EA040020B1 - AMINO ACID DERIVATIVES FUNCTIONALIZED AT THE N-TERMINAL, CAPABLE OF FORMING DRUG ENCAPSULATED MICROSPHERES - Google Patents

Description

and their salts, the use of these compounds or their salts and a polynucleotide in the manufacture of a pharmaceutical composition for delivering a polynucleotide into a cell, compositions for delivering a polynucleotide into a cell containing these compounds are also described, and the use of these compositions for the treatment of proliferative disorders, inflammatory disorders, autoimmune disorders and liver diseases.

Related Applications

This application claims priority under U.S. Patent Law Section 35 § 119(e) based on U.S.S.N. Provisional Application. 61/552423, subject on 27 October

2011, which is incorporated herein by reference.

Governmental support

The present invention was made with government support under grant No. R37 EB 000244, provided by the US National Institutes of Health. The government has certain rights to the present invention.

Background of the invention

In 1998, Mello and Fire reported the possibility of gene silencing using RNA interference. See Fire et al, Nature (1998) 391:806-811. Since then, scientists have rushed to take advantage of the enormous therapeutic potential of targeted gene knockdown. This is supported by the fact that the first report of small interfering RNA (siRNA) mediated RNAi in humans was published only twelve years after the phenomenon was described in Caenorhabditis elegans. See Davis et al, Nature (2010) 464:1067-1070. It is well understood that the development of genetic drugs is being slowed down by the inability to efficiently deliver nucleic acids in vivo. When unprotected, genetic material introduced into the bloodstream can be degraded by DNases and RNases, or if it is not degraded, the genetic material can elicit an immune response. See, for example, Whitehead et al., Nature Reviews Drug Discovery (2009) 8:129-138; Robbins et al, Oligonucleotides (2009) 19:89-102. The intact siRNA must then enter the cytosol, where the antisense strand is inserted into the RNA-induced silencing complex (RISC) (Whitehead supra). RISC binds to complementary mRNA sequences and destroys them, thereby preventing translation of the target mRNA into a protein, i.e., it performs gene silencing.

To overcome delivery challenges, nucleotides have been complexed with various delivery systems, including polymers, lipids, inorganic nanoparticles, and viruses. See, for example, Peer et al. Nature Nanotechnology, (2007) 2:751-760. However, despite promising data from ongoing clinical trials for the treatment of respiratory syncytial virus and types of liver cancer (see, e.g., Zamora et al., Am. J. Respir. Crit. Care Med. (2011) 183:531538), the clinical application of siRNA still requires the development of safer and more efficient delivery systems. To this end, numerous lipid-like molecules have been developed, including poly-β-amino esters and amino alcohol lipids. See, for example, PCT Application Publication Nos. WO 2002/031025; WO2004/106411; WO2008/011561; WO 2007/143659; WO 2006/138380 and WO 2010/053572. Amino acid-, peptide-, polypeptide-lipids (APPL) have also been studied for a variety of applications, including applications as therapeutics, biological surfactants, and nucleotide delivery systems. See, for example, Giuliani et al., Cellular and Molecular Life Sciences (2011) 68:2255-2266; Ikeda et al, Current Medicinal Chemistry (2007) 14: 111263-1275; Sen, Advances in Experimental Medicine and Biology (2010) 672:316-323; and Damen et al., Journal of Controlled Release (2010) 145:33-39. However, there remains a need to research and develop new APPL systems with improved properties, such as new and improved APPL nucleotide delivery systems.

Brief summary of the invention

This document describes the compounds and compositions of the present invention, in certain embodiments, characterized by the conjugation of various groups, such as lipophilic groups, with the amino group or amide group of an amino acid, a linear or cyclic peptide, a linear or cyclic polypeptide, or their structural isomer to obtain compounds according to of the present invention, collectively referred to herein as APPL. Such APPLs are believed to be useful for a variety of applications such as, for example, improved nucleotide delivery.

Exemplary APPLs include, but are not limited to, compounds of formula (I), (II), (III), (IV), (V), and (VI), as well as their salts, as described herein:

(III)

(V) ^(VI )

- 1 040020 where m, n, p, R', R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , Z, W, Y and Z are defined in this document, provided that

APPL contains at least one representative of the group of formulas (i), (ii) or (iii):

(i) (ii) (iii) where each R' is independently hydrogen or optionally substituted alkyl;

X is O, S, NRX, where R ^x is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

Y is O, S, NR ^Y , where R ^Y is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

^RP is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; And

R ^l is optionally substituted C _1-50 alkyl, optionally substituted C ₂₅₀ alkenyl, optionally substituted C _2-50 alkynyl, optionally substituted heteroC _1-50 alkyl, optionally substituted heteroC _2-50 alkenyl, optionally substituted heteroC _2-50 alkynyl, or polymer.

In certain embodiments, a group of formula (i) is a group of formula (i-a) or a group of formula (i-b):

In certain embodiments, a group of formula (i-a) is a group of formula (i-a1) or a group of formula (i-a2):

In certain embodiments, a group of formula (i-b) is a group of formula (i-b1) or a group of formula (i-b2):

In certain embodiments, at least one representative of R ¹ is a group of the formula:

where L is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted heteroalkylene, an optionally substituted heteroalkenylene, an optionally substituted heteroalkynylene, an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, or an optionally substituted heteroarylene, and

R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group;

provided that at least one representative of R ⁶ and R ⁷ represents a group of the formula:

- 2 040020

where each representative of R' independently represents hydrogen or optionally substituted alkyl;

X is O, S, NR ^X where ^RX is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

Y is O, S, NRY, where RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;

^RP is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, a sulfur protecting group when attached to a sulfur atom, or a nitrogen protecting group when attached to a nitrogen atom; And

RL is optionally substituted C _1-50 alkyl, optionally substituted C _2-50 alkenyl, optionally substituted C ₂ . ₅₀ alkynyl, optionally substituted heteroC _1-50 alkyl, optionally substituted heteroC _2-50 alkenyl, optionally substituted heteroC _2-50 alkynyl, or polymer.

In certain embodiments, each R' is hydrogen.

In certain embodiments, L is an optionally substituted alkylene.

In certain embodiments, the group of formula (iv) is the formula:

where q is an integer from 1 to 50 inclusive.

In certain embodiments, each R ¹ is a group of formula (iv).

An exemplary APPL of the present invention is the compound (cKK-E12):

or its salt.

In another aspect, compositions containing APPL or a salt thereof are provided.

For example, in certain embodiments, a composition is provided comprising APPL or a salt thereof and optionally an excipient, wherein APPL is an amino acid, a linear or cyclic peptide, a linear or cyclic polypeptide, or a structural isomer thereof, and wherein the amino group or amide group of APPL is conjugated to a group of formula (i), (ii) or (iii). In certain embodiments, a group of formula (i), (ii), or (iii) is attached to an amino group located on the APPL backbone. In certain embodiments, the composition is a pharmaceutical composition, a cosmetic composition, a nutraceutical composition, or a composition that is not used in medicine. In certain embodiments, the non-medicinal composition is an emulsion or emulsifier useful as a food ingredient, fire fighting, surface disinfection, or oil refining.

In certain embodiments, the composition further comprises an agent. In certain embodiments, the agent is an organic molecule, an inorganic molecule, a nucleic acid, a protein, a peptide, a polynucleotide, a targeting agent, an isotopically labeled chemical, a vaccine, an immunological agent, or an agent suitable for biotechnology, such as intracellular protein production. In certain embodiments, the agent is a polynucleotide and the polynucleotide is DNA or RNA. IN

- 3 040020 certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, the agent and APPL are not connected by a covalent bond, for example, for example, the agent and APPL are not complexed by a non-covalent bond. However, in certain embodiments, the agent and APPL are linked by a covalent bond.

In certain embodiments, the composition is in the form of a particle. In certain embodiments, the implementation of the particle is a nanoparticle or microparticle. In certain embodiments, the particle is a micelle, liposome, or lipoplex. In certain embodiments, the particle encapsulates the agent, such as the agent to be delivered.

In another aspect, a method is provided for delivering a polynucleotide to a biological cell, comprising providing a composition comprising APPL or a salt thereof and a polynucleotide, and exposing the composition to the biological cell under conditions sufficient to facilitate delivery of the polynucleotide into the biological cell; where APPL is an amino acid, a linear or cyclic peptide or a linear or cyclic polypeptide, or a structural isomer thereof, wherein the amino group or amide group of APPL is conjugated to a group of formula (i), (ii) or (iii). In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is RNA/, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, upon delivery of RNA to a cell, the RNA is capable of interfering with the expression of a particular gene in a biological cell.

In yet another aspect, screening methods are provided. For example, in one embodiment, a method is provided for screening a library of compounds, the method comprising obtaining a plurality of different APPLs or salts thereof, and conducting at least one assay with the compound library to determine the presence or absence of a desired property; where APPL is an amino acid, a linear or cyclic peptide, or a linear or cyclic polypeptide, or a structural isomer thereof, wherein the amino group or amide group of APPL is conjugated to a group of formula (i), (ii) or (iii). In certain embodiments, the desired property is solubility in water, solubility at various pH values, ability to bind polynucleotides, ability to bind heparin, ability to bind small molecules, ability to bind protein, ability to form microparticles, ability to increase transfection efficiency, ability to support cell growth, ability to support cell attachment, the ability to support tissue growth, and/or intracellular delivery of APPL and/or an agent complexed or attached to it to aid biotechnology.

In yet another aspect, methods of using the APPL of the present invention are provided for the treatment of various diseases, disorders, or conditions. For example, in certain embodiments, a method of treating a disease, disorder, or condition afflicted by a subject is provided, comprising administering to a subject in need thereof an effective amount of APPL or a salt thereof, wherein APPL is an amino acid, a linear or cyclic peptide, or a linear or cyclic polypeptide , or a structural isomer thereof, wherein the amino or amide group of APPL is conjugated to a group of formula (i), (ii) or (iii).

A detailed description of one or more embodiments of the present invention is set forth in this document. Other features, objects and advantages of the present invention will be apparent from the detailed description, figures, examples and claims.

Definitions Chemical definitions

Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are listed according to the Periodic Table of the Elements, CAS version in the Handbook of Chemistry and Physics, 75 ^th Ed., on the flyleaf, and specific functional groups are generally defined as described herein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and MarchMarch's Advanced Organic Chemistry, ^5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, ^3rd Edition, Cambridge University Press, Cambridge, 1987.

The compounds described herein may contain one or more centers of asymmetry, and thus may exist in various isomeric forms, such as enantiomers and/or diastereomers. For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures using methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be obtained by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses the compounds, both individual isomers, essentially free of other isomers, and mixtures of different isomers.

When listing a range of values, it is assumed that it covers each value as well as a subrange within the range. For example, C _1-6 alkyl is meant to encompass C 1 , C ₂ , C ₃ , ^C 4 , C 5 , ^C 6 , C 1-6 , ^{C 1-5 , C} 1-4 , ^C 1-3 , ^C 1-2 , C2-6, ^C2-5 , ^C2-4 , ^C2-3 , ^C3-6 , ^C3-5 , ^C3-4 , ^C4-6 , C4-5 ^and C5-6 ^alky l.

As used herein, alkyl refers to the radical of a straight or branched chain saturated hydrocarbon group containing from 1 to 50 carbon atoms (C1 ₃₀ alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 40 carbon atoms (C _1-40 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 30 carbon atoms (C _1-30 αkil). In some embodiments, the implementation of the alkyl group contains from 1 to 20 carbon atoms (C _1-20 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 10 carbon atoms (C _1-10 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 9 carbon atoms (C _1-9 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 8 carbon atoms (C _1-8 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 7 carbon atoms (C _1-7 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 6 carbon atoms (C _1-6 αkil). In some embodiments, the implementation of the alkyl group contains from 1 to 5 carbon atoms (C _1-5 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 4 carbon atoms (C _1-4 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 3 carbon atoms (C _1-3 alkyl). In some embodiments, the implementation of the alkyl group contains from 1 to 2 carbon atoms (C 1-2 alkyl). In some embodiments, the implementation of the alkyl group contains 1 carbon atom (C1 alkyl). In some embodiments, the implementation of the alkyl group contains from 2 to 6 carbon atoms (C ₂ - ₆ alkyl). Examples of C _1-6 alkyl groups include methyl (C1), ethyl (C ₂ ), n-propyl (C ₃ ), isopropyl (C ₃ ), n-butyl (C ₄ ), tert-butyl (C ₄ ), sec-butyl (C ₄ ), iso-butyl (C ₄ ), n-pentyl (C ₅ ), 3-pentanyl (C ₅ ), amyl (C ₅ ), neopentyl (C ₅ ), 3-methyl-2-butanyl (C ₅ ), tertiary amyl (C ₅ ), and nhexyl (C ₆ ). Additional examples of alkyl groups include n-heptyl (C ₇ ), n-octyl (C ₅ ), and the like. Unless otherwise indicated, each representative of an alkyl group is independently unsubstituted (unsubstituted alkyl) or substituted (substituted alkyl) with one or more substituents. In certain embodiments, the alkyl group is unsubstituted C _1-50 alkyl. In certain embodiments, the alkyl group is a substituted C _1-50 alkyl.

As used herein, heteroalkyl refers to an alkyl group, as defined herein, which additionally contains at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus, within the main chain (ie, inserted between adjacent carbon atoms) and/or placed in one or more end positions of the main chain. In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 50 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-50 alkyl). In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 40 carbon atoms and 1 or more heteroatoms within the main chain (heteroC1-40alkyl). In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 30 carbon atoms and 1 or more heteroatoms within the main chain (heteroC1 ₃₀ alkyl). In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 20 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-20 alkyl). In certain embodiments, a heteroalkyl group refers to a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-10 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-9 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-8 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-7 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the main chain (heteroC _1-6 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the main chain (heteroC _1-5 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within

- 5 040020 main chain (heteroC _1-4 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the main chain (heteroC _1-3 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the main chain (heteroC1 ₂ alkyl). In some embodiments, the heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (heteroC1 alkyl). In some embodiments, the heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the main chain (heteroC _2-6 alkyl). Unless otherwise indicated, each member of a heteroalkyl group is independently unsubstituted (unsubstituted heteroalkyl) or substituted (substituted heteroalkyl) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC _1-50 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC1 ₅₀ alkyl.

As used herein, alkenyl refers to the radical of a straight or branched chain saturated hydrocarbon group containing from 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) (C ₂₅₀ alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 40 carbon atoms (C _2-40 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 30 carbon atoms (C _2-30 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 20 carbon atoms (C _2-20 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 10 carbon atoms (C _2-10 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 9 carbon atoms (C _2-9 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 8 carbon atoms (C _2-8 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 7 carbon atoms (C _2-7 alkenyl). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (C _2-6 alkenyl). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (C _2-5 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 4 carbon atoms (C _2-4 alkenyl). In some embodiments, the implementation of the alkenyl group has from 2 to 3 carbon atoms (C _2-3 alkenyl). In some embodiments, the implementation of the alkenyl group has 2 carbon atoms (C ₂ alkenyl). One or more carbon-carbon double bonds may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C _2-4 alkenyl groups include ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ), 2-butenyl (C ₄ ), butadienyl (C ₄ ), and so on. Examples of the C ₂₆ alkenyl groups include the aforementioned C _2-4 alkenyl groups, as well as pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (C ₆ ), and the like. Additional examples of alkenyl include heptenyl (C ₇ ), octenyl (C ₈ ), octatrienyl (C ₈ ), and the like. Unless otherwise indicated, each alkenyl group member is independently unsubstituted (unsubstituted alkenyl) or substituted (substituted alkenyl) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C _2-50 alkenyl. In certain embodiments, the alkenyl group is a substituted C _2-50 alkenyl.

As used herein, heteroalkenyl refers to an alkenyl group, as defined herein, which further contains at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus, within the main chain (ie, inserted between adjacent carbon atoms) and/or placed in one or more end positions of the main chain. In certain embodiments, a heteroalkenyl group refers to a group having 2 to 50 carbon atoms, at least one double bond, and 1 or more heteroatoms within the backbone (heteroC _2-50 alkenyl). In certain embodiments, a heteroalkenyl group refers to a group having 2 to 40 carbon atoms, at least one double bond, and 1 or more heteroatoms within the backbone (heteroC _2-40 alkenyl). In certain embodiments, a heteroalkenyl group refers to a group having 2 to 30 carbon atoms, at least one double bond, and 1 or more heteroatoms within the backbone (heteroC ₂₃₀ alkenyl). In certain embodiments, a heteroalkenyl group refers to a group having 2 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the backbone (heteroC _2-20 alkenyl). In certain embodiments, a heteroalkenyl group refers to a group having 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the backbone (heteroC _2-10 alkenyl). In some embodiments, the implementation of the heteroalkenyl group has from 2 to 9 carbon atoms, at least one double bond and 1 or more heteroatoms within the main chain (heteroC ₂₉ alkenyl). In some embodiments, the implementation of the heteroalkenyl group has from 2 to 8 carbon atoms, at least one double bond and 1 or more heteroatoms within the main chain (heteroC _2-8 alkenyl). In some embodiments, the heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within oc

- 6 040020 new chain (heteroC _2-7 alkenyl). In some embodiments, the implementation of the heteroalkenyl group has from 2 to 6 carbon atoms, at least one double bond and 1 or more heteroatoms within the main chain (heteroC _2-6 alkenyl). In some embodiments, the heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the backbone (heteroC _2-5 alkenyl). In some embodiments, the heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the backbone (heteroC _2-4 alkenyl). In some embodiments, the implementation of the heteroalkenyl group has from 2 to 3 carbon atoms, at least one double bond and 1 heteroatom within the main chain (heteroC _2-3 alkenyl). In some embodiments, the implementation of the heteroalkenyl group has from 2 to 6 carbon atoms, at least one double bond and 1 or 2 heteroatoms within the main chain (heteroC _2-6 alkenyl). Unless otherwise indicated, each member of a heteroalkenyl group is independently unsubstituted (unsubstituted heteroalkenyl) or substituted (substituted heteroalkenyl) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC _2-50 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC ₂₅₀ alkenyl.

As used herein, alkynyl refers to a straight or branched chain saturated hydrocarbon group radical containing from 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (eg 1, 2, 3 or 4 double bonds) (C _2-50 alkynyl). An alkynyl group that has one or more triple bonds and one or more double bonds is also referred to as en-ying. In some embodiments, the alkynyl group has 2 to 40 carbon atoms (C _2-40 alkynyl). In some embodiments, the alkynyl group has 2 to 30 carbon atoms (C _2-30 alkynyl). In some embodiments, the alkynyl group has 2 to 20 carbon atoms (C _2-20 alkynyl). In some embodiments, the alkynyl group has 2 to 10 carbon atoms (C _2-10 alkynyl). In some embodiments, the alkynyl group has 2 to 9 carbon atoms (C _2-9 alkynyl). In some embodiments, the alkynyl group has 2 to 8 carbon atoms (C _2-8 alkynyl). In some embodiments, the alkynyl group has 2 to 7 carbon atoms (C _2-7 alkynyl). In some embodiments, the alkynyl group has 2 to 6 carbon atoms (C _2-6 alkynyl). In some embodiments, the alkynyl group has 2 to 5 carbon atoms (C _2-5 alkynyl). In some embodiments, the alkynyl group has 2 to 4 carbon atoms (C _2-4 alkynyl). In some embodiments, the alkynyl group has 2 to 3 carbon atoms (C2 ₃ alkynyl). In some embodiments, the alkynyl group has 2 carbon atoms (C ₂ alkynyl). One or more carbon-carbon triple bonds may be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C _2-4 alkynyl groups include, without limitation, ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2-butynyl (C ₄ ), and etc. Examples of C _2-8 alkenyl groups include the aforementioned C ₂₄ alkynyl groups, as well as pentynyl (C ₅ ), hexynyl (C ₆ ), and the like. Additional examples of alkynyl include heptynyl (C ₇ ), octynyl (C ₈ ), and the like. Unless otherwise indicated, each representative of the alkynyl group is independently unsubstituted (unsubstituted alkynyl) or substituted (substituted alkynyl) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C _2-50 alkynyl. In certain embodiments, the alkynyl group is a substituted C _2-50 alkynyl.

As used herein, heteroalkynyl refers to an alkynyl group, as defined herein, which further contains at least one heteroatom (e.g., 1 to 25, e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus within the main chain (ie, inserted between adjacent carbon atoms) and/or placed in one or more end positions of the main chain. In certain embodiments, a heteroalkynyl group refers to a group having 2 to 50 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-30 alkynyl). In certain embodiments, a heteroalkynyl group refers to a group having 2 to 40 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-40 alkynyl). In certain embodiments, a heteroalkynyl group refers to a group having 2 to 30 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-30 alkynyl). In certain embodiments, a heteroalkynyl group refers to a group having 2 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-20 alkynyl). In certain embodiments, a heteroalkynyl group refers to a group having 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-10 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or no

- 7 040020 how many heteroatoms within the main chain (heteroC _2-9 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-8 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-7 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the backbone (heteroC _2-6 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the backbone (heteroC _2-5 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the backbone (heteroC _2-4 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the backbone (heteroC _2-3 alkynyl). In some embodiments, the heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the backbone (heteroC _2-6 alkynyl). Unless otherwise indicated, each member of a heteroalkynyl group is independently unsubstituted (unsubstituted heteroalkynyl) or substituted (substituted heteroalkynyl) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC _2-50 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC _2-50 alkynyl.

As used herein, carbocyclyl or carbocyclic refers to a radical of a non-aromatic cyclic hydrocarbon group containing from 3 to 10 carbon atoms in the ring (C _3-10 carbocyclyl) and no heteroatoms in the non-aromatic ring system. In some embodiments, the implementation of the carbocyclyl group has from 3 to 8 carbon atoms in the ring (C ₃₈ carbocyclyl). In some embodiments, the implementation of the carbocyclyl group has from 3 to 7 carbon atoms in the ring (C _3-7 carbocyclyl). In some embodiments, the implementation of the carbocyclyl group has from 3 to 6 carbon atoms in the ring (C _3-6 carbocyclyl). In some embodiments, the implementation of the carbocyclyl group has from 4 to 6 carbon atoms in the ring (C _4-6 carbocyclyl).

In some embodiments, the implementation of the carbocyclyl group has from 5 to 6 carbon atoms in the ring (C _5-6 carbocyclyl). In some embodiments, the implementation of the carbocyclyl group has from 5 to 10 carbon atoms in the ring (C _5-10 carbocyclyl). Illustrative C _3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ) , cyclohexenyl (C ₆ ), cyclohexadienyl (C ₆ ) and the like. Illustrative C _3-8 carbocyclyl groups include, without limitation, the aforementioned C _3-6 carbocyclyl groups, as well as cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), cycloheptatrienyl (C ₇ ), cyclooctyl (C ₈ ), cyclooctenyl (C ₅ ), bicycloheptanyl (C ₇ ), bicyclooctanyl (C ₅ ), and the like. Illustrative C _3-10 carbociyl groups include, without limitation, the aforementioned C _3-8 carbocyclyl groups, as well as cyclononyl (C ₉ ), cyclononenyl (C ₉ ), cyclodecyl (C ₁₀ ), cyclodecenyl (C ₁₀ ), octahydro-III- indenyl (C ₉ ), decahydronaphthalene (C ₁₀ ), spirodecanyl (C ₁₀ ) and the like. As shown in the examples above, in certain embodiments, the carbocyclyl group is either monocyclic (monocyclic carbocyclyl) or polycyclic (e.g., contains a fused, bridged, or spiro ring system such as a bicyclic system (bicyclic carbocyclyl) or a tricyclic system (tricyclic carbocyclyl) ) and may be saturated or may contain one or more carbon-carbon double or triple bonds. Carbocyclyl also includes ring systems where the carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the carbocyclyl ring and, in such cases, the number of carbon atoms continues to denote the number of carbon atoms in the carbocyclic ring system. . Unless otherwise indicated, each member of a carbocyclyl group is independently unsubstituted (unsubstituted carbocyclyl) or substituted (substituted carbocyclyl) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C _3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C _3-10 carbocyclyl.

In some embodiments, carbocyclyl or carbocyclic refers to a cycloalkyl, that is, a monocyclic saturated carbocyclyl group having 3 to 10 ring carbon atoms (C _3-10 cycloalkyl). In some embodiments, the implementation of the cycloalkyl group has from 3 to 8 carbon atoms in the ring (C _3-8 cycloalkyl). In some embodiments, the implementation of the cycloalkyl group has from 3 to 6 carbon atoms in the ring (C _3-6 cycloalkyl). In some embodiments, the implementation of the cycloalkyl group has from 4 to 6 carbon atoms in the ring (C _4-6 cycloalkyl). In some embodiments, the cycloalkyl group has 5 to 6 carbon atoms

- 8 040020 in the ring (C ₅ - ₆ cycloalkyl). In some embodiments, the implementation of the cycloalkyl group has from 5 to 10 carbon atoms in the ring (C ₅ - ₁₀ cycloalkyl). Examples of C ₅ - ₆ cycloalkyl groups include cyclopentyl (C ₅ ) and cyclohexyl (C ₅ ). Examples of C ₃ - ₆ cycloalkyl groups include the aforementioned C ₅ - ₆ cycloalkyl groups, as well as cyclopropyl (C3) and cyclobutyl (C ₄ ). Examples of C _{3 8} cycloalkyl groups include the aforementioned C ₃ - ₆ cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C ₈ ). Unless otherwise indicated, each member of a cycloalkyl group is independently unsubstituted (unsubstituted cycloalkyl) or substituted (substituted cycloalkyl) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C _3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C _3-10 cycloalkyl.

As used herein, heterocyclyl or heterocyclic refers to a radical of a 3-14 membered non-aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (3-14-membered heterocyclyl). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. The heterocyclyl group may be either monocyclic (monocyclic heterocyclyl) or polycyclic (e.g., fused, bridged, or spiro ring system, e.g., bicyclic system (bicyclic heterocyclyl) or tricyclic system (tricyclic heterocyclyl)), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may include one or more heteroatoms in one or two rings. Heterocyclyl also includes ring systems where the heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, where the point of attachment is either on the carbocyclyl ring or the heterocyclyl ring, or ring systems where the heterocyclyl ring as defined above is fused to one or more several aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring, and, in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Unless otherwise indicated, each heterocyclyl is independently unsubstituted (unsubstituted heterocyclyl) or substituted (substituted heterocyclyl) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (5-10-membered heterocyclyl). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring, where each heteroatom is independently selected from oxygen, sulfur , nitrogen, boron, silicon or phosphorus (5-8 membered heterocyclyl). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring, where each heteroatom is independently selected from oxygen, sulfur , nitrogen, boron, silicon or phosphorus (5-6-membered heterocyclyl). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (eg, 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments, the 5-6 membered heterocyclyl contains 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. Illustrative 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Illustrative 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Illustrative 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Illustrative 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Illustrative 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. illustration

- 9 040020 Active 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanil, oxepanil and thiepanil. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl. Иллюстративные бициклические гетероциклильные группы включают, без ограничения, индолинил, изоиндолинил, дигидробензофуранил, дигидробензотиенил, тетрагидробензотиенил, тетрагидробензофуранил, тетрагидроиндолил, тетрагидрохинолинил, тетрагидроизохинолинил, декагидрохинолинил, декагидроизохинолинил, октагидрохроменил, октагидроизохроменил, декагидронафтиридинил, декагидро-1,8-нафтиридинил, октагидропирролопиррол, индолинил, фталимидил , naphthalimidyl, chromanil, chromenyl, 1H-benzodiazepinyl, 1,4,5,7-tetrahydropyranopyrrolyl, 5,6-dihydro-4H-furopyrrolyl, 6,7-dihydro-5H-furopyranyl, 5,7-dihydro-4H-thienopyranyl , 2,3-dihydro-1H-pyrrolopyridinyl, 2,3-dihydrofuropyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolopyridinyl, 4,5,6,7-tetrahydrofuropyridinyl, 4,5,6,7tetrahydrothienopyridinyl, 1 ,2,3,4-tetrahydro-1,6-naphthyridinyl and the like.

As used herein, aryl refers to the radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a ring structure) containing 6-14 carbon atoms in the ring and providing for the presence of heteroatoms in the aromatic ring system (C ₆ - ₁₄ aryl). In some embodiments, the implementation of the aryl group has 6 carbon atoms in the ring (C ₆ aryl; for example, phenyl). In some embodiments, the implementation of the aryl group has 10 carbon atoms in the ring (C ₁₀ aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbons (C ₁₄ aryl; eg, anthracyl). Aryl also includes ring systems where the aryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, where the radical or point of attachment is on the aryl ring and, in such cases, the carbon number continues to denote the number of carbon atoms in the aryl ring system. Unless otherwise indicated, each aryl group member is independently unsubstituted (unsubstituted aryl) or substituted (substituted aryl) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C ₆ _ ₁₄ aryl. In certain embodiments, the aryl group is a substituted C ₆ _ ₁₄ aryl.

As used herein, heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 π electrons shared in a ring structure), having carbon atoms in the ring and 1 or more (for example, 1, 2, 3 or 4 heteroatoms in the ring) heteroatoms in the ring, provided in an aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (5-14 membered heteroaryl). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl includes ring systems where the heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, where the point of attachment is on the heteroaryl ring and, in such cases, the number of ring members continues to denote the number of ring members in the heteroaryl ring system. Heteroaryl also includes ring systems where the heteroaryl ring as defined above is fused to one or more aryl groups, where the point of attachment is on either the aryl or heteroaryl ring and, in such cases, the number of ring members indicates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups where one ring does not contain a heteroatom (for example, indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on any ring, i.e., or on a ring bearing a heteroatom (for example, 2-indolyl), or on a ring that does not contain a heteroatom (eg 5-indolyl).

In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring provided in the aromatic ring system, where each heteroatom independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (5-10 membered heteroaryl). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (5-8 membered heteroaryl). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having carbon atoms in the ring and 1 or more (e.g., 1, 2, 3, or 4) heteroatoms in the ring provided in the aromatic ring system, where each heteroatom independently selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus (5-6 membered heteroaryl). In some embodiments, the 5-6 membered heteroaryl has

- 10 040020 or more (eg 1, 2 or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon or phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, or phosphorus. Unless otherwise indicated, each member of a heteroaryl group is independently unsubstituted (unsubstituted heteroaryl) or substituted (substituted heteroaryl) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

Illustrative 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Illustrative 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Illustrative 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Illustrative 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Illustrative 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Illustrative 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzothiazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzothiadiazolyl, and indolylizinyl. Illustrative 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Illustrative tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The expression partially unsaturated is intended to encompass rings having multiple centers of unsaturation, but is not intended to include aromatic groups (eg, aryl or heteroaryl moieties) as defined herein.

As used herein, the term saturated refers to a ring fragment that does not contain a double or triple bond, that is, in the ring all bonds are single.

The addition of the suffix -ene to a group indicates that the group is a divalent moiety, for example, alkylene is a divalent alkyl moiety, alkenylene is a divalent alkenyl moiety, alkynylene is a divalent alkynyl moiety, heteroalkylene is a divalent heteroalkyl moiety, heteroalkenylene is a divalent heteroalkenyl moiety, heteroalkynylene is a divalent heteroalkynyl moiety, carbocyclylene is a divalent carbocyclyl moiety, heterocyclylene is a divalent heterocyclyl moiety, arylene is a divalent aryl moiety, and heteroarylene is a divalent heteroaryl moiety.

As is understood from the foregoing, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups as defined herein are, in certain embodiments, optionally substituted. Optionally substituted refers to a group that may be substituted or unsubstituted (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl group). In general, the expression substituted means that at least one hydrogen present in the group is replaced by a valid substituent, e.g., a substituent that, when substituted, produces a stable compound, e.g., a compound that does not spontaneously undergo transformation such as rearrangement, ring closure reaction , cleavage, or other reaction. Unless otherwise indicated, a substituted group has a substituent at one or more substitutable positions of the group, and if more than one position is substituted in any given structure, then the substituent at each position is the same or different. The expression substituted is intended to include the substitution of all permitted substituents for an organic

- 11 040020 cal compounds, any of the substituents described in this document, which leads to the formation of a stable compound. The present invention contemplates any and all such combinations to achieve a stable connection. For the purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein that satisfies the valences of the heteroatoms and results in the formation of a stable moiety.

Exemplary carbon substituents include, but are not limited to, halogen, -CN, -NO2, -N ₃ , -SO ₂ H, -SO ₃ H, -OH, -OR ^aa , -ON(R ^bb )2, -N( R ^bb )2,

-N(R ^bb )3 ⁺ X“, -N(OR ^cc )R ^bb , -SeH, -SeR ^aa , -SH, -SR ^aa , -SSR ^CC , -C(=O)R ^aa , -CO2H, -CHO, C(OR ^cc )2, -CO2R ^aa , -OC(=O)R ^aa , -OCO2R ^aa , -C(=O)N(R ^bb )2, -OC(=O)N(R ^bb )2, NR ^bb C(=O)R ^aa , -NR ^bb CO2R ^aa , -NR ^bb C(=O)N(R ^bb )2, -C(=NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa , OC(=NR ^bb )R ^aa , -OC(=NR ^bb )OR ^aa , -C(=NR ^bb )N(R ^bb )2, -OC(=NR ^bb )N(R ^bb )2 , NR ^bb C(=NR ^bb )N(R ^bb )2, -C(=O)NR ^bb SO2R ^aa , -NR ^bb SO2R ^aa , -SO2N(R ^bb )2, -SO2R ^aa , -SO2OR ^aa , - OSO2R ^aa , -S(=O)R ^aa , -OS(=O)R ^aa , -Si(R ^aa )3, -OSi(R ^aa )3 -C(=S)N(R ^bb )2, - C(=O)SR ^aa , C(=S)SR ^aa , -SC(=S)SR ^aa , -SC(=O)SR ^aa , -OC(=O)SR ^aa , -SC(=O)OR ^aa , -SC(=O)R ^aa , P(=O)2R ^aa , -OP(=O)2R ^aa , -P(=O)(R ^aa )2, -OP(=O)(R ^aa ) 2, -OP(=O)(OR ^CC )2, -P(=O)2N(R ^bb )2, OP(=O) ₂ N(R ^bb )2, -P(=O)(NR ^bb ) 2, -OP(=O)(NR ^bb )2, -NR ^bb P(=O)(OR ^cc )2, NR ^bb P(=O)(NR ^bb )2, -P(R ^cc )2, - P(R ^cc )3, -OP(R ^cc )2, -OP(R ^cc )3, -B(R ^aa )2, -B(OR ^CC ) ₂ , BR ^aa (OR ^cc ),

C1. ₅₀ alkyl, C ₂ . ₅₀ alkenyl, C ₂ . ₅₀ alkynyl, C ₃ . ₁₄ carbocyclyl, 3-14 membered heterocyclyl, C ₆ . ₁₄ aryl and 514-membered heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

or two geminal hydrogens on a carbon atom are replaced by the group ⁼⁰ ' ^=S ' =NN(R ^bb )2, =NNR ^bb C(=O)R ^aa , =NNR ^bb C(=O)OR ^aa , =NNR ^bb S( =O)2R ^aa , =NR ^bb or =NOR ^CC ;

each R ^aa is independently selected from C _1-50 alkylα, C ₂ . ₅₀ alkenyl, C ₂ . ₅₀ alkynyl, C ₃ . ₁₀ carbocyclyl, 3-14 membered heterocyclyl, C ₆ . ₁₄ aryl and 5-14 membered heteroaryl, or two R ^aa -groups are combined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

each R ^bb is independently selected from hydrogen,

-OH, -OR ³³ , -N(R ^cc )2,

-CN, -C(=O)R ^aa , -C(=O)N(R ^cc )2, CO'R ³³ . -SO2R ^aa , -C(=NR ^cc )OR ^aa , -C(=NR ^CC )N(R ^CC )2, SO2N(R ^CC )2, -SO ₂ R ^cc , -SO2OR ^cc , -SOR ³³ , - C(=S)N(R ^cc )2, -C(=O)SR ^cc , -C(=S)SR ^cc , P(=O)2R ^aa , -P(=O)(R ^aa )2, -P(=O) ₂ N(R ^CC )2, -P(=O)(NR ^cc )2,

From ₁ . ₅₀ alkyl, C ₂ . ₅₀ alkenyl, C ₂ . ₅₀ alkynyl, C ₃ . ₁₀ carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^bb groups combined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

each R ^cc is independently selected from hydrogen, C1. ₅₀ alkyl, C ₂ . ₅₀ alkenyl, C ₂ . ₅₀ alkynyl, C ₃ . ₁₀ carbocyclyl, 3-14-membered heterocyclyl, C ₆ . ₁₄ aryl and 5-14 membered heteroaryl, or two R ^cc groups combined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted 0, 1, 2, 3, 4 or 5 R ^dd groups;

each R ^dd is independently selected from halogen,

-CN, -NO ₂ , -N ₃ , SO ₂ H, -SO ₃ H, -OH, -OR ^ee , -ON(R ^ff )2, -N(R ^ff )2, -N(R ^ff )3 ⁺ X“, -N(OR ^ee )R ^ff , -SH, -SR ^ee , SSR ^ee , -C(=O)R ^ee , -CO2H, -CO ₂ R ^ee , -OC(=O)R ^ee , -OCO2R ^ee , -C(=O)N(R ^ff )2, OC(=O)N(R ^ff )2, -NR ^ff C(=O)R ^ee , -NR ^ff CO2R ^ee , -NR ^ff C (=O)N(R ^ff )2, -C(=NR ^ff )OR ^ee , OC(=NR ^ff )R ^ee , -OC(=NR ^ff )OR ^ee , -C(=NR ^ff )N(R ^ff )2, -OC(=NR ^ff )N(R ^ff )2, NR ^ff C(=NR ^ff )N(R ^ff )2,-NR ^ff SO2R ^ee , -SO2N(R ^ff )2, -SO2R ^ee , -SO2OR ^ee , -OSO2R ^ee , -S(=O)R ^ee , -Si(R ^ee )3, -OSi(R ^ee )3, -C(=S)N(R ^ff )2, -C( =O)SR ^ee , -C(=S)SR ^ee , -SC(=S)SR ^ee , -P(=O)2R ^ee , P(=O)(R ^ee )2, -OP(=O) (R ^ee )2, -OP(=O)(OR ^ee )2,

C1. ₅₀ alkyl, C ₂ . ₅₀ alkenyl, C ₂ . ₅₀ alkynyl, C ₃ . ₁₀ carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,

- 12 040020 aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^gg -rpynnaMu, or two geminal R ^dd substituents may be combined to form =0 or =S;

each R ^ee is independently selected from C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 carbocyclyl, C _6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R ^gg groups;

each R ^ff is independently selected from hydrogen, C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 carbocyclyl, 3-10 membered heterocyclyl, C _6-10 aryl, and 5-10- membered heteroaryl, or two R ^ff groups are combined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3 , 4 or 5 R ^gg groups; and each ^Rgg is independently a halogen,

-CN, -NO2, N ₃ , -SO ₂ H, -SO3H, -OH, -OCx-5oalkyl, -OMS | -5oalkyl) ₂ , -MC | -5oalkyl) ₂ , H (C1-5oalkyl) ^{3 +} X “, -HH (C1-5oalkyl)2 ⁺ X“ -XH2(Cx-5oalkyl) ⁺ X“, -MH3 ⁺ X“ X(OC1-5oalkyl) (C1-5oalkyl), -X (OH) (Cx-5oalkyl ), -NH(OH), -SH, -8Cx-5oalkyl, 88(C1-5oalkyl), -C(=0)(C1-5oalkyl), -CO2H, -C0 ₂ (Cx-5oalkyl), -OC( \u003d 0) (C1-5oalkyl), OS0 ₂ (Cx-5oalkyl), -C (=O)NH ₂ , -C (=0) H (Cx-5oalkyl) ₂ , -OC (=0) XH (C1- 5oalkyl), XHC(=0)(C1-5oalkyl), -X(C1-5oalkyl)C(=0)(C1-5oalkyl), -XHC0 ₂ (Cx-5oalkyl), XHC(=0)X(C1- 5oalkyl) ₂ , -XHC(=0)XH( _C1-5oalkyl ), -NHC(=O)NH ₂ , C(=XH)0(C1-5oalkyl),-OC(=XH)(C1-5oalkyl) , -OC(=XH)OCx-5oalkyl, C(=XH)X( _C1.50 alkyl) ₂ , -C(=XH)XH( _C1-5oalkyl ), -C(=NH)NH ₂ , OS( \u003d XH) X ( _C1.50 alkyl) ₂ , -OC (XH) XH (C1- ₅ oalkyl), -OC (NH) NH ₂ , XHC (XH) X (C1.5 oalkyl) ₂ , -NHC (= NH )NH ₂ , -XH80 ₂ ( _C1-5 oalkyl), -80 ₂ X( _C1-5 oalkyl) ₂ , 80 ₂ XH (Cx-5oalkyl), -80 ₂ XH ₂ , -80 ₂ Cx-5oalkyl, -80 ₂ OCx-5oalkyl, -O8O ₂ Cx-balkyl, SOCi-balkyl, -81(C1-5oalkyl)3, -O81(Cx-balkyl)3 -C(=8)X(Cx-5oalkyl) ₂ , C(= 8)XH( C1-5oalkyl), C(=S)NH ₂ , -C(=O)8(Cx-balkyl), -C(=8)8C1-balkyl, 8C(=8)8C1-balkyl, -P(=0 ) ₂ (C1-5oalkyl), -P(=0)(C1-5oalkyl) ₂ , -OP(=0)(C1-5oalkyl) ₂ , OP(=0)(OC1-5oalkyl) ₂ , Cx-5oalkyl, C ₂ -5 oalkenyl, C ₂ -5 o alkynyl, C3-ucarbocylyl, C6-uaryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl;

or two geminal R ^gg substituents can be combined to form ═O or ═S; where X is a counterion.

As used herein, the term halo or halogen refers to fluorine (fluorine, -F), chlorine (chlorine, -Cl), bromine (bromine, -Br), or iodine (iodine, -I).

As used herein, a counterion is a negatively charged group associated with a positively charged quaternary amine to maintain electron neutrality.

Exemplary counterions include halide ions (e.g., F ^- , Cl ^- , Br ^- , I ^- ), NO3 ^- , ClO4 ^- , OH ^- , H2PO4 ^- , HSO ₄ ^- , sulfonate ions (e.g. methanesulfonate, trifluorometasulfonate, ptoluenesulfonate, benzenesulfonate, 10-camphorasulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.) and carboxylate ions (for example, acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

Nitrogen atoms may be substituted or unsubstituted as valency permits and include primary, secondary, tertiary and quaternary nitrogen atoms. Illustrative nitrogen substituents include, but are not limited to, hydrogen,

-OH, -OR ^aa , -N(R ^CC )2, -CN, -C(=O)R ^aa , -C(=O)N(R ^CC )2, -CO ₂ R ^aa , -SO2R ^aa , C(=NR ^bb )R ^aa , -C(=NR ^cc )OR ^aa , -C(=NR ^CC )N(R ^CC )2, -SO ₂ N(R ^CC )2, -SO2R ^CC , -SO2OR ^CC , -SOR ^aa ,

-C(=S)N(R ^CC )2, -C(=O)SR ^cc , -C(=S)SR ^cc , -P(=O)2R ^aa , -P(=O)(R ^aa ) 2, -P(=O)2N(R ^CC )2, P(=O)(NR ^CC ) ₂ ,

C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 514 membered heteroaryl, or two R ^cc groups attached to the N atom, combine to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd - 13 040020 groups, and where R ³³ , R ^bb , R ^cc and R ^dd are defined above.

Nitrogen atoms may be substituted or unsubstituted as valency permits and include primary, secondary, tertiary and quaternary nitrogen atoms. Illustrative nitrogen substituents include, but are not limited to, hydrogen,

-OH, -OR ^aa , -N(R ^CC )2, -CN, -C(=O)R ^aa , -C(=O)N(R ^CC )2, -CO ₂ R ^aa , -SO2R ^aa , C(=NR ^bb )R ^aa , -C(=NR ^cc )OR ^aa , -C(=NR ^CC )N(R ^CC )2, -SO ₂ N(R ^CC )2, -SO2R ^CC , -SO2OR ^CC , -SOR ^aa , -C(=S)N(R ^cc )2, -C(=O)SR ^cc , -C(=S)SR ^cc , -Ρ(=Ο^, -P(=O)( R ^aa )2, -P(=O) ₂ N(R ^CC )2, P(=O)(NR ^CC )2,

C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl or two R ^cc groups attached to the nitrogen atom combine to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd -groups, and where Raa, R ^bb , R ^cc and R ^dd are defined above.

In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to,

-HE,

-OR ^aa , -N(R ^cc )2, -C(=O)R ^aa , -C(=O)N(R ^cc )2, -CO ₂ R ^aa , -SO2R ^aa , -C(=NR ^cc )R ^aa , C(=NR ^cc )OR ^aa , -C(=NR ^cc )N(R ^cc )2, -SO ₂ N(R ^cc )2, -SO2R ^cc , -SO2OR ^cc , -SOR ^aa , C (=S)N(R ^cc ) ₂ , -C(=O)SR ^cc , -C(=S)SR ^cc ,

C _1-10 αkyl (e.g. aralkyl, heteroaralkyl), C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl groups, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups, and where R ^aa , R ^bb , R ^cc and R ^dd are defined in this document. Nitrogen protecting groups are well known in the art and include those detailed in Protecting Groups in Organic Synthesis, T. W. Greene and PGM Wuts, ^3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups, e.g., amide groups (e.g., -C(=O)R ^aa ) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl- 2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o(benzoyloxymethyl )benzamide.

Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR ^aa ) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2 ,7-dibromo)fluorenylmethylcarbamate, 2,7-di-tert-butylmethylcarbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teos), 2 -phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-mpem-BOC), 1, 1-dimethyl-2,2,2-trichloroethylcarbamate (TSBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate (Bros), 1-(3,5-di-tert-butylphenyl)-1methylethylcarbamate (t-Bumeoc ), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyos), 2-(N,N-dicyclocarboxamido) ethyl carbamate, tert-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidine ylcarbamate, alkyldithiocarbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p-nitrobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate , 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Reos), 2-triphenylphosphonioisopropylcarbamate (Ploc), 1,1-dimethyl -2-cyanoethylcarbamate, v-chloro-n-acyloxybenzylcarbamate, p(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, tert-amylcarbamate, S-benzylthiocarbamate, n-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, n-decylox ibenzylcarbamate, 2,2-dimethoxyacylvinylcarbamate, o(N,N-dimethylcarboxαmido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2 -furanylmethylcarbamate, 2-14 040020 iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1cyclopropylmethylcarbamate, 1-methyl-1-(3,5- dimethoxyphenyl) thylcarbamate, 1-methyl-1-(p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl) ethyl carbamate, phenyl carbamate, p(phenylazo) benzyl carbamate, 2.4, 6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups, for example, sulfonamide groups (for example, -S(=O) ₂ R ^aa ) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr ), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3- oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4 tetramethyldisylylasecyclopentane adduct (STABASE), 5-substituted 1, 3-dimethyl-1,3,5-triazacyclohexan-2one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, Nmethylamine, N-allylamine, N-methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N- di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N -ferrocenylmethylamino (Fcm), N-2-picolilamino-N'oxide, K-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-n-methoxybenzylide enamine, N-diphenylmethyleneamine, N-methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro -2-hydroxyphenyl) phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylboric acid derivative, N-amine, N-copper chelate, Zinc N-chelate, Nnitroamine, N-nitrosoamine, amine-N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkylphosphoramidates, dibenzylphosphoramidate, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2.4 -dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide and 3-nitropyridine sulfenamide (Npys).

In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to,

-R ^aa , -N(R ^bb )2, -C(=O)SR ^aa , -C(=O)R ^aa , -CO2R ^aa , -C(=O)N(R ^bb ) ₂ , -

C(=NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa , -C(=NR ^bb )N(R ^bb )2, -S(=O)R ^aa , -SO2R ^aa , -Si(R ^aa )3, -P(R ^CC )2, P(R ^CC )3, -P(=O)2R ^aa , -P(=O)(R ^aa )2, -P(=O)(OR ^cc ) 2, -P(=O)2N(R ^bb )2 and -P(=O)(NR ^bb ) ₂ , where R ^aa ,

R ^bb and R ^cc are defined herein. Oxygen protecting groups are well known in the art and include those detailed in Protecting Groups in Organic Synthesis, T. W. Greene and PGM Wuts, ^3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy )methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2 -(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-4-methoxypiperidine- 4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7 -methanobenzofuran-2-yl, 1ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1benzyloxy-2-fluoroethyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenium yl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halogenobenzyl, 2 ,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl-N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, n-methoxyphenyldiphenylmethyl,

- 15 040020 di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4' ,4-tris(levulinoyloxyphenyl)methyl, 4,4',4tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl) -1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl-S,S-dioxudo, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-nxylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (lev ulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenyl benzoate, 2,4,6-trimethyl benzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Reos), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl-n-methoxubenzyl carbonate , alkyl-3,4-dimethoxybenzyl carbonate, alkyl-o-nitrobenzyl carbonate, alkyl-p-nitrobenzyl carbonate, alkyl-S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyldithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4methylpentanoate , o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6dichloro-4-(1 ,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate tate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl-N,N,N',N'-tetramethylphosphorodiamidate, alkyl-H-phenyl carbamate , borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to,

-R ^aa , -N(R ^bb )2, C(=O)SR ^aa , -C(=O)R ^aa , -CO2R ^aa , -C(=O)N(R ^bb )2, -C(= NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa , C(=NR ^bb )N(R ^bb )2, -S(=O)R ^aa , -SO ₂ R ^aa , -Si(R ^aa ) 3, -P(R ^CC )2, -P(R ^CC ) ₃ , -P(=O) ₂ R ^aa , P(=O)(R ^aa ) ₂ , -P(=O)(OR ^cc )2 , -P(=O)2N(R ^bb )2 and -P(=O)(NR ^bb ) ₂ , where R ^aa , R ^bb , and R ^cc are defined herein. Sulfur protecting groups are well known in the art and include those detailed in Protecting Groups in Organic Synthesis, T. W. Greene and PGM Wuts, ^3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

As used herein, a leaving group is an expression understood in the art referring to a molecular moiety that leaves with an electron pair in a heterolytic bond cleavage, where the molecular moiety is an anion or a neutral molecule. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving groups include, but are not limited to, halogens (eg, chlorine, bromine, iodine) and sulfonyl-substituted hydroxyl groups (eg, tosyl, mesyl, besyl).

These and other illustrative substituents are described in more detail in the detailed description, examples, figures, and claims. It is contemplated that the present invention is not limited in any way by the above illustrative list of substituents.

Other definitions

As used in this document, the use of the phrase at least one representative refers to one representative, but also covers two or more representatives, for example, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 representatives and up to 100 representatives.

Amino acid refers to D/L alpha amino acids of natural and synthetic origin, as well as beta and gamma amino acids of natural and synthetic origin. A peptide refers to two amino acids connected by a peptide bond. A polypeptide refers to three or more amino acids linked by peptide bonds. An amino acid side chain refers to the group(s) dependent on the alpha carbon atom (in the case of an alpha amino acid), the alpha and beta carbon atom (in the case of a beta amino acid), or the alpha, beta and gamma carbon atom (in the case of gamma amino acids). Exemplary amino acid side chains are depicted herein; see, for example, table 1 in the examples.

As used herein, a polymer refers to a compound containing at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating entities. linked by a covalent bond.

Conjugated and attached refers to the addition of a group by a covalent bond,

- 16 040020 and they are used interchangeably in this document.

As used herein, lipophilic refers to the ability of a group to dissolve in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene. Typically, a lipophilic group refers to an unsubstituted n-alkyl or unsubstituted alkenyl group having 6 to 50 carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19 , 8 to 18, 8 to 17, 8 to 16 or 8 to 15 carbon atoms.

The use of the terms structural isomer, organic molecule, and inorganic molecule is intended to encompass the conventional meaning of each expression as is known in the art.

As used herein, a small organic molecule or small molecule refers to an organic molecule with a molecular weight of 800 g/mol or less (e.g., less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than 400 g/mol). mol, less than 300 g/mol, less than 200 g/mol, less than 100 g/mol, from 50 to 800 g/mol inclusive, from 100 to 800 g/mol inclusive, or from 100 to 500 g/mol inclusive). In certain embodiments, the small organic molecule is a therapeutically active agent such as a drug (e.g., a small organic molecule approved by the Food and Drug Administration as provided for in the Code of Federal Regulations (CFR)). A small organic molecule can also form a complex compound with a metal. In such a case, the small organic molecule is also called the small organometallic molecule.

As used herein, an organic macromolecule or macromolecule refers to an organic compound with a molecular weight greater than 800 g/mol (e.g., greater than 800 g/mol, greater than 900 g/mol, greater than 1000 g/mol, greater than 2000 g/mol, from 801 to 2000 g/mol inclusive, from 900 to 2000 g/mol inclusive, from 1000 to 2000 g/mol inclusive, or from 801 to 1000 g/mol inclusive). In certain embodiments, the organic macromolecule is a therapeutically active agent, such as a drug (eg, an organic macromolecule approved by the Food and Drug Administration as provided for in the Code of Federal Regulations (CFR)). An organic macromolecule can also form a complex compound with a metal. In such a case, the organic macromolecule is also called an organometallic macrocompound.

As used herein, a small inorganic molecule refers to an inorganic compound with a molecular weight of 800 g/mol or less (e.g., less than 700 g/mol, less than 600 g/mol, less than 500 g/mol, less than 400 g/mol, less 300 g/mol, less than 200 g/mol, less than 100 g/mol, from 50 to 800 g/mol inclusive, from 100 to 800 g/mol inclusive, or from 100 to 500 g/mol inclusive). In certain embodiments, the small inorganic molecule is a therapeutically active agent such as a drug (e.g., a FDA-approved small inorganic molecule as provided in the Code of Federal Regulations (CFR)).

As used herein, an inorganic macromolecule refers to an inorganic compound with a molecular weight greater than 800 g/mol (e.g., greater than 800 g/mol, greater than 900 g/mol, greater than 1000 g/mol, greater than 2000 g/mol, from 801 to 2000 g/mol inclusive, from 900 to 2000 g/mol inclusive, from 1000 to 2000 g/mol inclusive, or from 801 to 1000 g/mol inclusive). In certain embodiments, the inorganic macromolecule is a therapeutically active agent, such as a drug (eg, an inorganic macromolecule approved by the Food and Drug Administration as provided for in the Code of Federal Regulations (CFR)).

As used herein, the term "salt" or "pharmaceutically acceptable salt" refers to those salts that are medically suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic reaction, or the like, and correspond to an acceptable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from acceptable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid. acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods used in the art, such as ion exchange. Другие фармацевтически приемлемые соли включают адипатные, альгинатные, аскорбатные, аспартатные, бензолсульфонатные, бензоатные, бисульфатные, боратные, бутиратные, камфоратные, камфорсульфонатные, цитратные, циклопентанпропионатные, диглюконатные, додецилсульфатные, этансульфонатные, формиатные, фумаратные, глюкогептонатные, глицерофосфатные, глюконатные, гемисульфатные, гептаноат

- 17 040020 naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectin, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C ₁ _ ₄ alkyl) ₄ salts. Exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, if necessary, non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and arylsulfonate. Additional pharmaceutically acceptable salts include salts formed by quaternization of an amine with a suitable electrophile, such as an alkyl halide, to form a quaternary alkylated amine salt.

Brief description of graphic materials

In FIG. 1 depicts structure design and optimization using in vivo evaluation in mice. Single amino acid lipid derivatives were tested at 1 mg/kg in mice, with lysine being the most preferred amino acid. Lysine peptide and polypeptide lipid derivatives were then tested at the same dose. The match rate improved from 1.7% to 23% (including those compounds not screened due to particle instability or lack of siRNA uptake). whereupon sKK-E12 was chosen as the prototype compound. K-E12; K: abbreviation for lysine, E: epoxide, A: aldehyde, O: acrylate, 12: carbon tail length. sKK-E12; c: cyclic; Control, phosphate-buffered saline.

In FIG. 2 shows the biodistribution of free Cy5.5-labeled siRNA and Cy5.5-labeled siRNA-cKK-E12 composition in mice at 1 and 24 h.

In FIG. 3 shows the effect of apolipoproteins on cKK-E12 silencing in HeLa cells. Apolipoproteins included ApoA-I (recombinant human ApoA-I protein), ApoA-II (native human ApoA-II protein), ApoB (native human ApoB protein), ApoC-I (native human ApoC-I protein), ApoC-II ( native human ApoC-II protein), ApoC-III (native human ApoC-III protein), ApoE (native human ApoE protein), ApoE2 (recombinant human ApoE2 protein), ApoE3 (recombinant human ApoE3 protein), ApoE4 (recombinant human ApoE4 protein ), ApoH (native human protein ApoH).

In FIG. 4 shows the effect of ApoE on gene silencing and cell uptake. A). Effect of ApoE on silencing with cKK-E12, cKK-A12 and cKK-O12 in vitro (siRNA: 50 ng/well). When ApoE was added, the effect on silencing was in the following order: cKK-E12 > cKK-A12 > cKK-O12, which was in good agreement with in vivo activity. IN). Internalization of cKK-E12 with Alex-647 labeled siRNA by the cell after 3 h of incubation is shown by HT automated confocal microscopy. ApoE improved cellular uptake and release of hCK-E12 from endosomes; scale bar: 20 µm.

Detailed Description of Certain Embodiments of the Present Invention

This document describes the compounds and compositions of the present invention, certain embodiments of which include the conjugation of various groups, such as lipophilic groups, with the amino group or amide group of an amino acid, a linear or cyclic peptide, a linear or cyclic polypeptide, or their structural isomer to obtain compounds of the present invention, collectively referred to herein as APPL. Such APPLs are believed to be useful for a variety of applications such as, for example, improved nucleotide delivery.

In one aspect, a compound of the formula is provided

or its salt;

where Q is O;

each R ¹ is independently -H, -CH ₃ , -CH(CH ₃ ) ₂ , -CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ CH(CH ₃ ) ₂ ,

WVW

- 18 040020

or a group of formula (iv), provided that at least one representative of R ¹ is a group of formula

R ^{6 R?} (iv)

L is an unsubstituted C _1-50 alkylene;

R ⁶ and R ⁷ each independently represents hydrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii);

each R ² is independently hydrogen or a group of formula (iii);

formula (iii) is

where RL is C _6-20 alkyl optionally substituted with one or more fluoro substituents.

In certain embodiments, at least one of R ² is hydrogen.

In certain embodiments, at least one representative of R ² is a group of formula (iii).

In certain embodiments, each R ¹ is a group of formula (iv).

In certain embodiments, the group of formula (iv) is the formula:

where q is an integer from 1 to 50 inclusive. In certain embodiments, the compound is selected from the group consisting of

and its salts, where p is 1; and each R ¹ is independently -H, -CH ₃ , -CH(CH ₃ ) ₂ , CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ CH(CH ₃ ) ₂ ,

- 19 040020

where R ⁶ and R ⁷ each independently represents hydrogen, a protective group for nitrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii); and provided that at least one R ¹ is a group of formula:

In certain embodiments, the compound is selected from the group consisting of:

and its salts, where p is 1; and provided that at least one representative of R ² represents a group of formula (iii).

- 20 040020

In certain embodiments, the connection is

In yet another aspect, a composition is provided for delivering a polynucleotide to a cell, comprising a compound according to any one of claims 1-8, or a salt thereof, and a polynucleotide.

In certain embodiments, the composition is a pharmaceutical composition.

In certain embodiments, the composition further comprises cholesterol.

In certain embodiments, the composition further comprises a PEGylated lipid.

In certain embodiments, the composition further comprises a phospholipid.

In certain embodiments, the composition further comprises an apolipoprotein.

In certain embodiments, the polynucleotide is DNA.

In certain embodiments, the polynucleotide is RNA.

In certain embodiments, the RNA is RNA/, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

In certain embodiments, the composition is in the form of a particle.

In certain embodiments, the implementation of the particle is a nanoparticle or microparticle.

In certain embodiments, the particle is a micelle, liposome, or lipoplex.

In certain embodiments, the particle encapsulates a polynucleotide.

In yet another aspect, the composition is used to treat a disease, disorder, or condition from which a subject suffers, wherein the disease, disorder, or condition is selected from the group consisting of proliferative disorders, inflammatory disorders, autoimmune disorders, and liver diseases.

In yet another aspect, the use of a compound or salt thereof and a polynucleotide is contemplated in the manufacture of a pharmaceutical composition for delivering the polynucleotide to a cell.

In certain embodiments, the polynucleotide is DNA.

In certain embodiments, the polynucleotide is RNA.

In certain embodiments, the RNA is RNA/, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

In certain embodiments, the polynucleotide is RNA, and when the RNA is delivered to a cell, the RNA is able to interfere with the expression of a particular gene in a biological cell.

In certain embodiments, the implementation of the polynucleotide encodes a protein or peptide.

In certain embodiments, the implementation of the polynucleotide encodes a protein or peptide.

Various ^RL groups, for example lipophilic groups, can be attached to APPL by conjugation of the primary or secondary amino group or amide group of an amino acid, peptide or polypeptide precursor or structural isomer thereof, with an epoxide, thiirane or aziridine of formula (ix), Michael attachment to an α,β-unsaturated ester, thioester or amide of formula (ii-x), or reductive amination to an aldehyde of formula (iii-x) (Scheme 1).

Scheme 1

- 21 040020

Thus, in its broadest aspect, the present invention provides APPL, and in certain embodiments, compounds of the formula

^{R2 R1}

NQ=/

^R1

Various embodiments of the formula / ^Q

-n\ ²

are described in more detail in this document.

Thus, in one aspect, a compound of the formula is provided:

or its salt;

where Q is O;

each R1 is independently -H, -CH3, -CH( _CH3 ) ₂ , -CH( _CH3 )( _CH2CH3 ), -CH2CH(CH3) ₂ ,

or a group of formula (iv), provided that at least one representative of R1 is a group of formula:

NG

L-N

^R7 ( _iv );

L is an unsubstituted C _1-50 alkylene;

R ⁶ and R ⁷ each independently represents hydrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii);

each R ² is independently hydrogen or a group of formula (iii); formula (iii) is

where RL is C _6-20 alkyl optionally substituted with one or more fluoro substituents.

In certain embodiments, at least one of R ² is hydrogen.

In certain embodiments, at least one representative of R ² is a group of formula (iii).

In certain embodiments, each R1 is a group of formula (iv).

In certain embodiments, the group of formula (iv) is the formula

where q is an integer from 1 to 50 inclusive. In certain embodiments, the compound is selected from the group consisting of

- 22 040020

and its salts, where p is 1; and each R ¹ is independently -H, -CH ₃ , -CH(CH ₃ ) ₂ , -CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ CH(CH ₃ ) ₂ ,

where R ⁶ and R ⁷ each independently represents hydrogen, a protective group for nitrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii); and provided that at least one R ¹ is a group of formula:

r ⁷ ' ⁿ r ⁶

In certain embodiments, the compound is selected from the group consisting of

-23 040020 and salts thereof, where p is 1; and provided that at least one representative of R ² represents a group of formula (iii).

In certain embodiments, the connection is:

In yet another aspect, a composition is provided for delivering a polynucleotide to a cell, comprising a compound according to any one of claims 1-8, or a salt thereof, and a polynucleotide.

In certain embodiments, the composition is a pharmaceutical composition.

In certain embodiments, the composition further comprises cholesterol.

In certain embodiments, the composition further comprises a PEGylated lipid.

In certain embodiments, the composition further comprises a phospholipid.

In certain embodiments, the composition further comprises an apolipoprotein.

In certain embodiments, the polynucleotide is

DNA.

In certain embodiments, the polynucleotide is RNA.

In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

In certain embodiments, the composition is in the form of a particle.

In certain embodiments, the implementation of the particle is a nanoparticle or microparticle.

In certain embodiments, the particle is a micelle, liposome, or lipoplex.

In certain embodiments, the particle encapsulates a polynucleotide.

In yet another aspect, the composition is used to treat a disease, disorder, or condition from which a subject suffers, wherein the disease, disorder, or condition is selected from the group consisting of proliferative disorders, inflammatory disorders, autoimmune disorders, and liver diseases.

In another aspect, the use of a compound or salt thereof and a polynucleotide is contemplated in the manufacture of a pharmaceutical composition for delivering the polynucleotide to a cell.

In certain embodiments, the polynucleotide is DNA.

In certain embodiments, the polynucleotide is RNA.

In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA.

In certain embodiments, the polynucleotide is RNA, and upon delivery of the RNA to a cell, the RNA is capable of interfering with the expression of a particular gene in a biological cell.

In certain embodiments, the implementation of the polynucleotide encodes a protein or peptide.

In certain embodiments, the implementation of the polynucleotide encodes a protein or peptide.

Scheme 2

In certain embodiments, the epoxide, thiirane, or aziridine of formula (i-x) is chiral, i. characterized by (R) or (S)-stereochemistry. Chiral epoxides, thiiranes and aziridines can be obtained from a variety of sources that are well known to those skilled in the art of organic synthesis. In some embodiments, the chiral epoxide, thiirane, or aziridine is purchased from a manufacturer. In some embodiments, the chiral epoxide, thiirane, or aziridine is synthesized according to methods known to those skilled in the art, such as, but not limited to, Sharpless epoxidation of primary and secondary allyl alcohols to 2,3-epoxy alcohols (see, for example, Katsuki et al., J. Am. Chem. Soc. 1980, 102, 5974; Hill et al., Org. Syn., Coll. Vol. 7, p. 461 (1990), Vol. 63, p. 66 (1985 ); Katsuki et al, Org. React. 1996, 48, 1-300). In some embodiments, the implementation of the chiral epoxide, thiirane or aziridine is obtained by separation

- 24 040020 mixtures (eg racemic mixture) of epoxides, thiirans or aziridines. In some embodiments, the chiral epoxide, thiirane, or aziridine is obtained by separating enantiomers or diastereoisomers using chiral chromatography. Chirality can be assessed in various ways, for example, obtaining a crystal structure of a compound containing a heavy atom attached thereto, obtaining an optical rotation of a compound, and/or NMR analysis after chemical modification of an optically active compound with a chiral derivatizer are some methods suitable for chirality estimates.

In certain embodiments where the epoxide, thiirane, or aziridine of formula (i-x1) is chiral, the conjugation reaction is regioselective and the reaction produces a chiral mixture of APPL containing more APPL conjugated to a group of formula (i-a1) than formula (ib1), for example, the reaction mixture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 95% to about 100%, or from about 99% to about 100% of APPL combined with formula (i-a1) .

In other embodiments, where the epoxide, thiirane, or aziridine of formula (i-x2) is chiral, the conjugation reaction is regioselective and the reaction produces a chiral mixture of APPL containing more APPL conjugated to a group of formula (i-a2) than formula (i-b2), for example, the reaction mixture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, from about 60% to about 100%, from about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, or about 99% to about 100% APPL combined with formula (i- a2).

In certain embodiments, APPL is isolated as a salt. For example, in certain embodiments, APPL is reacted with an acid (eg, an organic acid or an inorganic acid) to form the corresponding salt. In other embodiments, tertiary amines are alkylated to form the quaternary ammonium salt APPL. Tertiary amines can be alkylated with any alkylating agent, for example alkyl halides such as methyl iodide can be used to form quaternary amino groups. The anion associated with the quaternary amine may be any organic or inorganic anion. In certain embodiments, the anion is a pharmaceutically acceptable anion.

The present invention also provides APPL libraries obtained using the methods of the present invention. For example, in certain embodiments, a method for screening a library of compounds is provided, the method comprising: obtaining a plurality of different APPLs or salts thereof; and conducting at least one study with a library of compounds to determine the presence or absence of the desired property. These APPLs can be generated and/or screened using high throughput methods including fluid dispensers, robots, microtiter plates, computers, and the like. In certain embodiments, APPLs are screened for their ability to transfect polynucleotides or other agents (eg, proteins, peptides, small molecules) into a cell. For example, in one embodiment, a method is provided for screening a library of compounds, the method comprising obtaining a plurality of two or more different APPLs and screening the library of compounds for a desired property.

In one embodiment, a library of different APPLs is obtained in parallel. A different precursor and/or conjugating reagent is added to each vial in a vial set or to each well of a multiwell plate that is used to prepare a library. The set of reaction mixtures are incubated at a temperature and for a period of time sufficient to ensure the formation of APPL. APPL can then be isolated and purified using methods known in the art. The APPL can then be screened using high throughput methods to detect an APPL with a desired property, for example, where the desired property is water solubility, solubility at various pH values, polynucleotide binding capacity, heparin binding capacity, small molecule binding capacity, protein binding, ability to form microparticles, ability to increase transfection efficiency, ability to support cell growth, ability to support attachment

- 25 040020 cell regeneration, the ability to support tissue growth, and/or the intracellular delivery of APPL and/or an agent complexed with or attached to it to aid biotechnology, for example for the production of proteins. In certain embodiments, APPL can be screened for properties or characteristics required for coatings, additives, materials and excipients for biotechnology and biomedical applications, such as coating medical devices or implants with films or multilayer films as antifouling agents, means for forming a microstructure; and means for encapsulating the cell. In certain embodiments, APPL can be screened for properties or characteristics required in gene therapy (e.g., the ability to bind polynucleotides and/or increase transfection efficiency), biotechnology (e.g., promote intracellular protein production), or administration and/or delivery of a therapeutic agent ( eg, polynucleotide, small molecule, antigen, drug, protein, peptide, etc.) in a subject, tissue, organ, or cell.

Exemplary compounds of the present invention

This document specifically refers to certain compounds of the present invention. For example, compounds containing unsubstituted alkyl RL groups containing 8, 9, 10, 11, 12, 13 and 14 carbon atoms are specifically contemplated. In certain embodiments, R ¹ of such compounds is an amino acid side chain as defined in Table 1 in the examples.

Illustrative amino acid, peptide and polypeptide compounds of formula (I) include, but are not limited to:

- 26 040020 about

and THEIR salts.

Exemplary cyclized compounds of formula (II) include, but are not limited to:

-27040020 and their salts.

Illustrative cyclic dipeptide and cyclic polypeptide compounds of formula (III) include, but are not limited to:

- 28 040020

and in particular the cyclic-KK and polycyclic lysine APPL formulas: but

- 29 040020

- 30 040020

- 31 040020

and their salts.

Compositions

The present invention provides APPL as a component of the composition. For example, in certain embodiments, a composition is provided comprising APPL or a salt thereof and an excipient, wherein APPL is an amino acid, a linear or cyclic peptide, or a linear or cyclic polypeptide, or a structural isomer thereof, and wherein the amino group or amide group of APPL is conjugated to the group formulas (i), (ii) or (iii). In certain embodiments, a group of formula (i), (ii), or (iii) is attached to an amino group located on the APPL backbone.

The compositions described herein containing APPL and any excipient may be suitable for a variety of medical and non-medical applications. For example, pharmaceutical compositions containing APPL and an excipient may be useful in delivering an effective amount of the agent to a subject in need thereof. Nutraceutical compositions containing APPL and an excipient may be useful in delivering an effective amount of a nutraceutical, such as a dietary supplement, to a subject in need thereof. Cosmetic compositions containing APPL and an excipient may be formulated as a cream, ointment, balm, paste, film or liquid, etc., and may be useful in the application of cosmetics, hair products, and as personal care materials. hygiene, etc. Compositions containing APPL and an excipient may be useful in non-medical applications, for example, as an emulsion or emulsifier, useful, for example, as a food ingredient, fire fighting, surface disinfection, oil cleaning, etc.

Peptides play important roles in endogenous cellular signaling and substance translocation pathways and offer great potential for maximizing these interactions to enhance delivery efficiency in systems incorporating peptide fragments. Thus, compositions containing APPL and an excipient may also be useful in biotechnology, such as cellular biotechnology of economically important chemicals or fuels. For example, intracellular delivery of APPL or an agent complexing it may be useful in biotechnology by maintaining cell viability and/or growth, for example, in the production of proteins.

A composition may contain one type of APPL, but may also contain any number of times

- 32 040020 personal APPL types, eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different APPL types.

In certain embodiments, the composition further comprises an agent as described herein. For example, in certain embodiments, the agent is a small molecule, an organometallic compound, a nucleic acid, a protein, a peptide, a polynucleotide, a metal, a targeting agent, an isotopically labeled chemical, a drug, a vaccine, an immunological agent, or an agent useful in biotechnology. In certain embodiments, the agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA or RNA. In certain embodiments, the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. In certain embodiments, the implementation of the polynucleotide and one or more APPL are not connected by a covalent bond.

In certain embodiments, one or more APPLs are in particulate form. In certain embodiments, the implementation of the particle is a nanoparticle or microparticle. In certain embodiments, one or more APPLs are in the form of liposomes or micelles. It is understood that, in certain embodiments, such APPLs self-assemble to form a particle, micelle, or liposome. In certain embodiments, the particle, micelle, or liposome encapsulates the agent. The agent to be delivered by the particle, micelle or liposome may be in the form of a gas, liquid or solid. APPL can be combined with polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. with the formation of particles. These particles can be further combined with an excipient to form a composition.

Excipients include any and all solvents, diluents or other liquid excipients, dispersion or suspension forming aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as selected in depending on the specific dosage form required. General formulation and/or manufacturing considerations can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjunct of any type. Some examples of materials that can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffer substances such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solution, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, preservatives and antioxidants may also be present in the composition, based on decisions of the specialist involved in the development of the composition. As will be understood by a person skilled in the art, excipients can be selected based on the purpose for which the composition is suitable. For example, in the case of a pharmaceutical composition or a cosmetic composition, the choice of excipient will depend on the route of administration, the agent being delivered, the time period for the agent to be delivered, etc., and it can be administered to humans and/or animals orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (eg, with powders, creams, ointments, or drops), buccally, or as an oral or nasal spray.

Illustrative diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dicalcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch , powdered sugar, etc., as well as combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, cassava starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, crosslinked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surfactants and/or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, Chondrux extract, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (eg, bentonite [aluminosilicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, macromolecular alcohols (eg, stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol ), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), fatty acid esters of sorbitol (e.g., By lyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), esters of polyoxylene , polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Solutol), sucrose fatty acid esters, fatty acid esters of polyethylene glycol (eg Cremophor), polyoxyethylene ethers (eg polyoxyethylene lauryl alcohol ether [Brij 30]), polyvinylpyrrolidone, diethylene glycol monolaurate, triethanolaminoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, etc. ., and/or combinations thereof.

Exemplary binders include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., gum arabic, sodium alginate, irish moss extract, panwar gum, ghatti gum, psyllium husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone, magnesium aluminum silicate (Veegum), and arabinogalactan alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and/or combinations thereof.

Illustrative preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerin, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium matabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, disodium calcium edetate, dipotassium edetate, and the like), citric acid and its salts and hydrates (e.g., citric acid monohydrate). acids), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Illustrative antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chlorxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal.

Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Illustrative alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroxymmesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium matabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

- 34 040020

Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid. , calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, bicarbonate sodium, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic solution, Ringer's solution, ethyl alcohol, etc. ., as well as their combinations.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl beganate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., as well as their combinations.

Exemplary natural oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, juniper berry oil, chamomile oil, canola oil, cumin oil, carnauba oil, castor oil, cinnamon oil , cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol, gourd oil, grape seed oil, hazelnut oil, oil Hyssopa, Isopropyl Myristate, Jojoba Oil, Lacquer Fruit Oil, Lavandin Oil, Lavender Oil, Lemon Oil, Cubeba Litzeum Oil, Macadamia Nut Oil, Mallow Oil, Mango Seed Oil, Meadowfoam Seed Oil, Mink Oil, nutmeg, olive oil, orange oil, icelandic beryx fat, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil butter, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, Sasquana oil, savory oil, sea buckthorn oil, sesame oil, shea seed oil, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver oil, walnut oil and wheat germ oil. Illustrative synthetic oils include, without limitation, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Additionally, the composition may contain a phospholipid. Illustrative phospholipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dilauroyl-sn-glycero-3-phosphocholine (dilauroylphosphatidylcholine, DLPC), 1,2-dimyristoyl-sn-glycero3-phosphocholine (dimyristoylphosphatidylcholine, DMPC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (dipentadecanoylphosphatidylcholine, DPDPC), 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (dipalmitoylphosphatidylcholine, DPPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-myristoyl-2palmitoylphosphatidylcholine, MPPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac- (1-glycerol)] (DMPG) and 1,2-dimyristoyl-3-trimethylammonium propane.

In addition, the composition may further contain a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulose-based polymers and copolymers, e.g., cellulose ethers such as methyl cellulose (MC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methyl hydroxyethyl cellulose (MHEC), methyl hydroxypropyl cellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, for example, sodium salt, hydroxyethyl carboxymethyl cellulose (HCMC) and its various salts, carboxymethyl hydroxyethyl cellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan and alginic acid and its various salts, carrageenan, various gums including xanthan gum, guar gum, gum arabic, karaya gum, ghatti gum, konjac gum and tragacanth gum, glycosaminoglycans and proteoglycans , such as hyaluronic acid and its salts, proteins such as gel atin, collagen, albumin and fibrin, other polymers such as polyhydroxy acids such as polylactide, polyglycolide, lactide-glycolide copolymer and epsiloncaprolactone-glycolide copolymer, carboxyvinyl polymers and their salts (e.g. carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid/acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, ethylene oxide-propylene oxide copolymer and Pluronic polymer, polyoxyethylene (polyethylene glycol), polyanhydrides, polyvinyl alcohol, polyethyleneamine and polypyridine, polyethylene glycol (PEG) based polymers, such as PEGylated lipids (e.g. PEG-stearate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-K-[methoxy(polyethylene glycol)-1000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye

- 35 040020 tylenglycol)-5000]), their copolymers and salts.

In addition, the composition may further contain an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, poly-K-vinylpyrrolidone and their copolymers, poloxamer non-ionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, ficoll, celluloses), non-cationic polymethacrylates, non-cationic polyacrylates , such as poly(meth)acrylic acid, and ester amides and their hydroxyalkylamides, natural emulsifiers (e.g. gum arabic, agar, alginic acid, sodium alginate, tragacanth, Chondrux extract, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (eg, bentonite [aluminosilicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (eg, stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomer s (for example, carboxypolymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulose derivatives (for example, sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), fatty acid esters of sorbitol (for example, polyoxyethylene sorbitan monolaurate [ Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), esters of polyoxyethylene [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Solutol), sucrose fatty acid esters, fatty acid esters of polyethylene glycol (e.g. Cremophor), polyoxyethylene ethers (e.g. polyoxyethylene ether lauryl alcohol [Brij 30]), polyvinylpyrrolidone, diethylene glycol monolaurate, triethanolaminoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, and sodium docusate .d., and/or combinations thereof. In certain embodiments, the emulsifying agent is cholesterol.

In addition, the composition may further comprise an apolipoprotein. Previous studies reported that apolipoprotein E (ApoE) was able to increase cellular uptake and gene silencing for certain types of materials. See, for example, Akinc, A., et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7): p. 1357-64. In certain embodiments, the apolipoprotein is ApoA, ApoB, ApoC, ApoE, or ApoH, or isoforms thereof.

Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups and tinctures. In addition to APPL, the liquid composition may contain inert diluents widely used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol, as well as their mixtures. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and flavoring agents.

Injectable compositions, for example injectable aqueous or oily suspensions, can be formulated according to the known art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Suitable excipients and solvents that can be used in pharmaceutical or cosmetic formulations include water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile fixed oils are traditionally used as a solvent or suspending medium. Any flavorless fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable dosage forms. In certain embodiments, the particles are suspended in a carrier fluid containing 1% (w/v) sodium carboxymethylcellulose and 0.1% (v/v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Compositions for rectal or vaginal administration may be in the form of suppositories, which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax that is solid at ambient temperature, but liquid at body temperature and thus melts in the rectum or vaginal cavity and releases particles.

Solid compositions include capsules, tablets, pills, powders and granules. In such solid compositions, the particles are mixed with at least one excipient and/or a) fillers or dry diluents such as starches, lactose, sucrose, glucose, mannitol and silicic acid,

b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and gum arabic,

c) wetting agents such as glycerin,

d) tablet disintegrating agents such as agar-agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retardants such as paraffin,

f) absorption accelerators such as quaternary ammonium compounds,

g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate,

h) sorbents such as kaolin and bentonite clays, and

i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof.

In the case of capsules, tablets and pills, the dosage form may also contain buffer substances. Solid compositions of a similar type can also be used as fillers in hard and soft filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of drug formulation. They may optionally contain opacifying agents and may also be formulated such that they release the active ingredient(s) only, or preferably in a certain part of the intestine, optionally with a delayed release. Examples of capping compositions that can be used include polymeric materials and waxes.

Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.

Compositions for topical or transdermal use include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The APPL is admixed with the excipient and any necessary preservative or buffer that may be required. Ophthalmic composition, ear drops and eye drops are also contemplated as being within the scope of the present invention.

Ointments, pastes, creams and gels other than APPL may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof .

Powders and sprays other than APPL may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and powdered polyamide, or mixtures of these substances. Sprays may additionally contain standard propellant gases such as chlorofluorocarbons.

Transdermal patches have the added benefit of providing controlled delivery of a compound to the body. Such dosage forms can be obtained by dissolving or dispersing microparticles or nanoparticles in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate can be controlled either by providing a rate controlling membrane or by dispersing the particles in a polymer matrix or polymer gel.

Facilities

The agents to be delivered by the systems described herein may be therapeutic, diagnostic, or prophylactic agents. Any chemical compound that needs to be administered to a subject can be delivered using the complexes, picoparticles, nanoparticles, microparticles, micelles, or liposomes described herein. The agent may be an organic molecule (e.g., therapeutic agent, drug), an inorganic molecule, a nucleic acid, a protein, an amino acid, a peptide, a polypeptide, a polynucleotide, a targeting agent, an isotopically labeled organic or inorganic molecule, a vaccine, an immunological agent, etc. .

In certain embodiments, the agents are organic molecules with pharmaceutical activity, such as a drug. In certain embodiments, the drug is an antibiotic, antiviral, anesthetic, steroid, anti-inflammatory, antineoplastic, anti-inflammatory.

- 37 040020 cancer, antigen, vaccine, antibody, decongestant, antihypertensive drug, sedative, contraceptive, pregnancy aid, anticholinergic, analgesic, antidepressant, drug that reduces the frequency, duration and manifestation of psychotic episodes, R -adrenergic blocking agent, diuretic, cardiovascular active agent, vascular drug, non-steroidal anti-inflammatory agent, nutrient, etc.

In certain embodiments of the present invention, the agent to be delivered may be a mixture of agents.

Diagnostic tools include gases; metals; commercially available imaging tools used in positron emission tomography (PET), computed tomography (CAT), single photon emission computed tomography, radiography, fluoroscopy and magnetic resonance imaging (MRI); as well as contrast media. Examples of suitable substances for use as MRI contrast agents include gadolinium chelates, as well as iron, magnesium, manganese, copper and chromium. Examples of substances suitable for CAT and X-ray examination include iodine-containing substances.

Therapeutic and prophylactic agents include, but are not limited to, antibiotics, dietary supplements, and vaccines. Vaccines may include isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically modified organisms and viruses, and cell extracts. Therapeutic and prophylactic agents can be combined with interleukins, interferon, cytokines and adjuvants such as cholera exotoxin, alum, Freund's adjuvant, etc. Prophylactic agents include antigens of such bacterial organisms as Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeugae, Streptococcus mutans , Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, etc.; antigens of viruses such as variola virus, influenza A and B, respiratory syncytial virus, parainfluenza virus, measles virus, HIV, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus , adenovirus, papillomavirus, polio virus, mumps virus, rabies virus, rubella virus, coxsackie virus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A, B, C, D and E and etc.; antigens of fungi, protozoa and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasm falciparum, Trypanosoma brucei, Entamoeba histolytica gondii, Trichomonas vaginalis, Schistosoma mansoni, etc. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

Targeting agents

Because it is often necessary to target a specific cell, cell aggregation, or tissue, APPL, as well as complexes, liposomes, micelles, microparticles, picoparticles, and nanoparticles made therewith, can be modified to include targeting agents or targeting sites. For example, the APPL backbone may include a targeting site. A variety of agents or sites that target specific cells are known in the relevant art. See, for example, Cotten et al, Methods Enzym. 217:618, 1993. Targeting agents may be included throughout the particle or may be present only on the surface. Targeting agents can be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, nucleic acid, and so on. The targeting agent may be used to target specific cells or tissues, or may be used to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialoglycoproteins, human immunodeficiency virus (HIV) envelope protein gp120, carbohydrates, receptor ligands, sialic acid, aptamers, and the like. If the targeting agent is included throughout the volume of the particle, the targeting agent may be included in the mixture that is used to form the particles. If the targeting agent is only on the surface, the targeting agent can be bound (i.e., via covalent bond, hydrophobic interaction, hydrogen bond, van der Waals, or other interactions) to the formed particles using standard chemical techniques.

Polynucleotide complexes

The present invention contemplates that APPLs are particularly useful when administering polynucleotides. For example, APPLs contain secondary or tertiary amines, and although these amines are hindered, they are capable of interacting non-covalently with a polynucleotide (eg, DNA, RNA, DNA and/or RNA synthetic analogs, DNA/RNA hybrids, etc.). Polinuk

- 38 040020 leotides or their derivatives are brought into contact with APPL under conditions suitable for the formation of a non-covalent polynucleotide/APPL complex. I believe that the interaction of APPL with polynucleotide at least partially prevents the destruction of the polynucleotide. Due to charge neutralization on the backbone of the polynucleotide, the neutral or weakly positively charged complex is also more readily able to pass through hydrophobic cell membranes (eg, cytoplasmic, lysosomal, endosomal, nuclear). In certain embodiments, the complex is weakly positively charged. In certain embodiments, the complex has a positive (ζ-potential. In certain embodiments, the ζ-potential is between 0 and +30.

In one aspect, a method is provided for delivering a polynucleotide to a biological cell, comprising providing a composition containing APPL or a salt thereof and a polynucleotide; and exposing the composition to the biological cell under conditions sufficient to facilitate delivery of the polynucleotide to the interior of the biological cell; where APPL is an amino acid, a linear or cyclic peptide, or a linear or cyclic polypeptide, or a structural isomer thereof, wherein the amino group or amide group of APPL is conjugated to a group of formula (i), (ii) or (iii). In certain embodiments, the method is an in vivo method. In certain embodiments, the method is an in vitro method.

APPL can be at least partially provided in the form of a salt (eg, protonated) so as to form a complex with a negatively charged polynucleotide. In certain embodiments, the polynucleotide/APPL complex forms particles that are useful for delivering polynucleotides to cells. In certain embodiments, multiple APPLs can bind to a polynucleotide molecule. For example, the complex may include 1-100 APPL, 1-1000 APPL, 10-1000 APPL, or 100-10000 APPL associated with a polynucleotide molecule.

An increase in the nitrogen:phosphate ratio has been shown to positively affect the delivery of genetic material due to increased binding of nucleic acids and negatively affect the delivery due to increasing toxicity. See, for example, Incani et al., Soft Matter (2010) 6:21242138. In certain embodiments, the nitrogen:phosphate ratio (i.e., the ratio between the amino groups present in the APPL and the phosphate groups present in the polynucleotide) is from about 10:1 to about 50:1, inclusive. In certain embodiments, the nitrogen:phosphate ratio is from about 10:1 to about 45:1, from about 15:1 to about 45:1, or from about 20:1 to about 40:1, inclusive. In certain embodiments, the weight ratio of APPL:polynucleotide is from about 10:1 to about 20:1, inclusive. In certain embodiments, the APPL:polynucleotide mass ratio is about 15:1. In certain embodiments, the APPL:polynucleotide molar ratio is from about 10:1 to about 400:1, inclusive. In certain embodiments, the APPL:polynucleotide molar ratio is from about 10:1 to about 350:1, from about 15:1 to about 300:1, or from about 20:1 to about 250:1, inclusive.

In certain embodiments, the complex may form a particle. In certain embodiments, the implementation of the particle diameter varies from 10 to 500 microns. In certain embodiments, the implementation of the particle diameter varies from 10 to 1200 microns. In certain embodiments, the implementation of the particle diameter varies from 50 to 150 microns. In certain embodiments, the particle diameter ranges from 10 to 500 nm, in certain embodiments, the particle diameter ranges from 10 to 1200 nm, and in certain embodiments, the implementation is from 50 to 150 nm. The particles can be associated with the targeting agent, as described below. In certain embodiments, the particle diameter ranges from 10 to 500 pm, in certain embodiments, the particle diameter ranges from 10 to 1200 pm, and in certain embodiments, the implementation is from 50 to 150 pm. The particles can be associated with the targeting agent, as described below. The film configuration is very precisely designed and can be adjusted with 1 nm accuracy in the range of 1 to 150,000 nm and with a certain understanding of its molecular composition.

The polynucleotide can be complexed, encapsulated with APPL, or included in a composition containing APPL. A polynucleotide can be any nucleic acid including, but not limited to, RNA and DNA. In certain embodiments, the polynucleotide is DNA. In certain embodiments, the polynucleotide is RNA. In certain embodiments, upon delivery of RNA to a cell, the RNA is capable of interfering with the expression of a particular gene in a biological cell.

In certain embodiments, the polynucleotide is an RNA that performs RNA interference (RNAi). The RNAi phenomenon is discussed in more detail, for example, in the following references: Elbashir et al, 2001, Genes Dev., 15:188; Fire et al., 1998, Nature 391:806; Tabara et al, 1999, Cell 99:123; Hammond et al, Nature, 2000, 404:293; Zamore et al, 2000, Cell 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morris and Rossi, 2006, Gene Ther., 13:553. In certain embodiments, the implementation of the polynucleotide is a dsRNA (double-stranded RNA). In certain embodiments, the polynucleotide is a siRNA (small interfering RNA). In certain embodiments, the polynucleotide is shRNA (short hairpin RNA). In certain embodiments, the implementation of the polynucleotide is a miRNA (micro-RNA). Micro-RNAs (miRNAs) are genome-encoded non-coding RNAs of approximately 21-23 nucleotides in length that help regulate gene expression, particularly during development. See, for example, Bartel, 2004, Cell, 116:281; Novina and Sharp, 2004, Nature, 430:161; and US Patent Publication 2005/0059005; See also reviews in Wang and Li, 2007, Front. Biosci. 12:3975; and Zhao, 2007, Trends Biochem. Sc. 32:189. In certain embodiments, the polynucleotide is an antisense RNA.

In certain embodiments, the polynucleotide may be provided as an antisense or RNA interference (RNAi) agent. See, for example, Fire et al, Nature 391:806-811, 1998. Antisense therapy is intended to include, for example, administering or providing in situ single- or double-stranded oligonucleotides, or derivatives thereof, that specifically hybridize, e.g., bind, to cell conditions, with cellular mRNA and/or genomic DNA, or mutants thereof, so as to inhibit the expression of the encoded protein, for example, by inhibiting transcription and/or translation. See, for example, Crooke Molecular mechanisms of action of antisense drugs Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke Evaluating the mechanism of action of antiproliferative antisense drugs Antisense Nucleic Acid Drug Dev. 10(2): 123-126, discussion 127, 2000; Methods in Enzymology volumes 313-314, 1999. Binding can be by standard base pair complementarity or, for example, in the case of double stranded DNA binding, specific interactions in the major groove of the double helix (i.e., forming a triple helix). See, for example, Chan et al., J. Mol. Med. 75(4):267-282, 1997.

In some embodiments, dsRNA, siRNA, shRNA, miRNA, antisense RNA, and/or RNAi can be designed and/or predicted using one or more of a large number of suitable algorithms. As a few examples, the following sources can be used to design and/or predict polynucleotides: Alnylum Online, Dharmacon Online, OligoEngine Online, Molecula Online, Ambion Online, BioPredsi Online, RNAi Web Online, Chang Bioscience Online, Invitrogen Online, LentiWeb Online GenScript Online, Protocol Online; Reynolds et al, 2004, Nat. Biotechnol., 22:326; Naito et al, 2006, Nucleic Acids Res., 34:W448; Li et al, 2007, RNA, 13:1765; Yiu et al, 2005, Bioinformatics, 21:144; and Jia et al., 2006, BMC Bioinformatics, 7:271.

The polynucleotides may be of any size or sequence and may be single or double stranded. In certain embodiments, the implementation of the polynucleotide has a length of more than 100 base pairs. In certain embodiments, the implementation of the polynucleotide has a length of more than 1000 base pairs and may be more than 10,000 base pairs. The polynucleotide is optionally purified and is essentially pure. In certain embodiments, the polynucleotide is greater than 50% pure, in certain embodiments greater than 75% pure, and in certain embodiments greater than 95% pure. The polynucleotide can be obtained by any methods known in the art. In certain embodiments, the implementation of the polynucleotide was designed using the technique of recombination. See, for example, Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989). The polynucleotide can also be obtained from natural sources and purified from contaminants commonly found in nature. The polynucleotide can also be obtained by chemical synthesis in the laboratory. In certain embodiments, the polynucleotide is synthesized using standard solid phase synthesis chemistry.

The polynucleotide can be modified by chemical or biological means. In certain embodiments, such modifications result in increased stability of the polynucleotide. Modifications include methylation, phosphorylation, addition of terminal functional groups, and so on.

Polynucleotide derivatives can also be used in the present invention. Such derivatives include modifications to the bases, sugars and/or phosphate bonds of the polynucleotide. Modified bases include, but are not limited to, bases found in the following nucleoside analogs: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-ioduridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)methylguanine and 2-thiocytidine. Modified sugars include, but are not limited to, 2'-fluororibose, ribose, 2'-deoxyribose, 3'-azido-2',3'-dideoxyribose, 2',3'-dideoxyribose, arabinose (2'-epimer of ribose), acyclic sugars and hexoses. Nucleosides may be linked by bonds other than the phosphodiester bond found in naturally occurring DNA and RNA. Modified bonds include, but are not limited to, phosphothioate and 5'-M-phosphoramidite bonds. Combinations of different modifications can be used in one polynucleotide. Such modified polynucleotides can be made by any means known in the art; however,

- 40 040020 As will be appreciated by those skilled in the art, modified polynucleotides can be made using in vitro synthetic chemistry.

The polynucleotides to be delivered may be in any form. For example, the polynucleotide may be a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, and so on.

The polynucleotide can be any sequence. In certain embodiments, the implementation of the polynucleotide encodes a protein or peptide. Encoded proteins can be enzymes, structural proteins, receptors, soluble receptors, ion channels, pharmaceutically active proteins, cytokines, interleukins, antibodies, antibody fragments, antigens, blood clotting factors, albumin, growth factors, hormones, insulin, and so on. The polynucleotide may also include regulatory regions to control gene expression. Such regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, the TATA box, ribosome binding sites, transcription stop sites, and the like. In certain embodiments, the implementation implies that the polynucleotide does not encode a protein. For example, a polynucleotide can be used to correct an error in the genome of a cell that has been transfected.

In certain embodiments, the polynucleotide to be delivered comprises a sequence encoding an antigenic peptide or protein. Nanoparticles containing such polynucleotides can be delivered to an individual to induce an immune response sufficient to reduce the possibility of subsequent infection and/or reduce the symptoms associated with said infection. The polynucleotide in such vaccines can be combined with interleukins, interferon, cytokines and adjuvants such as cholera exotoxin, alum, Freund's adjuvant, etc. A large number of adjuvant compounds are known; a useful directory directory of many such compounds has been prepared by the National Institutes of Health. See, for example, Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al, Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al, Vaccine 10:151-158, 1992.

The antigenic protein or peptides encoded by the polynucleotide may be derived from bacterial organisms such as

Streptococcus pneumoniae,

Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae,

Streptococcus mutans Pseudomonas aeruginosa Salmonella typhi Haemophilus parainfluenzae Bordetella pertussis Francisella tularensis Yersinia pestis Vibrio cholerae Legionella pneumophila Mycobacterium tuberculosis Mycobacterium leprae Treponema pallidum Leptospirosis interrogans from viruses such as variola virus, influenza A and B, respiratory syncytial virus, parainfluenza virus, measles virus, HIV, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus , papillomavirus, polio virus, mumps virus, rabies virus, rubella virus, coxsackie virus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A, B, C, D and E, etc. P.; and from such fungi, protozoa and parasitic organisms as

Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,

Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,

Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,

Trichomonas vaginalis, Schistosoma mansoni, etc.

be used to encapsulate agents, including, but not limited to, organic molecules (e.g., cholesterol, drugs), inorganic molecules, nucleic acids, proteins, peptides, polynucleotides, targeting agents, isotopically labeled organic or inorganic molecules, vaccines, immunological agents etc. Other illustrative means are described in more detail in this document. Such particles may include other substances such as polymers (eg synthetic polymers (eg PEG, PLGA), natural polymers (eg phospholipids)). In certain embodiments, APPL is mixed with one or more agents (eg, cholesterol) and/or one or more other substances (eg, polymers).

In certain embodiments, the implementation of the particle diameter varies from 1 to 1000 microns. In certain embodiments, the implementation of the particle diameter varies from 1 to 100 microns. In certain variants

- 41 040020 implementation the particle diameter varies from 1 to 10 microns. In certain embodiments, the implementation of the particle diameter varies from 10 to 100 microns. In certain embodiments, the implementation of the particle diameter varies from 100 to 1000 microns. In certain embodiments, the implementation of the particles vary from 1 to 5 microns. In certain embodiments, the implementation of the particle diameter varies from 1 to 1000 nm. In certain embodiments, the implementation of the particle diameter varies from 1 to 100 nm. In certain embodiments, the particle diameter ranges from 1 to 10 nm. In certain embodiments, the particle diameter ranges from 10 to 100 nm. In certain embodiments, the particle diameter ranges from 100 to 1000 nm. In certain embodiments, the particles range from 1 to 5 nm. In certain embodiments, the particle diameter ranges from 1 to 1000 pm. In certain embodiments, the particle diameter ranges from 1 to 100 pm. In certain embodiments, the particle diameter ranges from 1 to 10 pm. In certain embodiments, the particle diameter ranges from 10 to 100 pm. In certain embodiments, the particle diameter ranges from 100 to 1000 pm. In certain embodiments, the implementation of the particles range from 1 to 5 pm.

Particles can be obtained using any method known in the art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those skilled in the art. In certain embodiments, the particle production methods are a double emulsification and spray drying process. The conditions used in the preparation of particles can be varied to obtain particles with the desired size or property (eg, hydrophobicity, hydrophilicity, external morphology, adhesiveness, shape, etc.). The particle preparation method used and the conditions (eg, solvent, temperature, concentration, airflow rate, etc.) may also depend on the agent being encapsulated and/or the composition of the matrix.

Methods developed to obtain particles for the delivery of encapsulated agents are described in the literature. See, for example, Doubrow, M., Ed., Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et ah, Reactive Polymers 6:275-283, 1987; Mathiowitz et al, J. Appl. Polymer Sci. 35:755-774, 1988.

If the particles obtained by any of the above methods have a size range outside the desired range, the particles can be sorted by size, for example, using a sieve. The particle may also be coated. In certain embodiments, the particles are coated with a targeting agent. In other embodiments, the implementation of the particles are coated to achieve the desired properties of the surface (for example, a certain charge).

Micelles and liposomes

The present invention also contemplates the use of APPL in the preparation of micelles or liposomes. Any agent can be further included in a micelle or liposome. Micelles and liposomes are particularly useful in delivering hydrophobic agents such as hydrophobic small molecules. When a micelle or liposome forms a complex with (eg, encapsulates or coats) a polynucleotide, it is also referred to as a lipoplex. Many techniques are known in the art for preparing micelles and liposomes, and any such technique can be used in conjunction with APPL to produce micelles and liposomes.

In certain embodiments, liposomes are formed by spontaneous assembly. In other embodiments, liposomes are formed when thin lipid films or lipid precipitates are hydrated and stacks of lipid crystalline bilayers become fluid and swollen. Hydrated lipid sheets separate during shaking and self-close to form large multilamellar vesicles (LMVs). As a result, water does not interact with the hydrocarbon center layer of the two-layer structures at the edges. Once these particles have been formed, the particle size reduction can be controlled by applying sound wave energy (sonication) or mechanical energy (extruding). See, for example, Walde, P. Preparation of Vesicles (Liposomes) In Encylopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: Los Angeles, 2004; Vol. 9, pp. 43-79; Szoka et al, Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes) Ann. Rev. Biophys. Bioeng. 9:467-508, 1980; each of which is included in this document. The production of liposomes involves preparing APPL for hydration, hydrating the APPL with shaking, and fine-tuning the bubbles to size to achieve a uniform distribution of the liposomes. The APPL is first dissolved in an organic solvent to ensure a homogeneous APPL mixture. The solvent is then removed to form a polymer film. This polymer film is thoroughly dried to remove residual organic solvent by placing the vessel or vial under a vacuum pump overnight. The hydration of the polymer film is completed by adding an aqueous medium and shaking the mixture. When LMV suspensions are disrupted by sonic wave energy, small monolayer bubbles (SUVs) are typically formed with a diameter in the range of 15-50 nm. Lipid extrusion is a technique in which a lipid/polymer suspension is forced through a polycarbonate filter with a defined pore size to produce particles having a diameter close to the pore size of the filter being used. When extrusion

- 42 040020 Filtering through filters with pores of 100 nm, as a rule, large monolayer polymeric bubbles (LUV) with an average diameter of 120-140 nm are formed. In certain embodiments, the amount of APPL in the liposome varies from 30 to 80 mole %, in certain embodiments from 40 to 70 mole %, and in certain embodiments from 60 to 70 mole %. In certain embodiments, the APPL used is additionally complexed with an agent such as DNA and RNA. In such embodiments, the use of the liposome is the delivery of polynucleotides.

The following scientific publications describe other ways to obtain liposomes and micelles: Narang et al., “Cationic Lipids with Increased DNA Binding Affinity for

Nonviral Gene Transfer in Dividing and Nondividing Cells” Bioconjugate Chem. 16:156-68, 2005; Hofland et al., “Formation of stable cationic lipid/DNA complexes for gene transfer” Proc. Natl. Acad. sci. USA 93:7305-7309, July 1996; Byk et al., “Synthesis, Activity, and Structure—Activity Relationship Studies of Novel Cationic Lipids for DNA Transfer” J. Med. Chem. 41(2):224-235, 1998; Wu et al., “Cationic Lipid Polymerization as a Novel Approach for Constructing New DNA Delivery Agents” Bioconjugate Chem. 12:251-57, 2001; Lukyanov et al, “Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs” Advanced Drug Delivery Reviews 56:1273-1289, 2004; Tranchant et al., “Physicochemical optimization of plasmid delivery by cationic lipids” J. Gene Med. 6:S24-S35, 2004; van Balen et al., “Liposome/Water Lipophilicity: Methods, Information Content, and Pharmaceutical Applications” Medicinal Research Rev. 24(3):299324, 2004.

Methods of treatment

More than 10,000 human diseases have been found to be caused by genetic disorders, which are abnormalities in genes or chromosomes. See, for example, McClellan, J. and M.C. King, Genetic heterogeneity in human disease. cell. 141(2): p. 210-7; Leachman, S.A., et al., Therapeutic siRNAs for dominant genetic skin disorders includingpachyonychia congenita. J Dermatol Sci, 2008. 51(3): p. 151-7. Many of these diseases are lethal, such as cancer, severe hypercholesterolemia, and familial amyloidotic polyneuropathy. See, for example, Frank-Kamenetsky, M., et al., Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA, 2008. 105(33): p. 11915-20; Coelho, T., Familial amyloidpolyneuropathy: new developments in genetics and treatment. Curr Opin Neurol, 1996. 9(5): p. 355-9. Since the discovery by Fire and Mello of gene expression silencing by RNAi (Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p 806-11) a large amount of work has been done to develop therapeutic applications of RNAi in humans. See, for example,

Davis, M.E., The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic. Mol Pharm, 2009. 6(3): p. 659-68; Whitehead, K.A., R. Langer, and D.G. Anderson, Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discovery, 2009. 8(2): p. 129-138; Tan, S.J., et al., Engineering Nanocarriers for siRNA Delivery. Small. 7(7): p. 841-56; Castanotto, D. and J.J. Rossi, The promises and pitfalls of RNA interference based therapeutics. Nature, 2009. 457(7228): p. 426-33; Chen, Y. and L. Huang, Tumortargeted delivery of siRNA by pop-viral vector: safe and effective cancer therapy. Expert Opin

Drug Deliv, 2008. 5(12): p. 1301-11; Weinstein, S. and D. Peer, RNAi nanomedicines: challenges and opportunities within the immune system. Nanotechnology. 21(23): p. 232001; Fenske, D.B. and P.R. Cullis, Liposomal nanomedicines. Expert Opin Drug Deliv, 2008. 5(1): p. 25-44; and Thiel, K.W. and P.H. Giangrande, Therapeutic applications of DNA and RNA aptamers. Oligonucleotides, 2009. 19(3): p. 209-22. Currently, more than 20 clinical trials involving siRNA therapeutic agents are ongoing or have been completed and have shown promising results in the treatment of various diseases. See, for example, Burnett, J.C., J.J. Rossi, and K. Tiemann, Current progress of siRNAishRNA therapeutics in clinical trials. Biotechnol J. 6(9): p. 1130-46. However, efficient and safe delivery of siRNA is still a key issue in the development of siRNA therapeutic agents. See, for example, Juliano, R., et al., Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm, 2009. 6(3): p. 686- 43 040020

95.

Thus, in another aspect, methods of using APPL are provided, for example, to treat a disease, disorder, or condition that a subject suffers from. APPLs are expected to be useful in the treatment of a variety of diseases, disorders, or conditions, in particular, a drug delivery system will be useful in the treatment of such a particular disease, disorder, or condition. Disease, disorder, and condition are used interchangeably in this document. In certain embodiments, the disease, disorder, or condition from which the subject suffers is caused by an abnormality in the subject's gene or chromosome.

For example, in one embodiment, a method of treating a disease, disorder, or condition afflicted by a subject is provided, comprising administering to the subject in need thereof an effective amount of a composition comprising APPL or a salt thereof. The exemplary disease, disorder or conditions are intended to include, but are not limited to, proliferative disorders, inflammatory disorders, autoimmune disorders, disease states, liver diseases, and amyloid neuropathies.

As used herein, an active ingredient is any agent that elicits the desired biological response. For example, APPL may be the active ingredient in the composition. Other agents, such as therapeutic agents, as described herein, may also be classified as an active ingredient. In certain embodiments, the composition, in addition to APPL, further comprises a therapeutic agent useful in the treatment of a disease, disorder, or condition. In certain embodiments, APPL encapsulates another (therapeutic) agent. In certain embodiments, APPL and another (therapeutic) agent form a particle (eg, nanoparticle, microparticle, micelle, liposome, lipoplex).

In certain embodiments, the condition is a proliferative disorder, and in certain embodiments, the composition further comprises an anti-cancer agent. Illustrative proliferative diseases include, but are not limited to, tumors, benign neoplasms, premalignant neoplasms (epithelioma in situ), and malignancies (cancers).

Illustrative cancers include, but are not limited to, acoustic neuroma, adenocarcinoma, adrenal cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary tract cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (eg breast adenocarcinoma, papillary breast carcinoma, breast cancer, medullary breast cancer), brain cancer (eg meningioma; glioma, eg astrocytoma, oligodendroglioma; medulloblastoma), bronchial cancer, carcinoid, cervical cancer (eg, cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (eg, colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endothelial sarcoma (eg, Kaposi's angiomatosis, multiple idiopathic hemorrhagic sarcoma), endoma cancer tria (eg, uterine cancer, uterine sarcoma), esophageal cancer (eg, esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (eg, intraocular melanoma, retinoblastoma), familial hypereosinophilia, gallbladder cancer, gastric cancer (eg, adenocarcinoma stomach), gastrointestinal stromal tumor (GIST), head and neck cancer (eg, head and neck squamous cell carcinoma, oral cancer (eg, oral squamous cell carcinoma (OSCC), throat cancer (eg, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer , oropharyngeal cancer)), hematopoietic cell cancer (eg, leukemia such as acute lymphocytic leukemia (ALL) (eg, B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (eg, B-cell AML, T cell AML), chronic myelocytic leukemia (CML) (eg B cell CML, T cell CML) and chronic lymphocytic leukemia (CLL) (eg B cell CLL, T cell CLL); lymphoma, such as Hodgkin's lymphoma (HL) (eg, B-cell HL, T-cell HL) and non-Hodgkin's lymphoma (NHL) (eg, B-cell NHL, such as diffuse large cell lymphoma (DLCL) (eg, diffuse B-cell large cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), types of marginal zone B-cell lymphoma (e.g., types of mucosal lymphoid tissue lymphoma ( MALT), lymph node marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström's macroglobulinemia), malignant reticuloendotheliosis (HCL), immunoblastic large cell lymphoma, lymphoblastic progenitor cell lymphoma and primary central nervous system (CNS) lymphoma and T-cell NHL such as T-lymphoblastic lymphoma/T progenitor lymphoblastic leukemia, peripheral T-cell lymphoma (PTCL) (eg, cutaneous T-cell lymphoma (CTCL) (eg, mycosis fungoides, Cesari reticulosis), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, T-cell

- 44 040020 enteropathic type lymphoma, T-cell lymphoma of the subcutaneous tissue like panniculitis, anaplastic large cell lymphoma); a combination of one or more leukemias/lymphomas as described above; and multiple myeloma (MM)), heavy chain disease (eg, alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, types of inflammatory myofibroblastic tumor, immunocyte amyloidosis, kidney cancer (eg, nephroblastoma, also called Wilms tumor, hypernephroma), liver cancer (eg, hepatocellular carcinoma (HCC), malignant hepatoma), lung cancer (eg, bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), leiomyosarcoma (LMS ), mastocytosis (eg, systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorders (MPD) (eg, polycythemia vera (PV), essential thrombocythemia (ET), agnogenic myeloid metaplasia (AMM), also called myelofibrosis (MF ), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilia syndrome (HES)), neuroblastoma, neurofibre omoma (eg, neurofibromatosis (NF) type 1 or 2, schwannomatosis), neuroendocrine cancer (eg, gastrointestinal or pancreatic neuroendocrine tumor (GEP-NET), carcinoid), osteosarcoma, ovarian cancer (eg, cystadenocarcinoma, fetal ovarian cancer, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (eg, pancreatic adenocarcinoma, intraductal papillary mucinous tumor (IPMN), insulinoma), penile cancer (eg, Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (eg, prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (eg, squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)) , cancer of the small intestine (eg, cancer of the appendix), soft tissue sarcoma (eg, malignant fibrous histiocytoma (MFH), liposarcoma, peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous carcinoma, sweat gland carcinoma, synovioma, testicular cancer (eg, seminoma, fetal testicular cancer), thyroid cancer (eg, papillary thyroid carcinoma, papillary thyroid carcinoma (RTS), medullary thyroid cancer), urethral cancer, vaginal cancer, and vulvar cancer (eg, Paget's disease of the vulva).

Anti-cancer agents include biotherapeutic agents against cancer as well as chemotherapeutic agents.

Exemplary cancer biotherapeutic agents include, but are not limited to, interferons, cytokines (eg, tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal antibody serotherapy agents, immunostimulants, and/or immunomodulatory agents (eg, IL -1, 2, 4, 6, or 12), immune cell growth factors (eg, GM-CSF), and antibodies (eg, HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab ), RITUXAN (rituximab), BEXXAR (tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to, antiestrogen (eg, tamoxifen, raloxifene, and megestrol), LHRH agonists (eg, goscrcline and leuprolide), antiandrogens (eg, flutamide and bicalutamide), photodynamic therapy agents (eg, vertoporfin (BPD- MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA2-DMHA)), nitrogen mustards (eg, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (eg, carmustine (BCNU) and lomustine (CCNU) ), alkylsulfonates (eg busulfan and treosulfan), triazenes (eg dacarbazine, temozolomide), platinum-containing compounds (eg cisplatin, carboplatin, oxaliplatin), vinca alkaloids (eg vincristine, vinblastine, vindesine and vinorelbine), taxanes ( for example, paclitaxel or a paclitaxel equivalent such as paclitaxel nanoparticles bound to albumin (ABRAXANE), paclitaxel bound to docosahexaenoic acid (DHA-p aclitaxel, Taxoprexin), paclitaxel linked to polyglutamate (PG-paclitaxel, paclitaxel polyglumex, CT-2103, XYOTAX), tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 linked to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel linked to erbB2 recognition peptide EC-1) and glucose-conjugated paclitaxel, eg 2'-paclitaxel-methyl-2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllines (eg, etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), metabolic inhibitors, DHFR inhibitors (eg, methotrexate, dichlormethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (eg, mycophenolic acid, thiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (eg, hydroxyurea and deferoxamine), uracil analogs (eg, 5-fluorouracil (5-FU), floxuridine, doxyfluridine, raltitrexed, tegafur-uracil, capecitabine ), cytosine analogs (eg, cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (eg, mercaptopurine and thioguanine), vitamin D3 analogs (eg, EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (eg, lovastatin ), dopaminergic neurotoxins (for example, ion 1

- 45 040020 methyl-4-phenylpyridinium), cell cycle inhibitors (eg staurosporine), actinomycin (eg actinomycin D, dactinomycin), bleomycin (eg bleomycin A2, bleomycin B2, peplomycin), anthracycline (eg daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pyrarubicin, zorubicin, mitoxantrone), MDR inhibitors (eg verapamil), Ca ²⁺ ATPase inhibitors (eg thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (eg axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-3 54825), erlotinib (TARCEVA®), gefinitib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVAST IN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH® ), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF- 02341066, PF299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI- 930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (eg, bortezomib (VELCADE)), mTOR inhibitors (eg, rapamycin, temsirolimus (CCI-779), everolimus (RAD-001) , ridaphorolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe), and OSI-027 ( OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizi n, prednisolone, dexamethasone, campatecin, plicamycin, asparaginase, aminopterin, metopterin, porphyromycin, melphalan, leirozidin, leirosin, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethylmelamine.

In certain embodiments, the condition is an inflammatory disorder, and in certain embodiments, the composition further comprises an anti-inflammatory agent. The expression inflammatory disorder refers to those diseases, disorders or conditions that are characterized by signs of pain (dolor due to the formation of toxic substances and irritation of the nerves), fever (calor due to vasodilation), redness (rubor due to vasodilation and increased blood flow), swelling ( tumor due to excessive inflow or limited outflow of fluid) and/or loss of function (functio laesa, which may be partial or complete, temporary or permanent). Inflammation takes many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterating, parenchymal , reparative, productive, proliferative, diphtheritic, purulent, sclerosing, serofibrinous, serous, nonspecific, specific, subacute, inflammation with suppuration, toxic, traumatic and / or ulcerative inflammation.

Illustrative inflammatory disorders include, but are not limited to, inflammation associated with acne, anemia (eg, hypoplastic anemia, hemolytic autoimmune anemia), asthma, arteritis (eg, multiple arteritis, temporal artery arteritis, periarteritis nodosa, Takayasu's syndrome), arthritis (eg. , crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, dermatomyositis, diverticulitis, diabetes (eg, type I diabetes mellitus, type 2 diabetes mellitus), skin disorders (eg, psoriasis, eczema, burns, dermatitis, itching (itchy scabies)), endometriosis, syndrome Guillain-Barré, infection, coronary heart disease, Kawasaki disease , glomerulonephritis, gingivitis, hypersensitivity, headaches (eg, migraine headaches, tension headaches), intestinal obstruction (eg, intestinal obstruction after surgery and intestinal obstruction during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (syndrome of painful bladder), gastrointestinal disease (eg, selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal diseases (eg, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis , diarrhea, gastroesophageal reflux disease (GORD or its synonym GERD), inflammatory bowel disease (IBD) (eg, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, disconnected bowel colitis, Behçet's syndrome , undifferentiated colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosus

- 46 040020 roses, scleroderma annulare, myasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious anemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (for example, Parkinson's disease, Huntington's disease and Alzheimer's disease) ), prostatitis, radiation-associated chronic inflammation of the head, pelvic inflammatory disease, reperfusion injury, regional enteritis, acute rheumatism, systemic lupus erythematosus, scleroderma, Raynaud's disease, sarcoidosis, spondyloarthropathies, Sjögren's syndrome, thyroiditis, transplant rejection, tendonitis , trauma or injury (eg, frostbite, chemical irritants, toxins, scarring, burns, mechanical trauma), vasculitis, vitiligo, and Wegener's granulomatosis.

In certain embodiments, the inflammatory disorder is inflammation associated with a proliferative disease, such as inflammation associated with cancer.

In certain embodiments, the condition is an autoimmune disorder, and in certain embodiments, the composition further comprises an immunomodulatory agent. Illustrative autoimmune disorders include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthropathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjögren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behçet's disease, types of hemolytic autoimmune anemia , multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute shoulder syndrome, psoriatic arthritis, and Still's disease), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin disease (eg, psoriasis, eczema, burns, dermatitis, pruritus (itchy scabies )), enuresis, eosinophilic disease, gastrointestinal disease (eg, selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal diseases (eg, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic count ita), gastritis, diarrhea, gastroesophageal reflux disease (GORD or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, ruptured bowel colitis, Behçet's syndrome, undifferentiated colitis) and inflammatory bowel syndrome (IBS)) and disorders ameliorated by agents that improve gastrointestinal motility (eg, bowel obstruction, bowel obstruction after surgery, and bowel obstruction during sepsis; gastroesophageal reflux disease (GORD or its synonym GERD); eosinophilic esophagitis, gastric paresis such as diabetic gastric paresis; food intolerances and food allergies, as well as other functional disorders of the digestive tract, such as non-ulcer dyspepsia (NUD) and non-cardiac chest pain (NCCP, including costochondritis)).

In certain embodiments, the condition is a disease state, and in certain embodiments, the composition further comprises an analgesic. The disease state includes, without limitation, neuropathic pain (eg, peripheral neuropathic pain), central pain, deafferentation pain, chronic pain (eg, chronic nociceptive pain, and other forms of chronic pain such as postoperative pain, such as pain following reconstructive surgery on the hip). , knee or other types of reconstructive surgery), preoperative pain, irritation of nociceptive receptors (nociceptive pain), acute pain (for example, phantom and transient acute pain), non-inflammatory pain, inflammatory pain, pain associated with cancer, pain from a wound, pain from burns, postoperative pain, pain associated with medical procedures, pain resulting from itching, painful bladder syndrome, pain associated with premenstrual dysphoric disorder and / or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain, ass associated with preterm labor, pain associated with drug withdrawal symptoms, joint pain, arthritic pain (eg, pain associated with crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis, or Reiter's arthritis), lumbar sacral pain, musculoskeletal pain, headache, migraine, muscle pain, lumbago, neck pain, toothache, dental/maxillofacial pain, visceral pain, and the like. One or more of the disease states discussed herein may include combinations of the various types of pain provided above and herein (eg, nociceptive pain, inflammatory pain, neuropathic pain, etc.). In some embodiments, individual pain may predominate. In other embodiments, the implementation of the disease state includes two or more types of pain without the predominance of one. The skilled practitioner can determine the dosage needed to achieve a therapeutically effective amount for a particular subject based on the disease state.

In certain embodiments, the disease state is an inflammatory pain. In certain embodiments, the disease state (e.g.,

- 47 040020 inflammatory pain) is associated with an inflammatory disorder and/or an autoimmune disorder.

In certain embodiments, the condition is a liver disease, and in certain embodiments, the composition further comprises an agent used to treat the liver disease. Exemplary liver diseases include, but are not limited to, drug-induced liver injury (eg, acetaminophen-induced liver injury), hepatitis (eg, chronic hepatitis, viral hepatitis, alcohol-induced hepatitis, autoimmune hepatitis, steatohepatitis), non-alcoholic fatty liver disease , alcohol-induced liver disease (eg, alcoholic liver, alcoholic hepatitis, alcohol-related cirrhosis), hypercholesterolemia (eg, severe hypercholesterolemia), transthyretin-associated hereditary amyloidosis, cirrhosis of the liver, liver cancer, primary biliary cirrhosis, cholestasis , polycystic liver disease, and primary sclerosing cholangitis. In certain embodiments, liver diseases are associated with inflammation.

In certain embodiments, the condition is hereditary amyloid neuropathy, and in certain embodiments, the composition further comprises an agent useful in hereditary amyloid neuropathy.

The subject being administered is intended to include, but is not limited to, a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, adolescent) or an adult subject (e.g., a young adult, middle-aged adult or elderly adult)) and/or other non-human animals, e.g. , rats, hamsters, cattle, pigs, horses, sheep, goats, cats and/or dogs), and birds (eg birds of economic importance such as chickens, ducks, geese and/or turkeys). In certain embodiments, the subject is an animal other than a human. An animal other than a human may be male or female and be at any stage of development. An animal other than a human may be a transgenic animal.

As used herein, and unless otherwise indicated, the terms treat, process of treatment, and treatment contemplate an action that takes place while a subject is suffering from a specific disease, disorder, or condition, whereby the action lessens the severity of the disease, disorder, or condition. , or inhibits or retards the development of a disease, disorder, or condition (therapeutic treatment), and also involves an action that takes place before the subject begins to suffer from a particular disease, disorder, or condition (prophylactic treatment).

In general, an effective amount of an active ingredient is an amount sufficient to elicit the desired biological response. As will be appreciated by those skilled in the art, an effective amount of a compound of the present invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the active ingredient, the disease to be treated, the route of administration and the age, health and condition of the subject. An effective amount includes therapeutic and prophylactic treatment.

As used herein and unless otherwise indicated, a therapeutically effective amount of an active ingredient is an amount sufficient to provide a curative effect in the treatment of a disease, disorder, or condition, or to delay or minimize one or more of the symptoms associated with disease, disorder or condition. A therapeutically effective amount of an active ingredient means an amount of the active ingredient, alone or in combination with other agents or therapies, that provides a therapeutic effect in the treatment of a disease, disorder or condition. The term "therapeutically effective amount" may include an amount that improves the overall therapy, reduces or eliminates the symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

As used herein and unless otherwise indicated, a prophylactically effective amount of an active ingredient is an amount sufficient to prevent a disease, disorder, or condition, or one or more of the symptoms associated with the disease, disorder, or condition, or to prevent recurrence. . A prophylactically effective amount of an active ingredient means an amount of the active ingredient, alone or in combination with other agents or therapies, that provides a prophylactic effect in preventing a disease, disorder or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic.

The active ingredient can be administered in such amounts, at such times and in such a way as is considered necessary to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its route of administration, its mechanism of action, and the like. The active ingredient, either APPL alone or APPL in combination with an agent, is preferably formulated into a dosage unit for ease of administration and dosage consistency. It will be understood, however, that the daily use of the active ingredient will be determined by the attending physician as part of the results of a thorough medical evaluation. The specific therapeutically effective dose level for any given subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the active ingredient used; the particular composition used; age, body weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular active ingredient used; duration of treatment; medicinal products used in combination with or in conjunction with the particular active ingredient used; and similar factors well known in the medical field.

The active ingredient may be administered by any route. In some embodiments, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (as in powders, ointments, creams and / or drops), transmucosal, intranasal, buccal, intestinal, sublingual; by intratracheal infusion, bronchial infusion and/or inhalation; and/or as an oral spray, nasal spray and/or aerosol. In general, the most appropriate route of administration will depend on a number of factors, including the nature of the active ingredient (eg, its stability in the gastrointestinal tract), the condition of the subject (eg, whether the subject is able to tolerate oral administration), etc.

The exact amount of active ingredient needed to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the side effects or disorder, the hallmarks of the particular compound(s), route of administration, and the like. P. The amount to be administered, for example, to a child or adolescent, may be determined by the practitioner or one of skill in the art, and may be as low as or the same as that administered to an adult.

Examples

The following examples are set forth in order to be able to fully understand the present invention described in this document. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the present invention in any way.

Amino acid, peptide and polypeptide lipids (APPL) for drug delivery.

A highly active and selective siRNA delivery system with a wide therapeutic window has been developed to solve the problems associated with the delivery efficiency, specificity and toxicity of biological agents through the use of rational design and optimization of new amino acid derivatives of lipids.

Previously, our group performed a combinatorial synthetic approach to develop novel cationic lipids (lipidoids) for siRNA delivery. See, for example, Akinc, A., et al., A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol, 2008. 26(5): p. 561-9; Love Kevin, T., et al., Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 107(5): p. 1864-9; Siegwart, D.J., et al., Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery. Proc Natl Acad Sci USA. 108(32): p. 12996-3001. A number of these compounds have demonstrated significant silencing effects in vivo. See, for example, Leuschner, F., et al., Therapeutic siRNA silencing in inflammatory monocytes in mice. Nat Biotechnol. 29(11): p. 1005-10. In the course of earlier studies, key chemical and structural features, as well as methods for obtaining formulations for the development of new substances, were discovered. See, for example, Akinc, A., et al., Development of Hpidoid-siRNA formulations for systemic delivery to the liver. Mol Ther, 2009. 17(5): p. 872-9; Akinc, A., et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7): p. 135764; Semple, S.C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 28(2): p. 172-6. For example, active compounds have a tail of 12 or more carbon atoms, as well as several tails. See, for example, Love Kevin, T., et al., Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 107(5): p. 1864-9. In order to improve efficacy, tissue and cell type selectivity, and tolerability, new chemical backbones need to be developed and investigated.

Amino acids are the natural building blocks of peptides and proteins in nature. Amino acid derivatives can be metabolized in the human body; therefore, these substances are likely to be well tolerated and safe as therapeutic agents. In addition, peptides play important roles in membrane transport, endogenous cellular signaling and substance transport pathways, and offer great potential to maximize the use of these

- 49 040020 interactions to enhance delivery efficiency in systems in which peptide fragments are introduced. Because of their important physiological functions and safety in humans, amino acid-based substances such as insulin and trastuzumab have been widely used as supplements and medicinal products in the treatment of various diseases. Studies have shown that it is reasonable to use amino acid derivatives for gene delivery or siRNA delivery. See, for example, Prata, S. L., et al., Lipophilic peptides for gene delivery. Bioconjug Chem, 2008. 19(2): p. 418-20; Adami, R.C., et al., An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA. Mol Ther. 19(6): p. 1141-51; Margus, H., K. Padari, and M. Pooga, Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery. Mol Ther. 20(3): p. 525-33. Combining the benefits of both the natural properties of amino acids and the structural features of lipidoids, a structure optimization strategy was applied through the use of iterative screening methods and a range of lipid amino acid derivatives was obtained through rational design. The development, synthesis and biological evaluation of this novel series of lipid amino acid derivatives is reported. This efficient and rational strategy resulted in the prototype substance cKK-E12. Its delivery efficiency, tissue and cell type selectivity, tolerability and mechanism of action have been systematically investigated. The current results indicate that this delivery system represents a new vehicle for efficient, selective and safe siRNA delivery, which shows great potential for various diseases.

General ways.

Method 1 Preparation of a compound of formula (I)-(III). Conjugation with formula (i).

A mixture of amino acids, peptides or polypeptides and a conjugating agent (epoxide, thiirane or aziridine) (the ratio of conjugating reagent to amine ranged from 1.5:1 to 3:1) in EtOH was exposed to radiation in a microwave oven at 150°C for 5 h. The reaction mixture was purified using flash column chromatography. If the amino acids, peptides or polypeptides were in salt form, triethylamine was added to the solution and stirred for 30 minutes at room temperature before exposure to radiation.

Scheme A

Scheme B

Scheme C

Scheme D

Scheme E

Method 2 Preparation of compounds of formula (I)-(III).

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Conjugation with formula (ii).

A mixture of amino acids, peptides or polypeptides and a conjugating agent (acrylate or acrylamide) (the ratio of acrylates or conjugating reagent to amine ranged from 1.5:1 to 3:1) in ethanol (EtOH), isopropanol (iPrOH) or acetonitrile was heated to 90° C and stirred for 2 hours to 2 days. The reaction solution was concentrated with silica gel and purified by flash column chromatography.

Scheme F

Scheme G

Scheme H

Scheme I

Scheme J

Method 3 Preparation of compounds of formula (I)-(III).

Conjugation with formula (iii).

Sodium triacetoxyborohydride (NaBH(OAc) ₃ ) was added at rt. The reaction mixture was stirred for 3 days at rt. The reaction solution was concentrated with silica gel and purified by flash column chromatography.

Scheme K

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Scheme L

Scheme M

Scheme N

Method 4 Preparation of compounds of formula (IV).

Compounds of formula (IV) can be obtained by condensation of 1,2-diamine with activated oxalic acid, where X ¹ represents a leaving group, such as bromine, chlorine or iodine, to obtain a cyclized product. Groups of formulas (i), (ii) or (iii) can be introduced after the ring-closing reaction, for example, in particular, by adding to R ¹ , the amino acid side chain, or to the nitrogen atom of the imino groups of R ⁴ Other groups on the backbone, for example, R ² groups can be introduced before the ring closure reaction. In particular, R ² may be a group of formula (i), (ii) or (iii) introduced prior to the ring closure reaction.

Scheme P

Method 5 Preparation of compounds of formula (V).

Compounds of formula (V) and (VI) can be obtained by condensation of 1,1-diamine with activated malonic acid, where X ¹ represents a leaving group, such as bromine, chlorine or iodine, to obtain a cyclized product. The groups of formulas (i), (ii) or (iii) can be introduced after the ring closure reaction, for example, in particular by adding to R ¹ , the side chain of an amino acid, or to the nitrogen atom of the imino groups RQ. Other backbone groups, such as R ² groups, can be introduced prior to the ring closure reaction. In particular, R ² may be a group of formula (i), (ii) or (iii) introduced prior to the ring closure reaction.

Scheme Q

Method 6 Preparation of compounds of formula (VI).

Compounds of formula (VI) can be obtained by condensation of hydrazine with activated succinic acid, where X ¹ represents a leaving group, such as bromine, chlorine or iodine, to obtain a cyclized product. Groups of formulas (i), (ii) or (iii) can be introduced after the closure reaction

- 52 040020 ring, for example, in particular, by adding to R ¹ the side chain of the amino acid, or to the nitrogen atom of the imino groups RQ. Other backbone groups, such as R ² groups, can be introduced prior to the ring closure reaction. In particular, R ² may be a group of formula (i), (ii) or (iii) introduced prior to the ring closure reaction.

Scheme R

hydrazine derivative (VI) of succinic acid

Illustrative predecessors

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* R ⁶ and R ⁷ are hydrogen in the precursor, and, after conjugation, are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom, or a group of formula (i), (ii) or (iii).

- 54 040020

linear arginine arginine- linear R- R HN^N^ NH h,n' ^x >< ^N >^ ^x or ^A4 •\N HjN^ H Nh hh ₂ 0 cyclic arginine arginine- cyclical R-R Η γ^ΝΗ H^ ^N \^ ^NH 0 hh ₂ hn^nh ₂ NH polyarginine (5000-15000) R-(R)n-R PR-5000 h ₂ nK< ⁿ IAof ^L 0 ^J _n L A4 ^NH n=27-85 NH linear histidine histidine- linear H- H N=\ Λ/ ^ΝΗ Ύϊ 0 ^or ^A4 T^NH N^/ 0II cyclic histidine histidine- cyclical N-N HN- ^21UN w 0 polyhistidine (5000-25000) linear glycine- glycine cyclic glycine- glycine linear arginine- lysine N-(N)p-N RN-5000 linear GG cyclical G-G linear AK N^\ o ^J _n C n = 32-161 H h ₂ n 0 0 AV 0 ^H2N \\ 0 ^or ^A4 V^NH N^/ 0 0 A _0H linear lysine cysteine- linear SC .SH nh ₂ A linear aspartic acid lysine linear DK ,CO ₂ H 1 nh ₂ 0 •CH I nh ₂

- 55 040020

linear glutamic acid lysine linear EC CO ₂ H Λ νη ₂ linear phenylalanine-lysine linear FK < ^Ph О Λ νη ₂ linear glycine- lysine linear GK Η ίί η ₂ ν^ ^ν τ ^οη Λ νη ₂ linear isoleucine- lysine linear PS V. «Λ νη ₂ linear leucine- lysine linear LK Λ „ Λ νη ₂ linear methionine- lysine linear MK SMe Λ νη ₂ linear proline- lysine linear RK /---\ ο η π τ ^0Η Λ νη ₂ linear glutamine- lysine linear QK conh ₂ Η2Ν^^ ^Ν ^^οη Λ νη ₂ linear serine-lysine linear SK ^Η °. θ A νη ₂

- 56 040020

Table 3. Conjugating reagents Name Structure EY E11 E12 E13 E14 E15 E16 AYu AN A12 A13 A14 O10 OH 012 013 014 N10 Nil N12 N13 N14 0 H'^//^///// 0 0 H-^/////////^/ 0 H''^//////////'/' 0 H'^ /^/^//////^/ 0 0 Ζ^/// ⁴ // ⁴ / ⁴ o 0 ^^N^/^ZX/Xz® n 0 _n '^/ZZXZX/XZ Η o Nz ^n/XzXXsz^/vx®^® Η 0 ^=^ ⁴ Ν ^//////////// Η 0 ^==^ Ν ^//////////// ^ Η

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Methods of synthesis.

Example 1 Synthesis of APPL.

Schemes A-R show the general routes for the synthesis of APPL formulas (I) - (VI) according to the present invention.

The use of such methods results in a variety of APPLs depicted in Tables 4 and 5.

The compounds were named by combining the designation of the amino acid, aldehyde (A), acrylate (O), amide (N) or epoxide (E) and carbon chain length. For example, K-E12 is the reaction product of lysine with 1,2-epoxydecane.

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- 59 040020

- 60 040020

- 61 040020

- 62 040020

- 63 040020

* the reaction may give a mixture of two or more APPL; also included are compounds where 1 or more tails (R ^l ) are replaced by hydrogen

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Table 5 Compound Chemical formula Calculated. Observation Number of tails A-E12 C27H54NO3+ 440.4098 440.4336 2 C-E12 C27H54NO3S+ 472.3819 472.4303 2 D-E12 C28H54NO5+ 484.3997 484.4327 2 E-E12 C29H56NO5+ 498.4153 498.4117 2 F-E12 C33H58NO3+ 516.4411 516.4332 2 G-E12 C26H52NO3+ 426.3942 426.3772 2 H-E12 C42H80N3O4+ 690.6143 690.6016 3 1-E 12 C30H60NO3+ 482.4568 482.4461 2 K-E12 C42H85N2O4+ 681.6504 681.6009 3 L-E12 C30H60NO3+ 482.4568 482.4771 2 M-E12 C29H58NO3S+ 500.4132 500.4471 2 N-E12 C40H79N2O5+ 667.5984 667.5894 3 R-E12 C17H32NO2+ 282.2428 282.2585 1 Q-E12 C29H57N2O4+ 497.4313 497.4268 2 R-E12 C54H109N4O5+ 893.8392 893.8400 4 S-E12 C27H54NO4+ 456.4047 456.3891 2 T-E12 C28H56NO4+ 470.4204 470.4186 2 V-E12 C29H58NO3+ 468.4411 468.4259 2 W-E12 C35H59N2O3+ 555.4520 555.4510 2 Y-E12 C33H58NO4+ 532.436 532.4149 2 CKG-E12 C32H64N3O4+ 554.4891 554.4852 2 SKT-E12 C34H68N3O5+ 598.5153 598.5179 2 CYK-E12 C39H70N3O5+ 660.5310 660.5350 2 CLK-E12 C36H72N3O4+ 610.5517 610.5556 2 CDK-E12* C36H70N3O6+ 640.5259 640.5316 2 SMK-E12 C35H70N3O4S+ 628.5082 628.5072 2 CKV-E12 C35H70N3O4+ 596.5361 596.5330 2 SAK-E12 C33H66N3O4+ 568.5048 568.4992 2 SSK-E12 33H66N3O4S+ 600.4769 600.6143 2 CQK-E12 C35H69N4O5+ 625.5262 625.4733 2 SRK-E12 C35H68N3O4+ 594.5204 594.5169 2 CFK-E12 C39H70N3O4+ 644.5361 644.5301 2 CWK-E12 C41H71N4O4+ 683.5470 683.5367 2 SEC-E12 C35H68N3O6+ 626.5103 626.5053 2 HK-E12 C36H72N3O4+ 610.5517 610.5501 2 CSK-E12 C33H66N3O5+ 584.4997 584.5029 2 sKK-EY C52H105N4O6+ 881.8029 881.8042 4 SKK-E12 C60H121N4O6+ 993.9281 993.9224 4 sKK-E14 C68H137N4O6+ 1106.0533 1106.0709 4 SKK-E16 C76H153N4O6+ 1218.1785 1218.2002 4

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A-A12 C27H56NO2+ 426.4306 426.4244 2 C-A12 C27H56NO2S+ 458.4026 458.3857 2 D-A12 C28H56NO4+ 470.4204 470.4188 2 E-A12 C29H58NO4+ 484.4360 484.4319 2 F-A12 C33H60NO2+ 502.4619 502.4560 2 G-A12 C26H54NO2+ 412.4149 412.4107 2 H-A12 C30H58N3O2+ 492.4524 492.4503 2 I-A12 C30H62NO2+ 468.4775 468.4714 2 K-A12 С54Н111N2O2+ 819.8640 819.8657 4 L-A12 C30H62NO2+ 468.4775 468.4752 2 M-A12 C29H60NO2S+ 486.4339 486.4318 2 N-A12 C28H57N2O3+ 469.4364 469.4328 2 R-A12 C17H34NO2+ 284.2584 284.2512 1 Q-A12 C29H59N2O3+ 483.4520 483.4543 2 R-A12 C42H87N4O2+ 679.6824 679.6783 3 S-A12 C27H56NO3+ 442.4255 442.4225 2 T-A12 C28H58NO3+ 456.4411 456.4398 2 V-A12 C29H60NO2+ 454.4619 454.4544 2 W-A12 C35H61N2O2+ 541.4728 541.4724 2 Y-A12 C33H60NO3+ 518.4568 518.4543 2 KK-A12 C84H171N4O3+ 1284.3346 1284.3458 6 KKK-A12 C114H231N6O4+ 1748.8051 1748.8340 8 SKK-A12 C60H121N4O2+ 929.9484 929.9445 4 A-O12 C18H36NO4+ 330.2639 330.2582 1 C-012 C33H64NO6S+ 602.4449 602.4426 2 D-O12 C19H36NO6+ 374.2537 374.2492 1 E-O12 C20H38NO6+ 388.2694 388.2672 1 F-O12 C24H40NO4+ 406.2952 406.2896 1 G-O12 C17H34NO4+ 316.2482 316.2423 1 H-O12 C36H66N3O6+ 636.4946 636.4969 2 I-O12 C21H42NO4+ 372.3108 372.3054 1 K-O12 C66H127N2O10+ 1107.9485 1107.9417 4 L-O12 C21H42NO4+ 372.3108 372.3052 1 M-O12 C20H40NO4S+ 390.2673 390.2628 1 N-O12 C19H37N2O5+ 373.2697 373.2668 1 R-O12 C20H38NO4+ 356.2795 356.2779 1 Q-O12 C20H39N2O5+ 387.2853 387.2831 1 R-O12 C21H43N4O4+ 415.3279 415.3235 1 S-O12 C18H36NO5+ 346.2588 346.2521 1 T-O12 C19H38NO5+ 360.2744 360.2733 1 V-O12 C20H40NO4+ 358.2952 358.2905 1 W-O12 C26H41N2O4+ 445.3061 445.3010 1 Y-O12 C24H40NO5+ 422.2901 422.2868 1 KK-O12 C87H167N4O13+ 1476.2524 1476.2533 5 KKK-O12 C123H235N6O18+ 2084.7652 2084.7650 7 sKK-012 C72H137N4O10+ 1218.0329 1218.0880 4

* the formation of an ethyl ester. Compounds derived from poly-L-lysine are not included.

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Exemplary compounds from Table 5:

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S-A12 T-A12 V-A12

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Example 2 Alternative Synthesis of Compound 23 (sKK-E12)

Synthesis of compound B. Compound A (487 mg, 1.02 mmol) was loaded into a 10 ml vessel and trifluoroacetic acid (TFA, 1.3 ml) was added dropwise at 0°C. The reaction mixture was warmed to room temperature and stirred for 30 minutes. Solvents were evaporated under reduced pressure and TFA salts in DMF (3.5 ml) were added dropwise to pyridine (100 ml) at 0°C. The reaction mixture was slowly warmed to room temperature and stirred overnight. The solvents were evaporated under reduced pressure and the white solid was washed with EtOAc to give pure B in 69% yield. MS: m/z 525 (M+H ⁺ ); 'H NMR (500 MHz, DMSO, ppm): δ 1.29-1.40 (m, 8H, CH ₂ CH ₂ ), 1.611.68 (m, 4H, CH ₂ ), 2.97 (dd, J = 6.0, 12.5 Hz, 4H, NCH ₂ ), 3.79 (br, 2H, COCH), 7.22 (t, J= 5.5 Hz, 2H, aromatic), 7.33-7 .37 (m, 8H, aromatic), 8.10 (s, 2H, NH).

Synthesis of Compound C To an opaque solution of compound B (95 mg, 0.18 mmol) in 50% acetic acid/CH ₂ C12 (6 mg) was added Pd on charcoal (10 wt%, 36.5 mg). The black suspension was degassed for 5 minutes. and introduced hydrogen gas. The reaction mixture was stirred at rt overnight and then filtered through a pad of celite, which was washed several times with MeOH. The combined filtrates were concentrated to give a yellow viscous oil which was solidified with EtOAc. The solid was washed with ethyl acetate to give Compound C in 90% yield. MS: m/z 257 (M+H ⁺ ); 'n NMR (500 MHz, D ₂ O, ppm): δ 1.39-1.52 (m, 4H, CH ₂ ), 1.67-1.71 (m, 4H, CH ₂ ), 1.84 -1.88 (m, 4H, CH ₂ ), 2.99 (t, J= 7.5 Hz, 4H, NCH ₂ ), 4.14 (t, J= 5.0 Hz, 2H, COCH).

Synthesis of compound 23 (sKK-E12). To a mixture of compound C (169.2 mg, 0.45 mmol) and 1,2 epoxydecane (523 mg, 2.7 mmol) in EtOH was added triethylamine (182 mg, 1.8 mmol) which was stirred for 30 min. at rt. The reaction mixture was then exposed to microwave radiation at 150° C. for 5 hours. The mixture was purified by flash column chromatography to give compound 23 (52% yield) as a light yellow oil. MS: m/z 993 (M+H ⁺ ); *H NMR (500 MHz, DMSO, rrsh): δ 0.87 (t, J = 7.0 Hz, 12H, CH ₃ ), 1.21-1.39 (m, 80H, CH ₂ ), 1, 64-1.67 (m, 4H, CH ₂ ), 2.25-2.44 (br, 12H, NCH ₂ ), 3.44 (br, 4H, CHOH), 3.79 (br, 2H, COCH ), 4.21 (d, J = 3.0 Hz, 2H, CHOH), 4.27 (d, J = 3.0 Hz, 2H, CHOH), 8.11 (br, 2H, CONH).

Example 3 Synthesis of Compound D

This putative compound D can be synthesized by reaction 23 with Lawson's reagent in dry toluene.

Example 4 Synthesis of compound E.

This putative compound E can be synthesized by reacting 23 with hydroxylamine hydrochloride or other substituted amines in methanol.

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biological methods.

Compositions with siRNA.

Composition A.

APPL, distearoylphosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG were dissolved in 90% ethanol at a molar ratio of 50:10:38.5:1.5. siRNA (against firefly luciferase or fVII) was dissolved in 10 mM citrate buffer, pH 3, at a concentration of 0.4 mg/ml. An ethanolic lipid solution and an aqueous siRNA solution were pumped with a syringe pump through a microfluidic mixing chamber to spontaneously form lipid nanoparticles containing siRNA. Lipids were combined with siRNA at a total lipid to siRNA ratio of 7:1 (w:w). These formulations were dialyzed against PBS to remove ethanol and exchange buffer.

Composition B.

APPL was formulated with cholesterol (Sigma-Aldrich), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Avanti), mPEG2000-DMG (synthesized by Alnylam), and siRNA using a microfluidic mixer. See, for example, Chen, D., et al., Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation. J Am Chem Soc. The formulations were then dialyzed against PBS in 3500 MWCO dialysis cassettes (Pierce) overnight. Particles were assessed using a modified Ribogreen assay (Invitrogen) for siRNA capture and dynamic light scattering (ZetaPALS, Brookhaven Instruments) for mean particle size. cKK-E12 formulations were prepared from cholesterol, DSPC and mPEG2000-DMG using a similar method at a molar ratio of 50:10:38.5:1.5. This formulation produced a particle diameter of 60-70 nm with approximately 65% siRNA uptake.

In vitro silencing of luciferase genes.

HeLa cells stably expressing firefly luciferase and Renilla luciferase were plated (14,000 cells/well) in each well of an off-white 96-well plate (Corning-Costar) and allowed to adhere overnight in growth media. The culture medium was composed of 90% DMEM free of phenol red, 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen). Cells were transfected with LNP formulated with anti-luciferase siRNA by adding the formulated particles to the culture medium. Transfection was performed in quadruplicate. Cells were allowed to grow for 1 day at 37° C., 5% CO ₂ and then analyzed for luciferase expression. Control experiments were performed with Lipofectamine 2000 as described by the manufacturer (Invitrogen). Firefly and Renilla luciferase expression was analyzed using Dual-Glo assay kits (Promega). Luminescence was measured using a Victor3 luminometer (Perkin Elmer).

In vivo silencing of factor VII genes in mice.

C57BL/6 mice (Charles River Labs) were used for siRNA silencing experiments. Before injection, formulations were diluted in PBS at siRNA concentrations (SEQ ID NO 1 (sense siFVII): 5'GGAucAucucAAGucuuAcT*T-3'; SEQ ID NO 2 (antisense): 5'-GuAAGAcuuGAGAuGAuccT*T-3') such that each mice were dosed with 0.01 ml/g body weight. The formulations were administered intravenously by tail vein injection. After 48 or 72 hours, body weight gain/loss was measured and mice were anesthetized with isoflurane inhalation to collect blood samples by retroorbital sinus blood collection. Serum was separated using serum separation tubes (Falcon tubes, Becton Dickinson) and factor VII protein levels were analyzed using a chromogenic assay (Biophen FVII, Aniara Corporation). A standard curve was built using samples from mice injected with PBS, and the relative expression of factor VII was determined by comparing the treated groups with the untreated PBS control.

Biodistribution of Cy5.5-labeled siRNA-cKK-E12 formulations in mice.

The mice mentioned above were systemically injected with formulated Cy5.5-labeled siRNA at a total siRNA dose of 1 mg/kg. Mice were sacrificed 1 or 24 hours after injection; then the pancreas, spleen, liver, kidneys, ovaries, uterus, heart, lungs and thymus were removed and visualized, as well as a fragment of adipose tissue and muscle tissue. Organs were examined using Caliper's Ivis imaging system using an excitation wavelength of 675 nm and an emission wavelength of 720 nm. Data was processed with Living Image software from Caliper. The signal intensity for individual organs was normalized with respect to the total signal intensity for all organs.

In Vitro siRNA transfection analysis and microscopy.

The effect of alipoproteins was assessed by in vitro analysis of siRNA transfection on cells

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HeLa, as previously reported. HeLa cells stably expressing firefly luciferase and Renilla luciferase were plated in a matte white 96-well plate (Corning-Costar) overnight. Cells were transfected with cKK-E12 formulated with 50 ng firefly-specific siLuc in quadruplicate. Apolipoproteins (Fitzgerald Industries) were incubated with cKK-E12 formulations for 5 minutes before being added to cells. After 24 h incubation at 37° C., 5% CO ₂ , cells were analyzed for expression using Dual-Glo assay kits (Promega). To visualize cell uptake, cKK-E12 was formulated with Alexa-Fluor 647 labeled siRNA and incubated with Hela cells for 3 h. Cells were then fixed in 4% paraformaldehyde, permeabilized with 0.1% saponin and stained with Hoescht. All images were acquired with an Opera spinning disc confocal system (Perkin Elmer) and data analyzed with Acapella software (Perkin Elmer).

Discussion.

To obtain APPL, single amino acids were reacted with aldehydes, acrylates, and epoxides. The newly synthesized single amino acid lipid derivatives were evaluated for their ability to silence liver genes in mice. A validated genetic target, factor VII (blood clotting factor), was chosen as a silencing marker. See, for example, Akinc, A., et al., A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol, 2008. 26(5): p. 561-9. New lipid derivatives were formulated with cholesterol, DSPC, PEGlipid, as well as siRNA using a microfluidic mixing technique. See, for example, Chen, D., et al., Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation. J Am Chem Soc. Formulations that were unstable in solution or did not have captured siRNA were not screened. Stable formulations were injected into mice by systemic administration at a dose of 1 mg/kg (FIG. 1). Based on this initial screening, KE12 was found to be more active than the others. The compliance rate (greater than 50% silencing) was one compound out of 60 (i.e. 1.7%, including compounds not screened due to particle instability or lack of siRNA uptake).

The increased efficacy of K-E12 has led to the development of a second set of lysine peptide and polypeptide lipid derivatives. Lysine dipeptides were reacted with epoxides to give diketopiperazine APPLs. Microwave radiation was used to prepare these backbones, which greatly reduced the reaction time from 3 days to 5 hours. In addition, to further confirm the chemical structure and improve the chemical accessibility for large-scale synthesis in the synthesis of cKK-E12, an alternative synthetic route was developed (example 2). Diamine 5 was synthesized according to the previously reported method (Bergeron, R.J., et al., Macromolecular Self-Assembly of Diketopiperazine Tetrapeptides. J. Am. Chem. Soc, 1994. 116(19): p. 8479-84; Kaur, N., et al., A Delineation of Diketopiperazine Self-Assembly Processes: Understanding the Molecular Events Involved in N-(Fumaroyl)diketopiperazine of L-Lys (FDKP) Interactions, Mol Pharmaceutics, 2008. 5(2): p. 294-315), it was reacted with 1,2-epoxydecane to give cKK-E12. Compound (C) was subjected to reductive amination or Michael addition reaction with dodecanalyl dodecyl acrylate to give cKK-A12 and cKK-O12. The reactions between lysine-lysine and poly-Llysine (molecular weight from 500 to 70000 g/mol) and aldehydes and acrylates were similar to those with single amino acids.

The next step was to evaluate the effects of silencing. Ten out of 43 compounds showed approximately 50% silencing at 1 mg/kg. The compliance rate of the second set of compounds was 23%, which is more than 10 times higher than the effectiveness of the first set of substances. The results suggest that this iterative screening method is an effective strategy for identifying prototype compounds. The results from the second set also showed that the epoxy derivatives were more active than the aldehyde and acrylate derivatives (such as cKK-E12 compared to cKK-A12 and cKK-012). Substances with the best fit were further tested at a lower dose of 0.1 mg/kg. Tail length significantly affected silencing, and tail lengths of 12-14 carbons were favored (cKK-E10, -E12, -E14 and -E16). cCC-E12 proved to be the most highly active substance and was selected for further study.

Biodistribution study.

The biodistribution study was performed with unprotected Cy5.5-labeled siRNA and in combination with cKK-E12. Without taking into account the contribution of free siRNA in cKK-E12, after 1 h, more than 80% of the particles were located in the liver, and most of the residual siRNA was excreted through the kidneys no later than 24 h (Fig. 2).

Effect of alipoproteins on cell uptake and gene silencing.

Previous studies have reported that apolipoprotein E (ApoE) is able to improve cellular uptake and gene silencing for certain types of substances. Akinc, A., et al., Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther. 18(7): p. 1357-64. In order to test the effect of various apolipoproteins on cell uptake and gene silencing, as well as to study the mechanisms of action, experiments were performed with cKK-E12 and 11 isoforms.

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ApoA, ApoB, ApoC, ApoE and ApoH. The results on Hela cells showed that most apolipoproteins affected cell viability, with the exception of ApoB. ApoA, ApoC and ApoH showed no significant effect on silencing compared to free cKK-E12 (Fig. 3). However, four different ApoE isoforms significantly increased luciferase silencing.

The activity of cKK-E12, cKK-A12 and cKK-O12 was compared with and without addition of apoE3 (apoE3 is the predominant isoform in humans; Figure 4A). Without the addition of ApoE3, cKKA12 was more active than cKK-E12 and cKK-O12. However, when ApoE3 was added, the effect on silencing was in the following order: cKK-E12 > cKK-A12 > cKK-O12, which was in good agreement with in vivo activity.

The results suggest that the ApoE-supplemented cell assay may serve as a practical and efficient model for pre-screening for silencing in liver hepatocytes. In addition, cell uptake of cKK-E12 formulated with AlexaFluor 647 labeled siRNA was visualized using automated confocal microscopy (FIG. 4B).

Other embodiments.

In claims, singular forms may mean one or more, unless otherwise indicated or the context clearly indicates otherwise. A claim or description that includes or between one or more members of a group is deemed to hold if one, more or all of the group of members are present in, involved in, or otherwise related to said product or method, unless otherwise noted or from context, the opposite is not obvious. The present invention includes embodiments in which exactly one member of the group is present in, involved in, or otherwise related to said product or method. The present invention includes embodiments in which some or all of the members of the group are present in, involved in, or otherwise related to said product or method.

Moreover, the present invention embraces all variations, combinations, and permutations in which one or more of the limitations, elements, parts, and descriptive expressions from one or more of the listed claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more of the restrictions specified in some other claim that is dependent on the same main claim. If the elements are presented as lists, such as in the Markush group format, each subgroup of elements is also expanded and any element(s) may be excluded from the group. It should be understood that, in general, if the present invention or aspects of the present invention are mentioned to include specific elements and/or features, then certain embodiments of the present invention or aspects of the present invention consist or essentially consist of such elements and/or features. For the sake of simplicity, such embodiments have not been specifically set forth in haec verba in this document. It should also be noted that the containing and consisting expressions are assumed to be open-ended and allow the inclusion of additional elements or steps. If ranges are given, their endpoints are included. In addition, unless otherwise indicated or the context is clear to the contrary, and as one skilled in the art understands, values that are expressed as ranges can take on any particular value or subrange within the stated ranges in various embodiments of the present invention, up to tenths of a unit of the lower range limit, unless the context clearly indicates otherwise.

This application refers to various issued patents, published patent applications, journal articles, books, manuals and other publications, all of which are incorporated herein by reference. If there is a conflict between any included reference and the present description, the description shall take precedence. In addition, any particular embodiment of the present invention that falls within the scope of the prior art may be expressly excluded from any one or more claims. Since such embodiments are intended to be known to those skilled in the art, they may be excluded, even if such exclusion is not expressly stated herein. Any particular embodiment of the present invention may be excluded from any claim, on any basis, whether or not it pertains to the existence of prior art.

Those skilled in the art will understand, or be able to ascertain, with just routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited by the foregoing description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this disclosure may be made without departing from the spirit and scope of the present invention as defined in the following claims.

Claims (29)

CLAIM 1. Compound formula or its salt; where Q is O; each R ¹ is independently -H, -CH ₃ , -CH(CH ₃ ) ₂ , -CH(CH ₃ )(CH ₂ CH ₃ ), -CH2CH(CH3)2, or a group of formula (N), provided that at least one representative of R ¹ is a group of formula L is an unsubstituted C _1-50 alkylene; R ⁶ and R ⁷ each independently represents hydrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii); each R ² is independently hydrogen or a group of formula (iii); formula (iii) is where R ^l represents C6. ₂₀ alkyl optionally substituted with one or more fluoro substituents. 2. Connection 3. Mule connection (iii). 4. A compound according to claim 1, wherein at least one R ² is hydrogen. according to claim 1, where at least one representative of R ² represents a forpo group of claims 1 or 2, where each R ¹ represents a group of formula (N). 5. A compound according to any one of claims 1 to 4, wherein the group of formula (N) is the formula where q is an integer from 1 to 50 inclusive. 6. A compound according to any one of claims 1 to 5, wherein the compound is selected from the group consisting of - 75 040020 o p is 1; and each R ¹ is independently -H, -CH3, -CH(CH3) ₂ , -CH(CH3)(CH ₂ CH3 ), -CH ₂ CH(CH3) ₂ , where R ⁶ and R ⁷ each independently represents hydrogen, a protective group for nitrogen or a group of formula (iii), provided that at least one representative of R ⁶ and R ⁷ represents a group of formula (iii); and provided that at least one R ¹ is a group of formula 7. The compound of claim 1, wherein the compound is selected from the group consisting of - 76 040020 and its salts, where p is 1; and provided that at least one representative of R ² is 8. A compound according to claim 1, wherein the compound is a group of formula (iii). 9. A composition for delivering a polynucleotide to a cell, comprising a compound according to any one of claims 1 to 8 or a salt thereof and a polynucleotide. 10. Composition according to claim 9, characterized in that the composition is a pharmaceutical composition. 11. Composition according to claim 9, characterized in that the composition additionally contains cholesterol. 12. The composition according to claim 9, characterized in that the composition further comprises a PEGylated lipid. 13. The composition according to claim 9, characterized in that the composition further comprises a phospholipid. 14. The composition according to claim 9, characterized in that the composition further comprises an apolipoprotein. 15. The composition according to claim 9, characterized in that the polynucleotide is DNA. 16. The composition according to claim 9, characterized in that the polynucleotide is RNA. 17. The composition according to claim 16, wherein the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. 18. Composition according to claim 9, characterized in that the composition is in the form of a particle. 19. The composition according to claim 18, characterized in that the particle is a nanoparticle or microparticle. 20. Composition according to claim 18, characterized in that the particle is a micelle, liposome or lipoplex. 21. The composition according to claim 18, characterized in that the particle encapsulates a polynucleotide. 22. The use of a composition according to any one of claims 9-21 for the treatment of a disease, disorder, or condition from which a subject suffers, wherein the disease, disorder, or condition is selected from the group consisting of proliferative disorders, inflammatory disorders, autoimmune disorders, and - 77 040020 liver diseases. 23. The use of a compound according to any one of claims 1 to 8 or a salt thereof and a polynucleotide in the manufacture of a pharmaceutical composition for delivering a polynucleotide to a cell. 24. Use according to claim 23, wherein the polynucleotide is DNA. 25. Use according to claim 23, wherein the polynucleotide is RNA. 26. Use according to claim 25, wherein the RNA is RNAi, dsRNA, siRNA, shRNA, miRNA, or antisense RNA. 27. The use according to claim 25, wherein the polynucleotide is RNA, and upon delivery of the RNA to the cell, the RNA is capable of interfering with the expression of a particular gene in the biological cell. 28. The composition according to claim 9, characterized in that the polynucleotide encodes a protein or peptide. 29. Use according to claim 23, wherein the polynucleotide encodes a protein or peptide.