of linkages (A) and (B) along its entire length. In addition to the carrier peptide, the oligomer may optionally include 5 'and / or 3' modifications, as described above.

  The oligomer is also considered to have a block of linkage (A) and a block of linkage (B). For example, the central block of linkage (A) may be located on the side by the block of linkage (B), and vice versa. In one embodiment, the oligomer has approximately equal lengths 5 ', 3; and a central region. The percentage of linkage (B) or (A) in the central region is higher than about 50% or (o) higher than about 70%. Oligomers used for antisense applications generally range in length from about 10 to about 40 subunits, more preferably from about 15 to 25 subunits. For example, the oligomer of the present invention having 19-20 subunits and having a length useful as an antisense oligomer is ideally 2 to 7, such as 4 to 6 or 3 to 5 The linkage (B) and the remaining linkage (A) may be included. An oligomer having 14-15 subunits may ideally have 2 to 5, for example, 3 or 4 linkages (B) and the remaining linkage (A).

  The morpholino subunits may be linked by non-phospho-based intersubunit linkages, as further described below.

  Other oligonucleotide analog linkages that are uncharged in their unmodified state but may also have pendant amine substituents can also be used. For example, the 5 'nitrogen atom on the morpholino ring can be used for a sulfamide linkage (or urea linkage if the phosphorus is replaced with carbon or sulfur, respectively).

  In some embodiments for antisense applications, the oligomer may be 100% complementary to the nucleic acid target sequence, or a heteroduplex formed between the oligomer and the nucleic acid target sequence. So long as the strand is sufficiently stable to resist the action of cellular nucleases and other modes of degradation that may occur in vivo, it may include, for example, mismatches to modulate mutations. If there is a mismatch, it becomes more unstable towards the terminal region of the hybrid duplex than in the middle. The number of mismatches allowed is based on well-understood principles in duplex stability, the length of the oligomer, the ratio of G: C base pairs in the duplex, and in the duplex It will depend on the location of the mismatch. Such antisense oligomers are not necessarily 100% complementary to a nucleic acid target sequence, but the target activity is such that the biological activity of the nucleic acid target, eg, expression of the encoded protein, is modulated. It is effective for stable and specific binding to the sequence.

The stability of the duplex formed between the oligomer and the target sequence is a function of the bound T _{m and} the sensitivity of the duplex to cellular enzymatic cleavage. The T _m of an antisense compound against a complementary sequence RNA is determined using conventional methods such as Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or Miyada C.I. G. and Wallace R.M. B. , 1987, Oligonucleotide hybridization technologies, Methods Enzymol. Vol. 154 pp. It can be measured as described in 94-107.

In some embodiments, each antisense oligomer has a binding T _m to complementary sequence RNA that is above body temperature, or in other embodiments, above 50 ° C. In other embodiments, T _m is in the range greater than 60-80 ° C. Based on well-known principles, the T _m of the oligomeric compound relative to complementation-based RNA hybrids can be determined by increasing the C: G to base ratio in the duplex and / or the heteroduplex (base pair Can be improved by increasing the length. At the same time, it may be useful to limit the size of the oligomer for the purpose of optimizing cellular uptake. For this reason, compounds that are 20 bases or less in length and that exhibit high T _m (higher than 50 ° C.) are generally in contrast to those requiring longer than 20 bases due to high T _m values. preferable. For some applications, oligomers that are longer, eg, longer than 20 bases, may have certain advantages. For example, in certain embodiments, longer oligomers may find particular utility for use in exon skipping or splice regulation.

  The targeting sequence base may be a normal DNA base or an analogue thereof, for example, uracil and inosine capable of Watson-Crick base pairing with the target sequence RNA base.

  The oligomer may include a guanine base instead of adenine when the target nucleotide is a uracil residue. This is useful when the target sequence varies across different viral species and the mutation at any given nucleotide residue is either cytosine or uracil. By using guanine as a variable moiety in the targeting oligomer, the well-known ability of guanine for base pairing with uracil (referred to as C / U: G base pairing) can be exploited. By including guanine at these positions, a single oligomer can effectively target a wide range of RNA target variability.

  Various isomers, for example, structural isomers (for example, compatible isomers) may exist in the compound (for example, oligomer, intersubunit linkage, terminal group). With respect to stereoisomers, the compounds may have chiral centers and exist as racemates, enantiomerically enriched mixtures, individual enantiomers, mixtures of diastereomers or individual diastereomers. May be. All such isomers, including mixtures thereof, are included within the present invention. The compounds may have axial chirality that can lead to atropisomers. Furthermore, some crystal forms in the compounds may exist as polymorphs, which are included in the present invention. In addition, some compounds may form solvates with water or other organic solvates. Such solvates are similarly included within the scope of the present invention.

  The oligomers described herein may be used in methods of inhibiting protein production or viral replication. Accordingly, in one embodiment, nucleic acids encoding such proteins are exposed to the oligomers disclosed herein. In a further embodiment of the foregoing, the antisense oligomer comprises either a 5 ′ or 3 ′ modified end group, or combinations thereof, as disclosed herein, and the base pair moiety B is A sequence effective to hybridize to a portion of the nucleic acid at a position effective to inhibit protein production is formed. In one embodiment, the position is the ATG start codon region of the mRNA, the pre-mRNA splice site, or the viral target sequence described below.

In one embodiment, the oligomer has a T _m greater than about 50 ° C. for binding to the target sequence and is taken up by mammalian cells or bacterial cells. In another embodiment, the oligomer can be conjugated to a transport moiety, eg, an arginine rich peptide, as described herein, to facilitate such uptake. In another embodiment, the terminal modifications described herein can function as a transport moiety to facilitate uptake by mammalian and / or bacterial cells.

  The preparation and properties of morpholino oligomers are described in more detail below and are described in US Pat. No. 5,185,444 and WO 2009/064471, which are hereby incorporated by reference in their entirety. Captured in the book.

D. Conjugate Formulation and Administration The disclosure also provides for the formulation and delivery of the disclosed conjugates. Accordingly, in one embodiment, the present disclosure relates to a composition comprising a peptide-oligomer conjugate disclosed herein and a pharmaceutically acceptable vehicle.

  Effective delivery of the conjugate to the target nucleic acid is an important aspect of treatment. Antisense oligomer delivery routes are not limited and include oral and parenteral routes, including various systemic routes including intravenous, subcutaneous, intraperitoneal and intramuscular, as well as inhalation, transdermal delivery and local delivery. It is done. The appropriate route can be determined by one skilled in the art to suit the symptoms of the subject under treatment. For example, a suitable route for delivery of antisense oligomers in the treatment of viral viral infections is topical delivery. On the other hand, delivery of antisense oligomers for the treatment of viral respiratory infections is by inhalation. The oligomer may be delivered directly to the site of viral infection or may be delivered to the bloodstream.

  The conjugate may be administered in any convenient medium that is physiologically and / or pharmaceutically acceptable. Such compositions may include any of a variety of standard pharmaceutically acceptable carriers used by those skilled in the art. For example, without limitation, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions such as oil / water emulsions or triglyceride emulsions, tablets and capsules. The selection of an appropriate physiologically acceptable carrier will vary depending on the mode of administration chosen.

  The compounds (eg, conjugates) of the invention can generally be used as the free acid or free base. Alternatively, the compounds of the present invention can be used in the form of acid or base addition salts. The acid addition salts of the free amino compounds of the present invention can be prepared by methods well known in the art and can be formed from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, Examples include mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid. Base addition salts include those salts formed with carboxylate anions, organic and inorganic cations such as alkali and alkaline earth metals (eg, lithium, sodium, potassium, magnesium, barium and calcium) and ammonium Ions, as well as salts formed with those selected from substituted derivatives thereof (eg, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.). Thus, the term “pharmaceutically acceptable salt” of structure (I) is intended to encompass any and all acceptable salt forms.

  In addition, prodrugs are also included within the context of this invention. Prodrugs are optionally covalently bonded carriers that release a compound of structure (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in such a way that the modification is cleaved, either by routine manipulation or in vivo, to yield the parent compound. Prodrugs include, for example, compounds of the invention where, when administered to a patient and cleaved to form a hydroxy, amine or sulfhydryl group, the hydroxy, amine or sulfhydryl group is attached to any group. It is done. Thus, typical prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups in compounds of structure (I). Furthermore, in the case of carboxylic acid (—COOH), esters such as methyl esters, ethyl esters and the like can be used.

  In some examples, liposomes can be used to facilitate uptake of the antisense oligonucleotide into cells (eg, Williams, SA, Leukemia 10 (12): 1980-1989, 1996; Lapalainen et al., Antinal Res. 23: 119, 1994; Uhlmann et al., Antisense oligoneotides: a new therapeutic principals, 4p, 4, 90, 4; Drug Carriers in Biology and Medicine, pp. 28 -341, see Academic Press, 1979). For example, as described in WO 93/01286, hydrogels may be used as a vehicle for administering antisense oligomers. Alternatively, the oligonucleotides may be administered in microspheres or microparticles (see, eg, Wu, G.Y. and Wu, CH, J. Biol. Chem. 262: 4429-4432, 1987). ) Alternatively, as described in US Pat. No. 6,245,747, the use of gas filled microbubbles conjugated with antisense oligomers can improve delivery to the target tissue. Sustained release compositions may also be used. These may comprise a semipermeable polymer matrix in the form of a shaped article, for example a film or microcapsule.

  In one embodiment, antisense inhibition is used to treat infection in a host animal by a virus by contacting the virus-infected cell with an antisense agent effective to inhibit replication of a particular virus. It is effective. The antisense agent is administered to a mammalian subject infected with a given virus, eg, a human or domestic animal, with a suitable pharmaceutical carrier. It is contemplated that the antisense oligonucleotide inhibits RNA virus growth in the host. With little or no adverse effects on the normal growth or development of the host, the RNA virus is reduced in number or eliminated.

  In one aspect of the method, the subject is a human subject, eg, a patient diagnosed with a local or systemic viral infection. Patient symptoms such as (1) immunodeficiency; (2) burn patients; (3) having an indwelling catheter, or (4) patients who have undergone surgery or have recently undergone surgery, Affects prophylactic administration of antisense oligomers. In a preferred embodiment, the oligomer is a phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and is delivered orally. In another preferred embodiment, the oligomer is a phosphorodiamidate morpholino oligomer contained in a pharmaceutically acceptable carrier and is delivered intravenously (iv).

  In another application of the invention, the subject is a livestock animal, such as a chicken, turkey, pig, cow or goat. Said treatment is either prophylactic or therapeutic. The present invention also includes livestock and poultry feed compositions comprising cereal supplemented with sub-therapeutic amounts of the above types of antiviral antisense compounds. In methods of raising livestock and poultry with cereals supplemented with sub-therapeutic levels of antiviral agents, cereals are supplemented with sub-therapeutic amounts of anti-viral oligonucleotide compositions as described above. Be considered.

  In one embodiment, the conjugate is administered in an amount and manner effective to provide a peak blood concentration of antisense oligomer of at least 200-400 nM. Typically, one or more doses of antisense oligomer are administered at regular intervals, generally over a period of about 1 to 2 weeks. A preferred dosage for oral administration is about 1-1000 mg of oligomer per 70 kg. In some cases, higher doses than 1000 mg oligomer / patient may be required. i. v. For administration, a preferred dosage is about 0.5 mg to 1000 mg of oligomer per 70 kg. The conjugate may be administered at regular intervals every day for a short period of time, for example within 2 weeks. However, in some cases, the conjugate is administered intermittently over an extended period of time. Administration may be followed by administration of antibiotics or other therapeutic treatment, or may be combined. The treatment regimen may be adjusted (dose, frequency, route, etc.) if indicated, based on the results of the subject's immunological assays, other biochemical tests, and physiological experiments under treatment.

  Efficacy in in vivo treatment regimes using the conjugates of the invention can vary based on the duration of administration, dosage, frequency and route and the symptoms of the subject under treatment (ie, prevention). Administration versus administration for local or systemic infection). Therefore, such in vivo therapies are often monitored under treatment with tests appropriate for a particular type of viral infection to achieve optimal therapeutic results, and the response in dosage or treatment regimen. You will need to make adjustments. Treatment may be monitored, for example, by general guidelines for disease and / or infection, such as complete blood count (CBC), nucleic acid detection methods, immunodiagnostic tests, virus culture or heteroduplex detection.

The effectiveness of the antiviral conjugates of the invention administered in vivo in inhibiting or eliminating the growth of one or more RNA viruses can be determined from a subject before, during and after administration of said antisense oligomer. It can be determined from a collected biological sample (tissue, blood, urine, etc.). Examples of such sample assays include (1) monitoring the presence or absence of heteroduplex formation with target and non-target sequences using techniques known to those skilled in the art, such as electrophoresis gel mobility assays. ; (2) monitor viral protein production as measured by standard techniques, eg, ELISA or Western blotting, or (3) measure effects on virus titer, eg, by Spareman-Carver method (Eg, Pari, GS, et al., Antimicrob. Agents and Chemotherapy 39 (5): 1157-1161, 1995; Anderson, K.P., et al., Antimicrob. Agents.
and Chemotherapy 40 (9): 2004-2011, 1996, Total, G. et al. E. (Ed) in: Manual of Standard Methods for Veterinary Microbiology, pp. 60-93, 1978).

E. Preparation of conjugates The morpholino subunits, the modified intersubunit linkages, and the oligomers containing them are described in the Examples and US Pat. Nos. 5,185,444 and 7, incorporated herein by reference in their entirety. 943,762, which can be prepared. The morpholino subunit can be prepared based on the general reaction scheme I below.

Reaction Scheme 1. Preparation of morpholino subunits

  Referring to Reaction Scheme 1, B represents a base pair moiety and PG represents a protecting group. The morpholino subunit can be prepared from the corresponding ribonucleoside (1) as indicated. Said morpholino subunit (2) may optionally be protected by reaction with a suitable protecting group precursor, for example trityl chloride. The 3 'protecting group is generally removed during solid phase oligomer synthesis, as described in more detail below. The base pairing moiety can be suitably protected during solid phase oligomer synthesis. Suitable protecting groups include benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloyloxymethyl for hypoxanthine (I). The pivaloyloxymethyl group can be introduced on the N1 position of the hypoxanthine heterocyclic base. Although unprotected hypoxanthine subunits may be used, the yield in the activation reaction is much better when the base is protected. Other suitable protecting groups include those disclosed in copending US patent application Ser. No. 12 / 271,040, which is incorporated herein by reference in its entirety.

Reaction of 3 with activated phosphorus compound 4 results in a morpholino subunit having the desired linkage moiety (5). The compound of structure 4 can be prepared using any number of methods known to those skilled in the art. For example, such compounds can be prepared by reaction of the corresponding amine with phosphorus oxychloride. In this regard, the amine starting material can be prepared using any method known in the art, for example, the methods described in the Examples and US Pat. No. 7,943,762. The above scheme shows the preparation of a linkage of type (B) (eg X is —NR ⁸ R ⁹ ), but the linkage of type (A) (eg X is dimethylamine) but in a similar manner. Can be prepared.

The compound of structure 5 can be used for solid phase automated oligomer synthesis for the preparation of oligomers containing said inter-subunit linkage. Such methods are well known in the art. That is, the compound of structure 5 can be modified at the 5 ′ end to include a linker to the solid support. For example, compound 5 can be attached to the solid support by a linker comprising L ¹ and / or R ¹⁹ . A typical method is shown in FIGS. In this way, the oligo can include a 5 ′ end modification after oligomer synthesis is complete and the oligomer is cleaved from the solid support. When supported, the 5 protecting group (eg, trityl) is removed and the unbound amine is reacted with the activated phosphorus moiety in the second compound of structure 5. This sequence is repeated until the desired length of oligo is obtained. The protecting group at the 5 'end may be removed or left if 5' modification is required. The oligo can be removed from the solid support using any number of methods or treatment of the example with a base to cleave the linkage to the solid support.

  Peptide oligomer conjugates comprise a desired peptide (prepared based on standard peptide synthesis methods known in the art) and an oligomer containing unbound NH (eg, 3 ′ NH of a morpholino oligomer). Can be prepared by binding in the presence of a suitable activating reagent (eg, HATU). The conjugate can be purified using a number of techniques known in the art, such as SCX chromatography.

  The preparation of modified morpholino subunits and peptide oligomer conjugates is described in more detail in the examples. Said peptide oligomer conjugate comprising any number of modified linkages is prepared using the methods described herein, methods known in the art and / or methods described in the references herein. obtain. What is described in the examples is an overall modification of the prepared PMO + morpholino oligomers as previously described (see, eg, WO2008036127).

F. Antisense activity of oligomers The present disclosure also provides methods of inhibiting protein production. The method includes exposing a nucleic acid encoding the protein to the peptide-oligomer conjugates disclosed herein. Accordingly, in one embodiment, nucleic acids encoding such proteins are exposed to conjugates as disclosed herein. The base-pairing moiety Pi forms a sequence effective to hybridize to a part of the nucleic acid at a position effective to inhibit the production of the protein. The oligomer may target, for example, an ATG start codon region of mRNA, a pre-mRNA splice site, or a viral target sequence described below.

  In another embodiment, the disclosure provides an antisense of a peptide oligomer conjugate comprising an oligonucleotide analog having a sequence of morpholino subunits and linked by intersubunit linkage and supporting a base-pairing moiety. A method of improving activity is provided. The method includes conjugating a carrier peptide described herein to the oligonucleotide.

In some embodiments, the increase in antisense activity is
(I) If the binding of the antisense oligomer to its target sequence is effective to interfere with the translation initiation codon for the encoded protein, compared to that provided by the corresponding unmodified oligomer A decrease in the expression of the encoded protein, or
(Ii) the corresponding unmodified if the binding of the antisense oligomer to its target sequence is effective to block an aberrant splice site in the pre-mRNA encoding the protein that is correctly spliced. Can be evidenced by an increase in the expression of the encoded protein compared to that provided by the oligomer. Suitable assays for measuring these effects are further described below. In one embodiment, the modification provides this activity in a cell-free translation assay, a splice correction translation assay in cell culture, or a splice correction gain of a functional animal model system as described herein. In one embodiment, the activity is improved by at least 2, at least 5 or at least 10 factors.

  Various exemplary uses for the conjugates of the invention are described below, including antiviral uses, treatment of neuromuscular diseases, bacterial infections, inflammation and polycystic kidney disease. This description is in no way meant to limit the invention, but serves to illustrate the range of human and animal disease symptoms that can be resolved using the conjugates described herein. is there.

G. Typical therapeutic use of conjugates The oligomers conjugated to the carrier peptide contain good efficacy and low toxicity, so that they are more than obtained with other oligomers or peptide-oligomer conjugates. Provides good therapeutic measures. The following description is not limiting and provides examples of typical therapeutic uses of the conjugates.

1. Targeting the stem-loop secondary structure of ssRNA viruses One class of typical antisense antiviral compounds has a sequence of 12-40 subunits and is located at the 5 'end of the positive sense RNA strand of the target virus. A morpholino oligomer described herein that targets a sequence complementary to a region associated with stem-loop secondary structure within 40 bases (eg, incorporated herein by reference, WO 2006/033933 or U.S. Patent Application Publication Nos. 200660269911 and 20050096291).

  Said method first comprises the step of identifying a region within 40 bases of the 5 'end of the positive strand in the infecting virus, as a viral target sequence, in which the sequence is capable of forming an internal stem-loop secondary structure. A morpholino oligomer having a targeting sequence of at least 12 subunits complementary to the virus-genomic region capable of forming an internal double-stranded structure is then constructed by stepwise solid phase synthesis. The oligomer is composed of the positive sense strand of the virus and the oligonucleotide compound, and is characterized by a divergence of Tm of at least 45 ° C., and the degradation of such a stem-loop structure. A chain structure can be formed. The oligomer is conjugated to a carrier peptide as described herein.

  The target sequence is identified by analyzing a 5 ′ end sequence, for example, 40 bases at the 5 ′ end, by a computer program capable of performing secondary structure prediction based on a survey on the minimum free energy state of the input RNA sequence. Can be done.

  In a related aspect, the conjugate has a single-stranded positive-sense genome and is one of the family of flavivirus, picornavirus, calicivirus, togavirus, arterivirus, coronavirus, astrovirus or hepevirus. Can be used in a method of inhibiting replication of infectious RNA viruses in mammalian host cells. The method is complementary to a region within 40 bases of the 5 ′ end of the positive-strand viral genome capable of forming an internal stem-loop secondary structure, as described herein, in an infected host cell. Administering a conjugate having a targeting sequence of at least 12 subunits. The conjugate is composed of (i) the positive sense strand of the virus and the oligonucleotide compound, and (ii) characterized by a Tm divergence of at least 45 ° C. and degradation of such a stem-loop secondary structure. Effective when administered to the host cell to form a heteroduplex structure. The conjugate can be administered to a mammalian subject infected with or at risk of infection with the virus.

  Exemplary targeting sequences targeting the terminal stem loop structures of Dengue virus and Japanese encephalitis virus are listed below as SEQ ID NOS: 1 and 2, respectively.

  Additional exemplary targeting sequences targeting the terminal stem loop structure of ssRNA viruses are described in US patent application Ser. No. 11 / 801,885 and WO 2008/036127, which are incorporated herein by reference. Can also be found.

2. Targeting the first open reading frame of an ssRNA virus A second class of typical conjugates is a first strand that encodes a polyprotein comprising a positive sense genome of less than 12 kb and a multifunctional protein. About use in inhibiting growth for viruses in the families of Picornavirus, Calicivirus, Togavirus, Coronavirus and Flavivirus having an open reading frame. In a particular embodiment, the virus is an RNA virus from the Coronaviridae family or the West Nile virus, Yellow fever virus or Dengue virus from the Flaviviridae family. The inhibitory conjugate has an anti-targeting base sequence that is substantially complementary to a viral target sequence spanning the AUG start site of the first open reading frame of the viral genome, as described herein. Includes sense oligomers. In one embodiment of the method, the conjugate is administered to a mammalian subject infected with the virus. See, for example, WO 2005/007805 and US Patent Application Publication No. 2003003235, which are incorporated herein by reference.

  A preferred target sequence is a region spanning the AUG start site of the first open reading frame (ORF1) of the viral genome. The first ORF generally encodes a non-structural protein, such as a polyprotein comprising a polymerase, helicase and protease. “Spanning AUG start site” means that the target sequence contains at least 3 bases on one side of the AUG start site and at least 2 bases of the other (total of at least 8 bases) To do. Preferably, the target sequence comprises at least 4 bases on the start site on both sides (a total of at least 11 bases).

  More generally, preferred target sites include targets that are conserved among various virus isolates. Other preferred sites include IRES (internal ribosome entry site), transactivation protein binding site and replication initiation site. Complexes and large viral genomes that can provide multiple unwanted genes can be effectively targeted by targeting host cell genes that encode for viral entry and host response to the presence of the virus.

  Various virus-genomic sequences are available from well-known sources, such as the NCBI Genbank database. The ORF1 AUG start site can also be identified by the gene database or references. Alternatively, the AUG start site of ORF1 can be found by scanning the sequence for the AUG codon in the region of the predicted ORF1 start site.

  The overall genomic organization for each of the four virus families is as follows, followed by obtaining typical target sequences for members (genus, species or strains) selected within each family .

3. Influenza virus targeting A third class of typical conjugates is used to inhibit the growth of viruses of the Orthomyxoviridae family and to treat viral infections. In one embodiment, the host cell is contacted with a conjugate as described herein. For example, the conjugate includes: 1) 25 bases at the 5 ′ or 3 ′ end of the negative sense viral RNA fragment; 2) 25 bases at the 5 ′ or 3 ′ end of the positive sense cRNA; 3) influenza virus 45 bases near the AUG start codon in mRNA; 4) a base sequence effective to hybridize to a target region selected from 50 bases near the splice donor or acceptor site of influenza mRNA affected by alternative splicing Including, for example, International Publication No. 2006/047683; US Patent Application Publication No. 20070004661; and International Patent Application No. 2010/056613 and US Patent Application No. See 12 / 945,081 )

^＊＊３’−ベンズヒドリル；^＊＋リンケージは、ＰＭＯプラスリンケージでアシル化されたトリメチルグリシンである；ＰＭＯｍは、３−窒素部位にメチル基を有するＴ塩基を表わす。 In this regard, exemplary conjugates include conjugates comprising an oligomer comprising SEQ ID NO: 3.

^{** 3'benzhydryl; *} + linkage is the acylated trimethylglycine with PMO positive linkage; PMOm represents a T nucleotide having a methyl group at 3-nitrogen moieties.

  Said conjugates are particularly useful for the treatment of influenza virus infection in mammals. The conjugate can be administered to a mammalian subject infected with or at risk of infection with the influenza virus.

4). Targeting Picornaviridae Viruses A fourth class of typical conjugates is used to inhibit the growth of Picornaviridae viruses and to treat viral infections. Said conjugates are particularly useful for the treatment of enterovirus and / or rhinovirus infection in mammals. In this embodiment, the conjugate is a targeting sequence that is complementary to a region related to the viral RNA sequence within one of two of the 32 conserved nucleotide regions in the 5 ′ untranslated region of the virus. Morpholino oligomers having a sequence of 12 to 40 subunits, including at least 12 subunits having (eg, WO 2007/030576 and 2007/030691, incorporated herein by reference or No. 11 / 518,058 and 11 / 517,757, copending and co-owned US patent applications). A typical targeting sequence is listed in SEQ ID NO: 6 below.

5. Targeting Flaviviridae Viruses A fifth class of typical conjugates is used for inhibiting flavivirus replication in animal cells. Exemplary conjugates of this class are between 8 and 40 nucleotides in length and are at least one of the 5 ′ cyclized sequence (5′-CS) or 3′-CS sequence of the positive strand flavivirus RNA. A morpholino oligomer having a sequence of at least 8 bases complementary to a region of the viral positive-strand RNA genome. A highly preferred target is the 3'-CS, and a typical targeting sequence for Dengue virus is listed below in SEQ ID NO: 7 (eg, incorporated herein by reference, WO 2005 / 030800, or co-pending and co-owned US patent application Ser. No. 10 / 913,996).

6). Targeting Nidoviridae Viruses A sixth class of typical conjugates is used for inhibition of Nidovirus replication in virally infected animal cells. Typical conjugates of this class contain between 8-25 nucleotide bases, between the transcriptional regulatory sequence (TRS) in the 5 ′ leader site of the positive strand viral genome and the 3 ′ subgenomic region of the negative strand. Morpholino oligomers having sequences capable of interfering with base pairing at (see, for example, WO 2005/065268 or US 20070037763, which is incorporated herein by reference).

7). Filovirus Targeting In another embodiment, one or more conjugates described herein are used to replicate Ebola virus or Marburg virus in a host cell, wherein said cell is a conjugate described herein. Contact with a gate, eg, a conjugate having a targeting base sequence complementary to a target sequence composed of at least 12 consecutive bases in the AUG start site region of the positive strand mRNA, as described further below Can be used in a method of inhibiting.

  The viral genome of filovirus is not segmented and is approximately 19,000 bases of single-stranded RNA in the antisense direction. The genome encodes seven proteins from monocistronic mRNA complementary to vRNA.

  The target sequence spans 30 bases immediately downstream (within 25 bases) or upstream (within 100 bases) of the selected Ebola virus protein or within the 3 ′ end of the minus-strand viral RNA, or A positive strand (sense) RNA sequence that is downstream or upstream or the base. Preferred protein targets are the viral polymerase subunits VP35 and VP24, but L, nucleoprotein NP and VP30 are also contemplated. Of these, the first protein is preferred, for example, VP35 is preferred for the latter expressed L polymerase.

  In another embodiment, one or more of the conjugates described herein provide for replication of Ebola virus or Marburg virus in a host cell, wherein said cell is an AUG of positive strand mRNA in the filovirus mRNA sequence. Can be used in a method of inhibiting by contacting with a conjugate described herein having a targeting base sequence that is complementary to a target sequence composed of at least 12 consecutive bases within the start site region (For example, WO 2006/050414 or US Pat. Nos. 7,524,829 and 7,507,196, as well as US patent application Ser. No. 12/402, incorporated herein by reference. 455; 12 / 402,461; 12 / 402,464; and 12/853 See pending application by 180 No.).

8). Targeting Arenaviruses In another embodiment, the conjugates described herein can be used in methods for inhibiting viral infection in mammalian cells by species in the Arenaviridae family. In one aspect, the conjugate can be used to treat a mammalian subject infected with the virus (eg, WO 2007/103529 or US Pat. No. 7, incorporated herein by reference). , 582,615).

Table 5 is a typical list of target viruses targeted by the conjugates of the invention when summarized by their Old World or New World Arenavirus classification.

  The arenavirus genome consists of two single-stranded RNA segments designated S (small) and L (large). In virions, the molar ratio of S-segment RNA to L-segment RNA is approximately 2: 1. The complete S-segment RNA sequence is determined for multiple arenaviruses and ranges from 3,366 to 3,535 nucleotides. The complete L-segment RNA sequence is also determined for multiple arenaviruses and ranges from 7,102 to 7,279 nucleotides. The 3 'terminal sequences in the S and L RNA segments are identical in 17 of the last 19 nucleotides. These terminal sequences are conserved among all known arenaviruses. The 19 'or 20 nucleotides at the 5' end at the beginning of each genomic RNA are each sufficiently incompletely complementary to the corresponding 3 'end. Because of this complementarity, the 3 'and 5' ends are considered base pairs and form a pan handle structure.

  Infected virions or viral RNA (vRNA) replication occurs in infected cells to form antigenomic, virus-complementary RNA (vcRNA) strands. Both vRNA and vcRNA encode complementary mRNAs. Therefore, arenaviruses are classified as ambisense RNA viruses, rather than negative sense or positive sense RNA viruses. The ambisense orientation of the viral gene is both the L-segment and the S-segment. The NP and polymerase genes are present at the 3 ′ ends of the S and L vRNA segments, respectively, and are encoded in a conventional negative sense (ie, they are expressed through transcription of vRNA or genomic complementary mRNA). ) The genes located at the 5 'end of the S and L vRNA segments, GPC and Z, are encoded in the mRNA sense, respectively, but there is no evidence of direct translation from the genomic vRNA. These genes are expressed by full-length complementary replication of genomic vRNA that functions as a replication intermediate rather than through transcription of genome-sense mRNA (ie, vcRNA) from the antigenome.

  A typical targeting sequence for an Arenaviridae virus is listed in SEQ ID NO: 8 below.

9. Respiratory Syncytial Virus Targeting Respiratory syncytial virus (RSV) is the single most important respiratory pathogen in infants. RSV-induced respiratory symptoms such as bronchiolitis and pneumonia often require hospitalization in children under one year of age. Children with cardiopulmonary diseases and premature babies are particularly prone to experience severe disability from this infection. RSV infection is also an important disease in the elderly and high-risk adults. RSV infection is the second most commonly identified cause of viral pneumonia in the elderly (Falsey, Hennessey et al., 2005). The World Health Organization estimates that RSV is responsible for 64 million clinical infections and 160,000 deaths worldwide every year. Currently, there are no vaccines available to prevent RSV infection. Although many major advances in our understanding of RSV biology, epidemiology, pathophysiology, and host immune responses have existed for decades, discussions on optimal management of infants and children infected with RSV have continued considerably. It has been. Ribavirin is the only approved antiviral agent for treating RSV infection, but its use is limited to high-risk or severe infants. The usefulness of ribavirin has been limited by its cost, available efficacy, propensity to develop resistant viruses (Marquardt 1995; Prince 2001). The current need for additional effective anti-RSV agents is well recognized.

It is known that peptide-conjugated PMO (PPMO) can be effective for RSV inhibition in both tissue culture and in vivo animal model systems (Lai, Stein et al., 2008). Two antisense PPMOs designed to target sequences containing the 5 'end region of RSV L mRNA and the translation start site region tested for anti-RSV activity in cultures of two human airway cell lines It was done. One of them (SRV-AUG-2; SEQ ID NO: 10) reduced the virus titer to> 2.0 log ₁₀ . Intranasal (in) treatment of BALB / c mice with RSV-AUG-2 PPMO prior to RSV inoculation resulted in a virus titer of 1 in lung tissue 5 days post infection (pi) .2 log ₁₀ and reduced pulmonary inflammation 7 days after infection. These data indicated that RSV-AUG-2 provided potent anti-RSV activity worthy of further study as a candidate for potential therapeutic use (Lai, Stein et al., 2008). As mentioned above, despite the success with RSV-AUG-2 PPMO, it is desirable to use the conjugates disclosed herein to resolve the toxicity associated with previous peptide conjugates. Thus, in another embodiment of the invention, one or more conjugates described herein may replicate RSV in a host cell and the cells described in the conjugates described herein, eg, As described further below, by contacting with a conjugate having a targeting base sequence complementary to the target sequence composed of at least 12 consecutive bases within the AUG start site region of the mRNA derived from RSV, It can be used in a method of inhibiting.

The L gene of RSV encodes an important component of the viral RNA-dependent RNA polymerase complex. In contrast to the sequence spanning the AUG translation start site codon of the RSV L gene mRNA, the antisense PPMO designed in the RSV-AUG-2 PPMO state is present at 13 nt in the coding sequence from the 5 ′ end of the L mRNA. It is complementary to the sequence from the “gene start” sequence (GS). Therefore, a preferred L gene targeting sequence is any 12 consecutive bases from 40 bases extending in the 3 ′ direction from the 5 ′ end of the L gene mRNA shown as SEQ ID NO: 9 in Table 6 below, Alternatively, it is complementary to 22 bases in the L gene coding sequence. Exemplary RSV L gene targeting sequences are listed as SEQ ID NOs: 10-14 in Table 6 below. Any of the intersubunit modifications of the present invention described herein are included in the oligomer to provide tissue-specific for improved antisense activity, improved intracellular delivery and / or improved therapeutic activity. May provide sex. Exemplary oligomer sequences comprising the intersubunit linkages of the present invention are listed in Table 6 below.

10. Neuromuscular Disease In another embodiment, a therapeutic conjugate is provided for use in the treatment of disease symptoms associated with neuromuscular disease in a mammalian subject. Antisense oligomers (eg, SEQ ID NO: 16) have been shown to have activity in the MDX mouse model for Duchenne muscular dystrophy (DMD). Exemplary oligomer sequences, including linkages used in some embodiments, are listed in Table 7 below. In some embodiments, the conjugate is
(A) as previously described (see, eg, US patent application Ser. No. 12 / 493,140; and WO 2006/086667, incorporated herein by reference); Antisense oligomer targeting human myostatin having a base sequence complementary to at least 12 consecutive bases in the target region of human myostatin mRNA specified by SEQ ID NO: 18 for treating muscle fatigue symptoms (Typical mouse targeting sequences are listed as SEQ ID NOs: 19-20); and
(B) as previously described (eg, WO 2010/048586 and 2006/000057, or US Patent Application Publication No. 09/061960, all of which are incorporated herein by reference). An antisense oligomer capable of generating exon skipping in the DMD protein (dystrophin) to restore the partial activity of the dystrophin protein to treat DMD, eg, from SEQ ID NOs: 22-35 Comprising an oligomer selected from PMO having a selected sequence.

  Several other neuromuscular diseases can be treated using the modified linkages and end groups of the present invention. Exemplary compounds for the treatment of spinal muscular atrophy (SMA) and myotonic dystrophy (DM) are described below.

  SMA is an autosomal recessive disease caused by a chronic loss of alpha-motor neurons in the spinal cord and affects both children and adults. Decreased expression of remaining motor neurons (SMN) is responsible for the disease (Hua, Sahashi et al., 2010). The mutation that causes SMA is located in the SMNI gene, but when expressed from an alternative splice that forms defective exon 7 (Delta7SMN2), the paralogous gene SMN2 can be made available by compensating for the SMN1 deficiency. Become. Intron 6, exon 7 and antisense compounds targeting intron 7 have all been shown to induce exon 7 including the degree of mutation. Antisense compounds that target intron 7 are preferred (see, eg, WO2010 / 148249, 2010/120820, 2007/002390 and US Pat. No. 7,838,657). Exemplary antisense sequences that target the SMN2 pre-mRNA and induce improved exon 7 inclusions are listed below as SEQ ID NOs: 36-38. It is contemplated that selected modifications of these oligomer sequences using the modified linkages and end groups described herein will have improved properties compared to those known in the art. Furthermore, any oligomer that targets intron 7 of the SMN2 gene and encompasses the features of the present invention would have the potential to induce exon 7 inclusions and provide therapeutic benefit to SMA patients. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominant genetic diseases caused by the expression of toxic RNAs that cause neuromuscular degeneration. DM1 and DM2 are associated with long poly CUG and poly CCUG repeats in the 3′-UTR and intron 1 regions of myotonic dystrophy protein kinase (DMPK) and zinc finger protein 9 (ZNF9), respectively (eg, , See WO 2008/036406). Normal individuals have as many as 30 CTG repeats, while DM1 patients have many repeats ranging from 50 to 1000. The severity of the disease and the age of onset correlate with the number of repetitions. Adult-onset patients show mild symptoms and have fewer than 100 repetitions. Younger-onset DM1 patients have as many as 500 repeats, and congenital cases usually have nearly 1000 CTG repeats. Extended transcripts containing CUG repeats form secondary structures that accumulate in the nucleus at the nuclear focus and capture RNA-binding protein (RNA-BP). Multiple RNA-BPs have been implicated in the disease, including musblind-like (MBNL) protein and CUG binding protein (CUGBP). The MBNL protein is homologous to the Drosophila muscleblind (Mbl) protein, which is required for photoreceptors and muscle differentiation. MBNL and CUGBP have been identified as transcript splicing regulatory genes that are affected by DM1, such as cardiac troponin T (cTNT), insulin receptor (IR) and muscle-specific chloride channel (ClC-1). It was.

  It is known in the art that antisense oligonucleotides that target extended repeats of the DMPK gene replace RNA-BP sequestration and reverse myotonic symptoms in an animal model of DM1 (WO No. 1). 2008/036406). It is contemplated that oligomers encompassing the features of the invention will provide improved activity and therapeutic potential for DM1 and DM2 patients. A typical sequence targeting the aforementioned poly CUG and poly CCUG repeats is listed below as SEQ ID NOs: 39-55, and is incorporated herein by reference in its entirety. Further described.

  Further embodiments in the present invention for treating neuromuscular disorders contemplate and include oligomers designed to treat other DNA repeat unstable gene disorders. Examples of these diseases include Huntington's disease, spinocerebellar ataxia, X-linked bulbar spinal muscular atrophy and spinocerebellar ataxia type 10 (SCA10), as described in WO2008 / 018795.

^* Dimerization indicates that the oligomer is dimerized by a linkage that joins the 3 'ends of the two monomers. For example, the linkage may be —COCH ₂ CH ₂ —S—CH (CONH ₂ ) CH ₂ —CO—NHCH ₂ CH ₂ CO—, or other suitable linkage. EG3 means triethylene glycol tail (see, eg, conjugates in Examples 30 and 31).

11. Antimicrobial Applications In another embodiment, the invention includes a conjugate comprising an antimicrobial antisense oligomer for use in treating a bacterial infection in a mammalian host. In some embodiments, the oligomer comprises between 10-20 bases and is comprised of an acyl carrier protein (acpP), gyrase A subunit (gyrA), ftsZ, ribosomal protein S10 (rpsJ), leuD, mgtC, It contains a targeting sequence of at least 10 contiguous bases complementary to the target region of the infected bacterial mRNA for the pilG, pcaA and cmal genes. The target region includes a translation start codon of bacterial mRNA, or a sequence that is within 20 bases in the upstream (ie, 5 ′) or downstream (ie, 3 ′) direction of the translation start codon. The oligomer binds to mRNA to form a heteroduplex, thereby inhibiting the bacterial replication.

12 Modulation of Nuclear Hormone Receptor In another embodiment, the present invention provides a pre-mRNA that encodes expression of a nuclear hormone receptor (NHR) from the nuclear hormone receptor superfamily (NHRSF) primarily for the receptor. The present invention relates to compositions and methods for regulating by controlling or altering splicing. Specific examples of NHR include glucocorticoid receptor (GR), progesterone receptor (PR) and androgen receptor (AR). In certain embodiments, the conjugates described herein result in enhanced expression of the receptor in a ligand-independent or other selected form, resulting in reduced expression of its inactive form.

  Embodiments of the present invention include exon sequences of selected NHRs, including oligomers, such as the “ligand binding exons” of NHRSF pre-mRNA and / or adjacent introns, among the NHR-domains described herein. Conjugates comprising oligomers complementary to intron sequences are included. The term “ligand binding exon” refers to an exon that is present in wild-type mRNA, but is removed from primary transcription (“pre-mRNA”) to produce ligand-independent mRNA. In certain embodiments, complementarity can be based on sequences in the sequence of the pre-mRNA spanning the splice site. The splice sites are not limited and include complementarity based on sequences spanning exon-intron bonds. In other embodiments, complementarity can be based solely on the sequence of the intron. In other embodiments, complementarity can be based solely on the sequence of the exon (see, eg, US patent application Ser. No. 13 / 046,356, incorporated herein by reference).

  NHR modulators may be useful for treating NHR-related diseases, including diseases related to the expression products of genes whose transcription is stimulated or repressed by NHR. For example, modulators of NHR that inhibit AP-1 and / or NF-κB have been described in inflammatory and immune diseases and disorders, such as osteoarthritis, joints, especially described herein and known in the art. It may be useful in the treatment of rheumatism, multiple sclerosis, asthma, inflammatory bowel disease, transplant rejection, graft-versus-host disease. Compounds that antagonize transcription promotion may be useful for treating metabolic diseases associated with improved levels of glucocorticoids, such as diabetes, osteoporosis and glaucoma, among others. Also, compounds that stimulate transcriptional promotion may be useful for treating metabolic diseases associated with the lack of glucocorticoids, such as, in particular, Addison's disease.

  Embodiments of the present invention are composed of the carrier protein and a morpholino subunit linked by a phosphorus-containing intersubunit linkage that binds the morpholino nitrogen of one subunit to the 5 ′ exocyclic carbon of an adjacent subunit. A method of modulating nuclear NHR activity or expression in a cell comprising the step of contacting the cell with a conjugate comprising an antisense oligomer. Said oligonucleotide comprises a targeting sequence of between 10 and 40 bases and at least 10 consecutive bases complementary to the target sequence. The target sequence is an NHR pre-mRNA transcript. This regulates NHR activity or expression. In one embodiment, the oligomer alters splicing of the pre-mRNA transcript and increases expression of a variant of NHR. In some embodiments, the oligomer induces full or partial exon skipping of one or more exons in the pre-mRNA transcript. In one embodiment, the one or more exons encode at least a portion of a ligand binding domain of NHR and the variant is a ligand-independent form of NHR. In one embodiment, the one or more exons encode at least a portion of the transcriptional promotion domain of NHR, and the variant reduces transcriptional activation activity. In one embodiment, the one or more exons encode at least a portion of the DNA binding domain of NHR. In one embodiment, the one or more exons encode at least a portion of the NHR N-terminal activation domain. In one embodiment, the one or more exons encode at least a portion of the carboxy-terminal domain of NHR. In certain embodiments, the variant binds to NF-κB, AP-1, or both and reduces transcription of one or more pro-inflammatory target genes.

  In one embodiment, the oligomer stimulates transcriptional activity of NHR transcriptional activity. In another embodiment, the oligomer antagonizes the transcriptional activity of the transcriptional activity of NHR. In one embodiment, the oligomer stimulates the transcriptional repressing activity of NHR. In another embodiment, the oligomer antagonizes the transcriptional repressive activity of NHR. In certain embodiments, the oligomer antagonizes the transcriptional activity of the transcriptional activity of NHR and stimulates the transcriptional repressive activity of NHR (eg, US Patent Application No. 61/313, incorporated herein by reference). (See 652).

  Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich-Fluka. Benzoyladenosine, benzoylcytidine and phenylacetylguanosine were obtained from Carbosynch Limited, UK.

  PMO, PMO +, PPMO and pending US patent applications that are known in the art for the synthesis of PMOs, including further linkage modifications described herein, are hereby incorporated by reference in their entirety. The methods described in Japanese Patent Application Nos. 12 / 271,036 and 12 / 271,040 and International Publication No. 2009/064471 were used.

  A PMO having a 3 'trityl modification is synthesized basically as described in WO2009 / 064471 except that the detrityl step is omitted.

Example 1
TERT-Butyl-4- (2,2,2-trifluoroacetamide) piperidine-1-carboxylate

To a suspension of tert-butyl 4-aminopiperidine-1-carboxylate (48.7 g, 0.243 mol) and DIPEA (130 mL, 0.749 mol) in DCM (250 mL) was added ethyl trifluoroacetate (35.6 mL). , 0.300 mol) was added dropwise with stirring. After 20 hours, the solution was washed with citric acid solution (200 mL × 3, 10% w / v aqueous solution) and sodium bicarbonate solution (200 mL × 3, concentrated aqueous solution), dried (MgSO ₄ ), silica (24 g ). The silica was washed with DCM and the combined eluates were partially concentrated (100 mL) and used directly in the next step. APCI / MS calc. 296.1 for C ₁₂ H ₁₉ F ₃ N ₂ O ₃ , found m / z = 294.9 (M−1).

(Example 2)
2,2,2-trifluoro-N- (piperidin-4-yl) acetamide hydrochloride

To a stirred DCM solution (100 mL) of the title compound in Example 1, a solution of hydrogen chloride in 1,4-dioxane (4M) (250 mL, 1.0 mol) was added dropwise. Stirring is continued for 6 hours, then the suspension is filtered and the solid is washed with diethyl ether (500 mL) to give the title compound (54.2 g, 96% yield) as a white solid. It was. APCI / MS calculated 196.1 for C ₇ H ₁₁ F ₃ N ₂ O, found m / z = 196.9 (M + 1).

Example 3
(4- (2,2,2-trifluoroacetamido) piperidin-1-yl) phosphonic dichloride

To a cooled suspension (ice / water bath) of the title compound in Example 2 (54.2 g, 0.233 mol) in DCM (250 mL), phosphorus oxychloride (23.9 mL, 0.256 mol) and DIPEA (121.7 mL, 0.699 mol) was added dropwise and stirred. After 15 minutes, the bath was removed and the mixture continued to stir to warm to ambient temperature. After 1 hour, the mixture was partially concentrated (100 mL), the suspension was filtered, and the solid was washed with diethyl ether to give the title compound (43.8 g, 60% yield) as a white solid. %). The eluate was partially concentrated (100 mL), the resulting suspension was filtered, and the solid was washed with diethyl ether to give additional title compound (6.5 g, 9% yield). . 1- (4-nitrophenyl) piperazine derivative _{C 17 H 22 ClF 3 N 5} O 4 P of ESI / MS calcd 483.1, found m / z = 482.1 (M- 1).

Example 4
((2S, 6S) -6-((R) -5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl) -4-tritylmorpholin-2-yl) methyl (4- (2,2,2-trifluoroacetamido) piperidin-1-yl) phosphonochloridate

To a stirred, cooled solution (ice / water bath) of the title compound from Example 3 (29.2 g, 93.3 mmol) in DCM (100 mL) was added Mo (Tr) T # (22.6 g, 46.7 mmol). DCM solution (100 mL), 2,6-lutidine (21.7 mL, 187 mmol) and 4- (dimethylamino) pyridine (1.14 g, 9.33 mmol) were added dropwise over 10 minutes. The bath was allowed to warm to room temperature. After 15 hours, the solution was washed with citric acid solution (200 mL × 3, 10% w / v aqueous solution), dried (MgSO ₄ ), concentrated, and the crude oil was loaded directly onto the column. Chromatography [SiO ₂ column (120 g), hexane / EtOAc eluent (gradient 1: 1 to 0: 1), repeated 3 times] fractions were concentrated to give the title compound (27.2 g as a white solid). Yield 77%). 1- (4-nitrophenyl) piperazine derivative _{C 46 H 50 F 3 N 8} O 8 P of ESI / MS calcd 930.3, found m / z = 929.5 (M- 1).

(Example 5)
((2S, 6R) -6- (6-Benzamido-9H-purin-9-yl) -4-tritylmorpholin-2-yl) methyl (4- (2,2,2-trifluoroacetamide) piperidine- 1-yl) phosphonochloridate

The title compound was synthesized in a manner similar to that described in Example 4 to give the title compound (15.4 g, 66% yield) as a white solid. ESI / MS calculated 1043.4, 1 / (4-nitrophenyl) piperazine derivative C ₅₃ H ₅₃ F ₃ N ₁₁ O ₇ P, found m / z = 1042.5 (M−1).

(Example 6)
(R) -Methyl (1-phenylethyl) phosphoramide dichloride

To a cooled solution (ice / water bath) of phosphorus oxychloride (2.83 mL, 30.3 mmol) in DCM (30 mL), 2,6-lutidine (7.06 mL, 60.6 mmol) and (R)-(+) A solution of -N, a-dimethylbenzylamine (3.73 g, 27.6 mmol) in DCM was added dropwise with continued stirring. After 5 minutes, the bath was removed and the reaction mixture was allowed to warm to room temperature. After 1 hour, the reaction solution was washed with citric acid solution (50 mL × 3, 10% w / v aqueous solution), dried (MgSO ₄ ), filtered through SiO ₂ and concentrated to a white foam. As the title compound (3.80 g). ESI / MS calculated 404.2 for the 1- (4-nitrophenyl) piperazine derivative C ₁₉ H ₂₅ N ₄ O ₄ P, found m / z = 403.1 (M−1).

(Example 7)
(S) -Methyl (1-phenylethyl) phosphoramide dichloride

The title compound was synthesized in a manner similar to that described in Example 6 to give the title compound (3.95 g) as a white foam. ESI / MS calculated 404.2 for the 1- (4-nitrophenyl) piperazine derivative C ₁₉ H ₂₅ N ₄ O ₄ P, found m / z = 403.1 (M−1).

(Example 8)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl methyl ((R) -1-phenylethyl) phosphoroamidochloridate

The title compound was synthesized in a manner similar to that described in Example 4 to give the title chlorophosphoramidate (4.46 g, 28% yield) as a white solid. Calculated ESI / MS value for C ₃₈ H ₄₀ ClN ₄ O ₅ P 698.2, found m / z = 697.3 (M−1).

Example 9
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl methyl ((S) -1-phenylethyl) phosphoroamidochloridate

The title compound was synthesized in a manner similar to that described in Example 4 to give the title chlorophosphoramidate (4.65 g, 23% yield) as a white solid. Calculated ESI / MS value for C ₃₈ H ₄₀ ClN ₄ O ₅ P 698.2, found m / z = 697.3 (M−1).

(Example 10)
(4- (Pyrrolidin-1-yl) piperidin-1-yl) phosphonic acid dichloride hydrochloride

To a cooled solution (ice / water bath) of phosphorus oxychloride (5.70 mL, 55.6 mmol) in DCM (30 mL) was added 2,6-lutidine (19.4 mL, 167 mmol) and 4- (1-pyrrolidinyl) -piperidine. (8.58 g, 55.6 mmol) in DCM (30 mL) was added and stirred for 1 hour. The suspension was filtered and the solid was washed with excess diethyl ether to give the title pyrrolidine (17.7 g, 91% yield) as a white solid. ESI / MS calculated 423.2 for the 1- (4-nitrophenyl) piperazine derivative C ₁₉ H ₃₀ N ₅ O ₄ P, found m / z = 422.2 (M−1).

(Example 11)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl (4- (pyrrolidine -1-yl) piperidin-1-yl) phosphonochloridate hydrochloride

To a stirred and cooled solution (ice / water bath) of dichlorophosphoramidate 8 (17.7 g, 50.6 mmol) in DCM (100 mL) was added Mo (Tr) T # (24.5 g, 50.6 mmol) in DCM ( 100 mL), 2,6-lutidine (17.7 mL, 152 mmol) and 1-methylimidazole (0.401 mL, 5.06 mmol) in DCM (100 mL) were added dropwise over 10 minutes. The bath was allowed to warm to room temperature while stirring the suspension. After 6 hours, the suspension was poured into diethyl ether (1 L), stirred for 15 minutes, filtered and the solid was washed with more ether to give a white solid (45.4 g). . The crude product was purified by chromatography [SiO ₂ column (120 grams), DCM / MeOH eluent (gradient 1: 0 to 6: 4)] and the combined fractions were diethyl ether (2.5 L). And stirred for 15 minutes, filtered and the resulting solid was washed with additional ether to give the title compound (23.1 g, 60% yield) as a white solid. ESI / MS calculated value 888.8 of 1- (4-nitrophenyl) piperazine derivative C ₄₈ H ₅₇ N ₈ O ₇ P, measured value m / z = 887.6 (M−1).

(Example 12)
3- (TERT-Butyldisulfanyl) -2- (isobutoxycarbonylamino) propanoic acid

To S-tert-butylmercapto-L-cysteine (10 g, 47.8 mmol) in CH ₃ CN (40 mL) was added K ₂ CO ₃ (16.5 g, 119.5 mmol) in H ₂ O (20 mL). After stirring for 15 minutes, iso-butylchloroformate (9.4 mL, 72 mmol) was slowly added. The reaction was carried out for 3 hours. The white solid was filtered through celite and the filtrate was concentrated to remove CH ₃ CN. The residue was dissolved in ethyl acetate (200 mL), washed with 1N HCl (40 ml × 3), brine (40 × 1) and dried over Na ₂ SO ₄ . The desired product (2) was obtained after chromatography (5% MeOH / DCM).

(Example 13)
TERT-Butyl 4- (3- (TERT-Butyldisulfanyl) -2- (isobutoxycarbonylamino) propanamide) piperidine-1-carboxylate

To the acid (Compound 2, from Example 12, 6.98 g, 22.6 mmol) in DMF (50 ml) was added HATU (8.58 g, 22.6 mmol). After 30 minutes, Hunig base (4.71 ml, 27.1 mmol) and 1-Boc-4-aminopiperidine (5.43 g, 27.1 mmol) were added to the mixture. The reaction was continued with stirring for another 3 hours at RT. DMF was removed under high vacuum and the crude residue was dissolved in EtAc (300 ml) and washed with H ₂ O (50 ml × 3). The final product (3) was obtained after ISCO purification (5% MeOH / DCM).

(Example 14)
Isobutyl 3- (TERT-butyldisulfanyl) -1-oxo-1- (piperidin-4-ylamino) propan-2-ylcarbamate

  To compound 3 prepared in Example 13 (7.085 g, 18.12 mmol) was added 30 mL of 4M HCl / dioxane. The reaction was complete after 2 hours at RT. The hydrochloride salt (4) was used in the next step without further purification.

(Example 15)
Isobutyl 3- (TERT-butyldisulfanyl) -1- (1- (dichlorophosphoryl) piperidin-4-ylamino) -1-oxopropan-2-ylcarbamate

To the compound 4 prepared in Example 15 (7.746 g, 18.12 mmol) in DCM (200 ml) at −78 ° C. under Ar was slowly added POCl ₃ (1.69 ml, 18.12 mmol), Subsequently Et ₃ N (7.58 ml, 54.36 mmol) was added. The reaction was stirred at RT for 5 hours and concentrated to remove excess base and solvent. After ISCO purification (50% EtAc / hexane), the product (5) was obtained as a white solid.

(Example 16)
Isobutyl 3- (TERT-butyldisulfanyl) -1- (1-chloro (((2S, 6R) -6- (5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)- Yl) -4-tritylmorpholin-2-yl) methoxy) phosphoryl) piperidin-4-ylamino) -1-oxopropan-2-ylcarbamate

  1-((2R, 6S) -6- (hydroxymethyl) -4-tritylmorpholin-2-yl) -5-methylpyrimidine-2,4 (1H, 3H)-in DCM (100 ml) at 0 <0> C To dione (moT (Tr)) (5.576 g, 10.98 mmol) was added lutidine (1.92 ml, 16.47 mmol) and DMAP (669 mg, 5.5 mmol) followed by 4 (6.13 g, 12.08 mmol) was added. The reaction was left stirring at RT for 18 hours. The desired product (6) was obtained after ISCO purification (50% EtAc / hexane).

(Example 17)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl hexyl (methyl) phospho Roamido chloridate

A solution of N-hydroxylmethylamine (4.85 ml, 32 mmol) in DCM (80 ml) was cooled to −78 ° C. under N ₂ . A solution of phosphoryl chloride (2.98 ml, 32 mmol) in DCM (10 ml) was added slowly followed by a solution of Et ₃ N (4.46 ml, 32 mmol) in DCM (10 ml). Stirring was continued while the reaction was allowed to warm to RT overnight. After ISCO purification (20% EtAc / hexane), the desired product (1) was obtained as a clear oil.

  To moT (Tr) (5.10 g, 10.54 mmol) in DCM (100 ml) at 0 ° C. was added lutidine (3.68 ml, 31.6 mmol) and DMAP (642 mg, 5.27 mmol) followed by 1 (4.89 g, 21.08 mmol) was added. The reaction was left stirring at RT for 18 hours. The desired product (2) was obtained after ISCO purification (50% EtOAc / hexane).

(Example 18)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl dodecyl (methyl) phospho Roamido chloridate

  The title compound was prepared based on the general procedure described in Examples 6 and 8.

(Example 19)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl morpholinophosphonochloridate

  The title compound was prepared based on the general procedure described in Examples 6 and 8.

(Example 20)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl (S) -2 -(Methoxymethyl) pyrrolidin-1-ylphosphonochloridate

  The title compound was prepared based on the general procedure described in Examples 6 and 8.

(Example 21)
((2S, 6R) -6- (5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-tritylmorpholin-2-yl) methyl 4- (3 4,5-trimethoxybenzamido) piperidin-1-ylphosphonochloridate

To 1-Boc-4-piperidine (1 g, 5 mmol) in DCM (20 ml) was added Hunig base (1.74 ml, 10 mmol) followed by 3,4,5-trimethoxybenzoyl chloride (1.38 g, 6 mmol) was added. The reaction was performed at RT for 3 hours and concentrated to remove the solvent and excess base. The residue was dissolved in EtAc (100 ml), washed with 0.05N HCl (3 × 15 ml), saturated NaHCO ₃ (2 × 15 ml) and dried over Na ₂ SO ₄ . The product (1) was obtained after ISCO purification (5% MeOH / DCM).

  To 7, 15 ml of 4N HCl / dioxane was added and the reaction was terminated after 4 hours. 8 was obtained as a white solid.

A solution of 8 (1.23 g, 4.18 mmol) in DCM (20 ml) was cooled to −78 ° C. under N ₂ . A solution of phosphoryl chloride (0.39 ml, 4.18 mmol) in DCM (2 ml) was added slowly followed by a solution of Et ₃ N (0.583 ml, 4.18 mmol) in DCM (2 ml). Stirring was continued while the reaction was allowed to warm to RT overnight. The desired product (9) was obtained after ISCO purification (50% EtAc / hexane).

  To moT (Tr) (1.933 g, 4.0 mmol) in DCM (20 ml) at 0 ° C., lutidine (0.93 ml, 8 mmol) and DMAP (49 mg, 0.4 mmol) were added followed by 9 ( 1.647 g, 4 mmol) was added. The reaction was left stirring at RT for 18 hours. The desired product (10) was obtained after ISCO purification (50% EtAc / hexane).

(Example 22)
Synthesis of cyclophosphoramide containing subunit ( ^CPT )

The moT subunit (25 g) was suspended in DCM (175 ml) and NMI (N-methylimidazole, 5.94 g, 1.4 eq) was added to give a clear solution. Tosyl chloride was added to the reaction mixture and the progress of the reaction was monitored by TLC until complete (about 2 hours). Aqueous treatment was performed by washing with 0.5 M citrate buffer (pH = 5) followed by brine. The organic layer was separated and dried over Na ₂ SO ₄ . The solvent was removed on a rotary evaporator to give a crude product. The crude product was used in the next step without further purification.

The moT tosylate prepared above was mixed with propanolamine (1 g / 10 ml). The reaction mixture was then placed in an oven overnight at 45 ° C. and subsequently diluted with DCM (10 ml). Aqueous treatment was performed by washing with 0.5 M citrate buffer (pH = 5) followed by brine. The organic layer was separated and dried over Na ₂ SO ₄ . The solvent was removed on a rotary evaporator to give a crude product. The crude product was analyzed and measured by NMR and HPLC and prepared for the next step without further purification.

The crude product was dissolved in DCM (2.5 ml DCM / g, 1 eq) and mixed with DIEA (3 eq). The solution was cooled with dry ice-acetone and POCl ₃ (1.5 eq) was added dropwise. The resulting mixture was stirred overnight at room temperature. Aqueous treatment was performed by washing with 0.5 M citrate buffer (pH = 5) followed by brine. The organic layer was separated and dried over Na ₂ SO ₄ . The solvent was removed on a rotary evaporator to give the crude product as a yellow solid. The crude product was purified by silica gel chromatography (crude product / silica = 1 to 5 ratio, gradient DCM to 50% EA / DCM) and fractions were pooled based on TLC analysis. The solvent was removed to give the desired product as a mixture of diastereomers. The purified product was analyzed by HPLC (NPP stop) and NMR (H-1 and P-31).

  The diastereomeric mixture was separated based on the following procedure. The mixture (2.6 g) was dissolved in DCM. This sample was loaded onto a RediSepRf column (80 g normal phase manufactured by Teledyne Isco) and eluted from 10% EA / DCM to 50% EA / DCM over 20 minutes. Fractions were collected and analyzed by TLC. Fractions were pooled based on TLC analysis and the solvent was removed on a rotary evaporator at room temperature. The diastereomeric ratio of the pooled fractions was determined by P-31 NMR and NPP-TFA analysis. If necessary, the above procedure was repeated until the diastereomeric ratio reached 97%.

(Example 23)
PMO plus global cholic acid modification

A succinimide activated cholic acid derivative was prepared based on the following procedure. Cholic acid (12 g, 29.4 mmol), N-hydroxysuccinimide (4.0 g, 34.8 mmol), EDCl (5.6 g, 29.3 mmol) and DMAP (1 g, 8.2 mmol) were placed in a round bottom flask. It was. DCM (400 ml) and THF (40 ml) were added and dissolved. The reaction mixture was stirred overnight at room temperature. Water (400 ml) was then added to the reaction mixture and the organic layer was separated and washed with water (2 × 400 ml) followed by saturated NaHCO ₃ (300 ml) and brine (300 ml). The organic layer was then dried with Na ₂ SO ₄ . The solvent was removed on a rotary evaporator to give a white solid. The crude product was dissolved in chloroform (100 ml) and precipitated into heptane (1000 ml). The solid was collected by filtration, analyzed by HPLC and NMR, and used without further purification.

  An appropriate amount of PMO plus (20 mg, 2.8 μmol) was weighed into a vial (4 ml) and dissolved in DMSO (500 μl). Activated cholic acid ester (13 mg, 25 μmol) was added to the reaction mixture based on the ratio of 2 equivalents of active ester per modification site, followed by overnight stirring at room temperature. The progress of the reaction was measured by MALDI and HPLC (C-18 or SAX).

  After the reaction was complete (determined by the disappearance of the starting PMO plus), 1 ml of concentrated ammonia was added to the reaction mixture when the reaction was complete. The reaction vial was then placed in an oven (45 ° C.) overnight (18 hours) followed by cooling to room temperature and diluting with 1% ammonia in water (10 ml). The sample was loaded onto an SPE column (2 cm) and the vial was rinsed with 1% ammonia solution (2 × 2 ml). The SPE column was washed with 1% ammonia in water (3 × 6 ml) and the product was eluted with 45% acetonitrile in 1% ammonia in water (6 ml). Fractions containing oligomers were identified by UV optical density measurements. The product was isolated by lyophilization. Purity and identity were measured by MALDI and HPLC (C-18 and / or SAX).

This same procedure is applicable for deoxycholic acid activation and conjugation to PMO ⁺ .

(Example 24)
Global guanidinylation of PMO plus

An appropriate amount of PMO plus (25 mg, 2.8 μmol) was weighed into a vial (6 ml). 1H-pyrazole-1-carboxamidine chloride (15 mg, 102 μmol) and potassium carbonate (20 mg, 0.15 mmol) were added to the vial. Water (500 μl) was added and the reaction mixture was stirred at room temperature overnight (about 18 hours). Completion of the reaction was determined by MALDI.

  When complete, the reaction was diluted with 1% ammonia in water (10 ml) and loaded onto an SPE column (2 cm). The vial was rinsed with 1% ammonia solution (2 × 2 ml) and the SPE column was washed with 1% ammonia in water (3 × 6 ml). The product was eluted with 45% acetonitrile in 1% ammonia in water (6 ml). Fractions containing oligomers were identified by UV optical density measurements. The product was isolated by lyophilization. Purity and identity were measured by MALDI and HPLC (C-18 and / or SAX).

(Example 25)
Global thioacetyl modification of PMO plus (M23D)

  An appropriate amount of PMO plus (20 mg, 2.3 μmol) was weighed into a vial (4 ml) and dissolved in DMSO (500 μl). N-succinimidyl-S-acetylthioacetate (SATA) (7 mg, 28 μmol) was added to the reaction mixture and allowed to stir overnight at room temperature. The progress of the reaction was monitored by MALDI and HPLC.

  When complete, 1% ammonia in water was added to the reaction mixture and stirred at room temperature for 2 hours. This solution was loaded onto an SPE column (2 cm). The vial was rinsed with 1% ammonia solution (2 × 2 ml). The SPE column was washed with 1% ammonia in water (3 × 6 ml). The product was eluted with 45% acetonitrile in 1% ammonia in water (6 ml). Fractions containing oligomers were identified by UV optical density measurements. The product was isolated by lyophilization. Purity and identity were measured by MALDI and HPLC (C-18 and / or SAX).

(Example 26)
Global succinic modification of PMO Plus

  An appropriate amount of PMO plus (32 mg, 3.7 μmol) was weighed into a vial (4 ml) and dissolved in DMSO (500 μl). N-ethylmorpholino (12 mg, 100 μmol) and succinic anhydride (10 mg, 100 μmol) were added to the reaction mixture and allowed to stir overnight at room temperature. The progress of the reaction was monitored by MALDI and HPLC.

  When complete, 1% ammonia in water was added to the reaction mixture and stirred at room temperature for 2 hours. This solution was loaded onto an SPE column (2 cm). The vial was rinsed with 1% ammonia solution (2 × 2 ml). The SPE column was washed with 1% ammonia in water (3 × 6 ml). The product was eluted with 45% acetonitrile in 1% ammonia in water (6 ml). Fractions containing oligomers were identified by UV optical density measurements. The product was isolated by lyophilization. Purity and identity were measured by MALDI and HPLC (C-18 and / or SAX).

The above procedure was further adapted to PMO plus glutaric acid (glutaric anhydride) and tetramethylene glutaric acid (tetramethylene glutaric anhydride) modifications.

(Example 27)
Preparation of oligonucleotide analogues containing modified end groups

A solution of 25 bases long PMO (27.7 mg, 3.226 μmol) containing unbound 3 ′ ends in DMSO (300 μL) was added to farnesyl bromide (1.75 μl, 6.452 μmol) and diisopropylethylamine ( 2.24 μL, 12.9 μmol) was added. The reaction mixture was stirred at room temperature for 5 hours. The crude reaction mixture was diluted with 10 mL of 1% aqueous NH ₄ OH and then loaded onto a 2 mL Amberchrome CG300M column. The column was then rinsed with 3 column volumes of water. The product was eluted with 6 mL of 1: 1 acetonitrile and water (v / v). The solution was then lyophilized to give the title compound as a white solid.

(Example 28)
Preparation of Morpholino Oligomers Preparation of trityl piperazine phenyl carbamate 35 (see FIG. 3):
To a cooled suspension of compound 11 in dichloromethane (6 mL / g 11) was added a solution of potassium carbonate (3.2 eq) in water (4 mL / g potassium carbonate). To this biphasic mixture was slowly added a solution of phenylchloroformate (1.03 eq) in dichloromethane (2 g / g phenylchloroformate). The reaction mixture was warmed to 20 ° C. The layers were separated upon completion of the reaction (1-2 hours). The organic layer was washed with water and dried over anhydrous potassium carbonate. Product 35 was isolated by crystallization from acetonitrile. Yield = 80%.

Preparation of carbamate alcohol 36:
Sodium hydride (1.2 eq) was suspended in 1-methyl-2-pyrrolidinone (32 mL / g sodium hydride). To this suspension was added triethylene glycol (10.0 equiv) and compound 35 (1.0 equiv). The resulting slurry was heated to 95 ° C. With the reaction complete (1-2 hours), the mixture was cooled to 20 ° C. To this mixture was added 30% dichloromethane / methyl tert-butyl ether (v: v) and water. Subsequently, the product-containing organic layer was washed with aqueous NaOH, aqueous succinic acid and saturated aqueous sodium chloride. The product 36 was isolated by crystallization from dichloromethane / methyl tert-butyl ether / heptane. Yield = 90%.

Preparation of tail acid 37:
To a solution of compound 36 in tetrahydrofuran (7 mL / g 36) was added succinic anhydride (2.0 eq) and DMAP (0.5 eq). The mixture was heated to 50 ° C. With the reaction complete (5 hours), the mixture was cooled to 20 ° C. and adjusted to pH 8.5 with aqueous NaHCO ₃ solution. Methyl tert-butyl ether was added and the product was extracted into the aqueous layer. Dichloromethane was added and the mixture was adjusted to pH 3 with aqueous citric acid. The product-containing organic layer was washed with a mixture of citrate buffer and saturated aqueous sodium chloride solution, pH = 3. The 37 dichloromethane solution was used in the preparation of compound 38 without isolation.

Preparation of 38:
N-hydroxy-5-norbornene-2,3-dicarboxylic imide (HONB) (1.02 eq), 4-dimethylaminopyridine (DMAP) (0.34 eq) are added to the solution of compound 37, Then 1- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC) (1.1 eq) was added. The mixture was heated to 55 ° C. With the reaction complete (4-5 hours), the mixture is cooled to 20 ° C. and subsequently 1: 1.
Washed with 0.2M citric acid / brine and brine. The dichloromethane solution was solvent exchanged with acetone and then with N, N-dimethylformamide. The product was isolated from acetone / N, N-dimethylformamide by precipitation into a saturated aqueous sodium chloride solution. The crude product was reslurried in water several times to remove residual N, N-dimethylformamide and salt. Yield to 38 from compound 36 = 70%. Introduction of the active “tail” onto the disulfide anchor resin was performed in NMP by the procedure used for subunit incorporation during solid phase synthesis.

Preparation of a solid support for the synthesis of morpholino oligomers:
This procedure was performed using a silanized, coated peptide with a glass frit of coarse air holes (40-60 μm), an overhead stirrer, and a three-way Teflon cock for bubbling or vacuum extraction of N _{2 with} the frit. Performed in containers (custom made by ChemGlass, NJ, USA). Temperature control was achieved in the reaction vessel with a circulating water bath.

The resin treatment / washing process in the following procedure consists of two basic operations: resin fluidization and solvent / solution extraction. For resin fluidization, the cock was positioned so that N ₂ flowed through the frit, and a specific resin treatment / wash was added to the reactor to allow it to fully wet the resin. Next, mixing was started and the resin slurry was mixed for a predetermined time. For solvent / solution extraction, mixing and N ₂ flow were stopped, the vacuum pump was started, and the cock was then positioned to drain from resin treatment / washing to waste. All resin treatment / washing capacities were 15 mL / g resin unless otherwise noted.

In a silanized, coated peptide container, aminomethylpolystyrene resin (100-200 mesh; approximately 1.0 mmol / g N ₂ substitution; 75 g, 1 equivalent, Polymer Labs, UK, part # 1464-X799) was added to 1-methyl- 2-Pyrrolidinone (NMP; 20 ml / g resin) was added and the resin was expanded while mixing for 1-2 hours. Subsequently, the swollen solvent is drained and the resin is washed with dichloromethane (2 × 1-2 min), 5% diisopropylethylamine (2 × 3-4 min) and dichloromethane (2 × 3 min) in 25% isopropanol / dichloromethane. 1 to 2 minutes). After draining the last wash, the resin is fluidized with a solution of disulfide anchor 34 (0.17M; 15 mL / g resin, approximately 2.5 equivalents) in 1-methyl-2-pyrrolidinone and the resin / reagent mixture is And heated at 45 ° C. for 60 hours. With the reaction completed, heating was completed, the anchor solution was discarded, and the resin was washed with 1-methyl-2-pyrrolidinone (4 × 3-4 minutes) and dichloromethane (6 × 1-2 minutes). . The resin was treated with a solution of 10% (v / v) diethyl bicarbonate in dichloromethane (16 mL / g; 2 × 5-6 minutes) and then washed with dichloromethane (6 × 1-2 minutes). Resin 39 (see FIG. 4) was dried to constant weight (± 2%) under N ₂ flow for 1-3 hours and then under vacuum. Yield: 110-150% of the original resin weight.

Measurement of charge of aminomethylpolystyrene-disulfide resin:
The charge of the resin (number of possible reactive sites available) was measured by spectroscopic assay for the number of triphenylmethyl (tolyl) groups per gram of resin.

  A known weight of dry resin (25 ± 3 mg) was transferred to a 25 ml volume silanized flask and about 5 ml of 2% (v / v) trifluoroacetic acid in dichloromethane was added. The contents were mixed by gentle stirring and then allowed to stand for 30 minutes. The volume was brought to 25 mL with 2% (v / v) trifluoroacetic acid in additional dichloromethane and the contents were mixed thoroughly. Using a positive displacement pipette, an aliquot (500 μL) of the trityl-containing solution was transferred to a 10 mL volumetric flask and the volume was made up to 10 mL with methanesulfonic acid.

The trityl cation content in the final solution is measured by UV absorbance at 431.7 nm and the charge of the resin is determined by the appropriate volume, dilution and absorbance coefficient (ε) in trityl groups (μmol / g) per gram of resin. : 41 μmol ⁻¹ cm ⁻¹ ) and resin weight. The assay was performed three times and the average charge was calculated.

  The resin charge procedure in this example will provide a resin with a charge of approximately 500 μmol / g. When the disulfide anchor incorporation step was performed at room temperature for 24 hours, a charge of 300 to 400 μmol / g was obtained.

Tail charge:
The tail can be introduced into the molecule using settings and volumes similar to the preparation of aminomethylpolystyrene-disulfide resin. For the ligation step, 38 solutions (0.2 M) in NMP with 4-ethylmorpholine (NEM, 0.4 M) were used in place of the disulfide anchor solution. After 2 hours at 45 ° C., resin 39 was washed twice with 5% diisopropylethylamine and once with DCM in 25% isopropanol / dichloromethane. A solution of benzoic anhydride (0.4M) and NEM (0.4M) was added to the resin. After 25 minutes, the reactor jacket was cooled to room temperature and the resin was washed twice with 5% diisopropylethylamine in 25% isopropanol / dichloromethane and 8 times with DCM. Resin 40 was filtered and dried under high vacuum. The charge for resin 40 was defined as that of the original aminomethylpolystyrene-disulfide resin 39 used for the tail charge.

Solid phase synthesis:
Morpholino oligomers were prepared on a 2 mL Gilson polypropylene reaction column (Part # 3980270) in a Gilson AMS-422 automated peptide synthesizer. An aluminum block with a flow channel for water flow was placed around the column so as to be located on the synthesizer. The AMS-422 will alternatively add reagent / wash solution, hold for a specified time, and use vacuum to discard the column.

  For oligomers in the range of about 25 subunits or less in length, aminomethyl polystyrene-disulfide resins having a charge near 500 μmol / g resin are preferred. For larger oligomers, aminomethyl polystyrene-disulfide resins having a charge of 300-400 μmol / g resin are preferred. If a molecule with a 5'-tail is desired, the resin charged by the tail is selected with similar charge guidelines.

The following reagent solution was prepared.
Detrityl solution: 10% cyanoacetic acid (w / v) in 4: 1 dichloromethane / acetonitrile; Neutralization solution: 5% diisopropylethylamine in 3: 1 dichloromethane / isopropanol; Ligating solution: 1,3- 0.18M (or 0.24M for oligomers with elongations longer than 20 subunits) activated morpholino subunit and 0.4M N ethyl in the desired base and linkage form in dimethylimidazolidinone Morpholine. Dichloromethane (DCM) was used as a temporary wash to separate the different reagent solution washes.

In the synthesizer, the block was set at 42 ° C., and 2 mL of 1-methyl-2-pyrrolidinone was added to each column containing 30 mg of aminomethylpolystyrene-disulfide resin (or tail resin) and brought to room temperature at 30 ° C. Let stand for a minute. After washing twice with 2 mL of dichloromethane, the following synthesis cycle was used.

  The sequence of individual oligomers was programmed into the synthesizer so that each column received the appropriate ligation solution (A, C, G, T, I) in the appropriate sequence. When the oligomer in the column has completed its last subunit incorporation, the column is removed from the block and the final cycle is 4-methoxytriphenylmethyl containing 0.89M 4-ethylmonophorin. Performed manually with ligation solution containing chloride (0.32 M in DMI).

Cleavage from the resin, removal of base and backbone protecting groups:
After methoxytritylation, the resin was washed 8 times with 2 mL of 1-methyl-2-pyrrolidinone. 1 mL of a cleavage solution consisting of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in 1-methyl-2-pyrrolidinone is added, the column is capped and allowed to stand at room temperature for 30 minutes. I put it. After that time, the solution was drained into a 12 mL Wheaton vial. The highly shrunk resin was washed twice with 300 μL of cleavage solution. To the solution is added 4.0 mL concentrated aqueous ammonia (stored at −20 ° C.), tightly capped vial (Teflon-lined screw cap), and mix the mixture to mix the solution Stir. The vial was placed in an oven at 45 ° C. for 16-24 hours to achieve base and backbone protecting group cleavage.

Early oligomer isolation:
The ammolysis solution in the vial was removed from the oven and allowed to cool to room temperature. The solution was diluted with 20 mL of 0.28% aqueous ammonia and passed through a 2.5 × 10 cm column containing Macroprep HQ resin (BioRad). A methoxytrityl-containing peak was eluted using a salt gradient (A: 0.28% ammonia and B: 1M sodium chloride in 0.28% ammonia; 0-100% B, 60 min). The combined fractions were pooled and further processed depending on the desired product.

Demethoxytritylation of morpholino oligomers:
The pooled fraction from the Macroprep purification was treated with 1M H ₃ PO ₄ at pH 2.5 or lower. After initial mixing, the sample was left at room temperature for 4 minutes. At that time, the sample was neutralized with 2.8% ammonia / water to pH 10-11. The product was purified by solid phase extraction (SPE).

Amberchrome CG-300M (Rohm and Haas; Philadelphia, PA) (3 mL) was packed into a 20 mL frit column (BioRad Econo-Pac chromatography column (732-1011)) and the resin was added in 3 mL of the following: 0.28 % NH ₄ OH / 80% acetonitrile; 0.5M NaOH / 20% ethanol; water; 50 mM H ₃ PO ₄ /80% acetonitrile; water; 0.5 NaOH / 20% ethanol; water; 0.28% NH ₄ Rinse with OH.

  The solution from demethoxytritylation was loaded onto the column and the resin was rinsed 3 times with 3-6 mL of 0.28% aqueous ammonia. A Wheaton vial (12 mL) was placed under the column and the product was eluted by two washes with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia. The solution was frozen with dry ice and the vial was placed in a lyophilizer to produce a fluffy white powder. The sample was dissolved in water and filtered using a syringe through a 0.22 micron filter (Pall Life Sciences, Acrodisc 25 mm syringe filter with 0.2 micron HT Tuffryn membrane). The optical density (OD) was measured with a UV spectrometer to determine the oligomeric OD units present and the dosing sample for analysis. The solution was then put back into a Wheaton vial for lyophilization.

Analysis of morpholino oligomers:
A MALDI-TOF mass spectrometer was used to determine the composition of the fractions in the purification and to provide evidence for the identity (molecular weight) of the oligomer. As a matrix, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), 3,4,5-trihydroxyacetophenone (THAP) or alpha-cyano-4-hydroxycinnamic acid (alpha-cyano) The sample was diluted with a solution of -4-hydroxycinnamic acid (HCCA).

Dionex ProPac SCX-10, 4 × 250 mm column (Dionex Corporation; Sunnyvale, Calif.), 25 mM pH = 5, sodium acetate 25% acetonitrile (buffer A) and 25 mM pH = 5, sodium acetate 25% Acetonitrile 1.5M potassium chloride (buffer B) (gradient B10-100% over 15 minutes) or 25 mM KH ₂ PO ₄ 25% acetonitrile (buffer A) at pH = 3.5 and pH = 3 Cation exchange (SCX) HPLC was performed using 25 mM KH ₂ PO ₄ 25% acetonitrile (buffer B) with 1.5 M potassium chloride at 0.5 (gradient B 0-35% over 15 minutes). . The former system was used for positively charged oligomers with no attached peptide. On the other hand, the latter was used for peptide conjugates.

Purification of morpholino oligomers by cation exchange chromatography:
Samples were dissolved in 20 mM sodium acetate, pH = 4.5 (buffer A), applied to a column of Source 30 cation exchange resin (GE Healthcare), 0.5 M sodium chloride pH = 20 mM sodium acetate and 40% acetonitrile = Elute with a gradient of 4.5 (buffer B). The pool fraction containing the product was neutralized with concentrated aqueous ammonia solution and applied to an Amberchrome SPE column. The product was eluted, frozen and lyophilized as described above.

(Example 29)
Typical conjugate preparation

The peptide sequence AcR ₆ G was prepared based on standard peptide synthesis methods known in the art. In DMSO (3 mL), the PMO (NG-05-0255, 3′-H: M23D: 5′-EG3, sequence binding to exon 23 of mdx mouse, 350 mg, 1 equivalent), AcR ₆ G (142 mg, 2 Eq.), HATU (31 mg, 2 eq) in solution was added diisopropylethylamine (36 μL, 5 eq) at room temperature. After 1 hour, the reaction was worked up and the desired peptide-oligomer conjugate was purified by SCX chromatography (elution by gradient: A: 20 mM NaH ₂ PO ₄ in 25% acetonitrile / H ₂ O, pH 7.0; B: 25 Purified with 1.5M guanidine HCl and 20 mM NaH ₂ PO ₄ , pH 7.0) in% acetonitrile / H ₂ O. The combined fractions were subjected to solid phase extraction (1M NaCl followed by elution with water). After lyophilization, the conjugate was obtained as a white powder (257 mg, yield 65.5%).

(Example 30)
Treatment of MDX mice with exemplary conjugates of the invention The MDX mice have been approved and well characterized as an animal model for Duchenne muscular dystrophy (DMD) containing a mutation in exson 23 of the dystrophin gene. The M23D antisense sequence (SEQ ID NO: 15) is known to induce exon 23 skipping and functional dystropin expression repair. MDX mice were administered once (50 mg / kg) by tail vein injection with one of the following conjugates.
1.5'-EG3-M23D-BX (RXRRBR) ₂ (AVI5225);
2.5'-EG3-M23D-G (R) ₅ (NG-11-0045);
3.5'-EG3-M23D-G (R) ₆ (NG-11-0009);
4.5'-EG3-M23D-G (R) ₇ (NG-11-0010); or
5.5'-EG3-M23D-G (R) ₈ (NG-11-0216)

を意味する。 M23D is a morpholino oligonucleotide having the sequence GGCCAAACCTCGGCTTACCTGAAAT and “EG3” is attached to the 5 ′ end of the oligomer via a piperazine linker (ie, structure XXIX):

Means.

One week after injection, the MDX mice were sacrificed and RNA was extracted from various muscle tissues. Endpoint PCR was used to measure the relative abundance of dystrophin mRNA containing exon 23 and exon 23-deficient mRNA by antisense-induced exon skipping. Percent exon 23 skipping is a measure of antisense activity in vivo. FIGS. 5 and 6 show the results from quadriceps (QC, FIGS. 5A and 6A), diaphragm (DT, FIGS. 5B and 6B) and heart (HT, FIGS. 5C and 6C) at 1 week after treatment, respectively. Show. The dose response between AVI-5225 and other conjugates was similar. Among the arginine series, R ₆ G peptide had the highest efficacy in quadriceps and diaphragm and was comparable to other arginine peptides in the heart.

(Example 31)
BUN levels and survival of mice treated with typical conjugates

Mice were treated with the conjugate described in Example 30 and KIM-1 levels, BUN levels and viability were measured based on general procedures described in Example 32 below and known in the art. . Surprisingly, FIG. 7A shows that all glycine-conjugated conjugates had significantly lower BUN levels than the XB-conjugated conjugate (AVI-5225). Furthermore, mice treated with the glycine-conjugated conjugate survived longer at higher doses than the XB-conjugated conjugate, R ₈ G conjugate, which is the minimal tolerance of arginine polymer (FIG. 7B). All mice treated with the R ₆ G conjugate (NG-11-0009) survived at doses up to 400 mg / kg (data not shown).

  Mice treated with glycine-conjugated conjugates had significantly lower KIM-1 (FIG. 8A) and clusterin (FIG. 8B) levels than mice treated with AVI-5225. This data shows that the conjugates of the present invention have lower toxicity than the conventional conjugates and do not reduce the effectiveness of the conjugates as shown in Example 30 above. Thus, the conjugates of the present invention have better therapeutic means than other known conjugates and are potentially better drug candidates.

(Example 32)
Exemplary conjugate toxicology Four exemplary conjugates in the present invention were tested for their toxicology in mice. The conjugate is as follows.
1.5'-EG3-M23D-BX (RXRRBR) ₂ (AVI5225);
2.5'-EG3-M23D-G (RXRRBR) ₂ (NG-11-0654);
3.5'-EG3-M23D-BX (R) ₆ (NG-11-0634); and
4.5'-EG3-M23D-G (R) ₆ (NG-11-0009)

を意味する。 M23D is a morpholino oligonucleotide having the sequence GGCCAAACCTCGGCTTACCTGAAAT and “EG3” is attached to the 5 ′ end of the oligomer via a piperazine linker (ie, structure XXIX):

Means.

  Eight-week-old female mice (C57 / BL6; Jackson Laboratories, 18-22 grams) were treated with the above conjugate formulated in saline. The mice were acclimatized for a minimum of 5 days before the start of the experimental procedure.

The animals were housed in clear polycarbonate microisolator cages with approved irradiated contact nests, no more than 3 per cage. The cage includes animal protection laws (including all amendments) and the Guide for the Care and
Use of Laboratory Animal, National Academy Press, Washington, D.M. C. , 2010 was followed.

  Animals were randomly selected into treatment groups based on the cage weights specified in the table below. Group assignments were recorded in the study record.

  The day of administration in the study was designated as study day 1. The conjugate was administered as a slowly pushing bolus dose (approximately 5 seconds) via the tail vein. All animals were dosed over 2 days. Groups 1-8 were administered on the first day and Groups 9-16 were administered on the second day. Treatment groups (TG) 13-16 were administered in the table above. Results from these TGs did not affect the progress over other TGs. The first 2TG of each conjugate was administered in the table above. If all animals in the 100 mg / kg group died, then the remaining TG for that test item would not be administered and the study would be terminated. If at least one animal survived 2 hours after administration in the 100 mg / kg group, then the 150 mg / kg group was administered. If all animals in the 150 mg / kg group died, then the study would be terminated without administering the remaining TG for that test item. If at least one animal survived 2 hours after administration in the 150 mg / kg group, then the 200 mg / kg group was administered.

  Animals were observed once daily for moribund and mortality. Any animals showing signs of distress, especially signs of death approaching, were humanely euthanized based on the Numira Biosciences Standard Operating Procedures. Body weights were recorded on arrival date, dosing date and dissection date. Detailed clinical observations were performed and recorded at 0, 15 and 2 hours post-dose to assess injection resistance.

  Blood samples (maximum volume, approximately 1 mL) were obtained from all animals by cardiac puncture prior to dissection after day 3 administration. Blood samples were collected in red plug microtainer tubes and kept at room temperature for at least 30-60 minutes prior to centrifugation. Samples were centrifuged at approximately 1500-2500 rpm for 15-20 minutes to obtain serum.

  Animals not expected to survive until the next regular observation were weighed and euthanized. Animals found dead were weighed and the time of death was estimated as closely as possible. Blood and tissue samples were not collected.

  On the third day (2 days after administration), all animals were humanely euthanized with carbon dioxide. Euthanasia was performed according to the approved American Veterinary Medical Association (AVMA) euthanasia guidelines June 2007.

  Some global dissections included a record of experiments and study results. All outer surfaces and openings were evaluated. All abnormalities observed during tissue collection were fully described and recorded. No further organization was needed.

  Left and right kidneys were collected. Tissue was collected within 15 minutes of euthanasia. All instruments and tools used were exchanged between treatment groups. All tissues were snap frozen and stored at <−70 ° C. as soon as possible after collection.

  Renal injury marker data was obtained as follows. RNA from mouse kidney tissue was purified using a Quick Gene Mini80 Tissue Kit SII (Fuji Film). That is, approximately 40 mg of tissue is added to 0.5 ml lysis buffer (5 μl 2-mercaptoethanol in 0.5 ml lysis buffer) in a MagnaLyser Green Bead vial (Roche), and MagNA Lyser (Roche) is added. Used to equalize 2 sets of 3 × 3800 RPM and 3 sets of 1 × 6500 RPM. Samples were cooled on ice for 3-4 minutes between each low speed set and each high speed run. The homogenate was centrifuged at 400 × g for 5 minutes at room temperature. The homogenate was immediately processed for RNA purification based on the Quick Gene Mini 80 protocol. Samples were DNA digested with DNase I (Qiagen) for 5 minutes on the column. Total RNA was quantified with a NanoDrop2000 spectrometer (Thermo Scientific).

  qRT-PCR was performed using Applied Biosystems reagent (1-step RT-PCT) and predesigned primer / probe sets (ACTB, GAPDH, KIM-1, clusterin-FAM reporter).

Each reaction contained the following (total 30 μl).
15 μl 2 × qRT-PCR buffer from ABI One-Step kit 1.5 μl primer / probe mix 8.75 μl nuclease free water 0.75 μl 40 × multiscript + RNase inhibitor 4 μl RNA template (100 ng / μl)
The qRT 1-step program was run as follows.
1.48C, 30 minutes 2.95C, 10 minutes 3.95C, 15 seconds 4.60C, 1 minute 5. Repeat steps 3 to 4 39 times for a total of 40 cycles

  Samples were performed in triplicate wells and averaged for further analysis. Analysis was performed using the ΔΔCt method. That is, ΔCt [Ct (target) −Ct (reference)] = ΔΔCt obtained from the experiment, subtracted by control ΔCt [Ct (target) −Ct (reference)]. The fold change range was calculated: 2 ^-(ΔΔCt + SD) to 2 ^-(ΔΔCt-SD). Control = (pooled) vehicle-treated animal group, target = KIM-1; reference = GAPDH; SD = Sqrt [(SD target 2) + (SD reference 2)].

The result of KIM data is shown in FIG. The conjugate containing the carrier peptide with terminal glycine was the lowest R ₆ G peptide and had a lower KIM concentration. It is clear that both the terminal G and the presence of unnatural amine acid (aminohexanoic acid) play a role in the toxicity of the conjugate.

  Frozen serum samples were sent on dry ice to IDEX Laboratories (West Sacramento, Calif.) For processing. Serum dilutions were performed according to the IDEXX standard operating procedure (SOP) as required. Blood chemistry results were analyzed. Blood urea nitrogen levels are shown in FIG. Again, the G-linked conjugate had a lower BUN level. It is clear that both terminal G and the entire peptide sequence play a role in the toxicity profile of the conjugate.

  Kidney tissue (approximately 150 mg) was accurately weighed into a 2 mL screw cap vial partially filled with ceramic beads. 5 volumes of tissue PE LB buffer (G Biosciences) containing 10 U / mL proteinase K (Sigma) was added to 1 part of tissue. Samples were homogenized with Roche MagnaLyser (4 × 40 seconds @ 7,000 rpm with cooling during operation) and incubated at 40 ° C. for 30 minutes. When necessary, tissue homogenates were diluted with BSAsal (3 mg / mL BSA + 20 mM NaCl) to a high sample concentration within the calibration range.

  Calibration samples were prepared by adding a solution of 3 mg / mL BSA in 20 mM NaCl containing a known amount of the appropriate analytical reference standard. Two sets of 8 samples were each prepared. ULOQ was 40 μg / mL and LLOQ was 0.065536 μg / mL. An internal standard (NG-07-0775) was added to all samples except some blank samples designated as double blanks (without drug and internal standard). Samples were extracted by vortexing 100 μL aliquots with 3 volumes of methanol.

  After centrifugation (15 minutes, 14,000 rpm), the supernatant was transferred to a new tube and dried on Speedvac. The dried sample was reconstituted with an appropriate amount of FDNA (5 'd FAM-ATTTCAGGTAAGCCGAGGTTTGGCC 3') in [10 mM Tris pH 8.0 + 1 mM EDTA + 100 mM NaCl] -acetonitrile (75-25).

  Samples were analyzed on Dionex UltimateMate 3000 HPLC using anion exchange chromatography (Dionex DNAPac 4 × 250 mm column). The injection volume was 5 μL. The mobile phase consisted of 20% acetonitrile and 80% water containing 25 mM Tris pH 8.0, and a gradient that increased the NaCl concentration. The flow rate was 1 mL / min and the run time was 10 minutes per sample. The fluorescence detector was set at EX494 nm and EM520 nm. Peak identity was based on retention time. The peak height ratio (analytical target: internal standard) was used for quantification. A calibration curve was calculated based on the average response factor of two calibration samples (one set was run at the start of the batch and the other set was run at the end of the batch). A linear curve fitting a 1 / x weighting factor was used. A blank sample (calibration sample without reference compound) and a double blank sample (internal standard addition) were used to confirm the absence of assay specificity and carryover.

  FIG. 12 shows that kidney concentrations were similar among the conjugates tested.

The above data shows that the conjugates of the invention have similar efficacy and improved toxicity compared to other conjugates. FIG 9A~D relates _{R 6} G conjugate (NG-11-0009), summarizes these results.

  The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent literature mentioned and / or listed in the application data sheet are hereby incorporated by reference in their entirety. This is incorporated herein. Aspects of the above embodiments can be modified if it is necessary to use various patent, application, and publication concepts to provide further embodiments. These and other changes can be made to the embodiments in terms of the above detailed description. In general, in the following claims, the terminology used should not be construed as limiting the scope of the claims to the specific embodiments disclosed in the specification and the claims. Such claims should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims (1)

Invention described in the specification.

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