AU2014200910B2 - Lipid containing formulations - Google Patents

Abstract

Compositions and methods useful in administering nucleic acid based therapies, for example association complexes s such as liposomes and lipoplexes are described. C:\NRPortbl\GHMatters\MICHELES\513091 11 .docx 19/02/14 co RON, * \A . '.*,'\ C

Description

Lipid containing formulations

TECHNICAL FIELD

This invention relates to compositions and methods useful in administering nuclei e acid based therapies, for example association complexes such as liposomes and lipoplexes.

BACKGROUND

The opportunity to use nucleic acid based therapies holds significant promise, providing solutions to medical problems drat could not be addressed with current, traditional medicines. The locati on and sequences of an increasing number of disease-related genes are being identified, and clinical testing of nucleic acid-based therapeutic for a vari ety of diseases is now underway.

One method of introducing nucleic acids into a cell is mechanically, using direc micramjeetioo. However this method is not generally effective for systemic administration to a subject

Systemic.delivery of a nucleic acid therapeutic requires distributing nucleic ae* to target ceils and then transferring the nucleic acid across a target cell membrane intae and in a form, that can function in a therapeutic manner.

Viral vectors have, in some instances, been used clinically successfully to administer nucleic acid based therapies. However, while viral vectors have the inheres ability to transport nucleic acids across cell membranes, they can pose risks. One such risk involves the random integration of viral genetic sequences into patient chromosomes, potentially·damaging the genome and possibly inducing a malignant transformation. Another risk is that the viral vector may revert to a pathogenic geaoiyp either through mutation or genetic exchange with a wild type virus. lipid-based vectors have also been used in nucleic acid therapies and have beet formulated in one of two ways, in one method, the nucleic acid is introduced into pre.font.ved liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. The complexes thus formed have undefined and complicated structures mid the transfection efficiency is severely reduced by the presence of serum. The second methc involves the formation of DNA complexes with mono- or poly-cationic lipids without the orescnce of a neutral lipid. These complexes are prepared in the presence of ethano and axe· not stable in water. Additionally, these complexes are adversely affected by serum (see, Behr, Ace. Cliem, Res. 26:274-78 (1993)).

SUMMARY

The invention features novel preparations that include a polyamine compound or a lipid moiety described herein. in some embodiments, the invention features a preparation comprising one or more compounds, each individually having a structure defined by formula (I) or & pharmaceutically acceptable salt thereof,

formula (I) wherein each Xs and Xb, for each occurrence, is independently alkylene; n is 0, 1,2,3,4, or 5; each R is independently H,

R# Rb Rc Ra R* wherein at least n f 2 of the R moieties· in at least about 50% of the-molecules of the compound of formula (I) in the preparation (e.g., at least about 55%, at least about. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at-least about 95%, at least about 98%, at least about 99%, or substantially all) are not H; m is 1,2,3 or 4; Y is O, NR2, or S;

Rs is alkyl alkenyl or alkynyl; each of which is options!!}'· substituted with one or more substituents; and R2 is H, alkyl alkenyl or alkynyl; each of which is optionally substituted each of which is optionally substituted wife one or more substituents; provided feat, if n ~ 0, then at least n + 3 of fee R moieties are not H.

In some embodiments, when R is not H, R is 11«, for example, when R is not H, R i s R« for each occurrence, in some embodiments, when R is not H, R is R-0, for example, when R is not B, R. is R&. for each.occurrence.

In some embodiments, when R is not H, R is R«, for example, whm R is not E, R is RCs for each occurrence.

In some embodiments, when R is not H, R is R, for example, when R is .not If R is Rd, for each occurrence.

In some embodiments, when R is not H, R is R*, for example, whenR is net H, R is R*, for each occurrence.

In some embodiments, n + 2 of the R moieties of formula (I) are not H. In some embodiments, n -f 3 ofthe R moieties of formula (I) are not H, In some embodiments, n 4- 4 of the R moieties of formula (I) are not H.

In some embodiments, ivt 1 of the R moieties of formula (1) are not H.

In some embodiments, n > 0, and at least one R of NR of formula (I) is H,

In some embodiments, at least one R of NR;» of formula (I) is H.

In some embodiments, at least 80% of themoleeuies are a single structural isomer. For example,·;»··** 2 of the R moieties of formula (I) are not H, or n t- 3 of the R moieties of formula (I) are not H, or n + 4 of the R moieties of formula <I) are not II.

In some embodiments, n is 2 or 0.

In some embodiments, X* and X1' are Ca alkvlene, in some enfoodhnenis, n. is O and Xfe is ethylene or propylene.

In some embodiments, n >1 and X8 varies with at least, one occurrence.

In some embodiments, when R not H, R is

For example, Y can be O or NR2. In some embodiments, m is 2. In some efobodiments, Y is 0 or NR2 and m is 2. In some embodiments, m is 1. In some embodiements, m is 1 and Y is O or NR".

In some ..embodiments, R1 for at least one occurrence is alkyl, for example. R‘ for each occurrence is alkyl. in some embodiments, R{ is alkyl and R2is H, for at least, one occurrence,· e.g„ for, each occurrence. ίη. some embodiments, R5 and R2 are alkyl for at least one xx^urrence, e.g., for each occurrence.

In some embodiments, R1 for at least one occurrence is alkenyl

In some embodiments, R1 for at least one occurrence is alkenyl

In some embodiments, when R is not H, R is Ra^ for at least one occurrence, e.g., for each occurrence, and Y is O or NH, In some embodiments, Y is O, Is som©^ embodiments, Y is Nil In some embodiments, R1 is alkyl, e.g., Cso-so alkyl or (¾ alkyl. I» some embodiments, n is 2. In some embodiments, Xa, for each occurrence is C? alkyl ene and Xb is Cl alkylene, In some embodiments, m is 2.

In some embodiments, n is 2 and R, when R is not H, is Ra, for at least one occurrence, e.g., for each occurrence. In some·embodiments* R.1 is alkyl, e.g., Ck^js alkyl or (¾ alkyl. In some embodiments, Y is 0 or Y is NH, In some embodiments, X®, for each occurrence is Q> alkylene and Xb is (¾ alkylene. la some embodiments, m is 2:. in some embodi ments, at least 1 R of NR. is H and R, when not H is !«, for at least one occurrence, e.g. for each occurrence, and Y is O or NH. In some embodiments, Y is 0 or Y is NH la some embodiments, Rf is alkyl, e,g,,Cjo-!s alkyl or Cn alky!,. In some embodiments, n is 2. In some embodiments, Xs, for each occurrence is Q alkylene and Xh is Cj alkylene. In some embodiments, m is 2.

In some embodiments, a is 2 and at least I R of NR. is H and when R is not H, R is.R», lor at least one occurrence, e.g. for each occurrence, and Y is 0 or NH. In some embodiments, R! is alkyl, e.g,, Gums «ΪΜ or Cj? alkyl In some embodiments, ¥ is 0 or Y is NH. In some embodiments, X3. for each occurrence is €? alkylene and X3 Is CN alkylene. In some embodiments, m is 2.

In some embodiments, at least 1 R of N Ra is H and R is R«, for at least one occurrence, e.g, for each occurrence, and wherein Y i.s 0 or NH. In some embodiments, Y is 0 or Y is NH, In some embodiments, R1 is alkyl, e.g,, Cseoo alkyl, (¾.¾¾ alkyl or Ct2 alkyl In some embodiments, a is 2. In. some embodiments, X*,- for each occurrence Is C% alkylene and Xb is €2 alkylene. In some embodiments, m is 2,

In some embodiments, n is 2 and at least I R of NR? is H and R is 11¾. for at least one occurrence, e.g. for each occurrence, and wherein Y is 0 or NH. In some-embodiments, R1 is alkyl, e.g,, Cjous alkyl or C12 alkyl. In sonic embodiments, Y is 0 or Y is NI L ]» some embodiments, Xa, for each occurrence is (¾ alkviene sod Xs is· C$ alkviene. In some embodiments, m is 2.

In some embodiments, tire preparation comprises one or a mixture of the formula below, wherein R. is not H unless specific in die formula below.

In same embodiments, die preparation consists essentially of one or a mixture of the formula below

in some embodiments, each R is

, In some embodiments, each R is

. In some embodiments, R! is Csa-Css alkyl (e,g., Cn alkyl), or CunCjo alkenyl.

In some embodiments, R is

In some embodiments. R* Is CnrC^ alkyl, e.g., C52 alkyl. In some embodiments, R3 is Cy> alkyl and R" is H.

In some embodiments, n is Q and X is propylene. In some embodiments, 1 R is H. In some embodiments, when R is not H, R is Ra, for at.least one occurrence, e.g. for each occurrence, in some embodiments, R1 is alkyl, e.g., €)0.30 alkyl or (¾ alkyl. In some embodiments, Y is O or Y is NH. hi some embodiments, m is 2,

In some embodiments, formula. (1) is

In some embodiments, R is

In some embodiments. R3 is CirCj* alkyl, or Cio-CiO alkenyl In some embodlmeMs, R is

In some embodiments, R* isCur-Cs* alkyl, orCjo-Cso alkenyl and R2 is a in some embodiments, "»is 2;. XV for each occummee is Qg alkylene and Xb is (¾ alkyiene; and wherein each R is H or

R«, for at least one occurrence, e.g. for each occurrence, mis 2; Y is NH or O; R' is Css alkyl. In some embodiments, at least S0% of the molecules of foe compound of formula (I) are a single structural isomer, in some embodiments, Y is NH, e.g., wherein at least 80% of the molecules of the compound of formula (I) are a single structural isomer. In some embodiments, R is R“, for 5 occurrences, in some embodiments, in at least S0% of foe molecules of foe compound of formula (I), R is RK, for 5 occurrences. In some embodiments, Y is NH.

In some embodiments, the compound of formula (I) is an inorganic or organic salt thereof, e.g., a Iiydrohalido salt thereof, such as a hydrochloride salt thereof, in some embodiments, foe hydrochloride salt ranges from a single equivalent of HCL, to rr;-2 equivalents of HCL in some embodiments, foe compound of formula (I) is salt of an organic acid, e.g,, an acetate, for example, foe acetate salt ranges from a single equivalent of acetate, to af2· equivalents of acetate or a formate, for example, the formate salt ranges from a single equivalent of acetate, to 0+2 ecgrivatents of formate.

In some embodiments, foe compound of formula (I) is in foe form of a hydrate.

In some embodiments, R1, for at least one occurrence, e.g., for each occurrence, comprises an alkenyl moiety, for example, R} comprises a cis double bond.

In one aspect, foe invention features a preparation including a compound of formula (1) and a nucleic acid (e.g., anRNA such as m siRNA or dsRNA or a DNA). In some embodiment* the preparation also includes an additional lipid such as a fusogenic lipid, or a PEO-lipid, in some embodiments, die preparation comprises less than 11%, by weight, of

formula (ill), wherein X and n are defined as in formula (Ϊ) above.

In some embodiments, the preparation comprises less than 90% by weight of

formula (IV) wherein Y and R’ are defined as in formula (1) above.

In some embodiments, the preparation comprises a plurality of compounds of formula (I),

In some embodiments, the preparation comprises a mixture of compounds of the formulas below:

formula (Γ) formula (I”) wherein in formula (P), five of the R moieties are R3. In some embodiments, formula (F) and (Γ) are present in a ratio of from about 1:2 to about 2;!.

In one aspect, the invention features a method of making a compound of formula

(HX

formula (II) wherein each Xa and Xb, for each occurrence, is independently €.·.« alkylese; n is 0,1,2,3,4, or 5; and wherein each R is hKiependently H or

R*; in is 2; Y is 0, NR2, or S; IIs is alkyl or alkenyl; R“ is H or € alkyl or alkenyl; the method comprising reacting a compound of formula (Of)

formula fill) with a compound of formula (IV),

formula (IV) in the presence of a promoter.

In one aspect , the invention features a method of making a compound of formula {%

formula (II) wherein each X* and Xb, for each occurrence, is independently €.^ alkylate; n is 0,1, 2, 3,4, or 5; and wherein each R is independently H or

R* m is 2;

YisO, NRa. or S;

Rs is alkyl or alkenyl; R~ is H or C alkyl or alkenyl; the method comprising reacting a compound of formula (III)

formula (III) with a compound of formula (IV),

formula (IV) in Ore presence of a quencher.

In one aspect, the invention features a method of making a compound of formula POf

formula (II) wherein each Xs and Xb, for each occurrence, is independently Cu, alkylene; a is 0, I, 2, 3,4. or 5; and wherein each R is independently H or

R* m is 2;

YisO, NR\ orS; U5 is alkyl or alkenyl; R2 is B or alkvl or alkenyl; the method comprising reacting a compound of formula (III)

formula (III) with a compound of formula (IV),

formula (IV) wherein the reaction mixture comprises from about OJ about 1.2 molar equivalents of a compound of formula (IH), with from about 3.S to about 6.5 molar equi valents of a compound of formula (IV).

In some embodiments, the reaction mixture comprises from about 0,8 about 1,2 molar equivalents of a compound of formula (HI), with from about 5.5 to-about 6.5 molar equivalents of a compound of formula (IV). In some embodiments, tire reaction mixture comprises about 1 molar equivalents of a compound of formula (1.11), with from about fi molar equivalents of a compound of formula (IV). In some embodiments, the reaction mixture comprises about 1 molar equivalents of a compound of formula (HI), with from about 5 molar equivalents of a compound of formula (IV), la one aspect, the invention features a method of making a, compound of formula CO),

formula (I!) wherdm each Xs and Xb, for-each occurrence, is independently €].* alkyleae; n is 0,1,2,3,4, or 5; and wherein each R is independently M or

Ra; mis 2; Y is O, NR2, or S; R5 is alkyl or alkenyl; R2 is II or alkyl or alkenyl; the method comprising a two step process of reacting a compound of formula (MI)

formula (10) with a compound of formula (IV),

formula (IV) in the presence of boric acid and water wherein, the first step process involving the reaction mixture comprises from about 0,8 about 1.2 molar equivalents of a compound of formula (III), with from about 3.8 to about 4.2 molar equivalents of :a compound of formula (IV) and: the second step process involving addition of about 0.8 to 1.2 molar equivalent of compound of formula (IY).

In one aspect, the invention, features a method of making a compound of formula (11),

formula (Π) wherein each X3 and X*, for each occurreace, is independently Q^alkylene; n is 0,1,2,3, 4, or 5; and wherein each R is independently H or

R3 in is 2; Y is 0, NR2, or S; IIs is alkyl or alkenyl; R~ is H or alkyl or alkenyl; the method comprising reacting a compound of formula (111)

formula (III) with a compound of formula (IV),

formula (IV) and separating at least one structural isomer of formula (11) from the reaction mixture to provide a substantially purihed preparation comprising a structural isomer of formula (11),

In some embodiments;, the structural isomer of formula (II) is separated from the reaction mixture using chromatographic separation, in some embodiments, the chromatographic separation is using flash silica gel for separation of isomers, in some embodiments, the chromatographic separation is gravity separation of isomers using silica gel In some embodiments, the chromatographic separation is using moving bed ebrnraatagraphy tor separation of isomers. In some embodiments, the chromatographic separation uses liquid cfcomatagraphy (LC) for separation of isomers. In some embodiments, the· chromatographic separation is normal phase HPLC ibr separation of isomers. In some embodiments, the chromatographic separation is reverse phase HPLC for separation of isomers.

In some embodiments, the substantially purified preparation comprises at least about 80% of the structural isomer of formula (II), e.g., at least about 90% of the structural isomer of formula (II), at least about 95% of the structural isomer of formula (II).

In mother aspect, the invention features a method of making a compound of formula (V) or a pharmaceutically acceptable salt thereof

formula (V) wherein each X8 and X**, for each occurrence, is independently C*.« alkylene; n is 0,1,2,3.4, or 5; and wherein each R Is independently H or

m is l; Y is O, NR2, or S; ll! is alkyl or alkenyl; \ll is H or alkyl or alkenyl; the method comprising reacting, a compound offonnula (III)

formula. (Ill) with a compound of formula (VI)*

Q ~ Cl or Sr or ? formula (VI) to provide a compound of formula (V) or a phanmeeuficaliy acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt thereofis a hydrochloride salt of the compound of. formula (V).

In one aspect, the invention features a compound of formula (X),

formula (X) wherein R! and R^-are each independently .H, CrQ, alkyl, optionally substituted with 1-4 R5, QrQ alkenyl, optionally substituted with 1*4 R5, or C{NR6){NR6)2: R-5 'and R4 are each independently alkyl, alkenyl, alfcynly, each of which is optionally substituted with fltioro, ehloro, bromo, or iodo;

Ls and If are each independently -~NR6C{0)-, -C(G)NRft-, -00(0)-, -0(0)0-, ~ S-S-, -N(R&amp;)G(0)N(R6)-, -0C{0)N(R6)-, -N<R*)C(0)0~? ~0-N»C-, OR, -O0(O)Hff-fsMC-, or ~NHC(Q)NH-N“€-, and lAR4 can be taken together to form an acetal, a ketal, or art rirthoester, wherein R3 and it4 are defined as above and can also be H or phenyl;

Rs is iluoro, ehloro, bromo, iodo, -OR7, -N{R*)(R9), -€N, SRi0, S(O)Ri0, S(0)2lK! ,R° is H, Cj-Cg alkyl, R' is H or CrCs alkyl; each R8 and R9 are independently H or CVCs alkyl;

Ri0 Is H or CrQ alkyl; mis 1,2, 3,4, 5, or6; nisO, 1,2,3,4, 5, or 6; and pharmaceutically acceptable salts thereof.

In some embodiments, the compound is an inorganic salt thereof, for example a faydrohalide salt thereof such as a hydrochloride salt thereof. In some embodiments, fee compound is an organic salt thereof.

In some embodiments, R5 and R2 are each independently Ci-G$ alkyl.

In some embodiments, Rs is methyl.

In some embodiments, R2 is methyl

In some embodiments, R5 and R2 are both methyl. in some embodiments;, RJ'is H, methyl ethyl, isopropyl, or 2-hydroxyethyl.

In some embodiments, R2 is H.

In some embodiments, R2 is methyl, ethyl propyl or isopropyl.

In some embodiments, R1 is Η» methyl, ethyl, isopropyl, or 2-hydroxyethyi and R2 is O, methyl, ethyl, propyl, or isopropyl.

In some embodiments, m is 1.

In some embodiments, n is L

In some embodiments, both m and n are 1,

In some embodiments. L1 is -”NR6C(QK or -C(G)NR^-.

In some embodiments, L* is ~0C(0)~ or -0(0)0-.

In some embodiments, L* is 8-S-.

In some embodiments, V is -NCR^CCGlNfll6)-.

In some embodiments, Ls is -0C<0)N(R6)- or -N(R°)C(0)0-.

In some embodiments, L1 is -0-N~C-.

In some embodiments, L} -0C(0)NH-N=C- or ~ΝΗ€(0)ΝΗ-Ν==€-.

In some embodiments, L~ is or -C(G)NR6-,

In some embodiments, I,2 is -0C(O)- or -0(0)0-.

In some embodiments, L2 is S-S-. in some embodiments, L2 is -N(R6)C(0)N(R6)··.

In some embodiments, L2 is -OC(Q)N(Rs)- or -N(R.*)C(0)0-.

«J

In some embodiments, L~ is -O-NKX

In some embodiments, L2 '0C{0)NH~N«C* or -NHC(0)NK~Nk::C~> 1¾ some embodiments, both L* and L2 are-~NR:6C{OK or ~C(Q)NR\

In some embodiments, both I,L and I2 are -0C(0>- or -0(0)0-, in some embodiments, both L1 and Lf are S-S-.

In some embodiments, both Ll and L2 are -N(R6)CXO)N(RaH in some embodiments, both L* and V are or ~H(Ra)€(Q)0-,

In some embodiments, V is-NRf>C(0)- and L·2 is --3-5-,

In some embodiments, Ll is -00(0)- and L2 is --S-S-. la some embodiments, V is -00(0)1^6) or -H(Ra)€(0)G- and L2 is -5-8-, in some embodiments, L1 is -N(R6)C(0)N(R!>)~ and L2 is -8--3-,

In some embodiments, Ll~R3 and 1,¾4 are taken together to form an acetal, a ketai, or an orthoester.

In some embodiments, each RJ and R4 are independently alkyl.

In some embodiments, both R3 ami R4 are CVCas alkyl in some embodiments, each L5 and L2 are independently-S-S-, ~0C(O)N(R6}~ or ^(1^)0(0)0-.

In some embodiments, R3 is alkyl.

In some embodiments, R4 Is alkyl In some embodiments, R3 is alkenyl In some embodiments, R4 is alkenyl

In some embodiments, each R5 aid R4 ard independently alkenyl for example, each R3 and R4 are- independently CrCso alkenyl or each R3 and R4 are the same alkenyl moiety.

In some embodiments, each R3 and R4 includes two doable bond moieties. In some embodiments, at least one of the double bonds have a Z configuration. In. some embodiments, both of the double bonds have a Z configuration. In some embodiments, at least one of RJ and R4 is provided in formula (II) below

formula (ll) wherein x is an integer from t to S; and •j ( v js ajj integer from 1-10. In some embodiments, both of R and ΙΓ are of fee formula (II). In some embodiments, at least one of the double bonds have an E configuration, eg- both of the double bonds have an E configuration. In some embodiments, at least one oHT and R2 is provided in formula (III) below

formula (Hi) wherein x is an integer from 1 to 8; and y is an integer from 1-10. V v

In some embodiments, each R and R~ includes three double bond moieties. In some embodiments, a t least one of the double bonds have a Z configuration. In some embodiments, at least two of the double bonds have a Z configuration. In some embodiments, all three of the double bonds have a Z configuration. In some embodiments, at least one of R* and R2 is provided in formula (IV} below

formula (IV) wherein x is an integer from 1 to 8; and y is an Integer from 1-10. in some embodiments, both of Rf and R2 are as provided in formula (IV), In some embodiments, at least one of fee double bonds have an E configuration. In some embodiments, at least two of the double bonds have an E configuration. In some embodiments, all three of fee double bonds have ait E configuration, in some embodiments, at least one of II5 and R2 Is provided in formula (IV) below

formula (V) wherein x is an integer from 1 to 8; and v is δη integer from 1-.10, In some embodiments, both oi'K' and R~ are as provided in formula (V),. in,some embodiments, R and R" are each CrCr, alky! (e.g., methyl), Li and LI are each -00(0)-, and R3 and R4 are each alkenyl, in some embodiments, R3 and R4 are the same. In some embodiments, R5 and R4 both include two double bonds (e.g., having cts linkages). In some embodiments RJ and R4 are provided in formula (Π) below

formula (II)

Wherein x is an integer from I to 8 e.g,, 5; and y is as integer-tom. 1-10 e,g<, 4,

In one aspect, the invention features a preparation including a compound of xormuia {X}-

In one aspect, the invention features a preparation including a compound of formula (X) and a nucleic acid (e.g., an RNA such as an siRNA or dsRNA or a DMA),

In some embodiment, the preparation also includes an additional lipid such as a fusogenic lipid, or a .PBG-lipid,

In one aspect, the invention features a method of making a compound of formula C?0>

formula. (X) wherein

Rf and R~ are each independently Cj-Q alkyl, optionally substituted with M R5; r3 is alkyl, alkenyl, alkynyl l) ia-00(0)- R5 is -OR?, -N(RS)(R9), -m, SR1® S(O)R!0, S(Q)2RH> R6 is B, CrC6 alkyl;

Rr is H or Cj-CX alkyl; each IIs and R9 are independently H or CrQ, alkyl;

Rie is H or CrC6 alkyl; m and n ai'e each independently 1,2,-3,4, 5, or 6, the method eoniprising reacting a compound of formula {Vi),

formula (VI) with a compound of formula {VII}

formula. (VXI) in. the presence of a coupling agent, thereby providing a compound of formula (X).

In some embodiments, the coupling agent is a carbodiimide such as EDO.

In one aspect, the invention features a compound of formula (XV) below

formula (XV) wherein; each if and L2 are independently -a bond or C(Q); each R! and R2 are independently alkyl alkenyl or alkyayl; each of which is optionally substituted with one or more substituents; X is-C(0)Nfl·, €(S)NH, -G(O}Gi.3alkyiC(0)NHs or -C(O)CMalkyie(0)O··; m is an integer from 0-.11 and a is an integer from 1-500.

In some embodiments, I-1 and L2 are both a bond.

In some embodiments. If and L2 are both C(D).

In some embodiments, each R3 and R2 are independently alkyl, for example CV Gas alkyl, e.g^,C«jrCi* alkyl, e.g., 0¾ alkyl,alkyl, Ca alkyl, or Cj$ alkyl,. In some embodiments, both R* and R2 are alkyl, e.g., straight chain, alkyl having the same length, «•g·, Cf;~C28 alkyl, e.g,,Cjo-Cis alkyl, e.g„ 0» alkyl, Cm alkyl, Ci5 alkyl,.or Ck alkyl, In some preferred embodiments, both R1 and R2 are Ow alkyl

In some embodiments, the. formula XV reperesents a racemic mixture

In some embodiments, the compound of formula XV has an enantiomeric excess of the E isomer, e»g„ at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. in some-embodiments"the foimuk XV represents esarstiomerically pure-tR’ isomer.

In some embodiments, the compound of formula XV has an enantiomeric excess of the S isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. In some embodiments the formula XV represents enantiommeally pure IV isomer.

In some mbodimpais, each R? and R2 are independently alkenyl, for example, each Rf and R2 are independently Cs-Cso alkenyl or each R5 and R* axe foe same alkenyl moiety. In some embodiments, each R1 and R2 includes a single double bond, for example a single double bond in the E or Z configuration.

In some embodiments, each R* and R2 includes two double bond moieties. In some embodiments, at least one of the double bonds has a Z configuration, In some· embodiments, both of the double bonds have a Z configuration. In .some embodiments, at least one of R5 and R' is provided in foimuk (II) below

formula (11) wherein x Is an integer from ί to 8; and y is an integer from 1 -10, In some embodiments, both of Rs and R2 are of the formula (II). In some embodiments, at least one of the double bonds has an E configuration, e.g., both of the double bonds have an 1 configuration, hi some embodiments, at least one of R5 and R2 is provided in formula (111) below

formula (III) wherein x is an Integer front 1 to 8; and y is an integer Irom 1-10.

In some embodiments, each. Rl and R2 includes three double bond moieties, In some embodiments, at least one of the double bonds has a Z configuration. In some embodiments, at least two of the double bonds have a Z configuration. In some embodiments, all three of the double bonds have a Z configuration, In some embodiments, at least one of R* and R2 is provided in formula (IV) below

formula (IV) wherein x is an integer from I to 8; and y is an integer from MO. hi some embodiments, both of R* and RA are as provided In formula (IV), In some embodiments, at least one of the double bonds has an B configuration. In some embodiments, at least two of the double bonds have an E configuration. In some embodiments, all three of the double bonds have an E configuration. In some embodiments, at least one of R! and R2 is provided in formula (IV) below

forjnula(V) wherein x is an integer from I to 8; and y is an integer horn 1-10. In some eralxKliments, both of RJ and R"* are as provided in formula (V).

In some embodiments, X is -C(0)MH-, providing a compound of formula (XV’) below:

formula (XV). In some embodiments, each R* and R2 are independently alkyl, for example€^-(¾ alkyl, e.g.,CirCis alkyl, e.g,, Co alkyl, €<4 alkyl, €15 alkyl, or Cj(; alkyl,. In some embodiments, both R! and R2 are alkyl, e.g., straight chain 'alky! having the siane.length, ©ig,,.Cg-Qs»-alkyl,. ©,g,,C|0-Cf$ alkyl, e.g., Cj3 alkyl, Ct* alkyl, Cu alkyl, or 0½ alkyl. In some preferred embodiments, both R' and R2 are Ch alkyl

In some embodiments, X is -C(G)C2..3alkylC(0}C>~,

In some embodiments, m is an Integer from 1-10, for example an integer from 2-4 or an Integer 2,

In some embodiments, n is an integer front 1 -500, for example an integer from 40-400, from 100-350, from 40-50 or from 42-47.

In some embodiments, the compound is a compound of formula (XV*),

formula (XV1), wherein both li and L2 are a bond. In some embodiments, each R' and R~ are independently alkyl, for example Q-Ag alkyl, e,g,,CurCfa alkyl, mg., C54 alkyl, C\$ alkyl, or Cm alkyl, lit some embodiments, both R‘ and are alkyl, e.g., straight chain alkyl having the same length, e,g., QAg alkyl, e.g.,Cj(rC)s alkyl, e.g., Cu alkyl, Cjj alkyl, orCh* alkyl, In some preferred embodiments, both R1 and R* are C\4 alkyl In some embodiments, in is an integer from 1-10, for example an integer from 2-4 or an integer 2 In some embodiments, n is an integer from 1-500, tor example an integer from 40-400, or from 40-50,

In some embodiments, the compound is a compound of formula (XV1), wherein LI and L2 are both bonds. R1 and R2 are both alkyl (e.g,, CrAs alkyl, «.g-Ao-C}* alkyl, preferrably Cm alkyl), and n is an integer from.about 40-400.

In some embodiments, the comoond has a formula (XVI) below;

formula (XVI), wherein the repeating REG moiety has an average ntol ocular weiglit of 2000 with n value between 42 and 47,

In some embodiments, the compound of formula XV has ah enantiomeric ex cess of the R isomer, e.g., at least about 653b, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. In some embodiments the compound of formul a XVI is 4 stereo isomer with preferred absolute configuration 7P.

In one aigsect, the invention features a PEG lipid conjugated to a cholesterol moiety. For example» the compound -offormula (XX) below:

formula (XX). X is -C(0}HH-, C(S)NH, -C(Q}€ 5,3 alkyl C(0)NHs or -C(0)C,-3allcyie(0)O-.; m is an integer item (M l and π is an integer from 1-500.

In some embodiments the 0 attached to the cholesterol in formula (XX) is part of the cholesterol moiety. in some preferred embodiments, X is ~C(0)NH~, or -0(0)0j .3aikylC{0)0-.

In some embodiments, the compound of formula (XX) is as provided below in formula (XX’)

Ibnnula (XX').

In one aspect, the invention features a PEG lipid bound to a targeting moiety* for example a sugar residue. For example, the compounds of formula (XV) or (XX) are modified at the GMe terminal end with a targeting moiety. In some embodiments, the targeting moiety is bound to the PEG moiety via a Sinker, Exemplary targeted PEG lipids are provided in formulas (XXI) and (XXII) below.

In one embodiment, the lipid is a compound of formula (XXI)

formula (XXI) wherein; each Lj ami I" are independently a bond or C(O); each R1 and R2 are independently alkyl alkenyl, or alkynyl; each of which is optionally substituted with one or more sabs'tftuefits; each X. and X* is independently -NHG{0} C(S)NH, £(S)Mi,, C(0}C-oalkylG(D}NH-; NHC(0)Cs.3alkyiC{0)-C{0)C?^alk^e(0)0-; NHC(O.K^,. ,5«lkyl-; orCroaik>lC(Q)NHs m is an integer from (Η 1 and n is an integer from 1-500 p is am integer from I -6, e.g.. 3; T is a targeting moiety such as a glycosyl moiety (e.g;, a sugar residue).

Exemplary targeting moieties include

In some embodiments, L1 and L2 are both a bond.

In some embodiments, L5 and L2 are both C(Q).

In some embodiments, each R1 and R2 are independently alkyl, tor example C$-Cbg alkyl, e,g.Ao“C« alkyl, e.g., Gm alkyl, C\s alkyl, orCss alkyl,. In some embodiments, both R1 and R1 are alkyl, e.g., straight chain alkyl having the same lengthy e.g., €6-(¾ alkyl, eg-Rhe-Cis alkyl e.g., Cw alkyl, Cjs alkyl, or Cf6 alkyl In some preferred embodiments, both R1 and R2 are Cm alkyl

In. some embodiments, the formula (ΧΧΪ) reperesents a racemic mixture In some embodiments, the compound of formula (XXI) has an. enantiomeric excess of the R isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. In some embodiments the formula (XXI) represents enantiomerieally pure 97 ’ isomer.

In some embodiments, the compound of formula (XXI) has an enantiomeric excess of the S isomer, e.g,, at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. In some embodiments the formula (XXI) represents enantiomerieally pure \T isomer.

In some embodiments, each il1 and R2 are independently alkenyl, for example, eadr R1 and R“ are independently C6-C3.3 alkenyl or each R* and R“ are the same alkenyl moiety. In some embodiments, each R} and R2 includes a single double bond, for example &amp; single double bond, in the E or Z configuration. hr some embodiments, each R5 and R2 includes two double bond moieties. In some embodiments, at least one of the double bonds has a Z configuration. In some embodiments, both of tire double bonds have a Z'configuration, hi same embodiments, at least one of R3 and R2 is provided in formula (II) below

formula (II) wherein x is an integer .from 1 to 8; and v is an integer from 140. In some embodiments, both ofR* and R2 are of the formula (II). In some embodiments, at least one of the double bonds Iras an E configuration, e.g., both of the double bonds have an E configuration. In some embodiments, at least one ofR3 and R2 is provided in formula (II!) below

formula (III) wherein X is an integer from 1 to -8j and y is an integer from l-l 0.

In some embodiments, each R1 and R2 includes three double bond moieties, hr some embodiments, at least one of the double bonds has a Z configuration. In some embodiments, at least two of the double bonds have a Z configuration. In some embodiments, ail three of tire double bonds have a Z configuration, In some embodiments, at least one of R3 and R2 is provided in formula (IV) below

formula (IV) wherein x is an integer from 1 to 8; and v is an integer from 1-10, In some embodiments, both ofR* and R2 are as provided in formula (IV), In some embodiments, at least one of the double bonds has an E configuration. In some embodiments, at least two of fee double bonds have an E configuration. In some embodiments, all three of the double bonds have an E configuration. In some embodiments, at least one of R1 and R* is -provided in formula (IV) below

formula (V) wherein x is an integer from I to 8; and y is an integer from I-10. In some embodiments, both ofR3 and R4 are as provided in formula (V).

In some embodiments, p is 3.

In some embodiments, L is NHC(0)C^ alkyl (e.g., NHC{0)C3aikyl}.

In some embodiments, fee compound of formula (XX!) is fee compound of (ΧΧΓ) below:

formula (XX Γ).

In one embodiment, fee lipid is a compound of formula (XXII)

formula (XXII) wherein; each X and X’ is independently ---C(0)'NH-, ~NHC(0) -, C(S)NH, C(S)NH, ~ C(0}Ci.3aIkylC(O)NH-; NHC(0)Cj.ndkylC(0)-C(0)Cs,3a!kylC(0)0-;.NHC{0)Cfo 3alky!-;orC1..3alkyIG(0)NH-; m is an integer from 041 and n is an integer from 1-500 p is ait integer from 1-6, e.g., 3; T is a targeting moiety such as a gJycosyl.«5oi«ty (e.g., a s»g^ ^due).

Bxamplary targeting moieties include

In some preferred embodiments,·· the compound of formula (XX>I) is the compound of (XXIF) as provided below:

formula (ΧΧ.1Γ)

In one aspect the invention features an association complex comprising a compound preparation comprising a compound described herein (e.g.f a compound of formula (I) or a compound of formula (X)) and a nucleic ado such as an RNA a single stranded or double stranded RMA (e.g,> siRNA or dsRNA ora DNA), in some embodiments, the association complex i s a Hpoplex or a liposome. In some embodi ments tire association complex includes one or more, additional components -such as a targeting moiety, a fusogenic lipid, a PEGyiated lipid, such as a PEG-lipid described herein such as a PEG-lipid having the formula (XY), ,(XVS) or (XVI) or a structural component. In some embodiments, tire PEG-lipid is a targeted PEG-lipid as described herein, e.g.s a compound of fonnuia (XXI), (ΧΧΓ), (XXII), or (XXIP).

In one aspect, the invention features amethod of forming a liposome comprising contacting a lipid prepaation comprising a compound described herein (e.g. a lipid described herein such as a compound of formula (I) or formula (X)) with a therapeutic agent in tire presence of a buffer, wherein said buffer; is of sufficient strength that substantially all amines of the molecules formula 1 are protonated; is present at between 100 and 300mM; is present at a concentration that provides significantly more protonation of than does the same buffer at 20 rnM, in one aspect, the invention features a liposome made by the method described herein. Ιο one aspect, the invention features a method of forming a liposome comprising contacting δ lipid preparation described herein (e.g., a lipid preparation comprising a compound of formula (1} or a compound of formula (X)} with a therapeutic agent in a mixture comprising at least about 90% ethanol and rapidly mixing the lipid preparation with the therapeutic agent to provide a particle having a diameter of less then about 200 uM. In some embodiments, the particle has a diameter of less than about 50 uM,

In one aspect, the invention features a method of forming a liposome comprising contacting a lipid preparation described herein (e.g., a lipid preparation comprising a compound of formula (1) or a compound of formula (X)} with a therapeutic agent in the presence of a buffer, wherein said buffer has a concentration .from about· 100 to about 30GmM,

In one aspect, the invention features liposome comprising a preparation described herein (e.g., a lipid preparation comprising a compound of formula (I) or a compound of formula (X)> and a nucleic acid. In some embodiments, the preparation also includes-a PEGylated lipid, for example a PEG-lipid described herein, such as a PEG-Iipid having the formula (XV), ,(XV’) or (XVI), In some embodiments, the PEG-lipid is a targeted PEG-lipid as described herein, e.g., a compound of formula (XXI), (ΧΧΓ), (XXH), or (ΧΧΙΓ).Ιη some embodiments, the preparation also includes a structural moiety such as cholesterol, in some embodiments foe-preparation· of association complex includes compounds offormaulae (I), (XV) and cholesterol. in some embodiments, said nucleic acid is an siRNA, for example said nucleic acid is an siRNA which has been modified to resist degradation, said nucleic acid is an siRNA which has been modified by modification of the polysaccharide backbone, or said siRNA targets the·; ApoB gene.

In some embodiments, the liposome further eompristes a structural moiety and a PEGylated lipid, such as a PBG~Iipid described herein, wherein the ratio, by weight, of preparation (e.g·, a lipid preparation comprising a compound of formula (I) or a compound of formula (X)), a structural moiety such as cholesterol, PEGylated lipid, and a nucleic acid, is 8-22:4-10:4-12:0.4-2.2. In some embodiments, the structural moiety is cholesterol. In some embodiments, die ratio Is 10-20:0.5-8.0:5-10:0.5-2.0, e.g., 15:0.8:7:1. In some embodiments, the average liposome-diameter is between 10 ran mid 750 am, e,g., the average liposome diameter is between 30 and 200 run or the average liposome diameter is .between 50 and 100'ran. In some embodiments, the preparation, is less than 15%, by weight, of unreaeied lipid. In some embodiments, the ratio of the preparation (e.g,, a lipid preparation comprising a compound of formula (I) or a compound of formula (X)), the structural moiety such as cholesterol, and the PEG lipid Is about 42/48/10 (molar ratio). In some embodiments, the total lipid to nucleic acid (e.g., siRNA) is about 7.5% by weight

In some embodiments an association complex described herein has a weight ratio of total excipients to nucleic acid of less than about 15:1, for example, about 10:1, 7.5:1 or about 5:1.

In one aspect, the invention features a method of forming m association complex comprising a plurality of lipid moieties and a therapeutic agent, the method comprising: mixing a -plurality of lipid moieties In ethanol and. aqueous NaOAc buffer to provide a particle: and adding the therapeutic agent to the particle, thereby forming· the association complex.

In some embodiments, the lipid moieties are provided in a solution of 100% ethanol

In some embodiments, tire plurality of lipid moieties comprise a cationic lipid.

In some embodiments, the oationie lipid is a lipid described herein, for example, the cationic lipid is a lipid of one of the following or a mixture thereof:

. hi some preferred embodiments® cationic lipid is

In some embodiments, the plurality of lipid moietfes comprise a PEG-lipid, for example, the PEG-lipid has the following stracture:

wherein; each L1 and L·2 are independently a bond or C(O); each R5 and R2 are independently alkyl alkenyl or alkynyl; each of whichis optionally substituted with one or more substituents; X is ~€«})NH~, C(S)NH, -C{0)Cj.3al^lC(0)NH-; or-€(0}e5^a:lky!e{O)Os m is an integer from 041 and n is an integer from 1-500.

In some preferred embodiments, the PEG-lipid 'is a PEG lipd of formula (ΧΥί), wherein the repeating PEG moiety has an average molecular weight of 2000, for example, with an n value between 42 and 47 or the lipid provided below:

In some embodiments, the plurality Of lipid moieties comprises a structural lipid, for example, the structural lipid is cholesterol.

In some embodiments, tire PEG-lipid is a targeted PEG-lipid as described herein, e.g„ a compound of formula (XXI), (XXF), (XXII), or (ΧΧΐρ). in same embodiments, the method includes further comprising extruding the lipid containing particles, for example, prior to addition of the therapeutic agent.

In some embodiments, the therapeutic agent is a nucleic acid, for example, an siRNA, such as an siRNA which has been modified to resist degradation, an siRNA-which has been modified by modification of the polysaccharide backbone, or an siRNA conjugated to a Lipophilic moiety, hi some embodiments, the siRNA targets the ApoB gene,

In some embodiments, the association complex comprises a cationic lipid, a structural lipid, a PEG-lipid and. a nucleic acid.. In some embodiments, the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 36-48:42-54:6-1.4, for example, 38-46:44-52:8-.12 or about 42:48:10, in some embodiments, the weight ratio of total exipient to nucleic acid is less than about, 15:1.,. for example, about. 10:1 about 7.5:1 or about 5:1. In some preferred embodiments, foe cationic lipid has the following structure;

the PEG-lipid is a PEG lipd offormula (XVI), wherein the repeating PEG moiety has an-average molecular weight of2000, for example, with an n value between 42 and 47 or has the .following structure:

;and foe structural lipid is cholesterol, for example, wherein foe molar ratio of the cationic lipid, structural lipid, is PBG-Jipid is 38-46:44-52:8-12, e.g., about 42:48:10. k some preferred embodiments, the weight ratio of total exipient to nucleic acid is less than about 15:1, e.g., about 10:1, about 7,5:1, or about 5:1. k another aspect, foe invention features an association complex made from &amp; method described herein.

In another aspect, the invention features association complex comprising .a cationic lipid, a structural lipid, a PEG-lipid and a nucleic add, wherein the cationic lipid is is a lipid of one of the following or a mixture thereof:

the PEG-iipid is a PEG iipd of formula (XVI)* fte repeating PEG moiety has an average molecular weight of2000, for example, with m n value between 42 and 47 or has thefollowing structure:

; sod the structural lipid is cholesterol In some preferred embodiments, the nucleic acid is: an siRNA. 1» some preferred embodiments, the cationic lipid has the following formula:

In some preferred embodimonts» the molar ratio of the cationic lipid preparation, structural lipid (e.g„ cholesterol), PEG-lipid and nucleic acid is 36-48:42-54:6-14, tor example, 38-46:44-52:8-12 or about 42:48:10. In some preferred embodiments, the weight ratio of total exlpient to nucleic add is less than about 15:1, for example, about 10; 1, about. 7,5:1, or about 5:1.

Is some embodiments, .an association complex described herein has a mean diameter or particle sixe of less than, about 25000 nm, e.g,, from .abbot 20 to 200 nm, about 60, or about 50 nm.

In some embodiments, a nucleic acid as administered in an association complex described herein, demonstrates a serum half life (e,g,, in vitro) for at least about 4 hours, e.g., at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 1 week, at least about 2 weeks, or at least about 3 weeks,

In one aspect, the invention features a pharmaceutically acceptable composition comprising the preparation described herein.

In one aspect, the invention features a pharmaceutically acceptable composition comprising a liposome described herein.

In one aspect, the invention features a method of treating a mammal comprising, administering to said mammal a therapeutic amount of a pharmaceutically acceptable composition, for example, an association complex such as a liposome described herein.

The present invention as claimed herein is described in the following items 1 to 42: 1. A compound of formula (XV)

formula (XV) wherein 1 2 each of L and L is a bond; each R and R are independently C6-C28 alkyl, or C6-C30 alkenyl; X is -C(0)NH-; m is an integer from 0-11 and n is an integer from 1-500. 2. The compound of item 1, wherein each R and R are independently C6-C28 alkyl. 1 2 3. The compound of item 2, wherein each R and R are independently Cio-Cis alkyl. 4. The compound of item 1, wherein both R1 and R2 are straight chain C14 alkyl or Ci6 alkyl having the same length. 5. The compound of item 4, wherein both R1 and R2 are C14 alkyl. 6. The compound of item 1, wherein formula XV represents a racemic mixture. 7. The compound of item 1, wherein formula XV represents enantiomerically pure 7?’ isomer. 8. The compound of item 7, wherein formula XV represents a compound having an enantiomeric excess of ‘R’ isomer. 9. The compound of item 8, wherein the enantiomeric excess of the ‘R’ isomer is at least about 95% ee or greater than 97% ee. 10. The compound of item 9, wherein the enantiomeric excess of the ‘R’ isomer is at least about 98% ee or 99% ee. 11. The compound of item 1, wherein formula XV represents enantiomerically pure ‘S’ isomer. 12. The compound of item 11, wherein formula XV represents a compound having an enantiomeric excess of ‘S’ isomer. 13. The compound of item 12, wherein the enantiomeric excess of the ‘S’ isomer is at least about 95% ee or greater than 97% ee. 14. The compound of item 13, wherein the enantiomeric excess of the ‘S’ isomer is at least about 98% ee or 99% ee. 1 2 15 The compound of item 1, wherein each R and R is the same C6-C30 alkenyl moiety. 1 2 16. The compound of item 15, wherein each R and R includes a single double bond. 1 2 17. The compound of item 16, wherein each R and R includes a single double bond in the E or Z configuration. 1 2 18. The compound of item 15, wherein each R and R includes two double bond moieties. 19. The compound of item 1, wherein m is an integer from 1-10. 20. The compound of item 19, wherein m is an integer from 2-4 or an integer 2. 21. The compound of item 1, wherein n is an integer from 1-500, from 40 - 400, from 100 - 350, from 40 - 50 or from 42 - 47. 22. The compound of item 1, wherein, the compound has a formula (XVI) below:

formula (XVI), wherein the repeating PEG moiety has an average molecular weight of 2000 with n value between 42 and 47. 23. The compound of item 22, wherein the compound of formula XVI is a stereo isomer with preferred absolute configuration ‘R’. 24. The compound of item 23, wherein the compound of formula XVI has an enantiomeric excess of isomer of 90%, 95%, 97%, 98%, or 99%. 25. An association complex comprising: a. a cationic lipid; b. a PEG-lipid of claim 1; c. a structural lipid; and d. a nucleic acid. 26. The association complex of item 25, wherein said cationic lipid is one of the following or a mixture thereof:

27. The association complex of item 26, wherein said cationic lipid is

28. The association complex of item 26, wherein said cationic lipid is

29. The association complex of claim 25, wherein said PEG-lipid has the structure wherein:

n is an integer from 1-500. 30. The association complex of item 25, wherein said PEG-lipid has an enantiomeric excess of the R isomer.

31. The association complex of item 30, wherein the enantiomeric excess of the R isomer is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. 32. The association complex of item 29, wherein said structural lipid is cholesterol. 33. The association complex of item 25, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is 36- 48: 42- 54: 6- 14. 34. The association complex of item 33, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is 38- 48: 44- 52: 8- 12. 35. The association complex of item 33, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is in particular 42: 48: 10. 36. The association complex of item 25, wherein the weight ratio of total lipids to nucleic acid is less than about 15:1. 37. The association complex of item 36, wherein the weight ratio of total lipids to nucleic acid is about 10:1. 38. The association complex of item 37, wherein the weight ratio of total lipids to nucleic acid is about 7.5:1. 39. The association complex of item 37, wherein the weight ratio of total lipids to nucleic acid is about 5:1. 40. The association complex of item 25, wherein said cationic lipid is

said structural lipid is cholesterol; and said PEG lipid is

wherein: n is an integer from 1-500. 41. A method of forming an association complex of item 25, wherein the method comprises: (a) mixing the cationic lipid, PEG lipid, and structural lipid in ethanol and aqueous NaOAc buffer to provide particles; and (b) adding the nucleic acid to the particle, thereby forming the association complex. 42. The method of item 41, step (a) further comprising extruding the particles.

Definitions

The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine,

The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C36 alkyl indicates that the group may have from 1 to 136 (inclusive) carbon atoms in it.

The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). The terms "arylalkyl" or "aralkyl" refer to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term "alkylene" refers to a divalent alkyl, eg -CH2-, -CH2CH2-, -CH2- CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2CH2-.

The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-36 carbon atoms and having one or more double bonds. Examples of alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-36 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, prppargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the poin t of attachment of the alkynyl substituent.

The term “substituents” refers to- a group ^substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycio&amp;lkenyi, aryl, or heteroary! group at any atom of that group. Any atom can be substi.tut.eii Suitable substi tuents include, without limitation,-alkyl(e.g.. Cl, 02, C3, C4, C5, C6, C7, €8,0¾ CIO, Cll, Cl 2 straight or branched chain alkyl), cycloalkyl, haioalkyl (e.g., perfluoroaiky! such as €F3), aryl, heteroary!., aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, cycloalkenyi heterocycloalkenyl, alkoxy, haloalkoxv (e.g., periluoroalkoxv such as OCFr), halo, hydroxy, carboxy; carboxylaie, cyano, nitro, amino, alkyl amino, SO3H, sulfate, phosphate, methyienedioxy (-O-CBi-O' wherein oxygens are attached to same carbon (geminal substitution) atoms), ethyicnedioxy, oxo, ihioxo (e.g., OS), imino (alkyl, aryl, aralkyl), 8(0),,alkyl (where n is 0-2), S(Q)0 aryl (where n is 0-2), S(0)R heteroary! (where n is 0-2), S(O)n heterocyclyl (where a is 0-2), amine (mono-, di-. alkyl, cydoalkyl, aralkyl, heieroaralkyi, aryl, heteroary!, and combinations thereot), ester (alkyl, aralkyl, heteroaralkyi, aryl, heteroaryl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, aryl, heteroary!, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyi, and combinations thereof). In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents. In another aspect, a substituent may itself be substituted with any one of the above substituents.

The term “structural isomer” as used herein refers to any of two or more chemical compounds, such as propyl alcohol and isopropyl alcohol, having the same molecular formtda but different stmctural formulas.

The term “geometric isomer” or “stereoisomer” as used herein refers to two or more compounds which contain the same number and types-of atoms, and bonds (i.e., fee connectivity between atoms is the same), but which have different spatial ammgements of the atoms, tor example eis and trans isomers of a double bond, enantiomers, and diasteriomers.

For convenience, the meaning of certain tens® and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in-this section, tire definition in this section shall prevail eG," !i€y' "A" and "IF each generally stand for a. nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that fire tenn ‘‘ribonucleotide” or ‘‘nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety, The skilled person is well aware that guanine, cytosine, adenine, and uraei! may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil Hence, nucleotides containing uracil, guanine, or adenine may be replaced in tire nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mllNA molecule formed during the transmption of the corresponding gene, including mRNA that is a product of ENA processing of a primary transcription product, A target region is a: segment in a target gene that is complementary to a portion of the RNAi agent.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term ”eom.piememtarys,f when used to describe a Erst nucleotide sequence in relation to a. second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the Erst nucleotide sequence to hybridize and form:a duplex structure under certain conditions with .-an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 inM NaCI, 40 mM PIPES pH 6,4,1 mM ΒΌΤΑ, 50°C or 70WC for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply, lire skilled personwill be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This Includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucieotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences-can be referred to as “hilly complementary·" with respect to each other herein. However, where a first sequence is referred to as “substantially complementary* with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than. 4,3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to thdr ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not he regarded as mismatches with regard to the determination of complementarity. For example, an oligonucleotide agent comprising one oligonucleotide'21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 2inudeotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary” for the purposes of the invention. '‘Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Waison-Criok base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary" and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the an tisense strand of an oligonucleotide agent, or between tire antisense strand of an oligonucleotide agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is' “substantially complementary to at least part of’ a messenger ENA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest·. For example, a polynucleotide is complementary to at least a paft of an ApoB mRNA if the sequence is substantially complrnientaryto a non-interrupted portion of a.mRNA encoding ApoB.

As used herein, an; “oligonucleotide agent” refers to a single stranded oligomer or polymer of ribonucleic acid (ENA) or deoxyribonucleic add (DM A) or both or modifications thereof, which is antisense with respect to its target. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intemudeoside (backbone) linkages as well as oligonucleotides having non-natural ly-occurring portions which function similarly. Such modified or substituted oligonucleotides axe often preferred: Over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced sffmfty for nucleic acid target and increased stability in the presence of nucleases.

Oligonucleotide agents include both nucleic add targeting (NAT) oligonucleotide agents and protein-targeting (PT) oligonucleotide: agents. NAT and FT oligonucleotide agents refer to single stranded oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof. This term includes oligonucleotides composed of naturally occurring nncleohascs, sugars, and covalent irimmeleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, and/or increased stabili ty in the presence of nucleases. NATs designed to bind to specific RNA or DNA targets have substantial complementarity, e>g., at least 70,80,90, or 100% complementary, with at least .10,20, or 30 or more bases of a target nucleic acid, and iudude antisense R.N As, raicroRNAs, antagomirs and other non-duplex structures which can modulate expression. Other NAT oligonucleotide agents include externa] guide sequence (EOS) oligonucleotides (oligozymes), DNAzymes, and ribozymes. The NAT oHgonncfeofide agents can target any nucleic acid, e.g.s. &amp; miRNA, a pre-raiRNA. a pre-niRNA, an mENA, or a DNA. These NAT oligonucleotide agents may or may not bind via Watson-Crick complementarity to their targets, PT oligonucleotide agents bind to protein targets, preferably by virtue of three-dimensional interactions, and modulate protein activity. They include decoy RNAs, aptaraers, and the like.

While net wishing to be bound by theory, an oligonucleotide agent may act by one or more of a number of mechanisms, including a cleavage-dependent or cleavage-independent mechanism,- A cleavage-based mechanism can be RNAse H dependent and/or can include RISC complex function. Cleavage-independent mechanisms Include occupancy-based translational arrest, such as can be mediated bymRNAs, or binding of fee oligonucleotide agent to a protein, as do aptamers. Oligonucleotide agents may also be used to alter the expression of genes by changing the choice of splice site in a pre-mENA. inhibi tion of splicing can also result in degradation of the improperly processed message, feus down-regulating gene expression.

The. term ^double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic add molecules, having a duplex structure comprising two antiparallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to in the literature as siRNA (“short interfering RNA”). Where the two strands are part of one larger molecule, and feereibre are connected by an uninterropied chain of nucleotides between the 3’-end of one strand and the 5’end of fee respective other strand forming the duplex structure, fee connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or“shRNAT Where fee two strands are connected covalently hv means other than an. uninterrupted chain of nucleotides between the 3’-end of one strand and the Stead of the respective other strand forming the duplex structure, the connecting structure is referred to. as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs feat axe present in fee duplex. In addition to fee duplex structure, a dsRNA may comprise one or more nucleotide overhangs, in addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

As used herein, a: “nucleotide overhang” refers to the. impaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA .when a -3-end of one strand of the dsRNA extends beyond the 5’-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i,e., no nucleotide overhang at either end of the molecule, For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3 ’ end or 5’ end of an siRNA are not considered in determining whether an siRNA has an overhang or is biuni ended.

The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence, As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions aid, if present,, are generally in a terminal region or regions, eg., within 6,5,4,3,. or 2 nucleotides of the 5’ anchor 3’ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of tire antisense strand.

The terms “silence” and “inhibit die expression of’, in as far as they refer to a target gene, herein refer to the at least partial suppression of the expression of the gene, as manifested by a reduction of the amount of mRNA transcribed from the gene which may be isolated from a first cell or group of cells in which the gene is transcribed and which has or have been treated such that the expression of the gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

Alternatively, the degree of inhibition may be given in terms of a. reduction of a parameter that is functionally linked to gene transcription, e,g, the amount of protein encoded by the gene which is secreted by a cell, or fee number of ceils displaying a certain phenotype, e.g apoptosis. In principle, gene silencing may be determined in any cell expressing the target, either constitutive! y or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRMA inhibits the expression of the gene by a certain degree arid therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as snob reference.

For example, in certain instances, expression of the gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, the gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.

As used herein, the terms "treat", "treatment", and the like, refer to relief from or alleviation of pathological processes which can be mediated by down regulating a particular gene. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes which can be mediated by down regulating the gene), die terms "treat”, "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

As used herein, die phrases "therapeutically effective amount” and "propliylactieaHy effective amount" refer to an amount that provides a therapeutic benefit in fee treatment, prevention, or management of pathological processes winch can be mediated by down regulating fee gene on or an overt symptom of pathological processes which can be mediated by down regulating the gene. The specific amount that, is therapeutically effective can be readily determined by ordinary medical practitioner, mid may vary depending on factors known in the art, such as, e.g. the type of pathological processes which can be mediated by down regulating fee gene, the patient’s history and age, fee stage of pathological processes which can be mediated by down regulating gene expression, and fee administration of other anti-pafeologieal processes which can. be mediated by down regulating gene expression. An effective amount, in fee context of treating a subject, is sufficient to produce a therapeutic benefit. The term "tfaerapeufie benefit" as used herein refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of the subject’s cell proliferative disease, A list of noneXhaustive examples of this includes extension of the patients 11¾ by any period of time; decrease or delay in the neoplastic development of the disease; decrease hi hyperprol iteration; reduction in tumor growth; delay of raetastases; reduction in the proliferation rate of a cancer cell, tumor ceil, or any other hyperproliferative cell; induction of apoptosis in any treated cell or in any cell affected by a treated cell; and/or a decrease in pain to the subject that can be attiibufedid the patient's condition.

As used herein, a ‘"pharmaceutical composition” comprises a pharmacologically effective amount of an oligonucleotide agent and a pharmaceutically acceptable carrier. As used herein, “pharaiacologically effective «mount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an R.NA effective to produce the intended phanhacologteal, therapeutic or preventive result, For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for die treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof and are described in more detail below, The term specifically excludes cell culture medium.

The details of one or more embodiments of the invention are set thrill in the-accompanying drawings and the description below. Other features, objects, and advantages of the invention .will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 depicts a bar graph comparing the efficacy of various ND98 compositions.

Fig. 2 depicts a bar graph comparing the efficacy of various ND9S compositions.

Fig, 3 depicts a bar graph demonsrating the efficacy of a 6-tailed isomer of ND98.

Fig, 4 depicts a bar graph comparing the efficacy of association complexes, prepared, «sing two different procedures.

Fig. 5 depicts various PEG lipid moieties, including those having various chain lengths.

Fig. 6 depicts a bar graph comparing the efficacy of association complexes.

Fig, 7 depicts a bar graph comparing the tolerability of various complexes as the ratio of lipid to siRNA Is reduced.

Fig. B is a flow chart of a process for-makings» association complex loaded with nucleic: acid.

Fig, 9 are bar graphs depicting the efficacy of siRN As with two targets, FVH and

ApoB.

Fig. 10 is a flow' chart of a process for making an association complex loaded with nucleic acid.

Fig. 11 is a bar graph depicting the effect of particle size of association complexes on the efficacy of a nucleic acid in a silencing assay. figs. 12a and 12b are bar graphs comparing the serum half life of nucleic acid therapeutics in imfomrotafed and formulated·forms.

Fig, 13 is a bar graph comparing the efficacy of association complexes having PB<3 lipids with varied chain lengths.

DETAILED DESCRIPTION

Lipid preparations and delivery systems useful to administer nucleic acid based therapies such as si RNA are described herein,

Cationic Lipid compounds and lipid preparations

Pofyxmifte lipid preparations

Applicants have discovered that certain polyamine lipid moieties provide desirable properties for administration of nucleic acids, such as siRNA. For example, in some embodiments, a lipid moiety is complex®! with a Factor VO-targeting siRNA and administered to an animal such as a mouse. The level of secreted serum Factor VH is then quantified (24 h post administration}, where die degree of Factor VI! silencing indicates the degree of in vivo siRN A delivery. Accordingly, lipids providing enhanced in vivo delivery of a nucleic acid shell as si RN A are preferred. In particular, Applicants have discovered, polyandries having substitutions described herein can have desirable properties for delivering siRNA, such as bioavailability, biodegradahikly, and tolerability. ία one embodiment, a lipid prepa'alion includes a polyamine moiety having a plurality of substituents, such .as acrylamide or acrylate substituents attached thereto.

For example, a lipid moiety can include a polyamine moiety as provided below,

where one or more of the hydrogen atoms are substituted, for example with a substituent including along chain alkyl, alkenyl, or alkynyl moiety, which in some embodiments is further substituted, XR and Xb are aikylene moieties. In some embodiments, Xs and Xb have the same chain length, for example Xs and Xb are both ethylene moieties, in other embodiments Xs and Xfo are of di ffering chain lengths. In some embodiments, where the polyamine includes a plurality of X* moieties, Xa can vary with one or more occurrences. For example, where the polyamine is spermine, Xs in one occurrence is propylene, X3 In another occurrence is butylenes, and Xb is propylene.

Applicants have discovered that in some instances it is desirable to have a relatively high degree of substitution on the polyamine. For example, in some embodiments, Applicants have discovered that polyamine preparations where at least 80% (e,g., at least about 85%, at least about. 90%, at least about 95%, at least about $17%, at least about 98%, at least about 99%, or substantially all) of the polyamines in the preparation have at least n + 2 of the hydrogens substituted with a substituent provide desirable properties, for example for use in administering a nucleic acid such, as siRNA,

In some instances it is desirable (preferably) to have one or more of hetero atoms present on the substituent on the nitrogen of polyamine

In some embodiments, a preparation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof,

formula (I) each Xs and Xb, for each occurrence, is independently Ci„^ alkylene; a is 0,1»2, 3, 4, 0r 5; each R is independently H,

Rs Rjv 1% Ra wherein at least n + 2 of the R .moieties in at least about 80% of the molecules of the compound of formula (I) in the preparation are not H; m is 1,2,3 or 4; Y Is O, NR* or S;..R1' is alkyl alkenyl or alkynyl; each of which is optionally substituted; and R2 is alkyl alkenyl or alkvnyl; each of which is optionally substituted; prodded that, if n ~ o, than at least n + 3 of the ll moieties are not H.

As noted above, the preparation includes molecules containing symmetrical as well as asymmetrical polyamine derivatives. Accordingly, X* is independent for each occurrence and Xb is independent of Xa. For example, where n is 2, Xs can either be the same tor each occurrence ox can be different for each occurrence or can he the same for some occurrences and different for one or more other occurrences. Xh is independent, of Xs regardless of the number of occurrences of X® in each polyamine derivative. X*s for each occurrence and independent of Xb, can be methylene, ethylene, propylene, butylene, pentyl ene, or hexylene. Exemplary polyamme derivatives include those polyamines derived from -(ethane-1 ,2-diyI)diefhane-1,2-diamine, ethane-1,2» diamine, propane-1,3-diaminc, spermine, spermidine* putreeme, and N5 -(2-Aminoethyl)-propane-1,3-diamine. Preferred polyamide derivatives ineladepropano-1,3-diamme and Nl,NJ -fethane-l,2-diyl)ciiethano-l,2“diaminq.

The polyamine of formula (1) is substituted with at least nT2 R. moieties that are not H. In general, each non-hydrogen R moiety includes an alkyl, alkenyl, or aikynyl moiety, which is optionally substituted with one or more substituents, attached to a nitrogen of the polyamine derivative via a linker. Suitable linkers include amides, esters, thioesters, suifones, sulfoxides, ethers, amines, and thioethers, In many instances, the linker moiety is bound to the nitrogen of the polyamine via an alkyien© moiety (e.g., methylene, ethylene, propylene, or butylene). For example, ait amide or ester linker is attached to the nitrogen of the polyamine through a methylene or ethylene moiety.

Examples of preferred amine substituents sreprovided heknv:

In. instances where the. amine is bound to the linken-R* portion via. an ethylene group, a ! ,4 conjugated precursor acrylate or acrylamide can be reacted with the polyamine to provide the substituted polyamine. in instances where the amine is bound to the linker-R! portion via a methylene group, an amide or ester including an alpha-halo substituent, such as an alpba-chla.ro moiety, can be reacted with the polyamine to provide the substituted polyamine. In preferred embodiments, R2 is B. R moieties Hat are not H, ail require an R* moiety as provided above. In general,, the R5 moiety is along chain moiety, such as Ct-Cn alkyl, CVC32 alkenyl, or CrC32 alkynyl.

In some preferred embodiments, R! is an alkyl moiety. For example R1 is €%-C{3 alkyl, such as C12 alkyl Examples of especially preferred R moieties are provided below.

The preparations including a compound of formula (I) can be. mixtures of a plurali ty of compounds of formula (1). For example, the preparation can incl ude a mixture of compounds of formula (1) having varying degrees of substitution on the polyamine moiety. However, the preparations described herein are selected such that at least n + 2 Of the R moieties in at least about. 80% (e.g,< at least about $5%, at least about 90%, at least about 95%, at least about 91%, at least about 98%, at least about 99%, or substantially all) of the molecules of the compound of formula, (I) in the preparation are not H.

In some embodiments, a preparation includes a polyamine moiety having two amino groups wherein in at least 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or substantially all) of the molecules of formula (1). in the mixture are substituted with three R moieties that are not 11 Exemplary compounds of formula (I) are provided below.

In some preferred embodiments R is

In some preferred embodiments, R1 isCfe-C*» alkyl, or Cfp-Cjo alkenyl.

In some embodiments·, a preparation includes a poiyamkte moiety having three or four (e.g., four) amino groups wherein at least n+2 of the R moieties in at least about 80% (e.g,, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or substantially all) of the molecules of formula ¢1) are· not H. Exemplary compounds of formula (1) having 4 amino moieties are provided below.

Examples of polyamine moiety where all (i.e., ft+4) R moieties are not H are below:

In somepreferred embodiments 11 is

In some preferred embodiments, R.1 isCje-Cjg alkyl (e.g,, C!2 alkyl), or Cio-Cso alkenyl.

Examples of pelyamine moieties where five (i.e,, a*3) R .moieties are not H are provided below.

In some .preferred embodiments R is

In some preferred embodiments, Rf isCjo-Cjg alkyl (e.g,. Cn alkyl), or Cio-C^, alkenyl.

Bxamples of polyamine moieties where four (he, h+2) It moieties are not H are provided below:

in some preferred embodiments R is

In'Some preferred embodiments, Rl isCte-Cjs alkyl (e.g,, C&amp; alkyl), or CtfrCw alkenyl,

In some preferred embodiments, the polyamine is a compound of isomer (I) or (2) below, preferably a compound of isomer (1)

isomer (1) isomer (2).

In some embodiments, the preparation including a compound of formula (I) includes a mixture of molecules having formula (1). For example, the mixture can include .molecules having the same polyamine core but differing R substituents* such as differing degrees of R substituents that are not H. in some embodiments, a preparation described herein includes a compound of formula (I) having a single polyamine core wherein each R of the polyamine core is either R or a single moiety such as

The preparation, therefore includes a mixture of molecules having formula (1), wherein the mixture is comprised of either polyamine compounds of formula (!) having a varied number of R. moieties that are- H and/or a polyamine compounds of formula (1) having a single determined n umber of R moieties that are not H where the compounds of formula (I) are structural isomers of the polyamine, such as the structural isomers provided above,

In some preferred embodiments the preparation includes molecules of formula (I) such that at least 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at. least about 97%, at least about 98%, at least about 99%, or substantially all) of tie molecules area single structural isomer.

In some embodiments, the preparation includes a mixture of two or more compounds of formula (I). In some embodiments, the preparation is a mixture of. structural isomers of the same chemical formula. In some embodiments, the preparation is a mixture of compounds of formula (I) where the compounds vary in the chemical nature of fire R substituents. For example, the preparation can include a mixture of the following compounds:

•formula (1) wherein n is 0 and each R is independently H or

and

formula (I) wherein n is 2 and each R is independently H or

In some embodiments, the compound of formula (I) is in the form of a salt, such as a pharmaceutically acceptable salt, A salt, for example·, can be formed between an anion and a positively charged substituent {e.g., amino) on a compound described herein. Suitable anions include fluoride» chloride, bromide, iodide, sulfate, bisulfate» nitrate, phosphate, citrate, meihanesulfonate, trifltioroacetate» acetate, fumaraie, oleate, valerate, maleate, oxalate, isouieotinate, laetate, salicylate, tartrate, fannate, pantothenate, bitartrate, ascorbate, succinate, gentisinate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, efoanesulfonate, beBzene$olf€mateSjp-toluensulfosate, and pamoate. In some preferred embodiments, the compound of formula (!) is a hydrohalide salt, such as a hydrochloride salt.

Compounds of .formula (l) can also be present in the-form of hydrates (e.g., (HjO)r) and solvates, which are included herewith in the disclosure.

Bhcleavahie cationic lipids

Applicants have discovered that certain cationic lipids that include one or more biocleavahle moieties can be used as a component in an association complex, such as a liposome, for the delivery of nucleic acid therapies (e.g., dsRNA). for example, disclosed herein are cationic lipids that are subject to cleavage in vivo, for example» via m enzyme such as an esterase, an amidase, or a disulfide cleaving enzyme. In some instances, the lipid is cleaved chemically, for example by hydrolysis of an acid'labile moiety such as an acetal or ketal. In some embodiments, the lipid includes a moiety that is-hydrolyzed in vitro and then subject to enzymatic cleavage by one or more of an esterase, amidase, or a disulfide cleaving enzyme, This can happen in vesicular compartments of the cell such as endosomes. Another acid sensitive cl eavabie linkage is p-thiopropianate linkage which is cleaved in foe acidic environment of endosomes (ieong et a!. Bio conjugate chem. 2(M)3,4,1426). la soi»» embodiments, (be invention features a. compound of fommla (X) or a pharmaceutically acceptable salt thereof wherein

formula (X) wherein R! and R" are each independently K, Cj-Q alkvi, optionally' substitute with 1-4 R\ CrCe alkenyl, optionally substituted with 1-4 R?, or CCNR^CNR6}^; R3 and R4 are each independently alkyl, alkenyl, alkynly, each of which is optionally substituted with jfluoro, eliioro, hroino. or iodo; V and L2 are each independently ~~NRftC(0)-, -C(0)NR% -0C(0>, -0(0)0-, -S-S-, -Ν(Κ&amp;)€.(0)Ν(Εδ)-, -0€(0)Ν(Κδ)-, -^)0(0)0-, -0-N-0-, 0R-0€(0}NH; or ΐΛίΟ and L3-R4 can be taken together to form an acetal or a ketal; R5 is fluord, chJo.ro, bromo, iodo, -OR7, -N(R*)(R9.), -CN, SRi0, S(0)Rw, 8(0)211Κΐ R5 is H, CrQ alkyl, R,! is H or CrC<i alkyl; each Rs and Rs are independently H or Ch-Cs alkyl;

Rs<) is II or C-rQ alkyl; rn is 1,2,3,4,5, or 6; n Is 0,1,2,3,4, 5, or 6; and pharmaceutically acceptable salts thereof.

In some embodiments, RJ is H, a lower alkyl, such as methyl, ethyl, propyl, or isopropyl, or a substituted alkyl, such as 2-hydroxyethyl.

In some embodiments, R2 is H or a lower alkyl, such as methyl, ethyl, propyl, or isopropyl.

In some embodiments, Rf or R2 form a quanadine moiety with tile nitrogen of formula. (X). L!-RJ and L2-R4 or the combination thereof provide at. least one moiety that is cleaved in vivo. In some embodiments, both L’-R* and L2-R4 are bioeleavable. For example, both Lf-R3 ami L-R4 are independently subject to enaymaitic cleavage (e.g„ by an esterase, amidase, or a disulfide cleaving enzyme). In some embodiments, both

Ls and L2 are the same chemical moiety such as an ester* amide or disulfide, fe other instances, L and if are different, for example, one of L or tr is m ester rm the other of L3 or l2 is a disulfide, la some embodiments, lAll3 and L2~R4 together form an acetal or ketal moiety, which is hydrolyzed in vivo.

In some embodiments, one of L’-R3 or L2-R4 is subject to enzymatic cleavage. For example, one oflZ-R3 or iAr4 Is cleaved in vivo, providing a free hydroxyl moiety or tree amine on the lipid, which' becomes'available to chemically react with the remaining iAr' or lAR* moiety, Exemplary embodiments are provided below:-

In some preferred embodiments, a carbamate or area moiety is included in 'Combination with an amide, ester or disulfide moiety. For example, the lipid includes an ester moiety, which upon cleavage (e.g., enzymatic cleavage) becomes available to chemically react with the carbamate or urea moiety. Some preferred combinations of L* and L·2 include two amides, two esters, an amide and an ester, two disulfides, an amide and a disulfide, -an ester and a disulfide, a carbamate and a disulfide, and a urea and a disulfide. Exemplary compounds are provided below;

Amide and ester linkages with Z configuration (two double bonds)

R' = H, Ms, Et, propyl, isopropyl or 2-hydroxyetftyl ana R“ «Η;!» 1 to€, m» 1-8, n ~ 1-10 R!« H, Ms, Et, propyl, isopropyl or 2-hydroxyethyl and R" - Me; I = 1 to 8, m - 1-8, ft =-1-10 R'» H, Ms, Et, propyl, isopropyl or 2~hydfoxyetnyl and R":: Et; I ~ 1 to 8, m = 1--8, n = 1-10 R' - H, Ms, Et, propyl, isopropyl or 2-hydroxyetriyI and R* = propyl; ι ~ i to 8, m « 1-8, n - 1-10 R1 = H, Ms, Et, propyl, isopropyl or 2-hydroxyethyl and R" - isopropyl; i - 1 to 6, m ~ 1-8, n = 1-10

Amide Ester linkage with Z configuration (three double bonds}

R‘ = H, Me, El, propyl, isopropyl or 2-hydroxyetftyi and R" = H; f = 1 to 6, ;rs = 1 -8, n = 1-10 R‘ = H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R* = Ms;! = 1 to 6, m = 1-θ, n = 1-10 R‘ = H, Ms, Et, propyl, isopropyl or 2-hydfoxyethyl and R” = Et I = 1 to 8, m » 1-8, rt = 1-10 R' = H, Me, Et, propyl, isopropyl or 2-hydroxyethyi and R° = propyl; i~ 1 to δ, m » 1-8; n « 1-10 , R' = H, Me, Et, propyl, isopropyl or 2-hydroxyethyi and R“ = isopropyl; I = 1 to 6, m ~ 1-8, rs = 1-10

Amides and ester linkages with E configuration (two double bonds)

R'« H, Me, B, propyl isopropyl or 2-hydroxyethyi and R" ~ H; 1= 1 to 6, m = 1-8, π - 1-10 R' = H, Me, Et, propyl, isopropyl or 2-ftydrcKyethyi and R" ~ Me; I = 1 to 6, m = 1-8, π = 1-10 R’ a H, Me, Et, propyl, isopropyl or-2-hyclroxyefhyi and R“ — Et; t ~ 1 to 6, m = 1-8. rs = 1-10 R' = H, Me, Et, propyl isopropyl or 2~hydroxyethyl and R" ® propyl; 1=1 to 0, fn = 1-8, r> =1--10 R* ^ H. Me, Et, propyl isopropyl or 2-hydroxyethyi and R" = isopropyl: l = 1to8,m ~ 1-8. n = 1-10

Amides and ester linkages with E configuration (three double bonds)

R' » H, Me, Et, propyl isopropyl or2-fcydroxyethyf and RM * H; i« 1 to 6, m «1-8, n “ 1-10 R' = H, Me, Et, propyl isopropyl or 2~ftydroxyeihyi and R" ~ Ms; i = 1 to 6. m = 1-8, n = 1-10 R*H, Sfe,Et propyl isopropyl or 2-trydroxye-thyi and R" = Et; I = 1 to 5, m« 1-8, n == 1-10 K ~ H, Ms. Et, propyi isopropyl or 2-hydroxyethy} and RK - propyi; ? * 1 to 6, m « 1-8, ή a 1-10 R ^ H, Me, Et, propyi isopropyl or 2-hydroxyethy! and R* *isopropyl; i = 1 to 6, m = 1*8, n ~ 1-10

Disulfide linkages

R’ = H, Me, Et, propyl, isopropyl or 2-hydr0xyetoyt and Ft" = Η; I = 1 to δ, m- 6-28 R’ = H, Me, Et, propyl. Isopropyl orS-nydroxystoyi and R*« Me; i«1 to 6, m ~ 8-28 R! ~ H, Me, Et, propyi isopropyl or2-pydfoxyeii!yl and R° = Et I -1 to 8, m * 8-28 R’« H,Me, Et, propyl Isopropyl or2-fijtoroxyethyi and R”~ propyl; t*'1 to6..m * 8-28 H’ - H, Me, Et, propyi isopropyl or 2-hydfOxyethyf and R” * isopropyl; i«1to8,m“ 6-28

Disulfide linkages with unsaturated alkyl chains, E and Z configuration

E = H, Me, ES, propyl, isopropyl or 2-hydroxyath)=l and R" ~ Η; I = 1 to 8, m = 1-8, rs = 1-10 E' = H, Me, Ei, propyl, isopropyl or 2-hydroxystbyi and R“ = Me; I ~ 1 to 6, m = 1-8, π ~ 1-10 R’s H, Me, ES, propyl, isopropyl or 2-hydrcxyethy! and R“ = Et; i = 1 to 6, m - 1-5, n =- 1-10 ,E' 3 h. Me, Ei, propyl, isopropyl of 2-hydroxyethyl and RK -= propyl; I = 1 to 6, rr, == 1-8, r == 1-10 E =: h, Me, E!, propyl, isopropyl or 2-PydrexyetPyl and R" = isopropyl;! ~ 1 So δ, m :== 1-8, n ··== 1-10

Amide and disulfide linkages with saturated and unsaturaied alkyl chains

ft' »'H, Me.. Et, propyi, isopropyl or 2-pydroxyethyl and R" = H; i = 1 to 6, m »6-2S R‘-H, Me.. Et, propyl,isopropylor2-hydroxyethyfandR“* Me; I = 1 to6, m = 8-28 R‘ - H, Me, Et. propyl, isopropyl or2-hydroxyetbyj and ft” * Et; I * 1 to 6, m - 6-28 R’« M, Ms, Et, propyi, isopropyl or2-hydroxyethy{ and ft" * propyl; f = lb 6. m * 6-28 R’* H, Ms, Et, propyl, isopropyl or 2-hydroxyeihyi and R" = Isopropyl; S ~ 1 to 8, m ~ 8-28

R! ~ H, Me, Et, propyl, Isopropyl or 2-bydroxyethyi and R“ = Η: t = 1 to 8, m = 1-8, n -1-10 R** H. Ms, Et, propyl, isopropyl or 2-hydrexyethyl and R“ * Me; i« lto8.pi* 1-8, n = 1-10 R‘ ~ H, Me, Et,, propyi, isopropyl sr2-hydroxyethyi and R" = Et; I ~ 1 to 8, m - 1-8, n * 1-10 R’« H, Me, Et. propyi. isopropyl or 2-hydroxyethy! and ft" = propyi; I - 1 to 8, m - 1-8, n - 1-10 R: - H, Me, Et, propyi, isopropyl or 2-bydroxyethyi and ft”»isopropyi; I * 1 to 8, m * 1-8, n « 1-10

Ester and disulfide linkages with saturated and utisaturated alkyl, chains

S’ ~ H, Me, Et, propyl isopropyl or Z-hydroxysthyl and R“ ~ Η; I = 1 to ¢, m « 6-28 E' -- H, Me, Et, propyl isopropyl or Z-hydroxy ethyl and RK = Me; I = 1 to 6, m = 8-28 R‘ = H, Me, 'Et, propyl Isopropyl or 2-hydroxyethyl and R” * Et; f» 1 to β, m « 6-28 R‘ = H, Me, £!, propyl Isopropyl or Z-hydrdxyethyl and R" = propyl; l ~ 1 to 6, m ~ 8-28 R' ® H, Me, Et propyl, Isopropyl or 2-hydroxyotftyi and R"«isopropyl I ~ 1 to 6, m * 6-28

R: ~ H, Me, Et, propyl isopropyl or 2-hydraxyetbyl and R" - Η; I = 1 to 8, m = 1-8, n a 1-10 R ~ H, Ms, Et, propyl, isopropyl or 2-bydroxyeihyl and R" * Me; I ~ 1 to 8, m = 1-8, n = 1-10 R·« H, Ms, Et, propyl, isopropyl or 2-bydroxyemyl and R” = Et, I = 1 to 8, rn ~ 1-8, n =-1-10 R· - Id, Ms, Et, propyl isopropyl or 2-hydroxyethyl and R" ~ propyl I = 1 to 8, m - 1-8, ft 1-10 Ft« H, Ms, Et, propyl Isopropyl or 2-hydroxyethyl and R“ = isopropyl; i * 1 Jo 8, m »1-8, n - 1-10

Carbamate or urea and disulfide linkages with alkyl chains

R' ~ H, Me, Ef, propyl, isopropyl or 2-hydroxyethy! and R" “ Η; I ~ 1 to 6, m ~ 8-28 R’ = H, Me, Et propyl, isopropyl or 2-hyt}roxyethyi and FT ~ Me; i ~ 1 to 6, m ~ 6-28 R’ = H, Me, Et, propyl, isopropyl or 2-hydroxyethyS and R" ~ Et; I ~ 1 to 6, m » 6-28 R* - H, Ms, Et, propyl, isopropyl or2-hydroxyethyl and R" = propyl; f = 1 fa 8, m - 6-28 R' = H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R" ~ isopropyl; I ~ 1 to 6, m ~ 8-28

Carbamate or urea and disulfide linkages with unsaturated alkyl chains

R' = Η» Ms, Et, propyl, isopropyl or 2-fiydroxyethyl and R" ~ H; f ~ 1 to 6, m ~ 3-26 R* ~ H, Me, Et, propyl isopropyl or 2-ftydraxystfty) and R” ~ Me; i = 1 to 6, m - 8-28 R‘" H, Me, Et, propyl isopropyl or 2-hydroxyethy1 and R” ~ Et; 1 = 1 to 8. m = 6-28 R‘ “ H, Me, Et, propyl, isopropyl or 2-ftydraxyethyj and R” = propyl; ί = 1 to 6, m == 6-28 R’ = H, Me, Et, propyl, isopropyl or 2-Pydroxyethyi and R” = isopropyl: I = 1 to 6, m = S-28

Carbamate or urea and disulfide linkages with unsaturated alkyl chains

R' = H, Me, Et, propyl, Isopropyl·or 2~Pydfoxyetbyt and R* = H;} = 1 to 8, m = 8-28 R'» H, Me, Et, propyl, isopropyl or Mydroxyethyi and R* - Me; i - 1 to 6, m = 8-28 R‘ * h, Me, Et, propyl, isopropyl or 2-hydroxyethyt and R" .= Et; i = 1 to 6, m s 6-28 FT = H, Me, Et, propyl, isopropyl or 2-hydroxyetftyi and FT = propyl; l~1 to 8, m = 6-28 R' = H, Me, Et, propyl, isopropyl or 2-nydmxy&amp;tftyi and R* = isopropyl·; 1 = 1 to 6, m ~ 6-28

Carbamate: and urea linkages with, ansaturated aikyl chains

R* = H Ms, Et, propyl, isopropyl or 2-hydroxyethy! and R* * Η; f = ftp-8, m-1-10, n ~ 1-10 R' ~ H Ms, Et propyi, isopropyl of 2-hydroxyeti^'i and R"« Mo; I»1 to 8, rrs = 1-10. n* 1-10 R! *· H, Me, Et propyl isopropyl or 2-hydroxyethyi and R" = Et; f «1 to 6, m “ 1-10, n ~ 1-10 R’ = H· Me, Et, propyl, isopropyl or 2-hydroxyethyi and R" * propyl; I = ί to B, m *1-10, n -1-10 R’ * H, Me, Et. propyl, isopropyl or 2-hydroxyethyl and R” = isopropyi; I«1 to 6, m ~ 1-10, n ~ 1-10 in some embodiments, the lipid .includes an oxime or hydmone, which can undergo acidic cleavage, R,:; and R4 are generally long chain hydrophobic moieties, such, as alkyl, alkenyl, or alkynyi, in some embodiments, R3 or R4 are substituted with a halo moiety, for example, to provide a perfiuoroalkyl by per.iluoroalken.y1 moiety, Bach of R3 and R* are independent of each other. in some embodiments, both of K3 and R4 are the same, In. soma embodi ments, R3 and R4 are different

In some embodiments R3 and/or R4 arc alkyl. For example one or both of R3 and/or R4 are Cg to Cyo alkyl, e.g., (¾ to Cu alkyl, Ci2 to C20 alkyl, or (¾ alkyl.

In some embodiments, R3 and/or R4 are alkenyl. In some preferred embodiments, R2 and/or R4 Include 2 or 3 double bonds. For example-R-* and/or R4 includes 2 double bonds or R3 and/or R4 includes 3 double bonds. The double bonds cars each independently have a Z or E configuration. Exemplary’ alkenyl moieties are provided below:

wherein -x is an integer from I to 8; and y is an integer from I -1Q. in some preferred embodiments.. R3 and/or R4 are €$ to C$o alkenyl, e.g., Cf<* to Cx, alkenyl, Go to'C20 alkenyl, or Cj7 alkenyl, for example having two double bonds, such as two double bonds wife Z configuration. R3 and/or R4 can be fee same or different. In some preferred embodiments, IG and R4 are fee same.

In some embodiments, R3 and/or R4 are alkynyl. For example C§ to 0¾ alkynyS, e.g,, Cio to Cu alkynyl, C12 to Cse-alkynyl. R3 and/or R4 can have from 1 to 3 triple bonds, for example, one, two, or three triple bonds.

In some embodiments, fee compound of formula (X) is in fee form of a salt, such as a pharmaceutically acceptable salt. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include fluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate, phosphate, citrate, rnethanesnlfonate, trifluoroacetate, acetate, fomarate, oleate, valerate, maleaie, oxalate, isonicotinate, lactate, salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate, succinate, geniisinate, gluconate, glucaromte, saecharate, formate, benzoate, glutamate, ethanesulfonate, beuzenesui innate, p~ toluensulfonate, and pamoate. In some preferred embodiments, fee compound of formula (X) is a hydrohalide salt, such as a hydrochloride salt.

Compounds of formula (X) can also be present in the- form of hydrates (e.g., (%0)«) and solvates, which are included herewith in the disclosure. FEG~lipid compounds

Applicants have discovered that certain PEG containing lipid moieties provide desirable properties for administration of a nucleic acid agent such as single stranded or double stranded nucleic acid, for example siRNA, For example, when a PEG containing lipid, such as a lipid described herein, is formulated into an association-, complex-wife a nucleic acid moiety, such as siRNA and administered to a subject, the lipid provides enhanced delivery of the nucleic acid moiety. Tins enhanced delivery can be determined, tor example, by evaluation in a gene silencing assay such as silencing of FVH. In particular. Applicants have discovered the PEG-Hpids of formula (XV) can have desirable properties for the delivery of siRNA, including improved bioavailability, diodegradabiliiy, and tolerability, to some embodiment, the PEG is attached via a Oak®· moiety to a structure Including two hydrophobic moieties, such as a long chanln alkyl moiety. Examplary FEG-lipids are provided above, for example, those encompassed by formula (XV), (XVs). and (XVI). In some preferred embodiments, the PECMipid has the structure below:

, wherein the preferred stereo chemistry of tire chiral center is VT and the repeating PEG moiety has a total average molecular weight of about 2000 dal torts. to some embodiments, a PEG lipid described herein is conjugated to a targeting moiety, e.g., a glyeosyl moiety such as a

in some embodiments, the targeting moiety is attached to the PEG lipid through a linker, for example a linker described herein. Exemplary targeted PEG lipid compounds ate compounds of formula (XXI), (XXF}».(XX[1), and (ΧΧΪΓ) described herein. Methods of making such lipids are described, for example, in Examples 42 and 43.

Methods of ma kin g cationic lipid compounds and cationic lipid containing preparations

The compounds described herein can be obtained from commercial sources (e.g., Asinex, Moscow, Russia; Bionet, Camelfbrd, England; ChemDiv, SanDiego, €A; Comgenex, Budapest. Hungary; Enamine, Kiev, Ukraine; IP Lab, Ukraine; Interbioscreen, Moscow, Russia; Maybridge, Tintagel, UK; Specs, lire Netherlands; Timtec, Newark, DE; Vitas-M Lab, Moscow, Russia) or synthesized by conventional methods as shown below using commercially available starting materials and reagents.

Methods of making polymnine lipids

In some embodiments, a compound of formula (1) can be made by reacting a polyaraine of formula (01) as provided below

formula (III) wherein Xs, Xs, and a are defined as above with a 1,4 conjugated system of formula (IV)

formula (IV) wherein Y and It’ are defined as above to provide a compound of formula (I).

In some embodiments, the compounds of formula (III) and (IV) are reacted together neat (i.e., free of solvent). For example, the compounds of formula (III) and (IV) are reacted together neat at elevated temperature (e.g., at least about 60 °C·, at least about 65 “Cj at least about 70 °C, at least about 7S °C, at least about 80 °C, at least about 85 °C, or at least about 90 °C), preferably at about 90 °C.

In some embodiments, the compounds of formula (III) and (IV) are reacted together with a solvent (e.g,, a polar aprotic solvent such as acetonitrile or DMF). For example, the compounds of formula (Hi) and (IV) are reacted together in solvent at an elevated temperature from about 50 C‘C to about 120 eC,

In some embodiments, the compounds of formula (III) and (IV) are reacted together in the presence of a radical quencher or scavenger (e.g,, hvdroquinone). The reaction conditions including a radical quencher can be neat or in a solvent e.g., a polar aprotic solvent such as acetonitrile or DMF. 'flic reaction can he at an elevated temperature (e.g,, neat,at an elevated temperature such as 90 °C or with solvent at an elevated Comperature such as from about 50 °C to about 120 °C). The term "'radical quencher” or ^radical scavenger1” as used herein refers to a chemical moiety that can absorb free radicals in a reaction mixture, Examples of radical quenefrers/seavengers include hydroquirione, ascorbic acid, cresols, thiamine, 3,5-Di-tCit«butyM*· hydroxy toluene, iert4%tyl~4~hyd.roxy&amp;nlsole and thiol containing moieties.

In. some embodiments., the compounds of formula (ΙΠ) and (IV) are reacted together m the presence ofa.reaction promoter (e.g., water or a Michael addition promoter such as acetic acid, boric acid, citric acid, benzoic acid, tosic add, pentai1uorophe.mil,.picric acid aromatic acids, salts such as bicarbonate, bisnlphate, mono and di-hydrogen phophates, phenols, perhalophenols, nitrophenols, sulphonic acids, FITS, etc.}, preferably boric acid such as a saturated aqueous boric acid. The reaction conditions including a reaction promoter can he neat or in a solvent e.g., a polar aptotic solvent such as acetonitrile or DMF. The reaction can he at an elevated temperature (e.g., neat at an elevated temperature such as 90 °C or with solvent at an elevated temperature such as .from about 50 °C to about 120 "C). The term “reaction promoter*’ as used herein refers to a chemical moiety that, when used in a reaction mixture, acederates/enhanees the rate of reaction.

The ratio of 'compounds of formula (ΙΠ) ία formula (IV) can be varied, providing variability in the substitution on the polyamine of formula (III). In general, polyamines having at least about 50% of the hydrogen moieties substituted with a non-hydrogen moi ety are preferred. Accordingly, ratios of compounds of formula (OI)/formula (IV) are selected to provide for products having a relatively high degree of substitution of the tree -amine (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%j, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about.97%, at least about 99%, or substantially all). In some preferred- embodiments n is 0 in the polyamine of formula (III), and the ratio of compounds of formula (Hi) to compounds of formula (IV) is from about 1:3 to about I ;5, preferable about 1:4. In some preferred embodiments, n is '2 in the polyamine of formula (III), and the- ratio of compound of formula (III) to compounds of formula (IV) is front about 1:3 to about l :6, preferably about 1:5.

In some embodiments, the compounds of formula (ΠΙ) and formula (IV) are reacted in a two step process. For example, the first step process includes a reaction mixture having from about 0.8 about 1,2 molar equivalents of a compound of formula (III), with from about 3,8 to about 4.2 molar equivalents of a compound of formula (IV) and -the second step process includes addition of about 0.8 to 1,2" molar equivalent of compound of formula (.IV) to the reaction mixtee.

Upon completion of the'reaction, one or more products having formula (1) can be isolated from fee reaction mixture. For example, a compound of formula (1} can be isolated as a single product (e.g., a single structural isomer) or as a mixture of product (e.g., a plurality of structural isomers and/or a plurality of compounds of formula (1)).

In some embodiments, one or more reaction products can be isolated and/or purified using chromatography, such as flash chromatography, gravity chromatography (e.g., gravity separation of isomers using silica gel), column chromatography (e.g,, normal phase HPLC or RPHPLC), or moving bed chromatography. In some embodiments, a reaction product is purified to provide a preparation containing at least about 80% of a single compound, such, as a single structural isomer (e.g,, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least, about. 99%).

In some embodiments, a free amine product is treated wife an acid such as H€l to prove an amine salt of the product (e.g., a hydrochloride salt), in some embodiments a salt product provides improved properties, e.g,, for handling-and/or storage, relative to the corresponding free amine product in some embodiments, a salt product can prevent or reduce fee rate of formation of breakdown product such as N-oxide or N-carbonatc formation relative to the ..corresponding free amine. In some-embodiments, a salt product can have improved properties for use in a therapeutic formulation relative to the corresponding free amine.

In some embodiments, the reaction mixture is further treated, for example, to purity one or more products or to remove impurities such as unreacted starting materials. In some embodiments the reaction mix ture is treated with an immobilized (e.g., polymer bound) thiol moiety, which can trap umcaeted acrylamide. In some embodiments, an isolated product can be treated to further remove impurities, e.g., an isolated product can be treated with an immobilized thiol moiety, trapping unreaeted acrylamide compounds.

In some embodiments a reaction product can be treated with an immobilized (e.g,, polymer bound) isothiocyanate. For example, a reaction product including tertiary amines can be treated with an immobilized isofhiocyanate to remove primary and/or secondary'' amines from the product.

In some embodiments, a compound of formula (I) cm be made by reacting a polyamine of formula (01) as provided below

formula (HI) wherein Xs, Xb, and n are defined as above with a compound offonnula (VI)).

formula (VI) wherein Q is Cl, Br, or I, and Y and R5 are as defined above.

In some embodiments, the compound of formula (ID) and formula ( VI) are reacted together neat In some embodiments, the compound of formula (HI) and formula (VI) are reacted together in the presence of one or more solvents, for example a polar aprotie solvent such as acetonitrile or DMP. In some embodiments, the reactants (formula (HI) aid formula (VI)) are reacted together at elevated temperature (e.g,, at least about 50 CC. at least about 60 °C, at least about 70 °C, at least about BO °C, at least about 90 °C, at, least about 100 °C).

In some embodiments, the reaction mixture also includes abase, for example a carbonate such as K2CO3.

In some embodiments, the reaction mixture also includes a catalyst. in some embodiments, the compound offonnula (VI) is prepared by reactlngan amine moiety- with an activated add such as an acid anhydrate or add halide (e.g., acid chloride) to provide a compound of formula (VI).

The ratio of compounds of formula (Hi) to formula (VI) can be varied, providing variability in the substitution on the polyamine of formula (III). In general, polyamines having at least about 50% of the hydrogen moieties substituted with a non-hydrogen moiety are preferred. Accordingly, ratios of compounds of formula (III)/tormula (VI) arc selected to provide for products having a relatively high degree of substitution of the free ammo (e.g«, at least about 50%, at least-about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%. at least about 90%, at least about 95%, at least about. 97%, at least about 99%, or substantially all). In some preferred embodiments a is '0 in the polyamiae of formula (III), and the ratio of compounds of .formula (III) to compounds of formula (VI) is from about 1:3 to about 1:5, preferable about 1:4. In some preferred embodiments, n is 2 in the polyamiae of formula (III), and the ratio of compound of formula (III) to compounds of formula-(VI) is from about 1:3 to about 1:6, preferably about 1:5.

In some embodiments, the compounds of formula (III) and formula (VI) are reacted in a two step process. For example, the first step process includes a reaction mixture having from about 0.8 about 1.2 molar equivalents of a compound of formula (01), with from about 3,8 to about 4.2 molar equivalents of a compound of formula (VI) and; the second-step process includes addition of about 0.8 to 1.2 molar equivalent of compound of formula (VI) to the reaction mixture.

In some embodiments, one or more amine moieties o f formula (III) are selectively protected using a protecting group prior to reacting the polyamine of formula (III) with a compound of formula (IV) or (VI), thereby providing improved selectivity its the synthesis of the final product. For example, one or more primary amines of the polyamine of formula (111) can be protected prior to reaction with a compound of formula (IV) or (VI), providing selectivity for the compound of'formula- (IV) or (VI) to react with secondary amines, Other protecting group strategies can be employed to provide for selectivity towards primary amines, for example, use of orthogonal protecting groups that can be selectively removed.

Upon completion of the reaction, one or more products having formula (I) can be isolated from the reaction mixture. For example, a compound -of formula (1) cm be Isolated as a single product (e.g., a single structural isomer) or as a mixture of product (s.g., a plurality of structural isomers and/or a plurality of compounds of formula (1)). in some embodiments, on or more reaction products can be isolated and/or purified using chromatography, such as flash chromatography, gravity chromatography (e,g., gravity 'separation of isomers using silica gel), column chromatography (e.g,, normal phase HPLC or RFHP.LC), or moving bed: chromatography. In some embodiments, a reaction product is purified to provide a preparation -containing at least about 80% of a single compound, such as· a single structural Isomer (e.g,, at least about 85%> at least about 90·%, at least about 95%, at least about 9/%, at least about 99%).

In some embodiments, a free amine product is treated with an acid such as HC1 to prove an amine salt of the product (e.g., a hydrochloride salt). In some embodiments a salt product provides improved properties* e.g., lor handling and/or storage, relative to the corresponding tree amine product. In some embodiments, a salt product· can prevent or reduce the rate of formation of breakdown product such as N-oxicteor N-carbonate formation relative to the corresponding tree amine. In some embodiments, a salt product can have improved properties for use in a therapeutic formulation relative to the corresponding free amine.

In some embodiments, a polyamine cationic lipid can be made in using a regiosdective synthesis approach. The regioselective synthetic approach provides a convenient way to make site 'specific alkylation on nitrogen(s) of the polyamide backbone that leads to synthesis of sped lie alkylated derivatives of interest. In general, a compound of formula (1) is initially reacted with-a reagent that selectively reacts wife primary amines or terminal amines to block them from reacting or interfering with further reactions and these blockages could be selectively removed at appropriate stages during the synthesis of &amp; target compound. After blocking terminal amines of a compound of formula (I), one or more of the secondary amines could be selectively blocked with an orthogonal amine protecting groups by using appropriate molar ratios of the reagent and reaction conditions. Selective alkylations, followed by selective deprotection of the blocked amines and further alkylation of regenerated amities and appropriate repetition of the sequence of reactions described pro vides specific compound of interest. For example, terminal amines ofhiethyicnetetrarmnefi) is selectively blocked with primary amine specific protecting groups (e.g., trill uoroaeetamide) under appropriate reaction conditions and subsequently- reacted with excess of orthogonal amine protecting reagent [(Boc) ?0, for e.g.)] in the presence of a base (for e.g., diisopropylethylamine) to block all internal amines (e.g,, Boe). Selective removal of the terminal protecting group and subsequent alkylation of the terminal amines, for instance with an acrylamide provides a folly terminal amine alkylated derivative of compound 1. Deblocking of the internal amine protection and subsequent alkylation with calculated amount of an acrylamide for instance yields a partially alkylated product 7. Another approach to make compound 7 is to react terminally protected compound 1 with calculated amount of an orthogonal amine protecting reagent [(ΒοφΟ, for e.g.}1 to obtain a partially protected derivatives of compound 1. Removal of the teroiinal amine protecting groups of partially and selectively protected 1 and subsequent alkylation of all unprotected amines with an acrylamide, for instance, yields compound 7 of interest.

Methods of making lipids having a hiocleavahle moiety

In some embodiments, a compound of formula (X) can be made by reacting a compound of formula

formula (XI) with a compound of formula (XII)

formula (XII) wherein RJ, R2, and R3 am as defined above.

In some etnbodiments, the compounds of formulas (XI) and (XII) are reacted in the presence of a coupling agent such, as a carbodiimide (e,g.s a water soluble carbodihnide such as EDO).

Other chemical reactions and starting materials, can. be employed to provide a compound of formula (X) having two linking groups L! and ΙΛ For example, the hydroxyl moieties of formula (XI) could be replaced with amine moieties to provides precursor to amide or urealinking groups.

Upon completion of the reaction, one or more products having formula (X) ear be isolated from the reaction mixture. For example, a compound of formula (X) can be isolated as a single product (e.g., a single structural isomer) or as a mixture of product (e.g., a plurality of structural isomers and/or a plurality of compounds ofifonnula (X)}, In some embodiments, on or more reaction products can be isolated and/or purified' using clnomatography, such as flash chromatography, gravity'· djromatography (e.g., gravity separation of isomers using silica gel), column chromatography (e.g., normal phase HFLC or RfiHPLC), or moving bed chromatography. In some embodiments, a reaction product Is purified to provide a preparation containixig at least about 80% of a single compound, such as a single structural isomer (e,g., at least about 85%, at least about 90%, at least about 95%, atleast about 97%, at least about 99%),

In some embodiments, a foe amine product is treated with ad acid sued as 1T€1 to prove an amine salt of the product (e.g,, a hydroeMonde salt). In some embodiments a salt product provides improved properties, e.g., for handling and/or storage, relative to the corresponding foe amine product In some embodiments, a salt product can prevent or reduce the rate Of formation of breakdown product such as N-oxide or bi-carbonate formation relative to the corresponding free amine, In some embodiments, a salt product can have improved properties for use in a therapeutic formulation relative to the corresponding foe amine.

Methods of making PEG~I*pids

The PEG-lipid compounds can be made, for example, by reacting a glyceride moiety (e,g,, a dimyristyl glyceride,: dipalmityl glyceride, or distearyl glyceride) with an activating moiety under appropriate conditions, for example, to provide an activated intermediate-that could be subsequently reacted with a PEG component having a reactive moiety such-as' an-amine or a hydroxyl group to obtain a PEG-iipid, For example, a dalkylglyceride (e.g.,.dimyristyl glyceride) is initially reacted with Ν,Ν'1-dlsuecminudyl carbonate in the presence of a base (for e.g,, trietkylaroine) and subsequent reaction of the intermediate formed with a PEG-amlne (e.g,, mPBG2000-Nib) in foe presence of base such as pyridine affords a PEG-lipid of interest Under these conditions the PEG component is attached to the lipid moiety via a carbamate' linkage. In another instance a PEG-lipid can be made, for example* by reacting a glyceride moiety (e,g„ dimyristyl glyceride, dipalmityl glyceride, distearyl glyceride, dimyristoyl glyceride, dipalmitoy! glyceride or distearoy! glyceride) with succinic anhydride and subsequent activation of the carboxyl generated followed by reaction of the acti vated intermediate with a PEG component with an amine or a hydroxyl group, for 'instance,· to obtain a PEG-lipid·' In one example, dimyristyl glyceride is reacted with succinic anhydride: in the presence of a base such as DMA? to obtain a hemi-succinate. The foe carboxyl, moiety of foe bend-succinate thus obtained is activated using.standard carboxyl activating agents such as BBTU and diisopropylethylamine and subsequent reaction of the activated carboxyl with mPEBSQOQ-Nlfo, for instance, yields a PEG- lipid, in .this-approach the PEG component is linked to foe lipid component via a succinate bridge.

Association complexes

The lipid compounds and lipid preparations described herein can be used as a component in an association complex.,for example a liposome or a Bpopiex. Such association complexes can be used to administer a nucleic acid based therapy such as an RNA, for example a single stranded or double stranded RNA such as dsRNA.

The association complexes disclosed herein can be useful for packaging an oligonucleotide agent capable of modifying gene expression by''targeting·and binding to a nucleic add. An oligonucleotide agent can be single-stranded or double-stranded, and can include, e.g., a dsRNA, aa pre-fnRNA, an mRN A, a mieroR'NA (miRNA), a miRNA precursor (pre-miRNA), plasmid or DNA, or to a protein. An oligonucleotide agent featured in the invention can be, e.g., a dsRNA. a microRN A, antisense 'RNA. antagomlr, decoy RNA, DMA, plasmid and aptamcr.

Association complexes can include a plurality of components. In some embodiments, an association complex such as a liposome can include an active ingredient such as a nucleic acid therapeutic (such as an oligonucleotide agent, e.g., dsRNA.), a cationic lipid such as a lipid described herein. In some embodiments, the association complex can include a plurality' of therapeutic agents, for example two or three single or double stranded nucleic acid moieties targeting more than one gene of di fferent regions of foe same gene. Other components can also be included in an association complex, including a PEG-lipid such as a PEG-lipid described herein, or a structural component, such as cholesterol. In some embodiments foe association complex also includes a fusogenic lipid or component and/or a targeting molecule. In some preferred embodiments, the association complex is a liposome including an oligonucleotide agent such as dsRNA, a lipid described herein such as a compound of formula (I) or (X), a PEG-lipid such as a PEG-lipid described herein (e.g,, a PEG-lipid of formate (XV), and a structural component such as cholesterol

Single Stranded rihonucleid acid

Oligormcleotide agents include microRNAs (miRNAs), MicroRNAs are small noncoding-RNA molecules that are capable of causing post-transcriptional silencing of specific genes io cells such as by the inhibition of translation or through degradation of the targeted mllNA. An miRNA can be completely complementary or can have a region ofnoncomplemmtadty with a target nucleic acid, consequently resulting in a “bulge” at the region ofnon-cemplcmeatanty. The region of nor, complementarity (the bulge) can be flanked by regions of sufficient (minplemeiitarity* preferably complete complemehtayity to allow duplex ihnnatiem. Preferably,, the regions of complementarity are at least 8 to 10 nucleotides long 8.9, or 10 nucleotides long). A miRNA can inhabit gene expression-by repressing translation, such as when the microRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The invention also can include double-stranded precursors of miRNAs that may or may not form a bulge when bound to their targets.

In a preferred embodiment an oligonucleotide agent -featured in the invention can target an endogenous miRNA or pre-mtRNA The oligonucleotide agent featured in the invention can include naturally occurring nucleobases, sugars, and covalent intemueieoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions that function similarly. Such modified Or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity fpr the endogenous miRNA target, and/or increased stability in the presence of nucleases. An oligonucleotide agent designed to bind to. a specific endogenous miRNA has substantial complementarity, e.g,, at least TO, 80,90, or 100% complementary, with at least 1.0,20, or 25 or mote bases of the target miRNA. A miRNA or pre-miRNA can be 18-100 nucleotides in length, and more preferably from 18-80 nucleotides in length. Mature. miRNAs can have a length of 19-30 nucleotides, preferably 21 -25 nucleotides, particularly 21,-22,23, 24, or 25 nucleotides. MicroRNA precursors can have a length of 70-100 nucleotides and have a hairpin conformation. MicroRNAs can be generated in vivo -from pre-miRNAs by enzymes called Dicer and Drosha that specifically process long pre-miRNA into functional miRNA. The microRNAs or precursor mi-RNAs featured in the invention can be synthesized in vivo by a cell-based system or can be chemically synthesized, MicroRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic add, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Modifications can also increase sequence specificity, and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greater detail below.

Given a sense strand sequence (eg., the sequence of a sense strand of a eDNA molecule), an miRNA can be designed according to the rules of Watson and Crick base pairing. The miRNA can be complementary to a portion of an RNA, e.g., a miRNA, a pre-miRNA, a pre-mRNA or an mRNA. For example, the miRNA can be complementary to the coding region or noneodixtg region of an mRNA or pre-mRNA, e.g., tire region surrounding the translation start site of a pre-mRNA Or mRNA, such as the 5’ UTR. An miRNA oligonucleotide-can be, for example, from about 12 to 30 nucleotides in length,preferably about 15 to 28 nucleotides in length (e.g., 16,17,18, 19, 20. 21,22,23,24, or 25 nucleotides in length).

Ln particular, aumiRNA or a pre-miRNA featured in the invention can have a chemical modification on a nucleotide in an internal (/. ¢., non~terra.inai) region having noncomplementarity with the target nucleic acid. For example, a modified n ucleotide can be incorporated into the region of a miRNA that forms a bulge. 'Hie modification can include a ligand attached to the miRNA, e.g,, by a linker (e.g., see diagrams QT-I through. OT-IV below). The modification can, for example, improve phannacokinetics or stability of a therapeutic miRNA, or improve hybridisation properties {e.g., hybridisation· themrodynamics) of the miRNA to a target nucleic acid, la some embodiments, it is preferred that the orientation of a. modification or ligand incorporated into or tethered to the bulge region of a miRNA is oriented to occupy the space in die bulge region. For example, the modification can include a modified base or sugar on the nucleic acid strand or a ligand that functions as an intercalator. These are preferably located in the bulge. The intercalator am be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2,3, or 4 fused rings. The universal bases described below can. be incorporated into the miRNAs. In some embodiments, it is preferred that the orientation, of a modification or ligand incorporated into or tethered to the bulge region of a miRNA is oriented to occupy the space in the 'bulge region. This orientation, facilitates the improved hybridization properties or an otherwise desired characteristic' of the miRNA.

In one mibodiment, an miRNA or a pre-miRNA can include an aminoglycoside ligand, which can cause the miRNA to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polytysine; galactosylaied polylysine; neomycin B; tobramycin; kanmnyein A; and acridine conjugates of aminoglycosides, such as Neo~N-acridine, Neo-S~acridhief Neo-C-acridine, Tobra-N-aeridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affimty for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-S-acridine has air increased affinity for the ΗΓ/ Rev-response element (RRE). In some embodiments the guanidine analog (the guamdinoglycoside) of an aminoglycoside ligand is tethered to an oligonucleotide agent In a guanidinoglycoside. the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an oligonucleotide agent.

In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. Preferably, foe cleaving group is tethered to the miRNA in a manner such that it is positioned in the bulge region, where it can access and cleave the target RNA, The cleaving group can be, for example, a bleomycin (eg·., bleomycin-A*-,bleomycin-A?„ or bleomyein-B;?}, pyrene, phermnlhroline (c,g. , O-phenanthroline), a polyamine, a tripeptide (e.gti lys-tyr-lys tripeptide), or metal ion chelating group. The metal Ion chelating group can include. e.g,t m Lu(lII) or EU(III) maerooydic complex, a Zn(H) 2,9-4imethy1phman.throKae derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(HI), lit some embodiments, a peptide ligand can be tethered to a miRNA or a .pre-mi. RNA to promote cleavage of the target RNA, eg., at the bulge region. For example, 1,8-dirnefch.yl-1,3,6,8,10,13-hexaaxacyclotetradeeane (eyefain) can be conjugated to a peptide (eg., bv m amino acid derivative) to promote target RNA cleavage. The methods and compositions featured in the invention include miRNAs that inhibit target gene expression by a cleavage or ηοη-deavage dependent mechanism,

Afl nxiRNA. or a pre-miRNA can be designed and synthesized to include a region of noncomplementarity {eg., a region that is 3,4, 5, orb nucleotides long) Hanked by regions of sufficient complementarity to form a duplex (eg,, regions that are 7, 8,9. 10, or 11 nucleotides long).

For increased nuclease resistance and/or binding affinity to the target, the mIRNA sequences can include 2!-0-methyl, SMltiorine, E'-Q-me&amp;oxyetliyl, 2’-Q~ aminopropyl 2’~amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (ENA), 2-thiopyrhnidines (&amp;g., 2-tMo-U), 2-aroino-A, .<3-clamp modifications, and ethylene nucleic acids (BNA), e.g., 25-4i-ethylene-bridged nucleic acids, can also increase binding affinity to the target. The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic -cleavage. An miRMA or a pre-miRNA can be limber modified by including a 3* cationic group, or by inverting the nucleoside at the S’-termimis with a 3-3'linkage. In another alternative, the3-terminus can be blocked with an. aminoalkyl group, e.g:, a 3s C5-aninoalkyl d'T. Other 3’ conjugates em inhibit 3.V5- exonucleclylic-cleavage, White not being bound by theory, a 3s conjugate, such as naproxen or ibuprofen, may inhibit exonueleolytlc cleavage by sterically blocking the exonuclcase from binding to the 3’ end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deokyribose,· glucose etc.) can blocks’-S’-exonucleases.-

The 5' -terminus can be blocked with an aminoalkyl group, e.g:, a 5-O-alkyl amino substituent Other 5’ conjugates can inhibit 5’-3’ exonudeolvtic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ihuprofes, may inhibit exonudeolytic cleavage by sterically blocking the exoauclease from binding to the 3' end of oligonucleotide. Even small alkyl chains, ary! groups, ot.heterocyclic conjugates or modified sugars (D-ribose? deoxyribosty glucose etc,) can block 3’~5'~ exonudeases, in one embodiment, an mIRNA or a pre-miRNA includes a modiflcation.ihat improves targeting, e.g. a targeting modification described herein. Examples of modifications that -target miRNA molecules to particular cell types include carbohydrate sugars such as galactose, N-acetylgalactosamine, mannose; vitamins such as. folates; other ligands such as RGDs and ROD mimics; and small molecules including naproxen, ibuprofen or other known protein-binding molecules.

An miRNA or a pre-miRNA can be amsiracted using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example,..an miRNA or a pre-miRNA can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase tire physical stability of the duplex formed between the miRNA or a pre~miRNA and target nucleic acids, eg., phosphorothioate derivatives and acridine- substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the miRNA or pre-miRNA. nucleic acid can be produced biologically using an expression vector into'Which a nucleic add has been subcloned in an antisense orientation (/.«., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

Antisense-Arne Oligonucleotide Auents

The single-stranded oligonucleotide agents featured in tire invention include antisense nucleic acids. An "antisense" nucleic acid includes a nucleotide sequence that is complementary to a "sense” nucleic acid encoding a gene expression product, t\g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an RNA sequence, e.g., a pre-roRNA, roRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target,

Given a coding strand sequence (e.g., the sequence of a. sense strand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be cornplenlemary to a portion of the coding or noncoding region of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisense oligonucleotide can be complementary to the region swounding the translation start' site of a pre-mRNA or mRNA, e.g., the 5’ Iff R. An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length {e.g., 11.12, 13,14,15, 16,18,19, 20,21,22,23, or 24 nucleotides in length). An antisense oligonucleotide can also be complementary to a miRNA or pre-miRNA.

An antisense nucleic- add can be constructed using· chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic add (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides .designed to increase the biological stability of tire molecules or to increase the physical stability of the duplex formed between tire antisense and target nucleic acids, e.g., phosphorothioaie derivatives and acridine substituted nueleotides can be used. Other appropriate nucleic add modifications are described herein. Alternati vely, die antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subeloned in an antisense orientation (/,«., RNA transcribed from fee inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

An antisense agent can include ribonucleotides oidy, deoxyrihonneleofides only (c,g, , oligodooxynucleotides). or both deoxyribonucleoiides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA, and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyrihonueleotides, or deoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked by RNA sequence· at the 5’ and 3? ends of the antisense agent, can hybridize to a complementary RNA, and the RNA target can be .subsequently cleaved by'aft enzyme, e.g., RNAse E. Degradation of the target RNA prevents translation. The flanking RNA sequences can include 2’-0~methylated nucleotides, and phosphorotbioate linkages, and the internal DNA sequence can include phosphorotliioate iniernucleotide linkages. The internal DNA sequence is preferably at least five nucleotides in length when targeting by RNAseM activity is desired.

For increased nuclease resistance, an antisense agent can be further modified by inverting the nucleoside at tire o’-terminus with a 3-3’ linkage, in another alternative, the 3-temrinus can be blocked with an aminoalkyl group.

In one embodiment, an antisense oligonucleotide agent includes a modification that improves targeting, e.g. a targeting modification described herein.

Decoy-type Oligonucleotide Agents

An oligonucleotide «gent featured in the invention can be &amp; decoy nucleic acid, e..g., a decoy RNA. A decoy nucleic acid resembles a. natural nucleic acid, but is modified in such a way as to inhibit or interrupt the activity of the natural nucleic acid.

For example, a decoy RNA can mimic the natural binding domain for a ligand. The decoy ENA therefore competes with natural binding target for the binding of a specific ligand. The natural binding target can he an endogenous nucleic acid, e.g.. a pre-niiRNA, miRNA, ptemRNA, mRNA or DMA, For example, it has been shown that over-expression of HIV trans-activation response (TAR) ENA cast act as a "decoy" and efficiently bind HlVtai protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA,

In one embodiment, a decoy RNA includes a modification that improves targeting, e.g. a targeting modification described herein.

The chemical modifications described above for miRN As and antisense RNAs, and described elsewhere herein, are also appropriate for use in decoy nucleic acids.

Apianon-ivne Oligonucleotide Agents

An oligonucleotide agent featured in the invention can be an aptamer. An aptamer binds to a non-nudeie acid ligand, such as a small organic molecule or protein, e.g,, a transcription or translation factor, and subsequently modifies (e.g.. inhibits) activity. An aptamer can fold into a specific structure that directs foe recognition of the targeted binding site on the non-nucleie acid ligand. An aptamer can contain any of the modifications described herein. hi one embodiment, an aptamer includes a modification that improves targeting, e.g. a targeting modification described herein.

The chemical modifications described above for miRNAs and antisense RNAs, and described elsewhere herein, are also appropriate for use in decoy nucleic acids.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and foe description below·. Other features and advantages of the invention will be apparent -from foe description and drawings, and from the claims. Tins application incorporates· all died references, patents, and patent applications by references in their entirety for all purposes.

In one aspect, the invention features antagomirs. Antagomirs are single stranded, double stranded, partially double stranded and hairpin structured chemically modified oligonucleotides that target a microRNA.

An antagomir consisting essentially of or comprising at least 12 orrnore contiguous nucleotides substantially complementary to an endogenous miRNA and more particularly agents that include 12 or more contiguous nucleotides substantially complementary to a target sequence of an miRNA or pre-miRNA nucleotide sequence. Preferably, an antagomir featured' in tile invention includes a nucleotide sequence sufficiently complementary to hybridise to a miRNA target sequence of about 12 to 25 nucleotides, preferably about 15 to 23 nucleotides. More preferably, the target sequence differs by no more than 1»% or 3 nucleotides from a .sequence shown in Table 1, and in one embodiment, the antagomir is an agent shown in Table 2a-e. In one embodiment, the antagomir includes a non-nucleotide moiety, <?,#.* a cholesterol moiety. “The non-nudeoiide moiety can be attached, c.g., to the 3’ or 5 ’ end of the oligonucleotide agent.

In a preferred embodiment, a, cholesterol moiety is attached to the 3 ’ end of die oligonucleotide agent.

Antagomits are stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g,, a nucleotide modification. In another embodiment, the antagomir includes a phosphorothioate at at least the first, second, or third internudeolkie linkage at -the 5’ or 3’ end of the nucleotide sequence. In yet another exnbodiment, the antagomir includes a I’-modified nucleotide, e.g.„ a S’-denxy. 2!-deoxy^’-fluoto^^'-O-mediyl, 2’-0-methoxyethyL (2,»0~M0B), 2'-0-aminopropyl (2-0-A!5), 2-0-dimethyJanaiftoethyl (2-O-DMAOE), 2T-Q~dimethyiami.nopropyl (2!-0-DMAP), 2-0-dimethyIaminoeihyloxyeth.yl (2!~0~DMAB0E), or 2-0-Ν» mefhylacetamido (2^}-NMA), In a particularly preferred embodiment, the antagomir includes at least one x’-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the antagomir include a 25-O-methyl modification.

An antagomir that is substantially complementary to a nucleotide sequence of an miRNA cm be delivered to a cell or a human to inhibit or reduce the activity of an endogenous miRNA, such as when aberrant or undesired miRNA activity, or insufficient activity of a target mRNA that hybridizes to the endogenous miRNA, is linked to a disease or disorder. In one embodiment, an antagomir featured in the invention has a nucleotide sequence that is substantially complementary to mill-122 (see Table!), whichhybridiz.es to numerous RNAs, including aldolase A mRN A, N-mye downstram regulated gene(Ndrg3) mRNA, IQ motif containing GTPase activating protein-1 (fqgapl) mRNA, HMO-CoA-reduetase (fimger) mRNA, and citrate synthase mRNA and others. In a preferred embodiment, die antagomir that is substantially complementary to miR.422 is antagomir»i22 (Table 2a-e), Aldolase A deficie.tt.des have been found to be associated with a variety of disorders, 'including hemolytic anemia, mlhrogryposis complex congenita, pituitary ectopia, rhabdonivolysis, hyperkalemia. Humans suffering from aldolase· A deficiencies also experience symptoms that include growth and developmental retardation, nsidiaeiai hypoplasia, hepatomegaly, as Well as myopathic symptoms. Thus a human who has or who is diagnosed as having any of these disorders or symptoms is a candidate to receive treatment with 'an antagomir that hybridizes to miR-122.

Doi&amp;le-straaded ribonncieic acid (dsRNA)

In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA} molecule packaged in an association complex, such as a liposome, for inhibiting the expression of a gene in a cell or mammal, wherein the dsRNA comprises m antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said gene, inhibits the expression of said gene by at least 40%. The dsRNA comprises two ENA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally 'fully complementary, to a target sequence, derived from the sequence of «η. mRNA formed during the expression of a gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex, structure' when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most, generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target, sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and-most generally between 1.9 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded .nucleotide overhang(s). The dsRNA can be synthesized by standard methods known-in the art as further discussed below, e,g,, by use of an automated DNA synthesizer, such as are commercially available from, for example, Eiosearch, Applied Biosystems, Inc.

The dsRNAs suitable for packaging in the association complexes described herein can include a duplex structure of between 18 and 25 basepairs (e.g., 21 base pairs). In some embodiments, the dsRNAs include at least one strand that is at least 21nt long. M other 'embodiments, the dsRNAs include at least one strand that is at least 15, lb, 17,18,19,20, or more contiguous nucleotides.

The dsRNAs suitable for packaging in the association complexes described herein can contain one or more mismatches to the target sequence. In a preferred embodiment; the dsRNA contains no more than 3 mismatches. If the antisense strand of the dsRMA contains mismatches to a. target sequence, it is preferable that the area of .mismatch not be located in the center of tire region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it Is preferable dial the mismatch be restricted to 5 nucleotides from either end, for example 5,4, 3, .2, or 1 nucleotide from either the 5’ or 3* end of the region of complementarity, in one embodiment, at least one end of the dsRNA has a single-sounded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. Generally, the single-stranded overhang is located at the 3’-iennmai end of the antisense strand or, alternatively, at the 3 ‘-terminal· end. of the sense strand. The dsRNA may also have a blunt end, generally located at the 5’~end of the antisense strand. S uch dsRNAs have improved stability and inhibitory* activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA. has a nucleotide overhang at the 3'-end, and ilte 5*-end is blunt In another embodiment, one or more of the nucleotides in .the overhang is replaced with .a. nucleoside thiophosphate.

In yet another embodiment, a dsRNA packaged in an association complex, such as a liposome, is chemically modified to enhance stability. Such nucleic adds may be synthesized -and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid'.chemistry", Beauc-age, S.L ct.al. (Edrs.), John Wilev &amp; Sons, Inc., New York, NY, USA, which, is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2’ modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of aon-natora! bases into the oligonucleotide chain, covalent aflaehrnentto a ligand or chemical moiety, and replacement of iniemucleotide phosphate linkageswith -alternate linkages such, as thiophosphates. More than one such modification may be employed Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-khown techniques, for example by introducing·covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Such chemically linked dsRNAs are suitable for packaging in the association complexes described herein. Generally, the chemical groups that can. be used to modify the daRN A include, without limitation, methylene blue; bifunctional .groups, generally bis-{2-chloroethy!)amine; H~acetyI-N’-(p-glyoxyibejEoyl)eystamine; 4-thfouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethyiene glycol linker is incorporated according to standard methods (e.g., Williams, DJ,, and K.B. Hall, Bloehem, (11)96) 35:14665-14670). in a particular embodiment, the S’-end of the antisense strand and the 3-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least, one nucleotide of the dsRNA comprises a phosphoroihioate orphosphorodithioate groups. The chemical bond at theentis of the dsRNA is generally fonned by triple-helix bonds.

In yet another embodiment, the nucleotides at one or both of the two single strands may be modified· to prevent, or inhibit the degradation, activities of cellular enzymes, such as, for example, without limitation,·certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are knows in flic art including, but not limited to, 2-amino modifications, 2’-amino sugar modifications, 2S-F sugar modifications, 2 !-F modifications, 2-’-alkyl sugar modifications, 2’-0-alkoxyalkyl modifications like 2’-0-mctlioxyeihyl, uncharged and charged backbone modifications, morpholine modifications, 2’O-meihyi modifications, andphosphoramidale (see, e.g., Wagner, NaL Med. (.1995) 1:1.116-8). Urns, at least one 2’«hydroxyl group of the nucleotides on a'dsRNA is replaced by a chemical group, generally by a-2’-F or a 2’-0*tnethyl group. Also, at least one nucleotide may he modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2’*oxygen of ribose with the 4’--carbon'of ribose. Oligonucleotides coiharoing the locked nucleotide are described in Koshkin, A.A., et a!., Tetrahedron (1998), 54; 3607-3630) and Obika, S. et &amp;.vTeimhedronLett (1998), 39:5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D.A. and D.R. Corey, Ckem. Biol. (2001), 8:1-7)-

Conjugating a ligand to a dsRNA can enhance its cellular absorption, as well as targeting to a particular tissue or uptake by speci fic types of cells such as liver cells. In certain instances, a hycrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated eadocyiosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in conrpoundsfhai are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense &amp; Nucleic Acid Drug Development 2002,12,103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, l,3-bis-04hexadecyl)glyeero3? and menthol. One example of a ligand for receptor-mediated eudocytosis Is folic acid. Folic acid enters the cell by iblate-receptor-mediatcd cndocytosis, dsRNA compounds bearing folic acid would be efficiently transported into the coll via the folate-receptor-mediated endocytosis, Xi and coworkers report that attachment of folic acid to the 3 ’-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide,, Li, S.; DesiunuMi, Η. M.; Huang, L. Phartn. Res. 1998, 75,1540, Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate dusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol. Other chemical .modifications for siRNAs have been described in Manoharan, M. RNA interference and chemically modified .small interfering RNAs. Current Opinion in Chemical Biology (2004), 8(6), 570-579. la ceitain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylamnumium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to rnflNA when the cationic ligand was dispersed throughout, the oligonucleotide. See M. Manoharan Antisense &amp; Nucleic Acid Drug Development 2002,12,103-'and references foemin.

The ligand-conjugated dsRNA of the invention may be synthesized bv the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized hearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use Of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further he attached to a solid-support material Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some-preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with, a linking moiety located on the 5' position of a nucleoside or oligonucleotide. In certain instances,, a dsRN A bearing an aralkyl ligand attached to the 3 ’-terminus of the dsRNA is prepared by first'Covalently attaching a monomer building block to a controlied-poire-glass support via a long-chain aminoalkyl group. Then, nucleotides arc bonded via standard solid-phase synthesis techniques to the monomer buildiug-bfoek. bound to the solid support The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.

The dsRNA used in the conjugates of the invention maybe conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivati ves.

Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: TJ.S, Pat Nos, 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat, No, 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat.

Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modifled,backbones; U.S. Pat. No. 5,386,023, draw»-to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,101., drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. fiat. No. 5,459,255, drawn to modified nueleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S, Pat No. 5,554,746. drawn to oligonucleotides having β~ laotarn backbones; U.S, Pat. No. 5,571,902, drawn to methods and materials for tire synthesis of oligonucleotides.; U.S, Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorofhioate linkages of high chiral purity; U.S. Pat, No, 5,506,351, drawn to processes for the preparation of 2-O-alkyl gnanosine and related aunpounds, including 2,6-diaminopurine compounds; U.S. Pat. No, 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat, No. 5,587,470, drawn to oligonucleotides having 3-deazapurioes: U.S. Pat No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4-desmethyl nucleoside analogs; U.S, Pat. Nos, 5,602.240, mid 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods· of syntlissiring 2!-tluoro-oiigonucleotides,

In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleotides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or: non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety,· the synthesis of tire sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugate bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see

Manoharan et ai, PCI" Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside eonfugates in addition to the standard phoephoramidites and non-standardphosphorsntidites that are commercially available and .routinely used in oligonucleotide synthesis,

The dsRNAs packaged in the association complexes described herein can include one or mote modified nucleosides, e.g., a 2*-0-methyl, 2-O-efeyl,, 2:~0-propyL 2?-0-aIiyh 2'-C)-aminoalkyl or ^-deoxy-S'-fluoro group in the nucleosides:. Such modifications confer enhanced hybridization properties to tire oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, ihnctionalixed, linked nucleosides can be augmented to include either or both a phosphofothioate backbone or a 2-O-methyl, 2l-0~ethy!, 2i-G-propyl> 2--Q-amiftoalkyl, 2 ~0-a!lyl or 2’-deoxy-2'-fl.uofo group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCI'

Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences possessing an amino group at the 5-terminus are prepared using a DMA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in fee art. Representative active esters include N-hydrosueeimtnide esters, ietrafluorophenolic esters, pentafiuorophenolic esters and pentachlorophenolic esters. The reaction of fee amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the S-position through a linking group. The amino group at the S'-terromus can he prepared utilizing a S'-Amino-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the deposition by the use of a ligand-nucleoside phesphoramidite wherein fee ligand is linked to fee 5'~hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphorainkiites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5'-terminus.

Examples of modified intemucleoside linkages: or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoaikylphospbotriesters,· methyl and other alkyl phosphonates including 3-alkylene phosphonates and chiral phosphonates, phosphinates, phosphocajnidates including 3'-antmo phosphormnidate and aminoalkyiphosphoramidates, thionophosphoramidaies, thionodkylphosphonstes, thionoalkylphosphoiriesters, and boranophosphates having normal 3-51 linkages, 2*-5‘ linked analogs of these, and those having inverted polarity wherein die adjacent pairs of nucleoside units are linked 3-5’ to 5-3' or 2f-51 to 5%-2\ Various sails, mixed salts and tree-add forms are also included.

Representative United States Patents relating to the preparation ofihe above phosphoru.s~aioin-containi.ng linkages include, but are not limited to, U.S, Pat Nos, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897: 5,264,423: 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;.5,550,111; 5,563,253 ; 5,571,799; 5,587,361:5,625,050; and 5,697,248, each of which is herein incorporated by reference.

Examples of modified internucleoside linkages or backbones that do not include a phosphorus atom therein 0..6.,. oligonudeosides) have backbones that am .formed by short chain alkyl or cycloalkyl intersugar linkages, mixed, heteroatom and alkyl or cyeloalfcyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholmo linkages (formed in part, from the-sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; tbnnacetyl and thioibrmacetyi backbones; methylene fbnnacetyl and thiofonnacetyl backbones; alkene containing backbones; sulfamate backbones; methyieneimino and methyknehydrabino backbones; sulfonate and snlfbnamide backbones; amide backbones; and others having mixed N, 0, S and C% component parts.

Representative United States patents relating to the preparation of the above oligonudeosides ineinde, but are not. limited to, U.S. Pat. Nos, 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; $,264,562;5,264,564; 5,405,93¾ 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602:240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

In certain instances, an oligonucleotide included in an association complex, such, as a liposome, may be modified by anon-ligand. group, A number of non-ligand molecules have been conjugated to oligonucleotides in order to. enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations tire available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (tetsinger et al, Proc. Mall Acad. Scl, USA. 1989,86:6553), cholic acid (Manoharao et al. Bioorg, Med. Chem. Lett,, 1994,4;.1QS3), a thioether, e.g., hexyl-S-tritylthiol (Marioharan et ah, Ann. N.Y, Acad. Sei. 1992,660:306; Manoharan et al, Bioorg, Med. Chem. Let, 1993. 3:2765), a thiocholesterol (Oberttauser et al., Nucl, Acids Res., 1992·, 20:533), an aliphatic chain, e.g,, dodecandiol or undecy] residues (SakomBehrooaias et al., EMBO T, 1991,10:1.11; Kabanov et al, FEES Lett., 1990,259:327; Svinarehuk et al., Bioehimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyiammoaiiim 1,2-di-O-hexadecyi-fac-glyeero-S-H-phosphonaie (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al, Nucl. Adds Res,, 1990,18:3777),.3 polyamide or a polyethylene glycol chain (Manoharan et a!,, Nucleosides &amp; .Nucleotides, 1995,14:969), or adamantane acetic acid (Manoharaa et ah, Tetrahedron Lett, 1995,36:3651), a pahaityl moiety (Mishra et al, Biochim, Biophys. Acta, 199.5,1264:229), or an octadecyiamine or hexylamino-carbonyl-oxycholesteroLmQiety (Crooke et .al., J. Pharmacol Exp. Titer., 1996,277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an arnmoiirtk.et at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed· either with the oligonucleotide still bound to the solid support, or following cleavage of foe oligonucleotide in .solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.

The modifications described above are appropriate for use with an oligonucleotide agent as described 'herein.

Fusogenic Lipids

The term, "fusogenic” refers to foe ability of a lipid or other drug delivery system to fuse with membranes, of a ceil. The membranes can be either the plasma membrane or membranes surrounding' organelles, e.g., endosome, nucleus, etc. Examples of suitable fusogenic lipids include, but are not limited to dioieoylphospliaiidyletiumolamine (DOPE), DODAC, DOOM A, DOOAP, or DLinDMA. hi some embodiments, the association complex include a small molecule such as an imidzole moiety conjugated to a lipid, for example, for endbsoroal release. PBGorPEG-iipids

In addition to cationic and fusogenic lipids, the association complexes include a bilayer stabilizing component (BSC) such as an ATTA-lipid or a PEG-lipid, Exemplary lipids are as follows: PEG coupled to dialkyloxypropyis (PEG-PAA) as described in, e.g., WO 05/026372, PEG coupled to diacylgij'eerol .(PEG-DAG) as described in, o.g., U.S, Patent Publication Nos. 20030077829 and 2005008689), PEG coupled to phosphafidylethanolaroine (PE) (PEG-PE), Or PEG conjugated to eeramides, or a mixture thereof (see, UJ* Pat. No. 5,885,613), hr a preferred embodiment, tbs association includes a PEG-lipid described hetesior example a PEG-lipid of formula (XV), (XV’) or (XVI). In one preferred embodiment, the BSC is a conjugated lipid that inhibits aggregation of the SPLPs. Suitable conjugated lipids include, but are not limited to PEG-lipid conjugate, ATTA-Hpid conjugates, eationic-polymrerdipid conjugates (CPLs) or mixtures thereof In one preferred embodiment, the SPLPs comprise- either a PEG-lipid conjugate or an ATTA-lipid conjugate together wife, a CPL PEG is a polyethylene· glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl, groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, aid PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from.'Sigma Chemical Co. and other companies and include, for example, the following: monomefeoxypolyefeylene glycol (MePBG-OH), monomethoxypolyethyl ene glycol-succinate (MePEG-S), monometboxypolyethylene glycol-succiojmidyl succinate (MePEG-S-NHS), monomeihoxvpolyeihylene glycol-amine .(MePEG~MH,sub.2), monomefeoxypolyethylene giveol-tresylate (MePEG-TRES), ami moBomethOxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). In addition, rnonomethoxypoiyefeyleneglyeol-aeeiic acid. (MePEG-Ci:Esub.2€DOB), Is particularly useful, tor preparing fee PEG-lipid conjugates including, e.g.s PBG-DAA conjugates.

In a preferred embodiment, the PEG has an average molecular weight.of from about 550 daltons to about 10,000 daltons, more preferably of about 750 daltons to about 5,000 daltons, more preferably of about 1,000 daltons to about 5,000 daltons, more preferably of about .1,500 daltons to about 3,000 daltons and, even more preferably, of about 2,000 daltons, or about 750 daltons. The PEG can be optionally substituted by an alkyl, aSkoxy, acyl or aryl group. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester containing linker moiety. As used herein, fee term "noxx-ester containing linker moiety” refers to a linker moiety that does not contain a carboxylic ester bond (-00(0)--}. Suitable non-ester containing linker moieties include, but are not limited to, amid© (-C(O)NH-), amino (-HR-), carbonyl (--C(G)~~)S carbamate (--HEClOjO"), urea (-HliG(0)NH-), disulphide (-S-S-), ether (-Ο-), succinyl (-(0)CCH.sub,2CH.sub.2C(0)“), suecraamidyl (~-NeC(G)CB.sub.2CH,sub;2C(0~ )NH--), ether, disulphide, etc. as well as axmbmations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used to couple the PEG to the lipid. Suitable ester containing linker moieties include, e.g,, carbonate (— 00(0)0-), suecinoyl, phosphate esters (-O-(O)PQH-Q-), sulfonate esters, and combinations thereof.

Targeting agents

In some embodiments, the- association complex includes a targeting agent. For example, a targeting agent can be included in the surface of the association complex (e.g., liposome) to help direct the association complex top targeted area of die body.

An ex ample of targeting agents galactose, mannose, and folate. Other examples of targeting agents include small molecule reeeptors, peptides and antibodies, in some embodiments, the targeting agent is conjugated to the therapeutic moiety such as oligonucleotide agent. In some embodiments, the targeting moiety is attached'directly to a lipid component, of an association complex, in some embodiments, the targeting-moiety is attached directly to the lipid component via PEG preferably with PEG of average molecular weight 2000 amu. in some embodiments, tile targeting agent is 'uneont«gated, for example on the surface of the association complex.

In some -embodiments, the association complex includes one ormpre components that improves the structure of the complex (e.g,, liposome), In some embodiments, a. therapeutic agents such as dsRNA can be attached (e.g., conjugated) ίο a lipophilic compound such as cholesterol, thereby providing a lipophilic anchor to the dsRNA. in some embodiments conjugation of dsRNA to a lipophilic moiety such as cholesterol can improve the encapsulation efficiency of the association complex.

Properties of association complexes

Association complexes such as liposomes are generally particles' with hydrodynamic diameter ranging from about 25 nm to 500 nm. In some preferred embodiments, the association complexes are less than 500 nm, e.g., from about 25 to about 400 am, e.g., from about 25 nm to about. '300 urn, preferably about 120 nm or less.

In some embodiments, the weight ratio of total excipients within the association complex to RNA is less than about 20:1, for example about 15:1, In some preferred embodiments, tire weight ratio is less than 10:1, for example about 7,5:1.

In some embodiments the association complex has a pKa such that the association complex is protonated under endozomal conditions (e.g., facilitating the rupture of the complex), but is not protonated under physiological conditions.

In some embodiments, the association complex provides improved in vivo delivery of an oligonucleotide such as dsRNA. In vivo delivery of an oligonucleotide can be measured, using a gene silencing assay, for example an assay measuring the silencing of Factor V1L in vivo Factor VII silencing experiments CS7BL/6 mice received tail vein injections of saline or various lipid formulations. Lipid-formulated siRNAs are administered at varying doses in an injection volume of 10 μΐ/g animal body weight Twmiy~ibar hours after administration, smun samples axe collected by reiroorbital bleed. Serum Factor VII concentrations are determined using a ehromegenic diagnostic kit (Cbasct Factor VII Assay Kit DiaPharma) according to manufacturer protocols.

Methods of making association complexes

In some embodiments, an association complex is made by contacting a therapeutic agent such as an oligonucleotide with a lipid in the presence of solvent and a buffer. In some embodiments, a plurality of lipids are included in the solvent, for example, one or more of a cationic lipid (e.g., a polyamine containing lipid or a lipid including a biocleavable moiety as described herein), aPEG-lipid, a targeting Hpid or a fusogenic lipid.

In some embodiments, the buffer is of a strength sufficient to protonate substantially all amines of an amine containing lipid such as Hpid described herein, e.g., a lipid of formula (I) or ibrmttia (X).

In some embodiments, the buffer is an acetate buffer, such as sodium acetate (pit of about 5). In some embodiments, the buffer is present in solution at a concentration of from about. 100 rnM and about 300 m'M.

In some embodiments, the solvent is ethanol, For example, in some embodiments, the mixture includes at least about 90% ethanol, or 100% ethanol.

In some emhrHiiments, the method includes extruding the mixture to provide association complexes having particles of a size with hydrodynamic diameter less than about 500 nm (e.g., a size from about 25 ran to about 300 nro, for example is some preferred embodiments the particle sizes ranges from about 40-120 nm). In some embodiments, the method does not include extniskm of the mixture.

In one embodiment, a liposome is prepared by providing a solution of a lipid described herein mixed in a solution with cholesterol, PEG, ethanol, and a 25 MM acetate buffer to provide a mixture of about pH 5. The mixture is gently vortexed, and to the mixture is added sucrose. The mixture is then vortexed again until the sucrose is dissolved. To this mixture is added a solution of siRNAin acetate buffer, voriexing lightly for about 20 minutes, Themixture is then extruded (e.g., at least about 10 times, e.g.s 11 times or more) through at least one filter (e.g., two 200 nm filters) at. 40 °C, and dialyzed against PBS at pH 7,4 for about 90 minutes at R.T.

In one embodiment, a liposome is prepared without extruding tlie- Kpbsoiae mixture. A lipid described herein is combined with cholesterol, PEG, and sillNA m 100% ethanol, water, and an acetate buffer having a concentration front about. 100 roM to about 300 tsM (pH of about 5), The combi nation is rapidly mixed in 90% ethanol. Upon completion, the mixture- is dialyzed (or treated with ultrafiltraiion) against an acetate.buffer having a concentration from about 100 mM to about 300 mM (pH of about 5) to remove ethanol,- and then dialled (or treated with ultrafiltration) against PBS to change buffer conditions.

Association complexes cajr.be formed in the absence of a therapeutic- agent such as single or double stranded nucleic acid, and then upon formation be treated with one or more therpauetically active single or double stranded nucleic acid moieties to provide a loaded association complex, ie„ an association complex that is loaded with the therpaueitcally active nucleic acids. The nucleic acid can be entrapped within the association complex, adsorbed to the'surface of the association complex or both. For example, methods of forming association complexes such as l iposomes above can he used to form association complexes fits© of a therapeutic agent, such as a nucleic acid, for example a single or double stranded RNA such as siRNA. Upon formation of the association- complex, the complex can then be treated with the therapeutic agent such as siRNA to provide a loaded association complex.

In one embodiment, a mixture including cationic lipio such as a lipio described in formula (I), preferably a cationic lipid of the following formula

cholesterol, and a PEGdipid, for example a PBG-iiptd described herein, such as the PEG-lipid below,

axe provided in ethanol {e.g,,100% ethanol) and combined with an aqueous buffer such as aqueous NaOAc, to provide unloaded association complexes. The association complexes are then optionally extruded, providing a more uniform size distribution of the association complexes. The association complexes are then treated with the thereapeuiic agent such as siRNA in ethanol (e.g., 35% ethanol) to thereby provide a loaded association complex, hi some embodiments, the association complex is then treated with a process that removes the ethanol, such as dialysis.

Characterization of association complexes

Association complexes prepared by any of the methods above are characterised in a similar manner. Association complexes are-first characterized by visual inspection. In general, preferred association complexes are whitish translucent solutions tree from aggregates or sediment, .Particle size and particle size distribution of iipid-nanoparticles are measured by dynamiclight scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Preferred particles are 20-300 ran, more preferably, 40-100 am in size, in some preferred embodiments, the particle size distribution is unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-bindingdye Ribogreen (Molecular Probes) in the presence or absence-of a formulation disrupting surfactant, 0.5% Triton~X100. The total siRNA in the iommlation is determined by the signal horn 'the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free’* siRNA content (as measured by the signal 1« the absence of surfactant) from the total siRNA: content, Percent entrapped siRNA is typically >85%.

Methods of using association complexes and compositions Including the same

Pharmaceutical compositions comprising oligonucleotide agents

An oligonucleotide agent assembled in an association complex can be administered, e.g., to a cell or to a human, in a single-smmded or double-stranded configuration. An ol igonucleoti.de agent that-is in a double-stranded configuration is bound to a substantially complementary oligonucleotide strand. Delivery of an oligonucleotide agent in a double stranded configuration may confer certain advantages on the oligonucleotide agent, such as an increased resistance to nucleases.

In one embodiment,-the invention provides pharmaceutical compositions including, an oligonucleotide agent packaged in an association complex, snch as a liposome, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the packaged oligonucleotide agent is nscM tor treating a disease or disorder associated with the expression or activity of a target gene, such as a pathological process which can be mediated by down regulating gene expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that axe formulated for delivery to a specific orgaxi/hssue, such as the liver, via parenteral delivery.

Tire pharmaceutical compositions featured in the invention are administered in dosages sufficient to inhibit expression of» target gene.

In general, a suitable dose of a packaged oligormcleotide agent will be such that the oligonucleotide agent delivered is in the range of 0.01 to 5,0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the oligonucleotide agent may be administered as two, three, or more sub-closes at appropriate .intervals throughout foe day or even using continuous infusion, or delivery through a controlled release formulation. In that case, the oligonucleotide agentcontained in each sub*dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e,g., using a conventional sustained release formulation which provides sustained release of the packaged oligonucleotide agent over a several day period. Sustained release formulations are well known in the art.

The skilled artisan wil l appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a. therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo halFIives for the individual oligonucleotide agents packaged in the association complexes can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases. Such models are used for in vivo testing of oligonucleotide agents packaged in lipophilic compositions, as well as for detennininga therapeutically effective dose.

Any method can be used to administer an oligonucleotide agent packaged in an association complex, such as a liposome, to a mammal. For example, administration can be direct; oral; or parenteral (e.g., by subcutaneous, intraventricular, •intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection), or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations).

Ait .oligonucleotide agent packaged in an association complex can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or indaventrieular administration, an oligonucleotide agent can be formulated into compositions sueb as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration.enhancers, carrier compounds, and other pharmaceutically acceptable carriers).

The oligonucleotide agents packaged in art association complex can be formulated in a pharmaceutically acceptable carrier or diluent. A #pharmaceuti.caliy acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other porrinent transport mid chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e..g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyotbyiene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycqlate); and wetting agents (e.g., sodium laury! sulfate),

EXAMPLES

Example i: Syntheses and purifMtMftf^^ iricthvienetetramiiie under Michael addition condition - method 1. (Scheme 1)

Scheme Is

8 (i) 90 °C, Neat 5 days

In a 350 mL pressure bottle A-dodecylacrylamide 1 (84 g, 0.35 mol) [Slee, Deborah Hi; Romano, SuzaimeL; Yu, Jinghua; Nguyen, true N.; John» Judy K.;

Rahcja, Net! K.; Axe, Frank U.; Jones, Todd Κ.» RipJca, William C, Journal of Medicinal Chemistry'· (2001), 44(13), 2094-2107] was taken and the solid was melted under argon by gently heating the vessel. To this melt was added triethyleneietramine 2 (10.2 g, 0.07 mol) and the mixture was heated at 90 °C for 5 days. Michael addition of triethyienetetraniine 2 to the acrylamide 1 yielded two five and the sole six alkylated products along with minor amounts of low alkylated products under neat reaction condition, lire reaction mixture was analyzed by TLC using CHjChtMeOHtNfits (90:5:5) as the eluent. Hie TLC showed the near complete consumption of the starting acrylamide 1. Hie reaction mixture was dissolved in dicidoromethane (40 mL), loaded on a pre-packed column of silica gel and the mixture was separated using eluent CH>Cl2:MeOH:NEt3 (48; 1:1 to 8:1:1). In order to achieve complete separation, multiple columns using the same conditions were performed and the fallowing pure products were obtained. The required five addition products 3 and 4 were isolated along with the six addition product 5, In this, reaction .mixture soma of the lower addition products were also detected in the TLC ami the LC-MS of the crude reaction mixture, A^Bodeeyt“3~((2*dodecylearbaiBOj'I-eth>'i)-{2-|{2-dodeeylcaf1)amo>'l-eth>1)-2-|(2-dodecyIc3rhai.noyl-ethyIH2*(2-(lodecyIcai'bamoyi~eriiyiiiniiao)-etliyIl-affiii3ni-ethyI-ammo)i>ropionamide. One of the two 5-alkylatcd derivatives, compound 3 (isomer I), was isolated as light yellow foam (12 g, 13%). MS m/z 672 (M3-2B/2), 448 (M+3H/3), 5HNMR CPCb δ 0.87 (/,,/- 6.5¾ I SB), i.20-1.39 (m, 92H), i .46-157 (ro, 12B), 2,20-2.50¾ 1611),2.60-2.78¾ Ι0Η), 3,10-3.25 ¾ 12H), 6.98 (bs, 3H), 7.41 (bSj 1B), 7.63 {bs, 1H), 8.85 (bs, lid). BC NMR CDCk 5 14,33,22.90.27.37, 29.59, 29.67,29.88,29.89,29.92, 32,13,39.74, 172,77. (3-i(2-{2-l|2-Bis-(2-dodecykarbanioyI-'eihyl)-ani»noi-ethyl}-(2-dodeeyleari?anjoyI-ethyl)-amiaoj“ethyiamino}-etiiyl)-(2-dodeey!carbamoyt~ediyi)-aminof-A-dodecyl-propionainide). Second 5-alkylated derivative, compound 4 (.isomer II) was isolated as a white powder {13,7 g, 14%). MS m/z 672 (M+2H/2), 448 (M+3B/3). !H NMR. COCb δ 0.87 (/, J = 6,5¾ 1SH), 1.20-1.39 (m, 92H), 1.44-154 (m, !.2H), 2,30-2.45 (m, 8ΪΙ),2.46-2.54¾ 8H),2.55-2.85 (rn, 10B), 3.15-3.30¾ 12H), 6.98 {bs, 3H), 7,41 (bs, IB), 7.63 (bs, 1H), 8.85 (bs, IH). 13GNMRCDCR δ 14,33,22.89, 27.28,27.38,29.59,29.69, 29.88, 29-89, 29.92,32,13, 39.65, 39.74, 50.84, 172.63,172.75,172.81,

Along with this a pure mixture of compounds 3 and 4(11,6 g, 12%)m2;3 (3:4) ratio was also isolated. 3- [ (BIS“(2-dodecylearbaiaoyl-efhyi)-atnin o|-ethyi}-(2~ dodecyk*arbaiHoyl-etiiyI)-ainlii(>l~ethyl|-(2-dodeeylcarbantoyl-efliyI)-a«H«oJ~etbyI}-(2-dodreykari>amoy!-ethyl)-amino]-iV-dodecyI-propkmaniide. The six alkylated product 5 was isolated as a cream powder (16.3 g, 17%). MS m/z 792 (M+2H/2), 528 (M+3H/3). !B NMR DMSQ-4 δ 0.87 (/, J = 7Hz, 18H), U5-1.40 (m, i Z2H), 1.45-1.53 (m, 12H), 2,20-2.35 (m, 12H), 2.37-2.50 (m, 1211), 2.64-2.78 (m, 12B), 3.10-3.25 (m, 12H), 7.26 (bs, 4H), 7.64 (bs, 2H), °C NMR CD<% δ 14.32,22,89,27.34,27.38 29.59,29.69, 29.90,29.92,32.13, 39.77,50.85,172.80.

Example 2: Syntheses and purification of compounds 3.4 and 9: alkylation of triethvlenetctramine under Michael addition condition..-.r:.MCfeod 2 tScheme..!).

In. another experiment, .in order to prevent the polymerization of the starting acrylamide 1 at high temperature, a radical quencher benzoquinone was added to tire reaction mixture.

Scheme 2a

* (i) 90 t!C, Catalytic amount (1.5 mg} of benzoquinone, 5 days

In this method a similar reaction to that of Method I (Example 1) was performed except that, a radical quencher benzoquinone was added to the reaction mixture. In a 150 ml, pressure bottle N'-dodecylacrylamide 1 (24 g, 100 mmol) was taken and to this 15 mg of benzoquinone was-added and the solid acrylamide was melted under argon by gently heating the vessel. To this malt was added trietliylenetetramine 2 (2.9 g, 20 mmol) and the mixture was heated at 9Q°G· for 5 days. The reaction mixture was analyzed by TLC using CH2a2:MeOH:Nlfe (90:5:5) as the eluent. The TIC showed the near complete consumption of the starting acrylamide I , The reaction mixture was dissolved in dichloromethane (40 xnL) and tire desired products 3,4 and 5 were isolated as described in Example 1, In this case a slight increase in the amount of six addition product was observed.

Compound 3: The dive addition product, isomer 1,. was isolated as light yellow foam (3.4 g, 13%), The analytical and spectral data for this compound was identical to that of 3 obtained by Method I,

Compound 4: The five addition product, isomer U, was isolated as a white powder (3,9 g, 14%), Hie analytical and spectral data for this compound was identical to that of 4 obtained by Method 1. A pure mixture of isomers 3 and 4 (1.9 g, 7%) was also isolated.

Compound 5: The six addition, product was isolated as a cream powder (6.9 gs 26%). The analytical and spectral data for this compound was identical to that of 5 obtained by Method 1.

Example 3: Syntheses and purification of compounds 3,4 and 4: alkylation of triethvienetgtyamine under Michael addition condition - method 3 fScheme 3)

In this -method the Michael addition was .performed in the presence of a promoter like boric add (Chaodhuri, MihirK..; Hussain, Sahid; Kantam, M. Lakshmi; Neelima, B, Tetrahedron Letters ¢005), 46(48), 8329-8331,} in order to enhance the rate of the reaction.

Scheme 3a f (

8 (i) 90 %, aq. boric acid, 2 days in this method a similar-reaction to that of Method 1 (Example 1} was performed except that, a Michael addition promoter, saturated aqueous boric acid was added to the reaction mixture, Jh a 150 mL pressure bottle M-dodecybacrylamide 1 (24 g, 100 mmol) was melted under argon by gently heating die vessel and to this 3 ml, of aqueous boric acid was added. To this melt was added trieihyleneietramine 2 -(2.9 g, 20 mmol) and the mixture- was heated at 90 °C for 2 days. The reaction mixture was analyzed by TLC using CHjOjiMeOHzNEtj. (90:5:5) as the eluent The TLC showed the near complete consumption of the starting acrylamide .1. The reaction mixture was dissolved in dieMofotnetbaoe (100 mL) and the solution was stirred with solid sodium bicarbonate and the organic- layer was filtered and concentrated in a rotory evaporator. This crude product was purified by column chromatography (silica gel) using CH^C^MeOHMits (48:1:1 to 8:.1:1), hi order to achieve complete separation, multiple columns.using the same conditions were performed and the following pure products were obtained. Under this reaction condition an increase in yields of compound 4 (isomer Π) and six addition product 5 were achieved.

Compound 3:1'be five addition product3, isomer I, was isolated as light yellow foam (3.1 g, 11 %}. The analytical and spectral data tor this compound was identical to that of 3 obtained by Method 1,

Compound 4; The five addition product A isomer II, was isolated as a white powder (3.7 g, 20%). .The analytical and spectral data for this compound was identical to that of 4 obtained by Method !. A pure mixture of isomers 3 and 4 (2,1 g, 7%) wm aiso isolated.

Compound 5: 'Hie six addition product 5 was isolated as a.cream powder (7,6 gj, 28%). The analytical and spectral data for this compound was identical to that, of 5 obtained by Method 1.

Exaronie 4: Syntheses and nurification of compounds 3 and 4; alkviatien of ........ " ' .T^JVun............................. . ...Μ,....,.,.......· . triefevlenetetramine under Michael addition condition. - method 4 (Scheme 4) in another experiment, in order to minimize the formation of the six addition product 5, use of solvent was attempted.

Scheme 4a

s (i) 90 °C, acetonitrile or DMF, 5 days

In this method a similar reaction to that of Method I (Example 1} and Method 2 (Example 2) was performed except that, the reactions were performed in the presence of solvents at 90 °C with stirring, in a 150 raL pressure irottle Aktodecyl-acryiamide 1 (10 g, 41.8 .mmol) was dissolved in 20 mL of either acetonitrile or DMF. To this solution was added triethylenetetramine 2 (Ϊ g, 6.8 mmol) and the mixture was heated at 90 *0 for $ days. The reaction mixture was analysed by TLC using CHaCl^MeOJS :NE% (90:5:5) as foe eluent The TLC showed the formation of only minor amounts of the required .five addition product. The major product in this reaction was a mixture of four addition products'along with very polar lower addition products.

Example 5 : Separation of unreacted acrvlami.de from fee reaction mixture and/or the isolated products 3.4 and 5

To remove unreacted acrylamide I from the reaction mixture, the reaction mixture is diluted with ethyl acetate or DMF and stirred with polystyrene or polymer bound thiol (or mercaptan) to capture all the acrylamide. The immobiliKcd thiol was added to the solution and gently shaken at ambient temperature and filter offihe solid, Michael addition of immobilized thiol to acrylamide capture aftunreacted acrylamide. Traces of acrylamide- as contaminant after isolation of each desired isomer coodd also be completely removed under the same condition, Tire isolated product 3 (or 4-.or 5) is dissolved in DMF or ethyl acetate and gently shaken with the immobilized acrylamide quencher, filter and evaporation of the filtrate in vacua affords a pure compound 3 (or 4 or 5) free of acrylamide contamination.

Example 6: Separation of primary and secondary amine contaminant from compound $

After column chromatographic separation of compound 5, to remover traces of primary and secondary amine contaminants,.the compound is dissolved in ethyl acetate or DMF and stirred with solid bound or immobilized isothiocyanate at ambient temperature overnight. Filler off the solid and evaporation of the filtrate affords a pure compound 5 free of any primary or secondary amine contamination.

Example 7: Separation of primary amine contaminants from compound 3 and 4 After the completion of the reaction the reaction mixture is treated with teirachloraphihalic anhydride in the presence of triethyfamine in dichioromethane at room temperature and the solvent is evaporated and the residue stirred with ethyl acetate and the solid is filtered and the filtrate is concentrated to get the products which lacks the primary amine contaminant.

Table I

Methods of synthesizing products 3 and 4

Method Temperature Promoter Solvent Radical Remarks

Quencher 1 90 °C None Neat None Formation of 3 and 4 in a combined isolated yield of 39%. The six addition product S was isolated in 17%, Reaction took six days for completion. 2 90<SC None Neat Bonzoqainone Benzoqmaone was used to prevent the polymersnation of acrylamide 1, The combined yield of 3 and 4 was 34%. However 26% of 5 was also isolated.

Reaction, time same as Method 1, 3 90 °C Boric acid Neat None Reaction rate enhanced. The reaction was completed in two days. The combined yield of 3 and 4 was 38%. Additional 28% of 5 was also isolated. 4 80-120 °C None DMF None Reaction very sluggish.

Only lower addition products formed,

Example 8: Methods of preparation of the hydrochloride salts: ofthe products 3,. lj.M.5 la order to improve the ease of handling' and increase the stability of the compounds listed above, they were converted into: their corresponding hydrochloride salts 6,7 and 8.

Hydrochloride of compound 3 (6): The amine 3 (9,4 g) was dissolved in 100 m.L of hot anhydrous 1,4-dioxane and 100 mL of 4M HC1 in dioxane was added and the mixture was stirred at room temperature overnight. Nitrogen was bubbled into the reaction mixture for Ih to remove the excess HC1 and the remaining solution was concentrated to *40 .mL· To this heterogeneous mixture TOO mL of EtO Ac hexanes (1:1) was added and tire-precipitated product was .filtered, washed with ethyl, acetate (50 mL), hexan.es (.100 mL) and the resulting powder was dried under vacuum to get the pure product 6 (9.99 g, 96%) as a cream powder. NMR CBC% δ 0,83 (t, J ~· 6.5Hz, 15.H), 1.204.39 (m, 92H), '2,64-2.70 (m, 8H), 2.90-3.10 (m, 16H), 3,25-3,4$ (m, 12H), 3,46-3.64 (m, 4H), 5.20-6.0 (bs, 2H), 8.05-8.15 (ra, 5H), 10, (!>s, 3H), nC NMR CDClj δ 13.83,22.04,26,48,28.69,28.79,28.90,29,04,3.1.26,38.71,168.38,168.53. Elemental Analysis: Calcd. e*TiL63NAs-4HCi.3e20: C, 63.05; Η, 11.3«; N, 8.17; Cl, 9,19. Found: C, 63.13; Η, 11,06; N, 8.21; Cl, 9.21.

Scheme 5s

8 (i) 4M HC1 in 1,4-dioxane, rt., !2h

Compound 7

The amine 4 (13.7 g, 10,2 mmol) was converted to the corresponding HO salt 7 using a similar procedure used above for 3 to obtain 6. The tetrahydrochloride salt 7 was isolated as a white powder (14.6, 96¾). Ή NMR CDCl* δ 0.82 (t, J ~ 6.5Hz, 15H), 1,20-1.41 (m, 92H), 2.52-2.72 (m, 8H), 2.90-3.10' (m, 16H), 3.25-3.45 (m, 1211), 3.46-3,64 (m,4H), 5,20-6.0 (hs, 2H), 8,05-8.15 (m, 5H), 10. (bs,.3H). ,3CNMRCDCb δ 8,42,13.84,22.04,26:48,28.69,28.79,29.00,31,26,45.44., 168.53,168.60. Elemental

Analysis: Calcd: CsiHi63N?05-4H€L2!bO; 0,63,79; H> 1U0; N, 8,17; C% 9.34, Found: C, 63.78; R> :N> 8'40; Cl> 9·73*

Scheme 6:i

* (%) 4M BCl in .1,4-dioxane, H, !2h

Compound 8

The amine 5 (13.7 g, 1.2 mmol.) was converted'to the corresponding HCI 8 using a procedure similar to that, described above for tire salt 6. The tetrahydrodiiori.de salt 8 was isolated as a white, powder (1.3 g, 96%). *H NMR DMSO-4 5 0.87 (r, J - 71¾ 1SH), 1,13-1,30 (m, Π2Η), 1.35-1.53 (mv 121¾ 2.10-2,25 (m, 121¾ 2.30-2.40 (m, 12B)S 2.60-2.76 (mf I.2H), 3.10-3.25 (m, 121¾ 7.26 (bs, 4H), 7,64 (bs, 2H), 10,1 (hs5 4H),

Scheme T

s (i) 4M HC1 in 1,4-dioxane, rt.s 12h,

Example 9: Selective protection of amino groups on trieth vlenetetramlne for directed synthesis of compounds 3 . and 4

Step 1: Preparation of compound 10; Triethylenetetraraine, 2 (20.55 g, 140,52 mmol, purchased. Itotir Sigma-Aidrieh) in acetonitrile (500 mL) was cooled over an ice bath under constant stirring. Ethyl triduroacetate (35,20 mL,-295,09.mmol) was added to the stirring solution and stirred for 20h. Solvent and volatiles were removed under reduced pressure and dried'under high vacuum to get 9 as white solid (44.4 g, 94%). The product thus obtained could be used for die next reaction without farther purification (Wender F. A. et a?. Organic Letters, 2005 7, 4815) .

Crude compound 9 (23.70,70 mmol) was dissolved in acetonitrile (400 mL) and stirred over as ice bath. Ar-(Benzylox>'carbonyloxy) succinate (Z-OSu, 43,?3g, 175 mmol, purchased From Novabiochem) and triethyl amine. (23.40 mL, 210mmol) were added to the reaction, mixture and stirred overnight, Solvents were removed and the residue was extracted into dichloromethsne (DCM), washed successively with water (two times) and brine, dried over anhydrous sodium sulfate. Solvent teas removed tit vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution, 30*70% EtOAe/Hexanes) to obtain compound 10 as white solid (38,.2¾ 89%). 5H NMR (DMSO-d6,400MHz) δ « 9.60-9,50(10, 21-1:), 7,40-7,20(10, 10H), S,02(s, 4H), 3.40-3i20(m, 12H). MS: CW$»F«N4Qc Cal. 606.19, Found. 607.2(M',>.

Step 2: Preparation of compound 11: Compound 10 (12.60 g, 20.78 mmol) was suspended in methanol (MeOH, 150 mL) at ambient temperature and 8M solution of methylamine in ethanol (40 mi) was added to the suspension under constant stirring, Ainhe solids went into solution, after' stirring for 1 h at ambient temperature, tire mixture was wanned to SOX' and stirred For 8h. Reaction was monitored by TLC. All the solvents were removed under reduced pressure and the residue was purified by silica gel column chromatography (gradient elution, 10% MeOH/DCM to 10:10:80,

MeOB :TEA:DCM) to yield the product 11 (7.80g, 91 %) as pale yellow gummy liquid. *11 NMR (DMSO-46,400MHz) δ - 7.80>7.4O<m, 10H), 5,G2-4.94(m, 411% 3.45-3,05(m, 8H), 2.70-2.55(m, 4H), 2.20(hs, 4K). MS: QaHwN^ Cal 414.23, Found 41$.20(Ml

Step 3: Preparation of compound 13: Compound 12 was prepared from triethylenetelramine, 160 (10.25¾ 7O.09mmoi) as .described in.step .1 for the synthesis of compound 9 by reacting with 1.1 molar equivalent' of ethyl trifluoroacetate (S.SOm-L, 77.1()mmol), Crude 12 thus obtained was dissolved in anhydrous DCM (400tnl) and cooled to Ο X. (Bdc^Q (53.53 mmol, 245.31 mmol): and trie&amp;ylamine (48 ml, 350mmol) were added and reaction mixture was allowed to stir overnight, Progress of the reaction was monitored by TLC; 'Solvents were removed in vacuo and the residue was extracted into DCM, washed with water, brine and dried. DCM was removed and die residue was purified by silica gd chromatography (gradient elution 50%EiOAc/Bexane to· EtOAc)' to obtain the desired product 13 (34.20g, 92%) as white solid, *H NMR (DMSO-dS, 400MHz) δ « 9.5I-9.38(ra, 1H), 6.82(bs, 1 if), 3.30-3,OO(m. 12H), 1.58*1,30(s, 2711). MS: C^iF^O? Cal. 542.29, Found 543.4(KT),

Step 4; Preparation of 14: A solution of compound 13 (25g, 47.32 mmol) in MeOB (200 mL) was stirred with &amp;2CO3 (50g) in the presence of water (i mL) at 50 °C overnight Progress of die reaction was moni tored byTLC. Solid K2CO3 was filtered off, washed with MeOH> combined washing and solvents were removedin vacuo. Residue obtained was purified by silica gel column chromatography to yield the desired product 14 (10,2 g, .50%) as white solid. !H NMR (DMSCM6, 400MIfe) δ - 6.S3(bs, IB), 2.95-3.30(ro, 12H), 2.62-2.50(m, 2H)5 1.25-1.45{m, 27H). MS: QilfcaNA Cal 446.31, Found 447.4(M*).

Scheme Sa

a Selective protection of triethylenetetrarnine nitrogens.

Step 5; Preparation of compound 15: Compound 9 (23.0g, 68.02 mmol) was dissolved in a mixture of acetonitrile/dichlorom.ethaneil;!, 300mL) and cooled to tfC. Z-OSu (I7.G0g, 69 miitol) was added to the solution ami stirred lor 10 .minutes. Triethylmnine (23.40 ml, 210mmoi) was subsequently added'to the reaction mixture and allowed to stir overnight. Solvents and triethylamine were removed in vacuo" md the residue was extracted into DCM, washed with water (two times), brine and dried. After removing solvent, the residue was purified by silica gel column chromatography (eluted initially with 20-60"% EtOAe/Hexane, then with 5% MeOH/DCM) to obtain the desired product .15 (13,3g) as white solid along with side product 10 (S.5g>. *Η NMR. (DMSO-46,400MHz) 6 - 9.6Q{bs,.lH),9J0(bs, 1H), 7.40~7.2g(m, 5H), S-.01(s,;2H), 3-40-3.10(m, 81Ί), 2.70“2.50(m, 4H). MS: CiskhFeH^ Cal. 472,15, Found 473. UM*).

Step 6: Preparation of compound 16; Treatment of compound 15 (13.4g, 28.38 mmol) with methyl amine (50 ml, 8M solution in EtOH) as described in step 2 yielded a colorless liquid compound 16 (6.10g, 79%). The product thus obtained could be used for next reaction without further purification. NMR (DMSO-dS, 400MHz} δ - 7.45-7,200», 60), 5.07(s, 2H), 3.45-2.90(ra, 8H), 2.60«2.30(m, 48). MStCwH&amp;N^Ox Cal. 280.19 Found 28L2(M*).

Scheme Ψ

4 Selective blocking of single secondary nitrogen of iriethyknetetramine BxampteTQ: Synthesis of 5-alkvlated single isomer 4 - Method l Step If Reaction, of 11. with Λ’-dodecylacrylamide: Diamine 11 .(1,00g, 2.41 mmol) and Acdodecylaeiyiamide (3,47g, 14,50 mmol) were taken together in a pressure tube and heated at 90“C for 5 days. The reaction was monitored by TLC. Once the reaction is over, the mixture is dissolved in dieMoromeihane and purified by flash chrom atography to get the products 17,18 and 19.

Step 2; Preparation of compound 20; C-ompound 19 (2.00g, 1.46 mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2,15 ml) to that 2 eq, of acetic acid is added. The mixture is hydrogenated under pressure (50 psi) using palladiem/earboh (G.200g, I0%wt) as a catalyst to get the desired product 20,

Step 3- Preparation of single isomer 4: Compound 20 (L50g, 1-36 mmol) and the acrylamide X (0,325 mmol, 1.36 mmol) is dissolved In toluene (4mL) and heated at 90"C davs-to form compound 4, Progressof the reaction is-monitored by TLC. After . ^ completion of reaction» the mixture is cooled to room temperature, dissolved m DCM and purified by flash silica gel column chromatography to obtain the desired product 4, Schema 10

Fxanmfe 11: Synthesis of 5«alkyiated single isomer 4 -- Method!

Step 1: Preparation of compound 21; Compound 16 (LOg. 3,56mmoi) and N~ dodeeylaerylamide (6.00g, 7eq) are taken together in a pressure tube and heated to obtain compound 21. Progress of the reaction is monitored by TLC. After completion of the reaction the mixture is dissolved in DCM and purified by flash silica gel dmmratography to afford the desired compound 21.

Step 2; Preparation of compound 4 from 21: Compound 21 (2.00g* *·35 mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2,1.5 mi) to that 2 eq. of acetic acid is added. 1¾ mixture is hydrogenated under pressure (5:0 p®*> ovm palkdium-earbon (0,200g, 10%wt) to afford the desired single isomer 4.

Scheme 11

Sample 12: Synthesis of 5-alkylated single isomer 3 - Method i

Step 1; Preparation of compound 22: Compound .1.4 ($,06g. 11.30 mmol) and A*-dodeevlaejwlamide (2,94g, 12.43 mmol)· were taken in toluene and heated at 90'C for five days. TLC was cheeked and showed the formation of product. Tile reaction mixture was directly loaded on a pre-packed column of column silica gel and purified by Bash chromatography (5% MeOH/DCM) to afford compound 22 (4.82¾ 62%). TH 'NM.il (DMSO-dd, 400MHz) δ - 8.17(bs, 1H), 6,60(5¾ 1H),,3.30-2.95(¼ 12H), 2,70(1, 1-5.801¼ 2H), 2.60ft /=6.008¾ 2H), 2.18ft 3-6.40¾ 2B), 1.35{m, 291¾ 1.26-1.15(¼ 18ΙΊ), 0.83ft 3=6,00Hz, 3H). MS: CmHpNsOt Cal. 685.54, Found 686.50^.

Step 2: Preparation of compound 23: Compound 22 (4.75g, 6,92 mmol) was dissolved in dichloromeihane (lOOmL) and cooled to O’C. Z-OSu (2.59¾ l,5eq) was. added to foe solution and stirred for 10 minutes. The reaction mixture was subsequently stirred with triethylamino (2.82 mL, 20.76ramol) overnight, Solvent and tnethylamine were removed in vacuo and the residue was extracted into dichloromeihane, washed successively with water (two times) and brine, and dried over anhydrous sodium sulfate- After removing solvent the residue was purified by flash silica gel column chromatography (5- ί 0% MeOH/DCM) to obtain the desired compound 23 (5,33¾. 94%), 5B NMR (CDCIj, 400MHz) 6 =-7,49-7.25(¼ 5H), 5.1 l(s, 2H), 3.60-3,02(¼ 14H), 2.45-45{m, 4H), 1.50-1.35(¼ 27H), 1,24-1.20(¼ 18H), 0.87ft /=6.00¾¾ 3H). MS; C44H77N5O9 Cal. 819.57, Found 820,7(M'f).

Step 3: Preparation of compound 24:4M HC1 in dioxane (SO m.L) was added into a solution of compound 23 (530¾ 6,50 mmol) in dioxane (100ml). The reaction mixture was then allowed to stir overnight Product was precipitated out. during the course© f the reaction. Solvent and HCl were removed under vacuum to yield a while solid. The residue was taken inMeOH containing excess triefhylamine and the suspension was stirred fbr Ih to obtain a homogeneous solution. Solvents were removed in vacuo and the residue was triturated with EtOAe, filtered off the tridhyltoiae hydrochloride salt Combined filtrate was evaporated under vacuum to obtain a gummy liquid 24 (3.30g, 98%). 2H NMR (CDCJ3,400MHz) δ - 7.37-7.2S(m, 51% 5,05(¾ 2H), 3.60-3.20(ms 414),3.10-2.70(114 10H), 2,40~2.20(ms 414), 1.404,30(m, 211), 1,25-1,17(m, I8H), 0.81 (U =6.00¾ 3H), MS: GasHssNsOj Cal 519,41, Ftxmd 520.4(54% Step 4: Preparation of compound 25: Compound 24 (1.00¾ 1.925 mmol) and i^-dodecylaerylamide (3.70g, 8eq) are taken together in a pressure tube and heated at elevated temperature to form desired compound 25. Formation of the prod uct is monitored by TLC and is subsequently purified by flash silica gel column chromatography to afford a pure compound 25.

Step 5: Preparation of compound 3: Compound 25 (2.00¾ 1.35 mmol) is dissolved in a mixture of ethyl acetate and methanol (.1:2, .15 ml) to that 2 eq, of acetic acid is added. The mixture is hydrogenated under pressure (50 psi) over palladiura-earbdft (0,20(% 10%wt) to afford flic desired product 3.

Scheme 12

Example 13: Synthesis of S-alkvlated sin ale isomer 3 - Method 2 Step 1: Preparation of compound 26: Benzyl bromide (1.25 ml, 1.5sq) to a suspension of compound 22 (4.80g, 7.0Ctamoi) and K2CO3 (9,67g, lOeq) in DMF (100 mi.) and the mixture was stored overnight. Progress of the reaction was monitored by TLC. Solids were filtered off, washed with MeOH and ethyl acetate. Combined filtrate was concentrated under reduced pressure and the residue thus obtained was purified by silica gel column chromatography (50-100% EtOAc/Hcxane) to afford the desired compound 26 <3.3% 61%). *H NMR (DMSD-dii, ,400MHz) δ - 7.77(bs, 2H), 7.2$-7.23(tn, 5H), 6.$5-6.70(m, 1H), 3.59(s, 2H)> 3,20-2,20(11¾ 18H), L3S&amp;.27H), 130-1.23(1^-211¾ 1.20-L15(iii, 18H), 6.81(1, fo 6.00Hz, 3H). MS: QaH^NsO? Cal. 775.58, Found 7?6.5(M't)

Step 2; Preparation of compound 27: Compound 26 (3.30g, 4.25 mmol) in dioxane (50m!) was stirred with 4M HC1 (SO mi) in dipxane overnight. Formation of white precipitate was seen during the course of the reaction. So! vent and acid were removed under vacuum and white residue thus obtained was re-dissolved in methanol containing excess Methylarmne. The homogeneous solution was then evaporated under reduced pressure to obtain while residue. The residue was triturated with EtOAc and filtered off triethylamine hydrochloride salt. Filtrate was evaporated under vacuum to afford the desired compound 27 (2,36g, 99%) as gummy liquid. lH NMR (CDCls, 400MHz) δ - 8.05(¾ J« 5.5 Hz, 1H), 7.40-7.20(m, 5H), 3,58(s, 2H), 3.10-2.30(m, 18H), 1,40-i .30(m, 2H), 1.35-1 .15(m, 18H), 0.82(¾ fo 6.00¾ 3H). MS: CbB»NsO Cal 475.43, Found. 498.4(MTNa)

Step 3: Preparation of compound 28: Neat compound 27 (!.00g, 2,10 mmol) and Aklodecylacrylainide (4.0g, 8eq) are mixed in a pressure tube and heated to elevated temperature to form compound 28. Formation of 28 is monitored by TEC and LC-MS. After c-ompkiion of the reaction the product is isolated by chromatographic purification to afford pure compound 28.

Step 4: Preparation of compound 3 front compound 28: Compound 28 (2.00g, 1.40 mmol) is dissolved in a mixture of ethyl acetate and methanol (1:2, IS ml) to feat 6 eq. of acetic acid is added. The mixture is hydrogenated under pressure (SO psi) over palladium-carbon (G.200g, 10%\vt) to obtain compound 3

Scheme 13

Example .14: Convergent synthesis of isomer 3 Method 1

Step 3; Preparation of compounds 30» 31 and 32: Ethylencdiamine 29 (0.978ml, 14,63mm0l), Akiodeeylacryiamide (7.00¾ 29.26mmol) and boric add (lOOmg) were taken In 5 mL of water and heated at. 90*0 for lour days. Complete disappearance of acrylamide was ascertained· by TLC analysis. The:reaction mixture was dissolved in DCM, washed with water and bicarbonate and dried over sodium sulfate. DCM was removed and the residue was purified by silica gel cohrnm chromatography (2:2:96 to 10:10:80% MeOH/FEA/DCM) ίο get compounds 30 (L86g) lH NMR .{CBC1* 400MHz) 6 - ?.05(bs, 2H>, 3.21 (q, 3-6.30 Hz, 4H), 2.87(1, 3= 6.001¾ 4B), 2,73(¾. 4H), 2.34(t,J-d.0QHz, 4B),l.S7<bs, 2H), 1.49-L45(m, 4H), 1.28-1,19(m, 40H), 0.87(1, J- 6.8Hz, 6H) MS: Cj2H^N402 Cal. 538.52, Found 539.50(Μ+). 31 (3,50g) hi NMR (DMSO-<I6,400MHz) δ - 8.20(bs, 1H), 3.20-2.!5(m, 22H),U6~ L30(m, 6e)f 1.25-Ί J5(m, 30H), 0.81(ts 1= 6.00Hz, 9H),MS: C47H95N5O3 Cal. 777.74, Found 778.7{M+) and 32 (1.75g) Ή NMR (DMSO-d6,400MHz) d *» 3.23-2.15(m, 2SH), 1.35-i.45(m, SH), 1.26-1.15(m, 40H), 0.82(1,> 6.00Hz, 12H). MS: C62Hl24NA Cal. 1016.97, Found 1018.0(Μτ).

Step 2: Preparation of compound 33: Compound 31 (1.55g, 2mmol) and K2CO3 (2.761,20mmol) are takes in DMF. To that chloroaceiaidehyde dimethyl acetal (0.453 ml, 4.0Qmmol) is added and stirred for 24h. Reaction is monitored by TLC, filtered offKaCOs washed with MeOH. Solvents are removed under reduced-pressure and the residue is subjected to chromatographic purification to afford compound 33.

Step 3: Preparation of compound 34: Compound 33 (2.00g, 2.31 rmxsol) is taken in a mixture of MeOIl and DCM, to drat PTSA (2,0eq) is added and reaction mixture is stirred overnight. The solution is neutralized with, sodium bicarbonate solution and extract with DCM and dried. Compound is purified by chromatographic separation to afford the desired product 34.

Step 4: Preparation of single isomer 3 from 34; Compound 34 (2.00g, 2.43 mmol) and 30 (1.31 g, 2,43 mmol) are taken in DOM; to that activated molecular sieves is added and stirred for 3h. The reaction is monitored by TLC. Once the reaction is over solvents is removed. The residue is dissolved in THF and sodium triacetoxyb<>rohydride (5 eq.) and acetic acid are added and stirred overnight. Solvents are removed and. extracts with DCM, -cteomatographic separation of the residue affords pure isomer 3.

Scheme 8

Example 15: Cohveraent synthesis of isomer 3 - Method 2

Th« desired single isomer 3 is also prepared from compound 30 by selective protection of one of the nitrogen to obtain compound 35. Compound 35 is subsequently reacted with aldehyde 34 under reductive conditions to obtain compound 3d. Add treatment of 3d affords desired compound 3.

Scheme 15

Example 16; Convergent synthesis of isomer 3 - Method 3

The desired single isomer 3 is also prepared from ..monobenzyl ethylenediarnine 37» Alkylation of 37 with 1 affords a mixture of compounds 38,39 and 40, Compound 48 is reacted with aldehyde 34 under reductive conditions to obtain compound 41. Hydmgetioiysis of 41 affords the desired compound 3.

Scheme id

Example 17: Convergent synthesis of isomer 4 - Method i

Step 1: Preparation of compounds 43: in a 150 ml pressure bottle /V'-dodecyl acrylamide 1 (16.4 g, 68.8 mmol) was melted under argon by gently heating the vessel and to this 3 ml of aqueous boric acid was added. To Ms melt was added Boc protected ethylenediamine 42 (5 g, 31.2 mmol) and the mixture was heated at 90 *C overnight. The reaction mixtee was analyzed by TIC using CH3Cl2:MeOH:NBt;i .(90:5:5) as the eluent The TLC showed the near complete consumption of the starting acrylamide 1. The reaction mixture was dissol ved in dichloromethane (100 ml.) and the solution was stirred with solid sodium bicarbonate, and the organic layer was filtered; and concentrated m a rolory evaporator, this crude product was purified by column chromatography (silica gel) using CW3Ch:MeOH:NKt3 (48:1:1 to 8:1:1). The major product in this reaction is the double addition product 43. M inor amounts of mono adduct was also observed.

Step 2: Preparation of compound 44: Compound 43 (2.00¾ 3.13 mmol) is taken in dioxane (50 mL) to that MCI (20 mL, 4M solution in dioxane} is added and stirred overnight. Solvent, is removed to get the compound 44.

Step 3: Preparation of single isomer 4 from 34 and 44: Compound 34 (2.0(% 2.43 mmol) and 44 (1.31¾ 2.43 mmol) are taken in DCM: to that activated molecular sieves is added and stirred for 3h. The reaction, is monitored byTLC. Once the reaction is over solvents are removed. The residue is dissolved in T1T.F and sodium triaceioxy horohydride (5 eq.) and acetic acid are added and stirred overnight. Solvents are removed and extracts with DCM, chromatographic separation of the residue affords pure isomer 4.

Scheme 17

Example 1 g: Addition of A-dodecvlacrvIaroide to 1 subseqnent reduction of the amide to amine

In order to study the effect of number of charges in the cationic lipid the Michael adducts of acrylamide 1 with 1,3-diaminopropane 45 was investigated.

Scheme 18*

* (I) 90 T, aq, boric acid, I Oh; (ii) 4M HCI in l.^oxane, it, 12b and (in) Bib Step I; Synthesis of 40,47 and 46: hi a 150 mt pressure bottle N-άodecyl-aervlamide 1 (15.4 g, 64 mmol) was melted under argon by gently heating the vessel and to this 3 mh of aqueous boric add was added. To this melt was added 1,3-diaramopropaae44 (1,58 g, 2.1 .mmol) and the mixture was heated at 90 eC overnight. The reaction mixture was analyzed by TLC using CHsCbiMeOHiNEb (90:5:5) as the eluent. The TLC showed the near complete consumption of the starting 'aery! amide .1, T he reaction mixture was dissolved in dichloromethane (100 mL) and the solution was stirred with solid sodium bicarbonate and the organic layer was filtered and concentrated in a rotory evaporator. This crude product was purified by column chromatography (silica gel) using CTbOyMeOHrNEb (48:1:1 to 8:1:1).. Ihe major product in this reaction is the triple addition product 46. Minor amounts’of tetra adduct 47 and bis adduct 48 were also isolated, A''-|yedceyl-3»{(2~dodeeylcarbamoyl-ethyi)-|3*(2''dodeeyIcarbsnK?yi>' ethyl3mlao)-propyl}-ami«o}-propionan»de 46. 'Hie three addition product 46 was isolated ’as- a white powder (5.7 g, 35%). MS m/z 793 (MH ). !H MMRCDClj δ 0,87 (/, J«* 6.6Hz, 9H), 1.20-1,30 <m, 60H), 1.42-1.66 (m, 6M), 2,33 (t, J - 6Hz, 4H), ,2.3,8-2.46 (m, 4H}? 2.60-2,70 (m, 4H), 2.84 (t, 2H), 3.15-3.28 (m,.6H), 6.65 (bs, IH),. 6.99.{bs, 3H). 4“f{3-[Bis-(2“dodecylcarbaHJoyi-ethyl)-amiHo|-propyl}-(2-dedecylcarbamoyl-ethylJaminoJ-A'-dodecyl-btttyrainide 47. The four addition pro duo* 47 was also isolated in minor amounts. A--DodecyK3“|“C2-dodecykarbam.oyl-etl»ylani!iio)-propy!amirEo]-propionamlde 48. The diadduct 48 was.-isolated as a cream powder (1.6 g, 10%), MS rn/z 553 (Mlf). *H NMR CDC13 o 0.S9 (t, J* 6.6Hz, 6H), L10-L20 (my40B), 1.42» 1.66 (as, 4H), 2.20-(1, J- 6Hz, 4H), 2.55 (t, 4B), 2.60 (t, 4H), 3.00 (m, 4H), 8.00 (¼ 2H),

Step 2: Conversion of amines 4,35 and 36 ίο their corresponding hydrochloride salts 49, 50 and 51.

The amine 46 (5.5 g) was converted to the corresponding HCI49 using,a procedure similar to the described in Example 8 and the dihydmcMoride salt 49 was isolated as a white powder (5.73 g, 92%), *H NMR DMSO-tf6 δ 0,88 (/, J = 7¾ 9H), 1.17-1,30. (m, 66H), U5-L45 (m, 61), 2,10-2.25 {515,2¾ 2.55-2,70(¾ 6H), 2.95-3.15 (m, 10H), 3,20-3.35 (m, 6H), 8,16 (t, 1H), 8,24 (t, ΪΗ), 9J 5 (bs, 1% 10.6$ (bs, 1H),

In a similar procedure to that described in Example 8 the amine 47 is treated with 4M HCl to obtain the dihydrodtloride salt.50, hi a similar procedure to that described in Example 8 the amine 48 is treated with 4M HCl to obtain the di.hydrochSor.ide salt 51,

Step 3 : Reduction of amides 46* 47 and 48 to amines 52,53 and 54; Amine 46 is refluxed in 1T1F with excess of diborane overnight and subsequent treatment with 4M HCl affords hydrochloride salt of poly amine 52·. A similar treatment of amines 47 arid 48 affords the corresponding reduced product 53 and: 54 as their .respective hydrochloride salt.

Example 19: Reduction of polyamides 3.4 and 5 to the· corresponding polvamlne dendrimers

Compound 3 is refluxed with large excess of diborane in THE to obtain the corresponding reduced product 55. After completion, of the reaction, the reaction mixture is treated with 4M HCl prior to work-up and the product is isolated as its hydrochloride salt. Hydrochloride salts, of 56 and 57 are also obtained from the corresponding precursors 4 and 5 respectively.

Scheme 19a

3 (i) BHS;THF, reflux

Example 20: Folvaimno alkyl lipids - reduction of amides to amines

Preparation of poly amines 60 from 32: Compound 32 (1.02g, 1 mmol') is taken in THF (20 ml), to that B%.THF (60 ml, 1M in THF) is added and refluxed for two days. Reaction is monitored by TLC. Removal of THF gives a white residue, which is treated with 1M HCi and extracts into DCM. Chromatographic separation of the crude products, yields pure compound 60.,

Preparation of polyamines 58 and 59 from 30 and 31: Reduction of amides 30 and 31 tinder similar conditions described for the preparation 60 respectively affords 58 and 59,

Scheme 20

Example 21: Synthesis of nolvamido-polyarnino alkyls - alleviation of amines using alkv! halides

Step I: preparation of compound 62: A solution of chloxoacetyl chloride (10.31 mL,. '129.37 mmol) in DCM (200 raL) was cooled over up ice bath and to this a solution of dodecyiamine.(61,20.00g, 107.81 mmol) m dichlororsethane containing TEA (36.70 ml, 269.5 mmol) was added dropwise over a period of 1 hr. The reaction mixture tuned brownish-black, by tins time, continued the stirring for another hour at 072, The reaction mixture was filtered through a sintered funnel, washed with EtOAe, diluted with chloroform, washed successively with water, sodiutn.bicarbonate, solution,. 1M Hd and brine, Organic layer was dried over sodium sulfate. Solvents were removed and the residue was purified by silica gel column chromatography (5-50% EtOAc/Hexane) to afford compound 62 (26.00¾ .92%) as brown solid. rH KMR ,(CDCU> 400MHz) δ = 6.59(bs, 1H), 4.03(s, 2H), 3,25(¾ J-6.00Hz, 2H), 1.544.49(m, •2H), 1.45-U5(m, !EH), O.86(t,>6.00Mx, 3H). MS: CMH28CiNO Cal. 26U9, Found 262.2QQA*}.

Step 2: Preparation of 63,64 and 65; Triethylenetetraminc 2 (1.00¾ 6.83 mmol) and ehloroaeetamide 62 (10.00¾ 5.5 eq) are taken together in a mixture of CH3CN/DMF (1:3), to that K3CO3 (9.43 g, .10 eq) and ΚΪ (50 rog)are added and heated at 85 °C for three days. The reaction mixture is filtered to remove solids, wash with DCM, solvents are removed in vacuo and chromatographic separation of the crude .residue affords pore compounds 63, 64 and 65.

Scheme21

urine alkyl

Step x; Preparation of 67: Chloroacetyl chloride (4.05ffiL·, 51 mmol) was takes is DCM (100 tnL) and cooled down to OX. To this a diehloromethane solution ofUN~ didodccylamine (66,1 S.QGg, 42,41 iranol) and TEA (14.43 ml, 2,5 eq,j were added dropwise over a period of .1 hr. The reaction mixture tuned brownish-black by this time, after the addition die reaction mixture- was stirred for 24 h. at amhinci temperature. Γ he reaction mixture was filtered- through a sintered funnel, washed with. EtOAc, diluted with chloroform, washed successively with waisr, sodium bicarbonate solution* i-M HCi and brine. Organic layer was dried over sodium sulfate. Solvents were removed in vacuo and tire residue was purified by silica gel column chromatography (5-50% utOAc/riexane to obtiaa the required product 67 (12.5g, 69%) as brownish liquid, SH NMR <CDC%, 400MHz) 5 - 4.04{s, 2H), 3.30(m4 4H>, 1.50-1.45(mt 2«), l .40-1.20(m, 18H), 0.87((, J- 6.00Hz, 3H). MS: CzsHsjCINO Cal 430.15, Found 431.2(M').

Step 2: Preparation of 68,69 and 7(h Triethylenetetraniae 2 (O.S'OOg, 6,83 mmol) and chloroaoetamide 67 (8.I0g, 5.5 eq) are taken together in a mixture of CHaCNZDMF (1:3),10 that K2CO3 (4.72g, 10 eq) and Kl (30 mg) are added and heated at 85 °C for three days. The .reaction mixture was filtered to remove insoluble solids, wash with DCIvL solvents are removed and chromatographic separation of the residue affords 168, 6.9 and 7.0.

Seheme.22

Example 23: Addition of Ah¥-dialk\lacrylamide to poiyafflines In order to study the effect of adding more hydrophobic chains to the cationic lipids, didodecylamine was «serf as a precursor to ike acrylamide.

Schenk ^

4 Φ Aeryloyl chloride, -10-0*0, DIPEA, CI-LCL,4h, (a) 90 *0, Neat, 5 days and (id) HCI/Dioxane

Step i’ Synthesis ofLA^V-Bidodecylaerylainide 71

To a solution of dkiodecylatnine 66 (25 g, 70,7 mmol) and dlisopmpydethyimntne (IS g, 141. mraol) in anhydrous CH2CI2 (700 mL) at -10 *€, a solution of aeryloyl chloride (7,68 g, 85 mmol) in CHjCI> (100 ml.) was added dmp wvss over a period of 20 rain. After the completion of the addi tion the reaotkm mixture was stirred for 4 h at 0 °C after which the TLC of the reaction mixture showed the completion of the reaction. The reaction mixture was washed-with said, NaHCOs solution (200 mL), water (200 mL), brine (100 mL) and dried overNaSO^ Concentration of the organic layer provided the product 71 (28.4 g, 100%) which was used as such in the next step, *H NMR CDClj δ 0.94 (/,,/- 6.5Hz, 6It), l .05-1.69 (m, 40H), 3,15-3,60 (dt, 4H), 5.64 (d, IH), 6,36 (d, IH), 6.63 (m, 1H).

Step 2: Reaction of triethydentetramine 2 and 71

The acrylamide 71 is treated with the amine 2 and after usual work-up and column purification the Michael addition products 72,73 and 74 are isolated.

Step 3: Synthesis of hydrochloride salts 75,76 and 77: Each single compound obtained is tafcea in dioxane and 4M HC1 in dioxane is added to the solution and stirred as described in example 8 to yield the corresponding hydrochloride salt.

Ryffnple 24: Alkenvlatioa of polyamines usiajg-IB^.o^aatpmt^l^aa&amp;yl acrylamide ifflder Michael addition condition

In order to study the effect of double bond in the alky] chain oleylamioe was used as a precursor to the acrylamide 79.

Scheme 24d

8 (i) Acryloyl chloride, -10-0 °C, DIPEA, CH2CI2,4hf (ii) 90 tC, Heat, 5 days and (Hi) HCI/Dioxane S tep i: Synthesis of compound 79: To a solution of cleylamine 78 (26,75 g> 100 mmol) and trietbylamine (20 g, 200 mmol) in anhydrous C!rl2€% (200 ml) at -10 ®C,. a solution of acryloyl chloride (9.9 g, 110 mmol) in €1¾¾ (100 mL; was added dropwiso over a period of 20 min* After the completion oi the addition the reaction mixture was stirred for 4 h at 0 "C after which the TLC of the reaction mixture showed the completion of the reaction. The reaction mixture was washed with said. NaMCGr solution (2(H) mL), water (200 mL), brine (100 mt) and dried over NaSO*. Concentration of the organic layer provided tire product 79 (32 g, s 00%) which was used as such in the next step, jH NMR CDCis δ 0.91 (t, J ·* d.SHz, 3H), i .05-1.35 (m. 24«},. 1.42 (t, 2Η), 1,96 (m, 4Η), 531 (i, IΗ), 5,33-536 (hi, IH), 5,54 (dd, 1Ή), 6.02 (dd, IH), 6.18 (dd, IH), 3,03 (bs, IH).

Step 2: Reaction of compound 79 with triethylenetetramiae

The acrylamide 79 is treated with triethylenetetramine 2 and after usual work-up and column purification of the Michael addition products affords pure 'compounds 89, 81 and 82,

Step 3: Synthesis of hydrochloride salts 83» 84 and 85: Each single compound (89,81 or 82) obtained is taken in dioxaae.asd 4M HO in dioxane is added to the solution and stirred as described in example 8 to yield the corresponding hydrochloride salt,

Example 25: Alkcnvlatlon of diamines using mono unsaturated N-alkvl acrylamide under Michael addition condition

Scheme 25'*

3 (i) 90 “C, aq. boric acid, I6h and <ii) HGl/Dioxane hi a similar procedure to that of Example 24 die acrylamide 79 Is treated with the diamine 45 and alter usual work-up and column purification the Michael addition products!®, 87 and 88 are isolated. Treatment of the free amine dins obtained with MCI in dioxanc affords the corresponding hydrochloride salts 89,90 and 91 respectively.

Example 26: Alkenyl ation of polyamines using poiv on saturated N-alkyl acrylamide under Michael addition, condition

In order to study the effect ofpolyimsaturaiios in the alkyl chain iinokyiamine 92 was used as a precursor to the acrylamide 93. 24H), 1.42 (t,2H), 1.96 (in, 4H), 5.31 <i> 1H), 5.33-5.36 (m, IK), 5.54 (dd, 1H), 6.02 (dd, IH), (UB (dd, IK), 8.03 (bs, 1B).

Step 2 : Reac tion of compo und 79 with triethylenetetrarnlne The acrylamide 79 is treated with triefftylenstetrmmne 2 and after usual work-up and column purification of the Michael addition products affords pure compounds 8(h 81 and 82.

Step 3: Synthesis of hydrochloride salts 83,84 and 8$: Each single compound (SO, 8t or 82) obtained is taken in dioxane and 4M HO in dioxane is added to die solution and stirred as described in example 8 to yield the corresponding hydrochloride salt. 25: Alteavlaftan of diamines· usitognaofte unsaturat^'N-alky? aorvlamiflft under Michael addition condition Scheme 25*

8 (i) 90 °C, aq, boric acid, 16h and '(it) HCl/Dtoxaue

In a similar procedure to that of Example 24 the acrylamide 79 is treated with the diamine 45 and after usual work-up and column purification the Michael addition products^, 87 and 88 are isolated. Treatment of the free amine thus obtained with HCI in dioxane affords the corresponding hydrochloride salts 89,99 and 91 respectively, Example 26: Aikenvlatien of polvamlncs using poly unsatumtM.N-a&amp;yl acrylamide onder Michael addition condition

In order to study the effect of polyunsaturation in the alkyl chain linoleyiamine 92 was used as a precursor to the acrylamide 93.

Scheme 2#

* (!) Aerleyl chloride, -10-0 X, DIPEA, CH2C12, 4h, (ii) 90 X, Neat, 5 days and (ii?) HCI/Dioxaoe

Step 1: Compound 93: Linolylamine 92 is treated with acrvloyl chloride in a similar procedure to that of Example 24, step 1 and the corresponding acrylamide 93 is isolated.

Step 2;-Reaction of compound 93 with triethyieneteiramine

The acrylamide 93 is treated with triethylenetetramme 2 in the presence of boric acid as described in Example 3 and after usual work-up and co ton purification of the Michael addition products affords pure compounds 94,9$ and 96.

Step 3; Synthesis of hydrochloride salts 97,9.8 and 99: Each single compound (94,,95 or 96} obtained Is taken in dioxane and 4M HCl in dioxane is added to the solution and stirred as described in example 8 to yield' tire corresponding hydrochloride salt.

Example 27: Alkenylation of diamines using poly unsaturated N-aikyl acrylamide under Michael addition condition

Scheme 27*

»(j) % <Jc, aq. boric acid,. 16h and (ii) HCl/Dioxane m a similar procedure to that of Example 3 the acrylamide 93 is treated with the diamine 45 in the presence of boric add and after usual work-up and column purification the Michael addition products 100,101 and 102 are isolated. Treatment of the free amine thus obtained with HC1 in dioxane affords the. corresponding hydrochloride salts 103,104 and 105 respectively.

Example 28: Alkenvlation of pdlymines using alkyl acrylates under Michael addition, condition Scheme. 28*

* (i) Methanol-water, 40 °C or Methanol, water, boric acid, room temperature Method 1; n-Dod ecyl aery] ate (106) is stirred with triethyleneteiranune 2 in methanol-water at 40 °C to obtain compounds 107,108 and 1.09, The products are isolated by chromatographic separation.

Method 2: n-Dodecylaerylate (106) is stirred with hi ethyl enetetr amine 2 in the presence of boric acid in methanol-water at 40 Xl to obtain compounds 107,108 and 109, The products are isolated by chromatographic separation.

Example 29: Aikenylation of diamines using -alkyl acrylates under Michael addition condition;

Scheme- 2Ψ

* (i) Methanol-water, 40 °C- or Methanol, water, boric acid, room temperature

Method 1: «-Dodeeylacrylate (106) is steed -with' triethyimctetrsmfeo 2 .in methanol-wxher at 40 °C to obtain compounds ΓΙ0, 111 and .1.12. The products are isolated bv chromatographic separation.

Method 2: ?i-Dodecylacrylate (106) is stirred with trieihyleneietrsmine 2 in the presence of boric acid in methanol-water at 40 °C to obtain compounds 110,· 111 and 112, The products are isolated by chromatographic separation.

Example 30: Synthesis of Oetadeca-9.12-dignoic acid 3- dimethyl amino-2-octodetia-9.12-41 enovioxv-pronvl ester 3

To a solution of the linoleic acid (25 g, 89,1 mmol) in anhydrous DMP (60 mL), diisopropyl ethylamme (I? mL, 100 mini) was added at room temperature with stirring followed by '3-(dimethylaatino)“3,2-pfopanedioI (4.8 g, 40.5 mmol) and BDCI (17.25 g, 89 j mmol) and the mixture was steed at room temperature overnight. The TLC of the reaction mixture (eluent 20% BtOAc in hexanes) showed-the completion of the reaction. The reaction mixture was poured into ice water and extracted with ethyl acetate (2 x 100 mL). The combined· organic layers were washed with·water (100 mL), saturated NaH'COa (100 mL) and dried over NajS04, Concentration of the organic layer provided the crude product which was purified by column chromatography (silica gel}: eluent: 20% EtOAe in hexanes). The fractions containing pure product was pooled and concentrated. The pure ester was isolated as a clear liquid (5.7 g, 22%), MS m/z 645 (M+H). *H NMRCDCisδ0.88 (gJ = 6.3Hz,6H)} 1.204,39 {m,28H}, 1.61 (t, /-=4.9 Hz, 12H), 2,03*2.08 (mf 8H), 2.26-2.38 (m> 10H)* 2,44*2.56 (m, 2H), 2.76 (i,J- 6.3 Hz, 4 H), 4.09 (dd,/- 6.:1 Hz &amp; 11.9 Hz, 111). 4.36 (dd, /- 3.3 Sc 11.9 Hz, IH), 5.29-5.34 (m, 1H), 5.34-5.41 (m, 8H). °C NMR GDCI3 S 14.30,22.79, -25.08,2-5.10,25.83, 27,40, 29,26, 29.30,29.34,29.42, 29.55, 29.83, 31.73, 34.32, 34.58,46.01,59.37,64.02, 128.08, 128.24,130.21, 530.42, 173.39,173.65.

Example 31: Exemplary procedure for making a liposome using extrusion

Prepare stock solutions of ND98 (120 rag/ml), cholesterol (25 mg/nil), and C16-PEG-Cer~2000 (100 rng/ral) in 100% ethanol. Store at. -20°C. Warm in 37°C water bath prior to preparing formulations (up to 30 minutes is helpful - it takes a while for the cholesterol to dissolve completely). 2X2mlPrep

To a 15ml Falcon tube, add: 1) 125«! of lipid 2) 200ul of cholesterol

3) 70ul of PEG 4}5ul of 100% ethanol 5}600ui of 25 mM sodium acetate pH 5 6}Mix gently (setting 5) on a vortex 7}Add 20 mg sucrose SWoriex again until sucrose has dissolved 9) Add I ml of a freshly-prepared (in a new Falcon tube) 1 mghn! solution of siRNA in 25 mM sodium acetate (-100 ul of 10 rnghnl siRNA + 900 ul of 25 mM sodium acetate) 10) 'Vortex lightly (setting 1, with Falcon tube holder adapter) for 20 minutes IQAfrer 15 minutes (5 .minutes remaining), clean extruder

12) Extrude ] 1 times through two 200 nm filters at 40 °C 13) Dialyxe against PBS, pH 7.4 for 90 minutes at RT in.3,500 M WCO Pierce cassettes

Example 32: Exemplary procedure for making a liposome without using extrusion

Prepare stock solutions ofND98 (120 rag'ml), cholesterol (25 mg/ml), and Cl 6-PE<j~Cer~2000 (100 mgftnl) in 100% ethanol. Store at -2DCC. Warm in 37CC water bath prior to preparing formulations (up to 30 minutes is helpful - it takes a while for the cholesterol to dissolve completely).

To a 15ml Falcon tube., add: 1 )125ul of lipid 2}20Ool of cholesterol

3) 70ul of PEG 4) 495ul of 100% ethanol 5) 100ul of water 6) Prepare 1 ml of 1 rng/ml siRNA in 100-300 mM sodium acetate, pH -5 7) Rap?d!y mix lipids in 90% ethanol with siRNA in acetate buffer 8) Dialyxe (or use ultrafiltration) against 100-300 roM sodium acetate, pH -5 to remove ethanol 9) DiaIyze (or use ultrafiltration) against PBS to change buffer conditions

Example 33: Exemplary protocol for quantification of RNA in a liposome

The procedure below can be used to quantify (1) foe proportion of entrapped siRNA and (2) the total amount of siRNA in a liposome.

Materials;

RiboGrcen (Molecular Probes) 2 % Triton X-100 TB butler

Protocol (M-well plate format): 1, Dilute samples to be tested in TE buffer such that siRNA concentration is - 2 ugftnL (0,4 - 4 og/rnL). Note dilution of samples, 2. Array 50 uL of each sample into 2 -wells (e.g. samples arrayed into 2 rows of mieroplate} 3. Add 50 uL of TE buffer to one of each, of the 2 samples (e.g, tap row samples), Hus sample will be used to determine “free” siRNA, 4, Add 50 uL of 2% Triton X-100 to the remaining of the 2 samples (e.g. bottom ro.w samples). This sample will be used to determine “total” siRNA. 5, Prepare standard siRNA dilutions 'by using known amounts of the siRN A to be quantified. Start with 50 uL of 4 ug/mL, and do 2-fold dilutions, Add 50 uL of 2% Triton X-100 to each of the standard sample dilutions. 6, Incubate for 15 min at room temperature, 7. Add 100 uL of diluted RiboGreen to all of the samples, Diluted RiboGreen to be used at 1:100 dilution, 8. Read plate in iluorimeter (Victor2) using FfTC settings.

Calculations:

Final volume in wells will be 200 uL.

RiboGreen will be at 1:200 final dilution,

Triton X-IQG will be at 0,5%.

Standards will be dilutions storting from 1 ug/mL.

Plot Standard Curve, perfonn linear fit.

Determine Entrapment % = Ιϋ0*(1*-“δοε”signal/ “total”signal)

DeterminefsIENA]: First convert “total” signal to concentration using the standard curve, then multiply by dilution factor.

Example 34: Comparison of Lipid moieties as formulated into Liposomes The effectiveness of lipid compositions can be tested by detemnning the relative ability of a lipid to deliver an. siRNA moiety to a target. For example, the silencing of a target indicates that the siRNA is delivered into the cell Applicants have compared liposome complexes that include each of the following lipid moieties together with siRNA that is used to silence Factor V0 (FV11).

Initially unpuriSed reaction mixtures were used. Different ND98 reaction mixtures were generated by synthesizing product at different ND;98 .monomer ratios; KD;9S~ 1:1, 2:1,-3:1,4:1.,5:1, -and 6:1, ND98 is generated by reacting '3S!D> the structure of which is provided below:

, with amine 98, the structure of which is provided below

in the ratios provided above (ixv, ND:9S 1:1,2:1» 3:1,4:1, 5:1, and 6:1}, Liposomes wereformulated at ND98:cholesterol:FED20()0-CerCl6;siRNA « 3 5:0,8:7:1 (wt ratios). Liposomes prepared with ND:98 = 1:1 and 2:1 precipitated dring formulation and were not characterized further.

Table 1, below provides the average particle size and percent entrapment of the liposomes using the various monomer ratios (he, the number indicating the ratio of ND relative to 98),

Table 1;

Figure 1 provides the results of the F VIl siliendng assay tor the various monomer ratios using an experimental dosing of 2 mgdeg siRNA, The results suggest that the ND98 5 tail moiety and/or ND 98 6 tail moiety are the active species as these are the most ahundants species on the 3ND98 6:1 preparation. As described a 5 tail moiety indicates a compound where 5 of the hydrogens on the starting amine 98 have been reacted with a starting acrylamide moiety ND. A 6 tail moiety indicates a compound where 6 of the hydrogens on the starting amine 9δ have been reacted with an acrylamide moiety ND. Accordingly, the numer of “tails” indicates the number of reacted hydrogens on the starting amine.

Example 35: Determination of preferred lipid isomer

Applicants purified ND98 lipid products. ND98 lipid moieties are the lipid moieties resulting in the reaction of ND, tire structure of which is provided below;

. with amine .98, die structure of which is provided below

Applicants tested 4-tail mixed isomers of ND98 (i.e., where four of the amine hydorgens have been reacted with the ND acrylamide above), single structural isomers of 54ail ND98 (i.e. , where· for of the amine hydrogens have been reacted with the ND acrylamide above), Examples of the two 5 tail isomers are provided below:

Liposomes of the-purified ND98 products were formulated with the following components in the following ratios: ND9S:ttholesterol;PBO20()C}"€erClb:siRNA 15:5:7:1..(wt ratios).

Table '2, below provides the average particle shse and percent entrapment of the liposomes using the various monomer ratios (i.e, the number Micating the ratio of ND relative to 98).

Table 2:

For lire purposes of table 2 and Figure 2: ND98 1 5-tailed (isomer I); ND98 2 “ S-tailed (isomer I r II); MD98 3 ~ 5-tailed (isomer If); and ND9S 4::: 4-tai'ied.

The liposomes where administered with siRNA hi a does of 2,5 mg/kg* and evaluated, for the silencing of FVIL Figure 2 provides the- results of the 4 tailed isomer mixture, the single 5 tailed isomers (i.e., isomer land II) and the mixture of 5.tailed isomers (i.e., isomer I and II).

Example 3d: Determination of preferred N098 isomer A purified isomer of 6 tailed ND98 was prepared and purified. ND98 structure corresponds with those described in examples 34 and 35 above. The 6 tai l indicates that ail of the hydrogens of amine 98 have been reacted with the KD starting material. With this lipid starting material, liposomes were formulated at the following ratios: HD9S:eholesterol:FBG2()00-Cer€!6:siRNA::: 15:5:7:1 (wtratios). Figures demonstrates the effectiveness of the ND9S 6 tail isomer in delivery of siRNA, which effectively silenced FVI1.

Example 37: liposome particle size usina various ND98 Hold starting materials A plurality'of lipid starting materials having the ND98 structures (as provided in examples 34 and 35 above) were formulated into liposomes. The particle size of the liposomes were evaluated, the results of which are provided in table 3 below:

Example 38: Extrusion tree liposome formulation Liposome complexes were prepared using ND98 lipids, the formulations include the Mowing ratios: ND98:cholesterol:FEG200a-C;erC]6:stRNA- 15:5:7:1 (wt ratios). The liposomes were prepared without extrusion, as generally described in Example 32 above. Two samples-were prepared, a first sample having.the following: 100 mM ~ siRNA prepared in 100 aM sodium acetate with a first dialysis step in 100 mM acetate; and a second sample having 300 iaM -- siRNA .prepared in 300 mM sodium acetate with a first dialysis step in 300 mM acetate.

Figure 4 shows the results of an. FVll silencing assay, demonstrating the comparative activity of the formulations made using the- various processes

Example 3gyj*egfoselecttve synthesis of cationic lipid 7-- strategy ]

Scheme 31a

8 Regiosdectivc synthesis of cationic lipid 7 ~ Approach 1

Step L Preparation of compound 9: Triethylenetetramine, I (48.83 gs 0334 mol, purchased from Sigma-Aldrich} in anhydrous acetonitrile (500 mL) was cooled over an ice bath under constant stirring. Ethyl triflnroaeetate (79,6 mL, 0.668 mo!) was added to the solution and after completion of the addition the reaction mixture was allowed to warm to room temperature and stirred for 2Gh. Solvent and volatiles were removed under reduced pressure and the residue was dissolved in minimum amount of warm dichlcroraetfaane ¢100 mL) -and to it cold hexanes was added with stirring. The precipitated product was 'cooled in ice and filtered to get a:· white-solid (112.2 g, .#9%),

Step 2. Synthesis of (2-{fer/-hutoxycarhon.y!»|2-{2r2,2“trifluorO'-aeetylantino}ethyi]-aminioi'-2-{2,2,2-tr*ilHor()-acet)'Iantmo)ethyi|-carl)amie add iert-bafyi ester 113

The trlilnroaeeiamide 9 (112.2 g, 0,332 mol) was dissolved in CHaCVTBF (600 mL/100 mL) and to it diisopropyleihylamme (12.9,25 g, .1 mol) was added and stirred over an ice bath, Di-mri-btityi diearbonate (14-5 g, 0.664 mol,- purchased fern Sigma Aldrich) in CH2CI2 (100 mL) was added drop wise to the reaction mixture and stirred overnight Solvents were removed, and the residue was stirred with a saturated solution of NaHCCH (400 mL) and filtered, and washed with hexanes (100 mL) mid dried in vacuo at 4$ °C overnight to obtain the pure diboc compound as a white solid (167 g, 94%). *H NMR for 113 (DMS046, 400MHz) δ = 9.60~9.40(m, 2H), 3.35-3.15(ra, 1-21¾ 1.30(^, 18H) MS: Cal. 43-8.17, Found 439,20(M*) MS:

Cal, 538.22, Found 539.2-Q(M*).

Step 3.- Synthesis of {2-antin tiutoxycaiisoRyl-aniinoj-ethyijcarhaniic acid ferr-butyl ester

The acetamide .1.13 (167 g, 0.31 mol) was taken in a stainless steel pressure reactor and to it a solution of meth.ylamine (33% by wt) in ethanol (200 ml) was added. The mixture was warmed to 90*C and stirred for 24 k Reaction was monitored by mass spectra. All the solvents were removed under reduced pressure and the residue was subjected to high.- vacuum at 80 °C to yield the product 114 (103 g, 96 %) as gummy liquid and this compound could be used for tire next reaction with out further purification, Ή NMR (CDCL, 400MHz) 6 - 3.20-3.00(m, 4H), 2.62-2.38 (m5 811), 1,32(¾ 9H), MS; CnH^Qs Cal. 246.2-1, Found 246.20(M*)*

Step 4. Synthesis of Michael addition product 115

The diamine 114 (103 g. 0.297 mmol), Ar-dodecylaeiylamide (356 g, 1-487 mol) and saturated solution of boric acid in water (30 mL) were taken together in a pressure reactor and heated at 90“C lor 4 days. The reaction was monitored by TLC and Mass spectra. The reaction mixture was extracted into dichtoromeihane (PCM), washed successively with NaHCOj solution and brine, dried over anhydrous sodium sulfate. Solvent was removed in vacuo arid: residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 340% MeOH/DCM) to obtain 11.5 as a pale yellow solid (228 g, 59%). MS: C^HisoN$Q*.€al.. 1303.16, Found 1304.2G(M'S).

Step 5..Preparation of diamine! 16 4M HC1 in dioxane (500 mL) was added to a solution, of the diboe compound Π5 (228 g, 0-175 mol) in methanol (100 mL) and the mixture was stirred at room temperature for 2 days. The reaction was monitored by Mass spectra. Alter the complete disappearance of the starting diboe compound, the precipitated hydrochloride salt was filtered, washed with THF (100 mL) and dried to get the pure salt as a white powder (178 g, 93%). The above salt was treated with saturated NallGOj (1.L) and extracted with dichloromethane (3 x 600 ml). The combined organic extracts were dried and concentrated to isolate the tetramer as a white solid (164 g, 85%). MS: C&amp;HmNgO* Cal. 1103.05, Found 1104.1.()^),

Step 6, Synthesis of 117; Compound 1.16 (164 g, 149 mmol) , N-dodecylacryiamide (35.6 g, 149 mmol) and saturated solution of boric acid in water (30 mL) were taken together in a pressure reactor and heated at 90*C for 3 days. Progress of the reaction, was monitored by TLC and Mass spectra. The reaction mixture extracted into dlchlorome&amp;ane (DCM), washed successively wifc NaHCOr solution and brine, dried over anhydrous sodium sulfate. Solvent was removed in vacuo and residue thus obtained, was purified by silica gel (2 Kg) column chromatography (gradient elution-0:5:95-10:10:80% TEA/MeOH/DCM) to obtain 117 as a pale yellow’ solid (83.8 g, 42%). MS: C%Hf5<£SgO$ Cai 1303.16, Found 1304.20(M+). The material was compared with authentic sample TLC (qualitative), HPLC and Mass spectra. MS: OsjHt&amp;NsOs Cai. 1342.28, Found !343,30(M+).

Step 7. Synthesis of the hydrochloride salt 7

Tim amine 117 (54 g, 40 mmol) was dissolved ethanol (100 mL) and to it 200 mL of 2M HCI in ether was added and the mixture was stirred at room temperature overnight. Nitrogen was bubbled to the reaction mixture and tire outlet was passed through dryrite end to a 10% solution ofKOH. After 30 minute, the reaction mixture was concentrated to dryness and; Hie residue was re-dissolved in $00 mL ot anhydrous ethanol and the mixture was concentrated in a rotary evaporator. This process was again repeated once again and the. thus obtained residue was dried in a vacuum oven at 43 °C overnigltt. the pm produetwas isolated as a cream powder (59.5 g, 99%)..

Example 40: R^oseteetive synthesis of cationic lipid 7 -- strategy,!

Method 1

Step!: Iriethylenetetrainine, 1 (200g g? L37 mol, purchased from Sigma-Aldrich) in acetonitrile (2 L) in a 4 neck 5L flask with overhead stirrer was cooled over an. ice bath under constant stirring. -Ethyl triiluroacetate (cSS.5 g, 2.74 mol) was added to die stirring solution and stirred for 20h. Solvent: and volatiles were removed under reduced pressure; the residue was triturated with a mixtuie of PCM* Hexane anc Altered to get 101 as white solid (429 g, 93%). The product thus obtained could be used tor the next reaction without farther purification. MS: GjoHiiF^Ch Cal. 338,12, Found 339.0(Mi.

Step2: Crude compound 101 (427g, 1.26 mol) was dissolved in a mixture of solvents (3 L, THF/DCM (1:2)) and stirred over an ice-water bath. Di-feri-butyi diearbonate ((ΒοφΟ, 270 g, 1.26 moL purchased from Sigma Aldrich) and DIEA (500 ml, 2,86 mol) were added to the reaction mixture and stirred overnight Solvents were removed and the residue was extracted into dichloromcthane (DCM, 1000 mL), washed successively with HaHCOo solution (500 mL), water (500 mL x2) and brine, dried over anhydrous sodium sulfate. Solvents were removed in vacua and residue thus obtained 'was triturated with DCM/Hexane (2:1) and filtered. Solvents were removed and the residue was dried under hi gh vacuum to get the compound 102 as gummy liquid (523g).

Part of the compound 102 was purified by silica gel chromatography (gradient elution, Ethyl acetate, followed by 3-10% MeQH/DCM) to obtain compound 102 as gummy liquid (102.00g,). 1H NMR for 102 (DMSO-d6: 400MHz) δ - 9.60-9.10(m, 3H), 3,35"3.25(m, 4H), 3,25-3.20(2, 2H), 3.20-3,10(m; 2H), 2.68-2.S8(m} 4H), !.35(s, 9H). MS; O^F^Q*.Cal. 438.17, Found 439.20(^1^),

Step 3: Purified compound 102 (102,0g, 233.40 mmol) was dissolved in Ethanol'Meihyl amine (400 ml, 33 wt% methytarnine solution in EtOH) at ambient temperature in a pressure reactor. The mixture was warmed to 90’C and stirred for two days. Reaction was monitored by mass spectra. All the solvents were removed .under reduced pressure and the residue was subjected to high vacuum at 80 °C to yield the product 103 (58.00 g, 99 %) as gammy liquid and. this compound could be used for the next reaction with out further purification. ’H NMR (CDCis, 40OMHx) δ ™ 3.20-3.00(111, 411), 2.62-2,38 (m, SB), U2(s, 9Ή). MS: CuH^Qj Cal, 246.21, Found 247,20(M’;).

Step 4: Triamine 103 (56.00 g, 227,64 mmol), AMIodecylaerylamide (327,00 g, 1365 mmol) and saturated solution of boric acid in water (50 mL) were taken together in a pressure reactor and heated at 9()'C for 6 days. The reaction was monitored, by TLC and Mass spectra. The reaction mixture extracted into dichloromethaoe (DCM), washed successively with NaHCOj solution (400 mL) and dried over anhydrous sodium sulfate, Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 340% MeOB/DCM) to obtain 104 as a pale yellow solid (186 g, 57%), *H NM'R (CDGI3, 400MBz) $ = 7.20(l%\. IB), 7,05(bs, IB), 6.S5(bs, IH), 6.74(bs, 1H), 3,25-3.03¾ 12H), 2.80-2,60 (m, 8H), 2.55-2,21(m, I2B) 1.524.45¾ 10H), 1,4% 9H), 1.344.20¾ 100H), 0.87(t,> 6,5¾ 1SH). MS: CuHmKfh Cal. 1442.33, Found 1443.30(1^).

Step 5: 4M HC1 in dioxane (400 mL) was added into a solution of compound 105 (184,00 g, 127,23 mmol) in dioxane (300 mL). The reaction mixture was then allowed to stir for overnight The reaction was monitored by Mass spectra. Excess HCI was removed by passing nitrogen through the solution. Solvents were removed under vacuum and residue was co evaporated three times with ethanol (500 ml, X 3} to yield a pale yellow gummy solid 7 (lS6,Q0g , 98%) as tetra hydrochloride salt Hie material was compared with authentic sample TLC (qualitative), HPL-C and Mass spectra, MS: 0*5Β3«1%05 Cal. 1342.28, Found 1343.30(½1).

Method 2

Compound 102.was prepared as described in Method 1: steps 1 tied 2. The crude product obtained from step 2 of Method i was used for the next reaction without further purification.

Step 3: Compound 102 (103.45g, 238.90 mmol, crude compound from step 2. Method 1 was dissolved in Ethanol/Mefhyl amine (400 mL 33 wt% methylaminc solution in BtOH) at ambient temperature in a. pressure reactor. The mixture was warmed to 90’C and stirred for two days. Reaction was monitored by mass spectra. Ail the solvents were removed under reduced pressure and tire residue was subjected to high vacuum at 80 °C, over a water bath to yield the product 103' (63,50 g) as pale yellow gummy liquid and this compound could be used for the next reaction with out further purification.

Step 4: Triamine 103 (63.50 g, 238 mmol), iV-dodecyiacrylamide (320.00 g, 1338 mmol) and saturated solution of boric acid in water (50 mL) were taken together in a pressure reactor and heated at 9G“€ tor 6 days as described in step 4, Method 1. The reaction was monitored by TLC and Mass spectra, The reaction mixture extracted into dicMoromethane (DCM), washed successively with NaHCOj solution (400 mL) and dried ova anhydrous sodium sulfate. Solvent was.-removed in vacuo and residue thus obtained «'as purified by silica gel column chromatography (gradient elution- Ethyl acetate then 5-10% MeOH®€M) to obtain t()4 as a pale yellow solid'.(65.2 g, 20%),

Step S; 2M HC1 in ether (800 mL) was added to compound 105 (65.00 g, 4$ mmol). The reaction mixture was then allowed to stir for overnight. The reaction was monitored by Mass spectra, Excess HC1 was removed by passing nitrogen through the solution. Solvents were removed under vacuum and residue was co evaporated three times with ethanol (500 mL X 3) to yield a pale yellow gummy solid 7 (66g , 98%) as tetra hydrochloride salt. Hie material was compared with authentic sample TLC (qualitative), HPLC and Mass' spectra. MS: CgiH^NsOs C8L 1342.28, Found 1343.30fM+).

Method 3

Compound 102 was prepared as described in Method 1: steps I and 2. The crude produc t obtained from step 2 of Method 1 was used for the nex t reaction without further purification,

Step3: Compound 1(12 (105.20g, 240 mmol, crude compound from method I) was dissolved in Ethanol/Methyl amine (400 ml, 33 wt% methylamiue solution in EtOH) at ambient temperature in a pressure reactor. The mixture was wanned to 90?C and stirred fer two days. Reaction was monitored by mass spectra. All the solvents were removed under reduced pressure and the residue was subjected to high vacuum at 80 *C over a water bath to yield the product 103 (64.70 g) as pale yellow gummy liquid and this compound could be used for the next .reaction with out further purification,

Step 4: Triaxmne 103 (64.70 g, 240 mmol), Addodecylacrylamide (370.00 g, 1569 mmol) and saturated solution of boric acid in water (50 mL) were taken together in a pressure reactor and heated at 90T for <5 days. The reaction was monitored by TLC and Mass spectra. The reaction mixture extracted into dichlorometlrane (DCM), washed successively with NaHC03 solution (400 mL) and dried over anhydrous sodium sulfate. Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 3-10% MeOH/DCM) to obtain 104 as a pale yellow solid (192 g).

Step 5; The desired compound 7 was obtained as hydrochloride salt from compound 104 as described in step 5, Method 1 of' Example 40, Compound 7: 194g (98%) as tetra hydrochloride salt The material was compared with, authentic sample TLC (qualitative), HP.LC and Mass spectra. MS: CatHi&amp;N^Os Cal, 1342.28, Found 1343.30{M').

Example 41:___Comparison of activity of siRNA .formulated into various association complexes having differing FBG-lipid moieties:

The effectiveness of lipid compositions can be tested by determining the relati ve ability-of a lipid to deliver an siRNA moiety to a target. For example, the silencing of a target indicates that the siRNA is delivered into the cell Applicants have compared association complexes that Include one of 1.3 different PBG-iipitf moieties as provided in Figure 5, together with siRNA that is used to silence Factor VII (FVH), PEG-lipids f -13 were synthesized rising the following proceeurcs:

Scheme la

* Scheme .1: mFEG2000-l,2-Di-0'alky!^n3-carbomoylglyceride Preparatiott of compound 5: l,2-Di~Odetradecyl-jn-glyccride 1 (30 g, 61.80 mmol) and Ay^-suceinimidylcarboante (DSC, 23.76 g, l,5eq) were taken in diehioromethane (DCM, 500 ml) and. stirred over an ice water: mixture. Trieihylaniine (25.30 ml, 3ecj) was added to stirring solution and subsequently the reaction mixture was allowed to stir overnight at ambient temperature. Progress of the reaction was monitored· by TLC. The reaction mixture was diluted with DC-M (400 ml) .and the organic layer was washed with water (2X500 mL), aqueous NaHCOs solution (500 mL) followed 'by standard work-up. Residue obtained was dried at ambient temperature under high vacuum overnight After drying the crude carbonate 3 thus obtained was dissolved in dichlororoethanc (500 mL) and stirred over an ice bath. To the stirring solution mPEGaooirNHa (4, 103.00 g, 47.20 mmol, purchased from NGF Coloration, Japan) and anhydrous pyridine (89 mL, excess) were added under argon. The reaction mixture was then· allowed stir at ambient temperature overnight Solvents and volatiles were removed under vacuum and the residue was dissolved in. DCM (200 mL) and charged on a column of silica gel packed in ethyl acetate. The column was initially eluted with ethyl acetate and subsequently with gradient of 5-10 % methanol in diehloromeihane to afford the desired PBG-Lipid S as a white solid (105.30gs 83%). SH NMR (CDC13> 400 MHz) δ « 5.20-5.12(¾¾ 133),. 4.18~4.01{m, 2H), 3.8O-3,70(m, 2¾ 3.70-3.2000, -Ο-CHrCMrO-, PEG-CH,}, 2.1O-2.01(m, 2H), 1.70-1.60 (m, 2B), L56-L45{m, 4H), 1.3M.15(m,48H), 0.84(t, J- 6.5Hz, 6H). MS range found: 2660-2836,

Preparation of 4b: 1,2- Di -O-hexadecy 1-svt-glycerid e ife (1.00 g, 1.848 mmol) arid DSC (0.71-0 g, l.Seq) were taken together in diehloromeihane (20 mL) and-cooled down to 0eC in an ice· water mixture. Triethylanune (1.00 mL, 3eq) was added to that and stirred overnight The reaction was followed by TLC, diluted with DCM, washed with water (2 times), NaHCQ* solution and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue 2b under high vacuum overnight. Tins compound was directly used for the next reaction without further purification. MPEGadoo-NHs 3 (1.50¾ 0.687 mmol, purchased from N0F Corporation, Japan) and compound from previous step 2b (0.7.02¾ I ,Seq) were dissolved In dich.loromethane (20. mL) under -argon. The reaction was cooled to 0°C. Pyridine (1 tnL>- excess) was added to that and stirred overnight. The reaction was monitored by TLC; Solvents and· volatiles were removed' under vacuum and the residue was purified by chromatography (first Ethyl acetate then 5-10% MeQH/DCM as a gradient elution) to get the required compound 4b as white solid (l.46 g, 76 %). 5H NMR (CDCIj, 400 MHz) δ 5.17(1, j-5.5Hz, HI), 4.13(dd, 1= 4.00Hz, 11.00 Hz, 1H), 4.0$(dd, 5.00¾ 11.00 ΪΗ), 3,82-3.75(m, 2M), 3,7O-3.20(m, -0-CH2-CHrO-, PEG-CHZ), 2.05-L90(ms ,2H), 1.80- 1.70 (ro,,2H), l,6i-i,45{m, 6H), l,3'5-U7(ra, 56H), 0.85(6 J- 6.5Kz, 6H). MS range found: 2716-2892.

Preparation of4e.* i,2-Di-0-ootadeeyl-.m~glyceride le (4,00 g, 6,70 mmd) and DSC (2.58 g, l.Seq) were taken together in dichtoromeihane (60 ml.) and cooled down to 0°C in an ice water mixture. Triethyiamine (2,75 mL, 3eq) was added to that and stirred overnight, Hie reaction was followed by TLC, diluted with DGM, washed with water (2 times}, NaHCO* solution and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue under high vacuum overnight. This compound was directly used for the next reaction with further purification, MPEG^iOS" NHj 3 (1.50g, 0.68? mmol, purchased from NOP Corporation, Japan) and. compound from previous step 2c (0.760¾ 1.5eq) were 'dissolved in diehlorometliane (20 mL) under argon. The reaction was cooled to 0°C. Pyridine (1 mL, excess) was added to that and stirred overnight The reaction was monitored by TLC. Solvents and volatiles were removed under vacuum and the residue was purified by chromatography (first Ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to get the required compound 4 e as white solid (0,92 g, 48 %). !H NMR (CDClj, 400 MHz) § = 5.22-5,15(m, 1H), 4.16(64, J« 4,0011¾ II.OO Hz, JH), 4.06(dd, J~ 5,00Hz, 11.00 Hz, IB), 3.8!-3.75(m, 2H)> 3.7Q~3.20(m, -0-CH2«CH2-O, PE0-CH2)s 1.80-1.70 <m, 2H), 1.6iM.48fm, 4H)> I,31-LI5(m, 64E), 0.85(t, J~ 6.5142:, 61-1), MS range found: 2774-2948.

Scheme 2 s

Scheme 2: rnPEG2000~ 1, 2-Di -C7~alky 1.-,577.3-succiny 1gly ccride Preparation of compound 6a: l,2-Di~04etradeeyi-.Yn-g!yeeride la (LOG g, 2,06 mmol), succinic-anhydride (0.416 g, 2 eq) and DMAP {0.628 g, 2.5eq> we taken together in dichlommethmie (20 ml,) and stirred overnight The reaction was followed by TLC, diluted with OCM, washed with'cold dilute citric acid, water and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue under high vacuum overnight This compound was directly used for the next reaction with further purification, ΜΡΒΟ^-ΝΗ'ζ 3 (i.50g, 0,687 mmol, "purchased from NOP Corporation, Japan), compound from previous step 5a (0.66g, 1.12 eq) and BBTU (0.430g, 1,13 mmol) were dissolved in a mixture of diehioromethane/DMF (2:1,20 mL) under argon. DIEA (0.358 mL. 3 eq.) was added to that and stirred overnight lire reaction mixture was transferred to a. targe flask and removed the solvents and volatiles under reduced pressure. The residue was dried under high vacuum overnight and purified by chromatography (first ethyl acetate then 5*10% MeOB/DCM as a gradient elution) to get the required compound 6a as white solid (0.822g, 43 %). *H NMR (CDCls, 400 MHz) δ « 6.34~6.30(m, 1H), 4,t6{dd, J«A00Hz, Π.00 Hz, 1H), 4,G8(dd, 5.00Hz, 11.00 'Hz, 1H), 3.82-3.78(m, 2H), 3.70-3.30(m, -0~CHrCerQ-, PEG* C%), 2.64 (t, J* 7.00Hz, 2H)5 2.43((, 6.80Hz, 2Η),1.76-1.72(ιη, 2H), i.56~1.48(m,· 4H), 1.34-1.16(^,-481¾ 0,85((, 3= 6,5Hz, 6H). MS range found"2.644-2804.

Preparation of compound 6b: 1,2~Di-0~hexadecykv«~gIyceride lb (LOG g, 1,848 mmol), succinic· anhydride (0.0,369 g, 2 eq) and DMA? (0.563 g, 2.5eq) were taken together in dicMoromethane (20 mL) and stirred overnight. 'the reaction was followed by TLC, diluted with DCM, washed with cold dilute citric acid, water and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue under high vacuum overnight This compound was directly used for the next reaction with further purification. MPEG^»-NH;> 3 (l„50g, 0,687 mmol, purchased from NOF Corporation, Japan), compound from previous step 5b (Q.66g, ! .03 mmol) and HBTU (0.400g, 1,05 mmol) were dissolved in a mixture ofdichloromethane/DMF (2:1, 20 hit.) under argon, DIEA (0.358 mL, 3 eq.) was added to that and stirred overnight The reaction mixture was transferred to a large flask and removed tire solvents and volatiles under reduced pressure. 'Hie residue was dried under high vacuum overnight and purified by diromatography (first ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to get the required compound 6b as white solid (0.300g, 16 '%}. TT NMR (CDCfe, 400 MHz) 8 - 6.33-6.28(ra, IH), 4J8{dd, J- 4.00Hz, 11.00 Hz, IH), 4,Q8fdd, J:-: 5.00Hz, Π.00 Hz, IH), 3.82-3,76(m, 2H), 3.70-3.30(m, ~0-CHrCHrCk PEG-eH2)s 2,65 (t, J» 7.08Hz, 2H), 2.44(t, k 6.83Hz, 2H), 1,764.68 (m, 2B), 1.57-1.48(m,: 4B), 1.324,I7(m, 56H), 0.86(1, k 6.6Hz, 6H), MS range found; 2640-2822.

Preparation of compound 6c: l,2-Di-(?-oct.adecy!-jH-glycerMe 1c (5.00 g, 8,37 mmol), succinic anhydride (! .70 g, 2 eq). and DMAP (2.55 g, 2.5eq) were taken together in diohloromcthane (50 ml) and stirred overnight The reaction was followed by TLC, diluted with DOM, washed with cold dilute citric acid, water and dried over sodium sulfate. Solvents were- removed under reduced pressure and the residue under high vacuum ovemight, This compound was directly used for the next reaction with bather purification. MPEOsoeo-MHs 3 (1.50¾ 0.687 mmol, purchased from NOF Corporation, Japan), compound from previous step 5c (0.718g, 1.03 mmol) and. HBTU (O.410g, 1.08 mmol) were dissolved in a mixture of diohloromethane/DMP (2:1,20 ml.,) under argon. DIEA (0,350 mL, 3 eq.) was added to-that"and stirred ovemight, The reaction mixture was transferred to a large flask and removed the solvents and volatiles under reduced pressure. The residue was dried under high vacuum ovemight and purified by chromatography (first ethyl acetate then 5-1.0% M&amp;OWDCM. as a gradient elution) to get the required compound 6c as white solid (1,1 g, 56 %). -H NMR {CDC%, 400 MHz) 8 - 6J8-6.33(m5 IH), 4.19<dd, J= 4.00¾ 11.00 Ik, i H), 4.07(66, 5,001¾ 11.00

Ik, 1H), 3,8.1-3.74¾ 211), 3.70-3.20¾ -O-CHa-CHrO-, PBG~€H?), 2.63 (t, J® 7.03Hz,2H), 2.43(1,1-6.87¾ 2H), 1.76-1.68 (m, 2H), 1.57-1.48¾ 4H), 1,32« U?{m, 64H), 0.86(1, M 6.60Hz, 6H), MS range tad: 2680-2922

Schemed®

8 Scheme 3: tnPBG2000-l,2-Di“C>-alkyl~,y«3-suceinylgIyeeride

Preparation of compound 8a: l?2-0i-0-tetradeeyIwii-glycedde: la (0,300 g, 0.618 mmol), MPEG-Suceinate 7 (l.OOg, 0.476 mmol, purchased from NOF Corporation, Japan), DCC (0.127 g, 1.3eq) and DMA? (0.058 g, 0.476 mmol) were taken in dichkimmefhane (20 ml) under argon and stirred overnight. Reaction was monitored by TEG, The reaction mixture was cooled to 0°C after stirring overnight and filtered off the precipitated solid. Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first eluted with. EtOAc, followed by 5-10 % DCM/MeOH gradient elution) to get the· compound 8a as a white solid (0.590 g, 48%). *H NMR (CDCfe, 400 MHz) 8 - 4.25-4.18¾ 2H), 4.08(dd, 5.60Hz, 11.50 Hz, Hi), 3.80-3.73¾ 2B), 3.70-3.30¾ -0-CH2-CHrO, PEG-CH2), 1.56-1.47¾ 4H), 1.30-1.15¾ 48H), 0.85(t, )- 6.601¾ 60). MS range found: 2440-2708

Preparation of compound 8b: l,2-Di-0-hexadecyl-.vn-glyeedde lb 0,33.4 g, 0.61B mmol), MPEG-Succinaie 7(1. OOg, 0,476 mmol,, purchased from NOF

Corporation, Japan), DCC (0.127 g, l.3eq) and 'DMAP (0.058 g, 0.476 mmol) were taken in. dichbromethane (20 mL) under argon and stirred overnight. Reaction was monitored by TLC. The reaction mixture was cooled to CPC after stirring o vernight and filtered off the precipitated solid. Volatiles and solvents were removed under reduced pressure and die resulting residue was purified by chromatography (first eluted with Et'OAe, followed by 5-10 % DCM/MeOH gradient elution) to get the compound 8b as a white solid (0,930 g, 74%). *H NMR (CDC13,400 MHz) S = 4,25-4.17(m, 2H), 4.09(di j» 5.50Hz, 11.50 Hz, 1H), 3.81-3.73(m, 2H), 3,70-3,30(m, -0-CHrCHrCK PEG-CHg), 1.58-1.47(¾ 4H), 1,30-1.17(m, 56B), 0,86(1 J::: 6,60Hz, 6H). MS range found: 2452-2760;

Preparation of «impound 8c: RS-Di-O-oeiadecyl-^-giyceride le (0.369 g, 0.618 mmol), MPEG-Suecinate 7 (l.OOg, 0.476 mmol, purchased from NOP Corporation, Japan), DCC (0,127 g, lJetj) and DMAP (0.058 gf 0,476 mmol) were taken in dtehloromethane (20 mL) under argon and stirred Overnight Reaction, was monitored by TLC, The .reaction mixture was cooled to 0 °C after stirring overnight and Steed off tire precipitated solid. Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by ehroiuatographv (first eluted with EtOAc»· followed by 5-10 % DCWMeOH gradient elution) to get the compound Sc as a white solid (0.960 g, 75%). 'H NMR (CDCR, 400 MHz) δ - 4.27-4.20(m, 211), 4.i0(dd, 1- 5,80Hz, i.1.50 Hz, Iff), 3.83-3.?4(m, 2H)} 3.70-33S(m, -0-€Η2-€%~0-, PEG-CHf), 1.54-1.46(in, 4H), 1,30-1.17(¾ 64H), 0.86(1, Ϊ* 6.60Hz, 6K). MS range found: 2508-2816.

Scheme 4“

8 Scheme 4; mPEG2000-1524^i"0~acyi-i7?3-suceinyiglycerjde

Preparation of compound 10a: 1,2-DimyTistoyl-w-glycerol 9a (0.317 g, 0.618 mmol), MPEG-Suceinate 7 (l.OOg, 0.476 mmol, purchased from NOF (.Corporation, Japan), DCC (0,127 g, l,3eq) and DMAP (0.058 g, 0,476 mmol) were taken in dichloromelhane (20 mL) under argon and stirred ovemi^it Reaction was monitored by-TEC. 'Hie reaction mixture was cooled to 0°C after stirring overnight and filtered off the precipitated solid, Volatiles and solvents were removed under reduced pressure and die resulting residue was purified by chromatography (first eluted with EtOAo, followed by 5-10 % DCM/MeQH gradient el ution) to get the compound 10a as a white solid (0,960 g> 78%). sii NMR (CDCh, 400 MHz) 8 - 5,26-5.20(m, 1H), 4.30-4.0S(m, 6H), 3.81-3.73(m, 2K), 3.70-3,40(m, -G-CI lrCHrO-, PEG-CHz), 2,65-2.60(ms 4H), 2.35-2.28(m, 4H), 1,63-1,52(m, 4H), 1.30~1.15(m, 44H), 0.86(1, J™ 6,60ϊίζ» 6H), MS range found: 2468-2732,

Preparation of compound lOht 1,2-Dipa1mitcyl~i'»"glyeerol 9b (0.352 g, 0.61 E mmol), MPEG^Siicetuate 7 (I.OOg, 0.476 mmol, purchased tram NOF Corporation, Japan), DCC (0.127 g, 1.3eq) and DMAP (0,058 g, 0.476 mmol) were taken in dichloromethane (20 mL) under argon and stirred overnight. Reaction was monitored by TLC. The reaction mixture was cooled to 0°C after stirring overnight and filtered off the precipitated solid. Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first eluted with EtQAc, followed, by 5-10 % DCM/MeOH gradient elution) to get the compound 10b as a white solid (1,02 g, 81%), *H NMR (CDClj, 400 MHz) 6 = 5.26-5.19(m, 1H), 4.3<Μ.05(η% 61% 3.80-3,40(m, -O-CH2-CH3-O-, PEG-CH-i), 2.65-2.60(τη, 4H), 2.33-2.24(10,4H), 1..63-L50(m, 4Hi. 1.30-].15{m, 52HI 0.85ft, }'* 6.60H&amp; 6H). MS range found: 2524-2792.

Preparation of compound 1.0c: 1,2-Dlstearoyl^n-glycerol 9c (0337-¾ 0.618 foiriol), MPBG-Suceinate 7 (t.OOg, 0.476 mmol, purchased from NOE Corporation, Japan), DCC (0.127 g, 1,3eq) and DMAP (0.058 g, 0.476 mmol) were taken.in diehloromethane (20 mL) under argon and stirred overnight. Reaction was monitored by TLC. The reaction mixture-was cooled to 0 °C after stirring overnight and filtered off the precipitated solid. Volatiles and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first elated.with EtOAe, followed by 5-10 % DCM/'MeOH gradient elution) to get the compound 10c as a white solid (1.04 g, 80%), *H NMR (CDC13,400 MHz) δ «* 5:26-5.1.9(m, 1H), 4.30-4.Q5(m, 6H), 3.80~3.4O(m, -0-CH2-CHrO-5 PEG-CHa), 2.66~2,59(ra, 4H), 2.31-2.26(m, 4B), 1,63-1.52(m, 41:1), 1,304.15(¾¾ 52H), 0.85(1, J« 6.601¾ 6H). MS range found; 2540-2844.

Scheme 5ί!

i! Scheme 5: €hoiesteryl-mPEG20OO

Preparation of compound 13: tnPEGegoo-OFi 11 (6,00g, 3 mmol, purchased from Sigma-Aldrich), Cholesterol heniisuccinate 12 (1.50 g, 3.08 mmol mmol) and. HBTIJ (L23g, 3.23 mmol) were dissolved in a mixture of dichlommethane/DMF (2:1, 100 raL) under argon. DIE A (1.60 mL, 3 eq.) was added to dial and stirred overnight Solvents and volatiles were removed under reduced pressure. The residue was dried under high vacuum overnight and purified by chromatography (first ethyl acetate then 5-10% MeOH/DCM as a gradient elution) to get the inquired compound 13 as white solid (5.0$g, 68 %). lK WAR (CDCfi, 400 ΜΗζ)δ - 5.35-5.25(m, 1H), 4.60-4,50((11, 1H), 4.22-4.18(1», 211), 3.80-3.?6(m, 2H), 3.72-3,46(m, -G-CHa-CHa-G-, PBG-CHj), 2.64-2.56(tn, 411), 2.31-2,20(01, 311), 2,01-0.8(1¾ 44H).MS range found: 2306-2654.

Example 42: Targeted PEG-iipids

Preparation of 19;

Step X: Compound 14 (2.00 g, 1.01 mmol) and cholesterol chloroionrmte 15 (0.453 g, 1.01 mmol) were taken, together in dicidorornethane (20 mL). The mixture was cooled in an ice-water bath. Triethylamine (0.448 mi) was added and the reaction mixture was stirred overnight Reaction was monitored by TLC. Solvent was removed and the residue was purified by silica get chromatography (Ethyl acetate followed by 5~ 10% McOH/DCM) to get the desired compound 16 (1.10g, 45.40 %), lH NMR (CDClj, 400 MHz) S - 5.35(m, 1H)S 5.15(m, XH), 3.40-3.SS(m, O-CH.-CHi-O), 3.10<b25(m, 10H), 0.S0-2,38(m, 44H, Cholesterol). MS range found: 2220-2490.

Step 2: Compound 16 (l.OOg, 0.417 mmol), 17 (0.235¾ 0.542 mmol) and HBTU (0.1.90¾ 0.5 mmol) were taken in a mixfitre of DCM'DMF (20 mL, 2:1), To that. D1EA was added and stirred overnight Reaction was monitored by TLC, solvents were removed under reduced pressure and the residue was purified by chromatography (5-10% MeOH/»CM) to get the desired compound 18 (1.02¾ 87 %). lU NMR. (BMSG-d6s 400 MHz) 6 - 7.52(4, J- 8.06 Hz, 1H), 7.33(1, J* 7.02 Hz, XH), 7.25(1, J= 7,32 Hz, 1H), 5.27(m, XH), 5.1S(d, )-3.2¾ 1H), 4.92(dd, j- 3.17,11.23 Hz, IK), 4.43(m, 1H), 3.60-4.020x1,5«), 3,20-3.55(ffi, Q-Cik-CHrO), 2.90-3,10(m, 10H). 2,05(s, 3H), 1.96(s, 3H), 1.84(¾ 3B), 1,77(¾ 3H), 0.80-2,38(m. 44H, Cholesterol). MS range found: 2680-2990,

Step 3: Compound IS (L02g, 0.362 mmol) was dissolved In a mixture· of MeOH/DCM (10 mL) to that 0.5 M solution of NaOMe in methanol (excess) was added and stirred overnight. Progress of the reaction was monitored by TLC. The mixture was neutralized' with AcOH, Solvents were removed under vacuum and the residue was paritled by chromatography (5-10 % MeOH /DCM.) to get compound 19 (280 mg; 30%). SH mill (CDClj, 400MHz) S - 5.38(m, lift 4,02-4.06(m; 7H), 3.30-3.80^, O-CHrCHrO), 3.20-3.29(1% SB), 2.08(¾ 3H), 0.8O-2.38(m, 44H, Cholesterol). MS range found: 2600*2900.

Preparation of 23;

Step 1: Compound 14 (2,00 g, 1.01 mmol) and compound 20 (0.453 g> LOlmmoi) were taken together in dichlorotoethane (20 mL). Ihe mixture was cooled in an ice-water bath. Pyridine (1 mL, excess) was added and the reaction mixture %a&amp; stirred overnight Reaction was monitored by TLC Solvent was removed and the residue was pari lied by silica gel chromatography (Ethyl acetate followed by 5-10% MeOH/DCM) to get the desired compound 21 (400 mg, 15 %). !H 14MR (CDC13, 400 MBs) δ *» 5.20(m, 1H), 4.05-4.20(¾ 2H), 3.20-3.80(¾ 0-CBrCHrO}, L?0-L82(ms 4H), 1.50-1.61 (m, 2H), 3.18-1.3¾¾ 60H), 0,87(1, 6,30 Hz, SB). MS range found: 2400-2750.

Step 2: Compound 21 (0.415 g, 0.159 mmol), 17 (0.100¾ 1.3 eq) and liBTU (O.OOg, 1.15 eg) were taken in a mixture of DCM/DMF (20 mL, 2:1). To that DiEA ¢0.2 ml.) was added and stirred overnight Reaction was monitored by TLC, solvents were removed under reduced pressure and the residue was purified by chromatography (3-10% MeOH/DCM) to get the desired compound 22 (0.45% 94%). £H NMR {CDOh 400 MHz) δ - 6.21(4 J- SJO Hz, IH), 5.33(4 J= 2.70 Hz, 1H), 5,l>5.20(m, 2H), 4.55(4 1« 8.15 Hz, IH), 4.01-4.20(m, 4H), 3.20-3.90(m, Ο-0Η2-ά:Ι2~Ο)5 2.14(8, 3H), 2.03(8,3H), l.99(s, 3H), 1.93(¾ 3H), 1.70-1.82(¾ 4H)S 1.50-1.61(¾ 4H)} Ll7-1.38(m, 60H), 0.86(1, J~ 6.32 Hz, 6H). MS range found: 2800-3200.

Step 3; Compound 22 (0.450 g, 0.359 mmol) was dissolved in a mixture of MeOH/DCM (5 mL) to that 0,5 M solution of NaOMe in methanol (excess) was added and stirred overnight Progress of the reaction was monitored by TLC. The mixture was neutralized with AeOH. Solvents were removed under vacuum and the residue was purified by chromatography (5-10 % MeOH/DCM) to get compound 23 (365 mg, 85 %). !H NMR (CDClj, 400 MHz) δ =- 5.18(¾ IH), 4.05.-4,20(¾ 411), 3.20-3.90(¾ G-CHs-Clfe-O), 2.05(3, 3iI), 1.71-1.-80(¾ 4H), 1.50-1.61(¾ 4H), 1.17-1,3&amp;(m, 60H), 0.87(4 3* 6.32 Hz, 6H). MS range found: 2760-3000.

As provided in Figure 6, the formulations, when administered to a subject, provided a varying degree of silencing of EVIL For example, formulation 3 provided a relative high degree of silencing of FV1, as did formulation 5,6, and 12,

Example 44: Tolerability of formulation LNP01 as dosed in mice

Empty liposomes witlicomposition ND98:eholesteroI:PEG~Cl442:48:30 (molar ratio) were prepared as described in Example 45. Different amounts of siRNA were then, added to the predbnned, extruded empty liposome to yield formulations with initial total excipient:siRNA ratios of 30:1,20:1,15:1,10:1, and 5:1 (wtrwt).

Preparation of a formulation at a total exdpimt:siRNAratio of 5:1 results in an excess of sIRNA in the formolalton, saturating the lipid loading capacity. Excess «RNA was then removed by tangential--flow filtration using a 100,000 MWCO membrane against 5 volumes of PBS. The resulting formulations were then administered to C57BL/6 mice via tail vein injection at 10 mg/kg siRNA dose. Tolerability of the formulations was assessed by measuring the body weight gain of the animals 24 h and 4S h post administration of the formulation, the results of which are provided in Figure 7.

Example 45: Formation of association complexes by first ibnmng unloaded complexes and then treating the, unloaded complexes with siRNA and administration of association complexes including two therapeutic agents

Association complexes having two different nucleic acid moieties were prepared as follows. Stock solutions of N.D93, cholesterol, and FEG~C14 m ethanol were prepared at the following eonerntratfons: 133 mg/mL, 25 mg/mL, and 100 mg/mL lor ND98, cholesterol, and PBG-C14, respectively, The lipid stocks were then mixed to yield 3S!B9S:eho1esteroI:PEG-Cl4 molar ratios of42:48:10. This .mixture was then added to aqueous buffer resulting in the spontaneous formulation of lipid nanoparticles in 35% ethanol, .100 mM sodium acetate, pH 5, The unloaded lipid nanopartiejes were then passed twice through a 0,08 pm membrane (Whatman, Nueleopore) using an extruder (Lipex, Northern Lipids) to yield ummodal vesicles 20-100 nm in size. The appropriate amount of siRNA in 35% ethanol was then added to the pre-sized, unloaded vesicles at a total excipientsiRNA ratio of 7.5:1 (wr.wt). The resulting mixture was then incubated at 37 °C for 30 min to allow for loading of siRNA into Hie lipid, nanoparticles. After incubation, ethanol removal and buffer exchange was performed by either dialysis or tangential flow filtration against PBS. The final formulation was then sterile filtered through a 0.2 pm filter. A flow chart demonstrating the order of addition of exhipients and therapeutie agents is provided in Figure 8, A 1:1 mixture of siRNAS targeting ApoB and Factor VII were formulated as described in Example 44. Separately, the same ApoB- and Factor ΥΠ-targeting siRNAs were-individually formulated as described in Example 31, The three formulations were. the» administered at varying doses in an injection volume of 10 pL/g animal body weight Forty-eight hours after administration, serum samples were collected by reiroorbiiaJ bleed, animals were sacrificed, mid livers were harvested. Seram Factor Vi 1 concentrations-were determined using a chromogenie diagnostic kit (Coaset Factor YII Assay Kit, DiaPharma) according to manufacturer protocols. Li ver mRNA levels of ApoB and Factor VII were determined using a branehed-DNA (bDNA) assay (Quantigene, Panoplies), the results of which are provided in figure 9, No evidence of inhibition between the two therapeutic agents was observed. Rather, both of the therapeutic agents demonstrated, effectiveness when administered.

Example 46: Methods of making association complexes using preformed vesicles

Lipid Stock Preparation

Stock solutions of lipidoid ND984HC1 (MW 1487), cholesterol, and PHG-C14 were prepared in ethanol at the following concentrations; 133 mg&amp;nL, 25 mgtoiL, and 100 rag'mL for ND98, cholesterol, and PEG-C14, respectively. Stock solutions were warmed at,5042 to assist in bring lipids into solution.

Empty Vesicle Preparation

The lipid stocks were then mixed according to the volumes listed below to yield ND98;cholesferoI:PEG-C14 molar ratios of42:48:10. An aqueous mixture was also prepared according to the volumes listed in the table below.

The eihanotic Lipid Mixture was then added to the Aqueous Mixture while rapidly stirring on a magnetic stir plate. Upon mixing, lipidoid vesicles ibrmeti spontaneously. The resulting vesicles were then extruded- (2 passes) through a O.OB μ membrane (Whatman, Nueleopore) to size the empty vesicles. All manipulations were performed at room temperature.

Loading of Empty' Vesicles with siRNA

An siRNA stock solution was prepared by dissolving desalted duplex siRNA in 50 mM sodium acetate pH S at a concentration of 10 mgdnL, An appropriate volume of this siRN A stock was mixed with the appropriate volume of ethanol to yield a diluted siRNA solution in 35% (vol) ethanol (see table below). siRNA Dilution

277 mL of diluted siRN A solution was added to 623 ml, of empty vesicle mixture while rapidly stirring on a magnetic stir plate. The resulting combined mixture was then incubated at 3?°C for 30 min to allow for loading of siRNA.

Ultra filtration and Terminal 0,2 μ Filtration

Ate incubation, .the 900 mL loaded 'nanoparticle mixture was diluted Into 1.8 L of PBS to yield a 2,7 I.· diluted mixture, this- diluted mixture was then concentrated to ~ 1 L and diafiliered by tangential flow filtration against 10 volumes of PBS using a Sartarius TPF system utilizing two stacked 100,000 MWCO cartridges. No backpressure was applied to the cartridge and the pump speed was set to 300 rpm. Alter buffer exchange the resulting solution was concentrated to roughly 2 mgteT siRNA,

Terminal filtration was performed by passing the solution through a 0,2 μ filter capsule (Whatman, Polycap 36 AS), A Sow chart illustrating this process is shown in Figure 10.

Example 47: Comparison of particle size on efficacy

Association complexes were formed using the procedure generally described in 'Example 46. However, because the complexes were being evaluated based on daw, different extrusion membranes were used to produce particles having the following diameters: 150 nm, 85nm, 60 nm, and 50 nm. The siRN As loaded in the complexes targeted factor VII.

The particles were evaluated in a Factor VII silencing assay, demonstrating that the 50 nm paticiea were the most efficacious relative to the 150 nm, 85«m> and 60 nm particles. The results of die assay are depicted in .Figure 1:1.

Example 48: Comparison of half life of nucleic acid agents unformulated versus formulated into an association complex

The half life of siRNA formulated in association complexes was evaluated in vitro in human serum at 37 °C. The association complexes were prepared as in Example 46. For purposes of comparison, unformulated siRN A was also evaluated in vilro inhuman serum. The percent of full length product determined by I IPLC was evaluated for both the formulated and unfonmdated siRNA, As demonstrated in Figure 12, the formulated siRNA had a significantly improved half life in vitro in human scrum.

Example 49: Comparison of efficacy of association ha ving PEG lipids of varied chain length

Association complexes were prepared as in Example 46 with variation on the length of the alkyl chain of the-PIG lipid. Alkyl chain lengths of 10,11,12,13,14, 15, and 16 were evaluated and compared. for efficacy In a Factor VII silencing assay. As shown m Figure 13, chain, lengths of 13,14, and 15 demonstrated the most silencing as measured in die assay, A number of embodiments of the invention have been described. Nevertheless, it will, be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly other embodiments are within die scope of the folio-wing claims.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Claims (41)

1. CLAIMS:

   1. A compound of formula (XV)

   formula (XV) wherein 1 2 each of L and L is a bond; each R and R are independently C6-C28 alkyl, or C6-C30 alkenyl; X is -C(0)NH-; m is an integer from 0-11 and n is an integer from 1-500.
2. 2. The compound of claim 1, wherein each R and R are independently C6-C28 alkyl. 1 2
3. 3. The compound of claim 2, wherein each R and R are independently C10-Cis alkyl. 1 2
4. 4. The compound of claim 1, wherein both R and R are straight chain C14 alkyl or Ci6 alkyl having the same length. 1 2
5. 5. The compound of claim 4, wherein both R and R are C14 alkyl.
6. 6. The compound of claim 1, wherein formula XV represents a racemic mixture.
7. 7. The compound of claim 1, wherein formula XV represents enantiomerically pure ‘R’ isomer.
8. 8. The compound of claim 7, wherein formula XV represents a compound having an enantiomeric excess of ‘R’ isomer.
9. 9. The compound of claim 8, wherein the enantiomeric excess of the ‘7?’ isomer is at least about 95% ee or greater than 97% ee.
10. 10. The compound of claim 9, wherein the enantiomeric excess of the ‘7?’ isomer is at least about 98% ee or 99% ee.
11. 11. The compound of claim 1, wherein formula XV represents enantiomerically pure ‘S’ isomer.
12. 12. The compound of claim 11, wherein formula XV represents a compound having an enantiomeric excess of ‘S’ isomer.
13. 13. The compound of claim 12, wherein the enantiomeric excess of the ‘S’ isomer is at least about 95% ee or greater than 97% ee.
14. 14. The compound of claim 13, wherein the enantiomeric excess of the ‘S’ isomer is at least about 98% ee or 99% ee. 15 The compound of claim 1, wherein each R and R is the same C6-C30 alkenyl moiety. 1 2
15. 16. The compound of claim 15, wherein each R and R includes a single double bond. 1 2
16. 17. The compound of claim 16, wherein each R and R includes a single double bond in the E or Z configuration. 1 2
17. 18. The compound of claim 15, wherein each R and R includes two double bond moieties.
18. 19. The compound of claim 1, wherein m is an integer from 1-10.
19. 20. The compound of claim 19, wherein m is an integer from 2-4 or an integer 2.
20. 21. The compound of claim 1, wherein n is an integer from 1-500, from 40 - 400, from 100 - 350, from 40 - 50 or from 42 - 47.
21. 22. The compound of claim 1, wherein, the compound has a formula (XVI) below:

    wherein the repeating PEG moiety has an average molecular weight of 2000 with n value between 42 and 47.
22. 23. The compound of claim 22, wherein the compound of formula XVI is a stereo isomer with preferred absolute configuration ‘R’.
23. 24. The compound of claim 23, wherein the compound of formula XVI has an enantiomeric excess of ‘R’ isomer of 90%, 95%, 97%, 98%, or 99%.
24. 25. An association complex comprising: a. a cationic lipid; b. a PEG-lipid of claim 1; c. a structural lipid; and d. a nucleic acid.
25. 26. The association complex of claim 25, wherein said cationic lipid is one of the following or a mixture thereof:
26. 27. The association complex of claim 26, wherein said cationic lipid is
27. 28. The association complex of claim 26, wherein said cationic lipid is
28. 29. The association complex of claim 25, wherein said PEG-lipid has the structure wherein:

    n is an integer from 1-500.
29. 30. The association complex of claim 25, wherein said PEG-lipid has an enantiomeric excess of the R isomer.
30. 31. The association complex of claim 30, wherein the enantiomeric excess of the R isomer is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%.
31. 32. The association complex of claim 29, wherein said structural lipid is cholesterol.
32. 33. The association complex of claim 25, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is 36- 48: 42- 54: 6- 14.
33. 34. The association complex of claim 33, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is 38- 48: 44- 52: 8- 12.
34. 35. The association complex of claim 33, wherein the molar ratio of said cationic lipid, said structural lipid and said PEGlipid is in particular 42: 48: 10.
35. 36. The association complex of claim 25, wherein the weight ratio of total lipids to nucleic acid is less than about 15:1.
36. 37. The association complex of claim 36, wherein the weight ratio of total lipids to nucleic acid is about 10:1.
37. 38. The association complex of claim 37, wherein the weight ratio of total lipids to nucleic acid is about 7.5:1.
38. 39. The association complex of claim 37, wherein the weight ratio of total lipids to nucleic acid is about 5:1.
39. 40. The association complex of claim 25, wherein said cationic lipid is

    said structural lipid is cholesterol; and said PEG lipid is

    wherein: n is an integer from 1-500.
40. 41. A method of forming an association complex of claim 25, wherein the method comprises: (a) mixing the cationic lipid, PEG lipid, and structural lipid in ethanol and aqueous NaOAc buffer to provide particles; and (b) adding the nucleic acid to the particle, thereby forming the association complex.
41. 42. The method of claim 41, step (a) further comprising extruding the particles.