AU2014200915A1 - 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 fonndlations TECHNICAL FiELD This invention relates to compositions and methods useful in administering nucleic 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 that could not be addressed with current, traditional medicines. The location and sequences of an increasing number of disease related genes are being identified, and clinical testing of nucleic aci&bascd therapeutic 0 for a variety of diseases is now underay. One method of introducing nucleic acids into a cell is mechanically; using direc microinjection. However this method is not generally effective for systemic administration to a subject. Systemic delivery of a nucleic acid therapeutic requires distributing nuclei aci to target cells and then transferring the nucleic acid across a target cell membrane intact and in a fcrn that can fuction in a therapeutic manner. Niral vectors have, in some instances, been used cliniedly successfully to administer nucleic acid based therapies. However, while viral vectors have the inherer ability to transport nuclei acids across cell membranes, they can pose risks. One such 20 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 genoty either through mutation or genetic exchange with a wild type virus; Lipid-based vectors have also been used in nuleic acid therapies and have beei 25 formulated in one of two ways, In one method, the nucleic acid is introduced into prefonned hUposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. The complexes thus formed have undefmed and complicated structures and the transfection efficiency is severely reduced by the presence of serum, The second methx involves the ornation of DNA complexes with mono- or poly-cationic lipids without 30 the presence of a neutral lipid. These complexes are prepared in the presence of ethane and are not stable in water, Additionally, these complexes are adversely affected by scrum (see, Bea, Acc. Chem. Res. 26:27448 (1993)). SUMMARY The nvention features novel preparations that incUde a polyamine copniound or 5 a lipid moiety described herein, In some embodiments, the invention features a preparation comprising one or more compounds, each individually having a ttrcture defined by finnula(1) or a phamiaceutically acceptable salt thereof, XI X R2NAX XN 'NR? R 10 formula (1 wherein each X'and Xb, for each occurrence, is independently Cp 6 alkylene; n is 0, 1, 2, 3, 4, or 5. each R is independently H, . S 0 O o s, y- R Y' R or R Ra R RI) Ri R, wherein at least n 4 2 of the R moieties in at least about 50% of the molecules of the compound of formula (1) in the preparation (e.g, at least about 55%, at least about 20 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 0, NR 2 , or S; i is alkyl alkenyl or alkynyl; each of which is optionally substituted 25 with one or more substituents; and R2 is H1, alkyl alkenyl or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n 0, then at least n + 3 of the R moieties are not Hi. 2 In some enbodiments, when R is not H, R is R, for example, when R is not H, R is R, for each occurrence, in some embodiments, when R is not H, R is Ra, for example, when R is not H, R is Rb, for each occurrence, 5 In some embodiments, when R is not H, R is R, for example, when R is not H, R is R,, for each occurrence. In some embodiments, when R is not H, R is K 4 , for example, when R is not Hi, R is 1%, for each occurrence. in soenc embodiments, when R is not H, R is R, for example, when R is not H 10 . is R, for each occurrence, I some embodiments, n + 2 of the R moieties of formula (1) are not H. In some embodiments, n + 3 of the R moieties of formula (l) are not H, In some embodiments, n + 4 of the R moieties of formula (I) are not It in some embodiments, n + I of the R moieties of formula (i) are not H. 5 in some embodiments, n> 0, and at least one R of NR of forala (1) is H. in some embodiments, at least one R of NR; of fomala (I) is IH, In some embodiments, at least 80% of the molecules are a single structural isomer, For example, n + 2 of the R moieties of formula (I) are not H, or n + 3 of the R moieties of formula (1) are not H, or n + 4 of the R moieties of formula tl) are not H, 20 in sone embodiments, n is 2 or 0. In some enbodiments, X and Xb are C 2 alkylene. In some embodiments, n is 0 andNb is ethylene or propylene. In some embodiments, n>1 and X'varies with at least one occurrence, In some embodiments, when R not El, R is a ~iR, y . For example, Y can be 0 or NR 2 In some embodiments, m is 2. In some embodiments, Y is 0 or NR 2 and m is 2. In some embodiments, in is 1. In some embodiements, m is I and Y is ) or NHR. In some embodiments, R'for at least one occurrence is alkyl, ior example, Rfor each occurrence is alkyl, 30 ln some embodiments, R is alkyl and RAis H, for at least one occurrence, e.g., for each occurrence. 3 In some embodiments, RI and RC are alkyl for at least one occurrence, e.g., for each occurrence, in some embodimentsRI for at least one occurrence is alken yL In some embodiments, RI for at least one occurrence is alkenyL 5 In some embodiments, when R is not H, R is 1t for at least one occurrence, e,g., for each occurrence, and Y is 0 or NH. In some embodiments, Y is 0, In some embodiments, Y is NH, In some embodiments, R" is alkyl, e.g., Cw. alkyl or(C alkyl, in sio embodiments, n is 2. In some embodiments, X', for each occurrence is C, alkylene and X" is C- alkylene. In some embodiments, m is 2. Io in sone embodiments, n is 2 and R, when R is not H, is R,, for at least one occurrence, e.g,, for each occurrence. In some embodiments, R is alkyl, e.g, Cwis alkyl or Cj alkyl In some embodiments, Y is 0 or Y is Ni in some embodiments, XV, for each occurrence is C2 alkylene and XCis C2 alkylene. in some embodiments, i is2 i in some embodiments, at least I R of NR. is H and R, when not H is R<i, for at least one occurrence, eg. for each occurrence, and Y is 0 or NH. In some embodiments, Y is 0 or Y is NH. In some embodiments, R.' is alkyl, e.g,, Cwo~sakyi or Cn~ alkyL In some embodiments, n is 2. In some embodiments, X* for each occurrence is C2 alkylene and X isO, alkylene. In sonic embodiments, m is 2, 2o in some embodiments, n is 2 and at least I R of NR is H and vhen R is not H, R is R, for at least one occurrence, e.g. for each occurrence, and Y is O or NH. In some embodiments, R1 is alkyl, e,g., C10 alkyl or Ca alky In some eibodimnents, Y is G or Y' is NIL In some emodinents, X, for each occurrence is Q2 alkylene and X is CQ alkylene. In some embodiments, m is 2. in some embodiments, at least I R of NR1 2 is H and R is R, for at least one occurrence, e.g. for each occurrence, and wherein Y is 0 or NR. in some embodimlents, Y is 0 or Y is NH. In some embodiments. R1 is alkyl, eg, CWa alkyl, C~ot alkyl or C alkyL in some embodiments, n is 2. In some embodiments, X, for each occurrence is C2 alkylene and N 5 is C2 alkylene. in some embodiments, m is 2, 3o In some embodiments, n is 2 and at least I R of NR2 is H and R is R,, for at least one occurrence, e.g. for each occurrence, and wherein Y is 0 or NH. In some embodiments, R" is alkyl, e.g, CjhIS alkyl or C1 2 alkyl. In some embodiments, Y is 0 4 or Y is NL In some embodiments, X, for each occurrence is C2 alkylene and X is C 2 akylene, in some embodiments, m is 2. In some eroodiments, the preparation comprises one or a mixture of thc formula below, wherein R is not H unless specified in the formula below. R R H H R N N N . R and RNNNN N /R 5 R R R R in some enibodiments, the preparation consists essentialy of one or a mixture of the formula below H i H RNN N R and R'N NN N R R R R ,I some embodiments, each R is R1 0 ,' A N'R 1 1C 2 . In some embodiments, each R is In some embodiments, R is CjorCj, alkyl (e.g., C2 alkyl), or Coa-C alkenvi, in some embodiments, R is R1 R4 , In some embodiments, R1 is CfrC 8 alkyl, e.g., C, 2 alkyl, in soime embodiments. R is C 2 alkyl and RI is H. 15 In some embodiments, n is 0 and X is propylene, in some embodiments, I R is H. In some embodiments, when R is not H, R is R, for at least one occurrence, e.g. for each occurrence, hn some embodiments, R' is alkyl, e.g, CQo.o alkyl or Cu alkyl, hi sone embodiments, Y is Q or Y is NH. In some embodiments, m is 2. In some embodiments, formula (1) is 0 a N' or RI 2 R In some embodiments, R is in some embodiments, R' is CurCI alkyl, or C 1

-C

30 alkenyl. In sonie embodiments, R is 5 R' N'R

R

2 ,Insome embodiments, R' isCj-C1 alkyl, or C 0

~

3 alkenyl and R 2 is In somc embodiments, n is2; X, for each occurrence is C2 alkylene and Xb is C2 alkylene; and wherein each R is H or 0 1 y R, for at least one occurrence, e.g. for each occurrence, 10 m is 2; Y is NH or 0; R! is C2 alkyl. In some embodiments, at least 80% of the molecules of the compound of formula (1) are a single structural isomer. [n some embodiments, Y is NH, e.g, wherein at least 80% of the molecules of the compound of formula (1) are a single 5 structural isomer. In some embodiments, R is Ri, for 5 occurrences. In sone embodiments, in at least 80% of the molecules of the compound of formula (I), R is R2, for 5 occurreneK in some embodiments, Y is NH. In some embodiments, the compound of formula (I) is an inorganic or organic salt thereof, e.g., a hydrohalide salt thereof, such as a hydrochloride salt thereof In 2:0 somte embodiments, the hydrochloride salt ranges from a single equivalent of HCL, to n+2 equivalents of MHCL In some embodiments, the compound of formula (1) is salt of an organic acid, e.g, an acetate, for example, the acetate salt ranges from a single equivalent of acetate, to n+2 equivalents of acetate or a format, fr example, the form ate salt ranges from a single equivalent of acetate, to n+2 equivalents of format. 25 In some embodiments, the compound of formula (1) is in the form of a hydrate. In some embodiments, R, for at least one occurrence, e.g., for each occurrence, comprises, an. aikenyl moiety, for example, R1 comprises a cis double bond. In one aspect, the invention features a preparation including a compound of formula (I) and a nucleic acid (e.g. an RNA such as an siRNA or dsRNA or a DNA), In 6 some embodiment, the preparation also includes an additional lipid such as a fasogcnioc lipid, or a PEG-lipid In some embodiments, the preparation comprises less than I %, by weight. of x 'l HN 'N, 'NH2 wherein X and n are defined as in ftnnula (I) above. In some embodiments, the preparation comprises less than 90% by weigt of RI frimula (V) wherein Y and R' are defined as in formula (I) above, In some emabodiments, the preparation comprises a plurality of compounds of fonnula (I). in some embodiments, the preparation comprises a mixture of compounds of the 15 frnulus below: R R RI, Ra i H R" Ra and R R fomaula (I") formula (I") wherein in formula (I"), five of the R moieties are R'. In same embodiments, fbrnula (I) and (I") are present in a ratio of from about 1:2 to about 2:1. 20 in one aspect, the invention features a method of making a compound of formula R2NT 'N ' NRz R R formula (1I) 25 each X" and X', for each occurrence, is independently Ci alkylene; r is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or 0 RI R.; m is2 Y is 0, NR, oir S; R' is alkyl or alkenyl;

R

2 is H or C alkyl or adkenyl; the method comprising reacting a compound of formula (ill) F I

H

2 N Nx Nh2 Hi formula (F11) with a. compound of formula (IV), 0 fibmala (IV) in the presence of a promoter. in one aspect, the invention features a method of making a compound of formula (li), X" X R N- IN 'NR2 iR[ 20 ' formula (II) wherein each X" and Xl, for each occurrence, is independently Cs aikylene; nis 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or 8 0 'YRl a m is 2; Y is 0, NR 2 , or S; 5 R' is alkyl or alkenyl;

R

2 is H or C alkyl or alkenyl; the method comprising reacting a compound of fornnula (111) HN 'N 'NH2 H i formula (III) 10 with a compound of formula (IV), 0 F- gR 4 ftbmula (IV) in the presence of a quencher. 15 In one aspect, the invention features a method of making a compound of formula (ii), R2N) l' 'NR, cinn formula (II) 20 v/herein each X' and Xt for each occurrence, is independently C., alkylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or 0 9 Rj m is 2; Y Is 0, NR 2 or S; Rl is alkyl or alkenyl; R is H or akyl or alikenyl; the method comprising reacting a compound of formula (III) X* X H2N NZ NH 2 ' n formula (IIt) with a compound of fonnula (IV), 10 formula (IV) wherein the reaction mixture comprises from about 08 about 1,2 molar equivalents of a compound of formula (III), with from about 3,8 to about 6.5. molar 15 equivalents of a compound of fonnula (IV), In some embodiments, the reaction mixture comprises from about 0.8 about 1,2 molar equivalents of a compound of formula (liI), with from about 5.5 to about 6.5 molar equivalents of a compound of formula (IV), In some embodiments, the reaction mixture conprises about I molar equivalents of a compound of fbnnula (I), with from 20 about 6 molar equivalents of a compound of formula (IV), In some embodiments, the reaction mixture comprises about I molar equivalents of a compound of formula (III), with from about 5 molar equivalents of a compound of fonmula (IV). In one aspect, the invention features a method of making a compound of formula (R),, 25

R

2 N {N' 'XNR 2 R forula (II) 10 each X' and Xb, for each occurrence, is independent C 4 alkyliene; n is 0, 1, 2,. 4, or 5; and wherein 5 each R is independently H or P1 0 mis 2; Y is 0, NR 2 . or S; R' is alkyl or alkenyl; R2 is H or alkyl or alkenyl; the method comprising a two step process of reacting a compound of HN1 'N 1

'NH

2 IHi 15 formula (111) 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 U.8 about 1L2 molar equivalents of a compound of fonula (11), wih fom about 3.8 to about 4,2 molar equivalents of a compound of frmula (IV) and the second step process involving addition of about 0.8 to 1.2 molar equivalent of compound of formula (IV). In one aspect, the invention features a method of making a compound of formula (II1),

I

XI X R2N N[ 'NR2 R fonnula (11) wherein each Xa and XK for each occurrence, is independently Ca alkylene; S nmist0, 1,2,3,>4, or5; and wherein each R. is independently H1 or 'm'r 10 m is 2; Y is 0, NR2, or 5 RI is alkyl or akenyl;

R

2 is H or alkyl or alkenyl; the method comprising reacting a compound of frnnula (Il) Xa X HzN 'N 'NH2 formula (IIl) with a compmnd of formula (IV), 0 fornula (IV) 20 and separating at least one structural isomer of fonnula (I) from the reaction mixture to provide a substantially puriflied preparation comprising a structural isoner of ormnula (11). In some embodiments, the structual isomer of formula (II) i.s separated from the 25 reaction mixture using chronatographic separation. in some enbodiments, the chromnatographic separation is using flash silica gel for separation of isomers. In somc embodiments, the chromatographic separation is gravity separation of isomers using silica get In some embodiments, the chromMtographic separation is using moving bed chromatagraphy for separation of isomers. In some embodiments, the chromatographic separation uses liquid chromnatagraphy (LC) for separation of isomers, In some 5 embodiments, the chromatographic separation is normal phase HPLC for separation of isomers. In some embodiments, the chromatographic separation is reverse phase HIPLC for separation of isomers. In some embodiments, the substantially purified preparation comprises at least about 80% of the structure isomer of formula (HI), eg,, at least about 90% of the 1o structural isomer of formula (II), at least about 95% of the structural isomer of formula (II), in another aspect, the invention features a method of making a compound of formula (V) or a pharmaceutically acceptable salt thereof [X 14 RsN 'N NR 2 R formia (V) wherein each X" and Xt for each occurrence, is independently Cse alkylene; n is 0, 1, 2, 3, 4, or 5; and 20 wherein each R is independently H or R' m is 1, 25 Y is 0, NR 2 , or S; RI is alkyl or alkenyl; R is H or alkyl or alkenyl; the method comprising reactinga compound of fon'ula (111 13 r XM xb HNN 'N" 'NHz n fbnnula (IH1) with a compound of fomula (VI), 0 Q = C or Bor ! formula (VI) to provide a compound of formula (V) or a pharmaceutically acceptable sal thereof 10 In some embodiments, the phanmaceutically acceptable salt thereof is a hydrochloride salt of the compound of fonnula (V), in one aspect, the invention features a compound of formula (X), R R4

R

2 LW 15 formula (X) wherein Ri and R are each independently If, C 1 -C alkyl, optionally substituted with 1-4 R, C1 C6 alkenyl, optionally substituted with 1-4 R.3 o C(NR 6 )NRf) 2 ; R" and R4 are each independently alkvl alkeny1, alkynly, each. o is 20 optionally subtituted with fluoro, chloro, bromo, or iodo; .L and I are each independently -NR t C(0)-, -C(O)NR, -OC(C)-, -C.(lO-, S-S->N(R>)C(O)N(R%)~, -OC(0)N(R 6 ) -N(R*)C(0)Q-, -0-N=:C> OR, -OC(O)N N Ci r~NHC(O)NH-N='C L-R and L t

R

4 can be taken together to form an acetal, a keta, or an orthoester 25 wherein R and R 1 are defined as above and can also be H or phenyl;

R

3 is fluoro, chloro, bromo, iodo, -OR 7 , -N(Re)(R) -CN, SRt S(O)R s(0) 2R 14 R is I, C;C6 alkyl, R is H or CrC6 alkyl; each Rb and Rt are independently H or C, -C alkyl; R is H or C 1 C alkyl; m is 1, 2, 3, 4, 5, or 6; n is 0, , 2 3, 4, 5, or 6; and pharmaceuticaliy acceptable salts thereof In some embodiments, the compound is an inorganic salt thereof, for example a hvdrohalide salt thereof such as a hydrochloride salt thereof In some embodiments, the 10 compound is an organic salt thereof In some embodiments, R1 and R2 are each independently Cr C, alkyl, In some embodiments, I is methyl In some embodiments, RI is methyl, In some embodiments, R 1 and le are both methyl. 5 In some embodiments, R is H, nethyl, thyl, isopropyl, or 2-hydroxyethyl hn some embodiments, R2 is H. In some embodiments, R'? is methyl, ethyl, propyl, or isopropyl. In some embodiments, RI' is H, methyl, ethyl, isopropy, or 2-hydroxyethyi and 1 is BL, methyI, ethyl, propyl, or isopropyL 20 in some embodiments, in is 1. In some embodiments, n is L In some embodiments, both m and n are 1, In some embodiments, L' is --NR 6 C(O)-, or -C()NK In some embodiments, L is -OC(0)- or -C(0)0 25 In some embodiments, L' is S-S-. In some embodiments, L' is -N(R)C(O)N(Rt)-. In some embodiments, L' is -OC()N(R)- or -N(R)C()O In some embodiments, L' is -O-N'C In some embodiments, L' -OC(O)NI--N=C- or ~N HC(O)NH-NC 31 In some embodiments, I" is -NRC(0)-, or -C(OYNR In some embodiments, LU is -OC(0)- or -C(0)0. in some embodiments, L) is S-S 15 In some embodiments, C is -N(R)C(O)N(R) in some embodiments, [? is -OC(0)N(R 6 )- or -N(Rt)C(0)0-, In sonic embodiments, 1 is -0-NC In sonic embodiments, L. -OC(0)NHN=C~ or -NHC(O)NH-N=C In some embodiments, both iL and I are -NR 6 0(Q), or -C(O)NR> In some embodiments, both L and I are -OC(O)- or -C(0)0, In some embodiments, both Li and I! are S-S-. In some embodiments, both L 1 and L are -N(Rt)C(0)N(RY-. hi some embodiments, both L' and If are -OC(0)N(R )- or -N(R 6 )C(0)(-, 10 In somc embodiments, I] is -NR 6 C(0)- and L is -S-S, In some embodiments, L is -OC(0)- and C is -S-S. In some embodiments, C is -OC(O)N(R ) or -N(R)C(0)0- and I is -S., In some embodiments, Ll is -N(R)C(O)N(R)~ and L2 is $-S& hi some embodiments, L~RA and I-R 4 arc taken together to forn an acetal, a 15 ketal, or an orthoester. In soie embodiments, each R 3 and R4 are independently alkyl, In some embodiments, both R 3 and .4 are C 6 Ka alkyL InI some embodiments, each L' and L are independently ,S-SOC(0)N(R)~ or -NR)C(O)O 20 hi some embodiments, R 3 is alkyl. In some embodiments, R 4 is alkyL In some embodiments, R 3 is alkenyl. In some embodiments R is alkenyl In some embodiments, each R 3 and R 4 are independently alkenyl, for example, 25 each R 3 and R 4 are independently Ca alkenyl or each R 3 and RI are the same alkenyl moiety. In some embodiments, each R 3 and R4 includes two double bond moieties. In some embodiment, at least one of the dobe bonds have a Z configuration. In. some embodiments, both of the double bonds have a Z configuration. In some embodiments, so at least one of R3 and R' is provided in formula (II) below formula (1i) 16 wherein x is an integer from I to 8; and is an integer from 1-10, In some embodiments, both of R 3 and R are of the formula ( ). In soene embodiments, at least one of the double bonds have an E 5 configuration, e g, both of the double bonds have an E configuration. In some embodiments, at least one of R' and R' is provided in formula (1I) below formUla (I) wherein 10 x is an integer from 1 to 8; and y is an integer from I-10, In some embodiments, each R' and R 2 includes three double bond moieties, 1n soni embodinments, at least one of the double bends have a Z configuration, hi some embodinits, at last two of the double bonds have a Z configuration. hi some 15 embodinents, 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 20 x is an integer from I to 8; and y is an integer from I - 10, In some embodiments, both of R and R 2 are as provided in formula (N), In some embodiments, at least one of the double bonds have an E configuration, in some embodiments, at least two of the double bonds have an E configuration. In seme embodiments, all three of the double bonds have an E 25 configuration., In some embodiments, at least one of RI and R 2 is provided in fonula (IV) below 'Y formula (V) wherein 30 x is an integer from I to 8; and 17 y is an integer from -10, in some embodiments, both of Ri and R 2 are as provided in formula (V) fn some embodiments, R' and Ri are each CrC alkyl (e.g, methyl), LI and L I are each -0C(0)- and R 3 and R14 are each alkenyl, In some embodiments, R3 and R4 o are the same. In some embodiments, R 3 and R 4 both include two double bonds (e.g, having Cis linkages). In some embodiments R 3 and R 4 are provided in fonnula (11) below tornmla (11) to wherein x is an integer from I to 8 e.g, 5; and y is an integer from 1-10 eg, 4, In one aspect, the invention features a preparation including a compound of formula (X), 15 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 DNA). In some embodiment, the preparation also includes an additional lipid such as a fusogenic lipid, or a PBGlipid, In one aspect, the invention features a method of making a compound of formula 20 (X), R R,

R

2 LtR3 fonnula (X) wherein R' an R 2 arc each independently C-C6 alkyl, optionally substituted with 1-4 25 Ri; R' is alkyl, alkenyl, alkynyl LI is -0C(0) t is -OR., -N(R)(R), -CN, SR S(O)R S(OhRL IR is H, C-C( alkyl; 30 R' is H or 0'-C5 alkyl; 18 each R and R are independently H or C-C6 alkyl; R is H or C1-C alkyl; in and n are each independently 1, 2, 3, 4, 5, or 6, the method comprising reacting a compound of formula (VI), R 1 O H formula (VI) with a compound of forula (VIH) HO' R formula (VII) 10 in the presence of a coupling agent, thereby providing a compound of fmmula (X). In some embodiments, the coupling agent is a carbodilmide such as EDCL In one aspect, the invention features a compound of fornula (XV) below R- O O 01L2 'IM n 15 formula (XV) wherein: each L' and L 2 are independently a bond or C(O); each R' and R2 are independently alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; 20 X is -C(O)NH, C(S)NH, -C(O)C.

1 3 lkylC(O)NH-; or -C(0)Cj..:alkylC(0)O-; m is an integer from 0-11 and n is an integer from 1-500. In some embodiments, I; and I are both a bond. In some embodiments, LI and L2 are both C(0). 25 in some embodiments, each Rl and Ri are independently alkyl, tbr example C6 Cs aikyl, eg.,C alkyl, e.g., Cu aikyC 1 alkyl Ca alky, or Ca alky, In some embodiments, both R and R2 are alkyl, e~g., straight chain alkyl having the same length, 19 e.g.,C( setalkyl, e.g,CieCn alkyl, e.g, C1 alkyl, C 14 alky!, Cu alkyl, or C1 alkyL hn some preferred embodiments, both R' and R 2 are Ca alkyl. In some embodiments, the fonula XV reperesents a racemic mixture in some embodiments, the compound of formula XV has an enantiomerie excess 5 of the R isomer, e g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. in some embodiments the fbnmula XV represents enantiomerically pure 'R' isomer. in some embodiments, the compound of formula XV has an enantiomerie excess of the S isomer, e.g, at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, so or 99%. In some embodiments the formula XV represents enantionerically pure ' isomer. In some embodiments, each Ri and Re are independently alkenyl, for example, each RI and R- are independently C 6 ;Cyo alkenyl or each R and R are the same alkenyi moiety. in some embodiments, each R1 and R 2 includes a single double bond, for i5 example single double bond in the E or Z configuration. in some embodiments, each RI and I 2 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 R and R is provided in fbnnula (ii) below 20 mula. (I0 wherein x is an integer from I to 8; and y is an integer from I -10 In some embodiments, both of R' and R 2 are of the 25 formula (11). In sonie embodimens, at least one of the double bonds has an E configuration, e.g., both of the double bonds have an E configuration. In some embodiments, at least one of R' and R is provided in formula (111) below 1101 formula (III) 30 wherein x is an integer from 1 to 8; and 20 y is an integer from 1-10. In some embodiments, each R' and R 2 includes three double bond moieties, In some embodiments, at least one of the double bonds has a Z configuration, In some enbodiments, at least two of the double bonds have a Z configuration. In some 5 embodiments, all three of the double bonds have a Z configuration. In some embodiments, at least one of R' and R 2 is provided in formula tV') below tornula (IV) wherein t o x is an integer from I to 8; and y is an integer from 1~10. In some embodiments, both of R' and R are as provided in fornula (IV) In some embodiments, at least one of the doable bonds has an E 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 15 configuration. In some embodiments, at least one of R and R 2 is provided in formula (IV) below fonnula (V) wherein 20 x is an integer from I to 8; and y is an integer from I-I0. in some embodiments, both of R and R' are as provided in formula (V). In some embodiments, X is -C(O)NH-, providing a. compound of formula (XV') below: 0 R I' Lt9 N \ 0 0, L formula (XV'). In some embodiments, each R' and Ri are independently alkyl, fm example C6-C alkyl, e.g,C Cj aikyl, e.g., C11 akyl, CA aikyl, C alkyl, or alkyl, In some embodiments, both R' and R2 are alkyl, e.g, straight chain alkyl having :n the saie length, eg. C 6

C

28 aikyl, eg.,Cw-C alkyl, e.g., C 0 alkyl, C14 alkyl, CI alkyl, or C f alkyl In some preferred embodiments, both R1 and R 2 are C14 alkyl In sone embodinents X is -C(O)CualkYlC(0)O~. In some embodients, i is an integer front 1 -10, for example an intger from 2 S 4 or an integer 2, In some embodinents, n is an integer from 1-500, for example an integer from 40-400, from 100-350, from 40-50 or from 424'?, in some embodiments, the compound is a compound of formula (XV'), O ~< L,.< L.' 0 I , formula (XV'). wherein both L and L2 are a bond, In some embodiments, each R' and R- are independently alkyI for example C6-C2 alkyl, e.g.,CQCalkyi, e g., Cs alkyl, C1 alkyl, or C , alkyl. In some embodiments, both R3 and R are alkyl, e.g., straight chain alkyl having the same length, e.g., Cf,-Q' alkyl, e g.,CI-Cl alkyl, e.g., Ca alkyl, C5 '15 alkyl, or Co alkyl, In some preferred embodiments, both R and 2 R are C 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, for example an integer from 40-400, or from 40-50. In some embodiments, the compound is a compound of fomula (XV'), wherein 20 Li and L2 are both bonds, R1 and R2 are both alkyl (e.g,, Cf-Cs alkyl, e g.,Cw-Co alkyl preferraoly C4 alkyO), and is an integer from about 40-400, in some embodiments, the comound has a formula (XVI) below: 0 fornula (XVI), wherein the repeating PEG moiety has an average moleculat 25 weight of 2000 wih n value between 42 and 47. In some embodiments, the compound of formula XV has an enantiomerie excess of the R isomer, e.g., at least about 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 22 or 99%. In some embovdments the compound of formula XVI is a stereo isomer with preferred absolute configuration TR, In one aspect, the invention features a PEG lipid conjugated to a cholesterol moiety. For example, the compound of fornmla (XX) below; Cholesterobs X, 1 m n formula (XX). X is -C(Q)Nit C(S)NH, 4C(O)CojakyC(O)NH-; or -C(Q)C 3 alky1C()O; m is an integer from 0-1 and n is an integer from 1-500, 10 In some embodiments the ( attached to the cholesterol in formula (XX) is part of the cholesterol moiety, In some preferred embodiments, X is -C(0)NH-, or -C(O)C.; 3 alkylC(O)Ct In some embodiments, the compound of formula (XX) is as provided below in 'fannurla (XX') 0 formula (X(X'), In one aspect, the invention features a PEG lipid bound to a targeting moiety, for examnple a sugar residue, For example, the compounds of formula (XV) ar (XX) are modified at the Oly-e terminal end with a targeting moiety, In some emboadiments, the 20 targeting moie ty is bound to the PEG m~oiety via a Unker. Examplary targeted PEG lipids are pro-vided in formulas (XXI) and (XXU-) below. In (ne emnbodiment, the lipid is a compound of fornmla (XXI) L2 R2 fornib a (XXX1) 25 wherein; 23 each 1) and If are independently a bond or (O); each R' and R 2 are independently alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; each X and X' is independently -C(O)NH NHC(O -, C(S)NH, C(S)NIN, t C(O)C" 3 alkyC(O)NH-; NHC(O)C -alky1C(0) -C(O)C-aikyIC(0)0-; NHC(0)C *alkyl-; or C1.31a-kylC(O)NH-; m is an integer from 0-11 and n is an integer ftom 1-500 p is an integer ftom 1-6, eg. 3; T is a targeting moiety such as a glycosyi moiety (egg a sugar residue). OH 08 HO Eixamplary targeting moieties include AcHN In some embodiments, L' and I? are both a bond. Ia some embodiments, L' and I' are both C((0), In some embodiments, each R' and R2 are independently alkyl, for example C6 17Cz alky, e.gCiCa alkyl, esg, Cj alky1, Cu alkyl, or C6 alkyl, I some etmbodiments, both R' and R2 are alkyl, e.g., straight chain alkyl having the same length, e g, C6a-Q alkyl, c.g.,Cw-Cjs alkyl, e.g., C14 l 0 u alky, or C e al In some preferred embodiments, both R. and R are C4 alkyl In some embodiments, the fbrmula (XXI) reperesents a racemic mixture 20 in some embodiments, the compound of fonnula (XXI) has an. enantiomeric excess of the R isomer, eg., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, In some embxxdiments the formula (XXI) represents enantiomerically pure ' isomrer, in some embodiments, the compound of formula (XXI) has an enantiomeric 25 excess of the S isomer, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. in somi embodiments the formula (XXI) represents enantiomerically pure 'S' isomer, In some embodiments, each R' and R2 are independently alkenyl, for example, each Ri and R are independently CaCro alkenyl or each R' and P 2 are the same alkenyl 24 moiety, In some embodiments, each R' and R2 includes a single double bond, for example a single double bond in the R or Z configuration. in some embodiments, each RI and .R2 includes two double bond moieties In some embodiments, at least one of the double bonds has a Z configuration. In some 5 embodinwnts, both of the double bonds have a Z configuration. In some embodiments, at least one of R. and R 2 is provided in fonnula (11) below fonnula (II) wherein 10 x isa n integer from I to 8; and y is an integer from 1-10. In some embodiments, both of R' and R2 are of the formula (II), In some embodiments, at least one of the double bonds has an I configuration, e.g., both of the double bonds have an E configuration. In some embodiments, at least one of R' and R 2 is provided in formula (iIf below fonnula (I1) wherein x is an integer from I to 8; and y is an integer from 1 -10, 20 In some embodiments, each R' and R2 includes three double bond moieties, In some enbodiments, at least one of the double bonds has a Z configuration, In some embodmnts, 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. R is provided in formula (IV) below 25 N formula (IV) wherein x is an integer from I to 8; and y is an integer from 1-10. In some embodiments, both of R and R2 are as 30 provided in formula (IV), In some embodiments, at least one of the double bonds has an 25 E configuraion. In some embodiments, at least two of the double bonds have an E contiguratlon. I some embodiments, all three of the double bonds have an E configuration, In some embodiments, at least one of R' and R is provided in fonnula (IV) below 'formlPala (V) wherein. x is an integer from I to 8; and y is art integer from 1 -10. In some embodiiments, both of R? and R4 are as 10 provided in formiala (V). In some embodimnents, p is 3, in some embodiments, , is NHC(O)C_ alkyl (e.g., NHC(O)Csalkyl). In some embodiments, the compound of formula (XXI) is the compound of (XXI) below: OH CM o HO tr'2N-A 7 NX -'~ N O T AcHN H H 1C5 formula (XX ). In one embodiment the lipid is a compound of fonnula (XXII) Cholesterol X X frmiula (XXII) 20 wherein; each X and X' is independently -C(O)NH-, -NHC(O) -, C(S)NH, O(S)Nil, C(0)C 3 alkyIO(O)NH-; NHC()C.

3 alkylC(O)~; -C(O)C ,a-kylC(O)O-; NHC(O)C 1 a1ky!-; or 2C..akylC()NH; m is an integer from 0~11 and 25 ni is an integer from 1500 p is an integer from 1-6, e.g., 3; 26 ' is a targeting moiety such as a glycosyl moiety (e.g., a sugar residue) OH HO-6 Examplary targeting moieties include AcHN In some preferred embodiments, the compound of fonnula (XXII) is the compomd of (XXIP') as provided below: OH HO N AcH!N H H formula (XXIP) In one aspect, the invention features an association complex comprising a compound preparation comprising a compound described herein (e.g, a compound of formunla (I) or a compound of formula (X)) and a nucleic acid such as an RNA a single 10 stranded or double stranded RNA (e.g, siRNA or dsRNA or a DNA), In some embodiments, the association complex is a lipoplex or a liposome In some embodiments the association complex includes one or more additional components such as a targeting moietya fusogenic lipid, a PEGylated lipid, such as a ?EG-lipid described herein such as a PEG-lipid having the formula (XV), ,(XV') or (XVI) or a is structural component. In some embodiments, the PEG-lipid is a targeted PEG-lipid as described herein. e.g., a compound of fomla (XXI), (XXP), (XXII), or (XXIP). In one aspect, the invention features a method of forming a liposome comprising contactig a lipid preparation comprising a compound described herein (e.g. a lipid described herein such as a compound of fornula (1) or formula (X)) with a therapeutic 20 agent in the presence of a buffer, wherein said buffer: is of sufficient strength that substantially all amines of the molecules formula I are protonated; is present at between 100 and 300imM; is present at a concentration that provides significantly more protonation 25 of thandoes the same buffer at 20 mN. In one aspect, the invention features a liposomne made by the method described herein, 2'7 in one aspect, the invention features a method of forming a liposome comprising contacting a lipid preparation described herein (esg, a lipid preparation comprising a compound of fbrnula (I) 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 a with the therapeutic agent to provide a particle having a diameter of less than 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 to presence of a buffer, wherein said buffer has a concentration from about 100 to about 300mM. in one aspect, the invention features liposome comprising a preparation described herein (e.g, a lipid preparation comprising a compound of formmia (I) or a compound of formula (X)) and a nucleic acid. In some embodimentsthe preparation 15 also includes a PEGylated lipid, for example a PEG-lipid described herein, such as a PEG-lipid 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), (XX., (XXII) or (XXIP) In some embodiments, the preparation also includes a stuctural moiety such as cholesteroL In some embodiments the preparation of 20 association complex includes compounds of formaulac (I), (XV) and cholesterol. In some embodiments, said nucleic acid is an siRNA, for example said nucleie 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 thi ApoB gene. 25 In sone embodiments, the liposome further comprisies a structural moiety and a PEGylated lipid, such as a PEG-ipid described herein, wherein the ratio, by weight, of preparation (e.g, a lipid preparation comprising a compound of fornrula (I) or a compound of formula (X)), a structural moiety such as cholesterol, PEGylated lipid, and a nucleic acid, is 822410:442:0A-2.2, In some embodiments, the structural moiety is ao cholesterol. In sonim embodiments, the ratio is 0-20:05-8.0:5-10:052A e g, 15:0.8:7:1. In some embodiinents, the average liposome diameter is between 10 nm and 750 na, e.g, the average liposome diameter is between 30 and 200 Dnn or the average 28 liposome diameter is between 50 and 100 rnm. In some embodiments, the preparation is less than 15%, by weight, of unreacted lipid. In sonie embodiments, the ratio of the preparation (eg, a lipid preparation comprising a compound of formula (I) or a compound of fomimia (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 nudeic 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 an association complex comprising a plurality of lipid moieties and a therapeutic agent, the method comprising: mixing a pluralitv 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% eth anol, In soni embodiments, the plurality of lipid moieties comprise a cationic lipid, In some embodiments. the cationic lipid is a lipid described herein, fbr example, the cationic lipid is a lipid of one of the following or a mixture thereof; H H .1j or 29 0 H N N -O H H N N H ) N.NNANANYoX No 0 H H ,In some preferred embodiments, the cationic lipid is H H HH In some embodiments, the plurality of lipid moieties comprise a PEG-lipid, for example the PEG-lipid has the following structure: 0 O R L2O wherein; each L and I are independently a bond or C(O); each R and R 2 are independently alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; 5 X is C-C()NH-, C(S)Nl -C(O)CalkyC(G)NH-; or -C(O)CtalkylC(O)O-; m is an integer from 0-11 and n is an integer from 1-500. In some preferred embodiments, the PE G-lipid is a PEG lipd of formula (XV, 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: .~. NO 'N N~z ~ H \ /nf In some embodiments, the plurality of lipid moieties comprises a structural lipid, For example, the structural lipid is cholesterol, In some embodiments, the PEG-lipid is a targeted PEG-lipid as described herein, eg, a compound of fonnula (XXI), (XXP), (XXi)., or (XXIP). 30 In some 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., In some embodiments, the siRNA targets the ApolB 5 gene, In some embodiments, the association complex comprises a cationic lipid, a structural lipid, a PEG-ipid and a nucleic acid.. In some embodiments, the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nuclei acid is 36-48:42-54:6-14, for example, 38-46:44-52:8-12 or about 42:48:10, in some embodiments, ie 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:. LIn some preferred embodiments, the cationic lipid has the following H H J M H structure:; the PEG-ipid is a PEG lipd of formula (XVI), wherein the repeating PEG moiety has an average molecular weight of 2000, for example, with an n value between 42 and 47 or has the following structure: H and the structural lipid is cholesterol, for example, wherein the molar ratio of the cationic lipid, structural lipid, is PEG-lipid is 3846:44-52:812, e.g. about 42:4810. In some preferred embodiments, the weight ratio of total exipient to nucleic acid is less than about 15:1, eg, about 10:1, about 7.5:1, or about 5:1. In another aspect, the invention features an association complex made from a method described herein. i1 another aspect, the invention features association complex comprising a cationie lipid, a structural lipid, a PEG-lipid and a nucleic acid, wherein the cationic lipid is is a lipid of one of the following or a mixture thereof: 31 H H ~~ N~,.~r&r~ H H & N H Cr HH H H the PEG-lipid is a PEG lipd of formula (XVI), wherein the repeating PEG moiety has an average molecular weight of 2000, for example, with ann value between 42 and 47 or has the following structure 9 OoiN 2 H \ /n and the structural lipid is cholesterol. In some preferred embodiments, the nucleic acid is an siRNA I some preferred embodiments, the cationic lipid has the following formula: H H 0 H H In Some preferred embodiments, 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, for example, 38 46:44-52;8-12 or about 42:48:10. In some preferred embodiments, the weight ratio of total e< dpient to nucleic acid is less than about 15:1, for example, about 10:1, about 7.5:1, or about 5:1 hi some embodiments, an association complex described herein has a mean diameter or particle size of less than about 2500W m, e.g., fiom about 20 to 200 nma, about 60, or about 50 anm In some enibodiments, 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 hows, 32 e.g, a 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 i 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 phannaceutically acceptable composition comprising a liposomes described herein. 5 In one aspect, the invention features 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. Definiions The term "halo" or "halogen" refers to any radical of fhorine, chlorine, bromin 10 or iodine, The term alkyll" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, For exanipl, CC alkyl indicates that the group may have from I to 136 (inclusive) carbon atoms in it, The term. "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are 15 replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyb. The terms "arylalkyl" or "andlkyP 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, Fxamples of "arylalkyl" or "aralkyl" include benzyl, 2-phonylethyl, 3-phenylpropyl, 9 20 fluorenyl, benzhydryl, and trityl groups. The term "alkylene" refers to a divalent alkyl, eg -C1-, -CH 2 CU-, CIH-CILCHIr, - ClIZCHIACH 2 CHJ, -C1YCl 2

CH

2 CtCH 2 -, and CH2CHz2CHtCH2CICIr The term "alkeny]" refers to a straight or branched hydrocarbon chain contang 25 2-36 carbon atoms and having one or more double bonds. Examples of alkenyl groups include, but are not united to, allyl, propenyb, 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 33 bonds. Examples of alkynyl groups include, but are not limited to, etbynyl, propargyl, 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 "substitut-ed" on an alkyl, cycloalkyl, 5 alkenyl, aikynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at, any atom of that group. Any atom can be substituted. Suitable substituents include, without limitation, alkyl (e.g, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C)I, C.12 straight or branched chain alkyl), cycloalkyl, haloalkyl (e g. perfluoroalkyI such as CF3,), ayl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, 10 cycloalkenyi, heterocycloalkenyl, alkoxy, haloalkoxy (e,g, periluoroalkoxy such as OCF3), halo, hydroxy, carboxy; carboxylate, cyano, nitro, amino, alkyl amino, 50311, sulfate, phosphate, methylenedioxy (O-CH2~O- wherein oxygens are attached to same Carbon (gemi nal substitution) atoms), ethylenedioxy, oxo, thioxo (e.g,, C=S), imino alkyll, aryl, aralkyl), S(O),,alkyl (where n is 0-2), S(O), aryl (where n is 0-2) S(O),, 15 heteroaryl (where n is 0-2), S(O), heterocyclyl (where n is 0-2), amine (mono- di, alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof) 4 ester (alkyl, aralkyl, heteroaralkyl, aryl, hoteroaryl); amide (mono-, di-, alkyl, aralkyl, heteroaraiky!, aryl, heteroaryl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyli, heteroaralkyl, and combinations thereof). in one aspect, the substituents 20 on a group are independently any one single, or any subset of the aftrementioned 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 isopropyA alcohol, having the same 25 molecular fonnula but different structural 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., the connectivity between atoms is the sane), but which have different spatial amagements of the atoms, for example cis and trans isomers of a double bond, 30 enantiomners, and diasteriomers. For convenience, the meaning of certain terns and phrases used in the specification, examples, and appended claims, are provided below. If there is an 34 apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail. "O," "G "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively, However, it will be 5 understood that the term "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 uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide hearing such replacement moiety. For 10 example, without limitation, a nucleotide comprising inosine as its base may base pair with nuclotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine, Sequences comprising such replacement moieties are embodiments of the invention. 15 As used herein, "target sequence" refers to a contiguous portion ofthe nucleotide sequence of an mRNA molecule formed during the transcription of the corresponding gene, including mRNA that is a product of RNA processing of primary transcri ption product A target region is a segment in a target gene that is complementary to a portion of the RNAi agent, 20 As used herein, the tern "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 "complementaryj when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, 25 refers to the ability of an oligonucleotide or polynucleotide comprising the first 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 mM NaC, 40 mM 3o PIPES pH 6.4, 1 mM EDTA, 50"C or 70'C for 12-16 hours followed by washing, Other conditions such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions 35 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 polynucleotide comprising the 5 second nacieotide sequence over the entire length of the first and second nucleotide sequence; Such sequences can be referred to as "Uly 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 10 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, an oligonucleotide agent comprising is one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be refenqed to as "fully complementary" for the purposes of the invention. "Complementary" sequences, as used herein, may also include, or be formed 20 entirely from, non-Watson-Crick 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 "coniplementary", "fully complementary" and "suhstantiallv complementary" herein may be used with respect to the base matching between the 25 sense strand and the antisense stnd of an oligonucleotide agentor between the 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 RNA (mRNA) refers to a polynaeleotide which is 00 substantially complementary to a contiguous portion of the mRNA. of interest. For example, a polynucleotide is complementary to at least a part of an ApoB mRNA if the 36 sequence is substantially complementary to 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 (RNA) or deoxyribonucleic acid (DNA) or both or 5 modifications thereof, which is antisense with respect to its target. This term includes oligonucieOtides composed of naturalIy-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly, Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties 10 such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. Oligonucleotide agents include both nucleic acid targeting (NA.T) oligorncleotide agents and protein-targeting (PT) oligonucleotide agents. NAT and PT oligonucleotide agents refer to single stranded oligomers or polymers of ribonucleic acid 15 (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent intemucleoside (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 20 such as, for example, enhanced cellar uptake, enhanced affinity for nucleic acid target, and/or increased stability 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 include antisense RNAs, microRNAs> antagomirs and other non-duplex 25 structures which can modulate expression. Other NAT oligonucleotide agents include external guide sequence (EGS) oligonucleotides (oligozymes), DNAzymes, and ribozymes. The NAT oligonucleotide agents can target any nucleic acid, e.g., a miRNA, a pre-miRNA, a pre-mRNA, an riRNA, or a DNA. These NAT oligonucleotide agents may or may not bind via Watson-Crick complementarity to their targets, PT 3o oligonucleotide agents bind to protein targets, preferably by virtue of three-dimensional interactions, and modulate protein activity. They include decoy RNAs, aptamers, and the like. 37 While not wishing to be bound by theory, an oligonucleotide agent may act by one or more of a number of mechanisms, including a cleavage-depencent or eleavage independent mechanism. A cleavage-based mechanism can be RNAse I dependent and/or can include RISC complex function. (leavage-independent mechanisms include 5 occupancy-based translational arrest, such as can be mediated by miRNAs, or binding of the oligonuceotide 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 mRNA. inhibition of splicing can also result in degradation of the improperly processed message, thus down-regulating gene expression, 10 The.term "double-stranded RNA" or "dsRNA", as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti parallel and substantially complementary, as defined above, nucleic acid stands. 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, 15 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 therefore are connected by an uninterrupted chain of nucleotides between the 3-end of one strand and the 5'end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop", "short hairpin RNA" or "shRNA" Where the two 20 strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'end of the respective other strand fbrming the duplex structure, the connecting structure is referred to as a linerer, 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 25 minus any overhangs that are present in the duplex. In addition to the 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 nucleoddes and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in 30 an siRNA Type molecule, are encompassed by "dsRNA" for the purposes of this specification and claims. 38 As used herein, a "nucleotide overhang" refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3Tend 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 nacleotides at that end of the 5 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 arn siRNA has an overhang or is blunt ended. 10 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 15 sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, eg., within 6, 5, 4, 3. or 2 nucleotides of the 5' and/or 3' termims. 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 the antisense strand. 20 The terms "silence" and "inhibit the 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 front 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 tens of (mRNA in control cells) (mRNA in treated cells) (mRNA in control cells) Alternatively, the degree of inhibition may be given in terms of a reduction of a 30 parameter that is functionally linked to gene transcription, e.g, the amount of protein 39 encoded by the gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In principle, gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay, BHowever, when a reference is needed in order to determine whether R a given d.sRNA inhibits the expression of the gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such 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 o 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 sone embodiments, the gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide ofthe 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), the tenns "treat" "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or 20 reverse the progression of such condition. As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes which can be mediated by down regulating the gene on or an overt symptom of pathological 25 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, and may vary depending on factors known in the art, such as, eg. the type of pathological processes which can be mediated by down regulating the gene, the patient's history and age, the stage of pathological processes which can be mediated by down 30 regulating gene expression, and the administration of other anti-pathological processes which can be mediated by down regulating gene expression. An effective amount;,in the context of treating a subject, is sufficient to produce a therapeutic benefit. 'The term 40 "therapeutic benefit" as used herein refers to anything that promotes or enhances the welbbeing 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 life by any period of time; decrease or delay in the neoplastic development of 5 the disease; decrease in hyperproliferation; reduction in tumor growth; delay of metastases; reduction in the proliibration rate of a cancer cell, tumor cell, 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 attributed to the patient's condition. 10 As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an oligonucleotide agent and a pharmaceutically acceptable carrier. As used heren, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of an RNA effective to produce the inended pharmacological, therapeutic or preventive result, For example, if a given cinical 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 the 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 20 administration, of a therapeutic agent. Such carriers include, but are not limited to, saline, buffed saline, dextrose, water, glycerol, ethanol, and combinations thereof and are described in more detail below, The term specifically excludes cell capture medium, The details of one or more embodiments of the invention are set iorth in the accompanying drawings and the description below. Other features, objects, and 25 advantages of the invention will, be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF:DRAWINGS Fig. I depicts a bar graph comparing the efficacy of various ND98 compositions. Fig, 2 depicts a bar graph comparing the efficacy of various ND98 compositions. Fig 3 depicts a bar graph demonsrating the efficacy of a 6-taied isomer of ND98. 41.

Fig. 4 depicts a bar graph comparing the efficacy of association complexes prepared using two different procedures. Pig.S depicts varous PEG lipid moieties, including those having various chain lengths. 5 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. 8 is a flow chart of a process for making an association complex loaded with nucleic acid. 10 Fig, 9 are bar gtaphs depicting the efficacy of siRNAs with two targets, FVIl and ApoB. Pig. 10 is a flow chart of a process for making an association complex loaded with nucleic acid, Fig. ii is a bar graph depicting the effect of particle size of association 15 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 theranpeutics in unformiulated and formulated fnns. Fig, 13 is a bar graph comparing the efficacy of association complexes having PEG iipids with varied chain lengths. 20 DETAILED DESCRIPTION Lipid preparations and delivery systems useful to administer nucleic acid based therapies such as siRNA are described herein. Cationic Lipid compounds and lipid preparations Polyamn !ipidprparatin 25 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 complexed with a Factor VII-targeting siRNA and administered to an animal such as a mouse. The level of secreted serum Factor VII is then quantified (24 h post administration), where the degree of Factor VII silencing so indicates the degree of in vvo siRNA delivery. Accordingly, lipids providing enhanced in vivo delivery of a nucleic acid such as siRNA are preferred. In particular, Applicants 42 have discovered polyamines having substitutions described herein can have desirable properties for delivering siRNA, such as bioavailability, biodegradability, and tolerability [n one embodiment, a lipid preparation includes a polyamine moiety having a 5 plurality of substituents, such as acrylamide or acrylate substituents attached thereto. For example, a lipid moiety can include a polyamine moiety as provided below,

H

2 N N' h4 NH 2 H where one or more of the hydrogen atoms are substituted, for example wNith a substituent including a long chain alkyl, alkenyl, or alkyny moiety, which in some embodiments is 10 further substituted. X' and Xb are alkylene moieties. In some embodiments, Xa and Xb have the same chain length, for example Xa and Xb are both ethylene moieties, in other embodiments XV and Xb are of differing chain lengths, In some embodiments, where the polyamine includes a plurality of X* moieties, X'can vary with one or more occurrences. For example, where the polyamine is spermine, X" in one occurrence is i5 propylene, X" in another occurrence is butylenes, and X' 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 20 97%, 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 moreoflietero atoms 25 present on the substituent on the nitrogen of polyamine in some embodiments, a preparation comprises a compound of formula (1) or a pharmaceutically acceptable salt thereof, R,, N N NR R' 43 fonnula (1) each X" and Xb, for each occurrence, is independently Ci 6 alkylene; n is 0, 1, 2, 4, or 5; each R is independently H, RO S R O-r Y 0 R, R 5 R, Rd R, wherein at least n + 2 of the R moieties in at least about 80% of the molecules of the compound of formula (1) in the preparation are not H; m is 1, 2, 3 or 4; Y is 0, NRt, or 10 S; Ri1 is alkyl alkenyl or alkynyl; each of which is optionally substituted; and R2 is H, alkyl alkenyi or alkynyl; each of which is optionally substituted; provided that, if n =0, than at least n t 3 of the R 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 15 occurrence and X' is independent of X. For example, Where n is 2 X' can either be the same for each occurrence or can be different for each occurrence or can be the same for some occurrences and different for one or more other occurrences. X * is independent of Xregardless of the number of occurrences of XV in each polyamine derivative. Xa for each occurrence and independent of Xl, can be methylene, ethylene, propylene, 20 butylene, pentylene, or hexyl ene. Exemplary polyamine derivatives include those polyamines derived from N1N -(ethane-1,2-diyl)diethanei ,2-diamine, ethaneI2 diamine, propane-1,3-diamine, spermine, spermidine, putrecine, and N-(2 Amninoethyl-paropan-13-dianmine, Preferred polyamine derivatives include propane 1,3-diamine and N ,N-(ethane-l,2-diyl)diethane-1,2-diamine. 25 The polyamine of formula (Y) is substitute with at least n+2 R moieties that are not H In general, each non-hydrogen R moiely includes an alkyl, alkenyl, or lkynyl moiety, which is optionally substituted with one or more substituents, attached to a nitrogen of the polyamine derivative via a linker. Suitable linkers include aides, esters, thioesters, sulfones, sulfoxides, ethers, amines, and thioethers. In many ao instances, the linker moiety is bound to the nitrogen of the polyamnine via an alkylene moiety (emg methylene, ethylene, propylene, or butylene), For example, an amide or 44 ester linker is attached to the nitrogen of the polyamine through a methylene or ethylene moiety. Examples of preferred amine substituents are provided below: 0 0 5 N R 1RN and R in instances Where the amine is bound to the linkerR portion via an ethylene group, a 1,4 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 amnide or ester including an alpha-halo substituent, I such as an alpha-chloro moiety, can be reacted with the polyamine to provide the substituted polyamine, In preferred embodiments, R' is H R moieties that are not H, all require an R' moiety as provided above. In general, the R moiety is a long chain moiety, such as C'Ce alky, Cr03 alkenyl, or C'C1 alkynyt 15 fn some preferred embodiments, R' is an alkyl moiety, For example R is Cj Ca alkyl, such as Ca2 akyt Examples of especially preferred R moieties are provided below. NQ-,A H),,CH and (CH2,CH3 Ra The preparations including a compound of formula (I) can be mixtures of a 20 plurality of compounds of formula (I), For example, th e preparation can include a mixture of compounds of formula (I) 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 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 25 99%, or substantially all) of the molecules of the compound of formula (1) in the preparation are not IL In some embodiments, a preparation includes a polyamnine 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 45 substantially all) of the molecules of formula (1) in the mixture are substituted with tree R moieties that are not L Exemplary compounds of formula (I) are provided below. H N R and 1'N N'R i i H R R In some preferred embodirments R is 0 0 S or 1 tn some preferred embodiments, Ri isCwCia alkyl, or Cw o30 alkenyl. In some embodiments a preparation includes a polyamiine moiety having three or four (eg, 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 10 97%, at least about 98%, at least about 99%, or substantially all) of the molecules of fomula (I) are not . Exemplary compounds of fonula () having 4 amino moietie are provided below. Examples of polyamine moiety where all (i.e., n+4) R moieties are not H are below: R R R R i5 R R , In some preferred embodiments R is >4 R or O'Rk P IT some preferred embodiments, RI isCj)-C alkyl (e.g., C alkyl), or CwC alkenyl. 20 Examples ofpoiyamine moieties where five (i.e, n+3) R moieties are not H are provided below: P R H H N N and R'N . NA R R R RR . 1n some preferred embodiments R is 46 0 a A ~~N' W or 0K.'. R' In some preferred embodiments, R isCurC 15 alkyl (eg, C 12 alkyl), or Clo~C3 alkenyl. Examples of polyamine moieties where four (ie, n+2) R. moieties are not H are 5 provided below; H RR R' N N NH R N N'R R R H R H H i H R'N N N..R R N s-' -,N -- N'R H RR R R and i some preferred embodiments R is 'UWN R' or RI N" 10 In some preferred embodiments, R isCwOC alkyl (eg, C 2 alkyl), or Cl(rCae alkeniyl In some preferred embodiments, the polyamine is a compound of isomer (I) or (2) below, preferably a compound of isomer (1) H N o. H' isomer (1) N N' O N 15 A ~A'~. HP~N~~~ 4 N. isomer (2). 4'7 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. 5 In some embodiments, a preparation described herein includes a compound of fornnula (1) having a single polyanine core wherein each R of the polyamine core is either R or a single moiety such as R or R The preparation, therefore includes a mixture of molecules having fonula (I), wherein mo the mixture is comprised of either polyamine compounds of formula (1) having a varied number of R. moieties that are H. and/or a polyamine compounds of fonula (1) having a single detennined number of R moieties that are not H where the compounds of fonnula (1) are structural isomers of the polyamine, such as the structural isomers provided above, ms i some prefer-ed embodiments the preparation includes molecules of tormula (1) such that at least 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 97%, at lVast about 98%, at least about 99%, or substantially all) of the molecules are a single structural isomer. In some embodiments, the preparation includes a mixture of two or ire ac compounds of formula (I), In some embodiments, the preparation is a mixture of structural isomers of the same chemical fonula, In some embodiments, the preparation is a mixture of compounds of fornul a (I) where the compounds vary in the chemical nature of the R substituents, For example, the preparation can include a mixture of the fbllo-wing compounds: X" x*,

R

2 N Nj 'NR2 R, R 25n formula (I) wherein n is 0 and each R is independently H or F31 and 48

R

2 N1 N 'NR 2 R; formula (1) wherein n is 2 and each R is independently U or In soie embodiments, the compound of formula (1) is in the forn of a salt, such 5 as a pharmaceutically acceptable salt. A salt, for example, can be formed between an aion and a Positively charged substituent (e.g., amino) on a compound described herein. Suitable onions include fluoride, chloride, bromide, iodide, sulfate., bisulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, fumarate, oleate, valerate, maleate, oxalate, isonicotinate, lactate, salicylate, tatrate, tannate, 1 pantothenate, bitartrate, ascorbate, succinate, gentisinate gluconate, glucaronate, saccharate, fnnrate, benzoate, glutamate, ethanesulfonate, benzenesulfonate, p toluen-sulfonate, and pamoate. In some preferred embodiments, the compound of fornma (1) is a hydrohalide salt, such as a hydrochloride salt. Compounds of formula (1) can also be present in the form of hydrates (e.g, S (H2O)nI ) and solvates, which are included herewith in the disclosure. Biocleavable cationic lipidh Applicants have discovered that certain cationic lipids that include one or more biocleavable moieties can be used as a component in an association complex, such as a liposome, for the delivery of nucleic acid therapies (eg. dsRNA). For example, 20 disclosed herein are eationic lipids that are subject to cleavage in vivo, for example, via an enzyme such as an esterase, an ainidase, 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 25 esterase, anidase, or a disulfide cleaving enzyme. This can happen in vesicular compartments of the cell such as endosomes. Another acid sensitive cleavable linkage is p-thiopropionate linkage which is cleaved in the acidic environment of endosomes (Jeong et aC.~ioconjugate chem. 2003, 4, i426). 49 In. some embodiments, the invention features a compound of fbnmua (X) or a phannaceuticaly acceptable salt thereof, wherein RN R4 R2 LI forumila (X) S wherein R and R 2 are each independently H, CrCs alkyl, optionally substituted with 1-4 R, C 2 C alkenyl, optionally substituted with 1-4 R", or C(NR4)(NR 6 h; R- and RI are each independently alkyl, alkenyl, alkynly, each of which is optionally substituted with fluoro, chloro, bromo, or iodo; 10 L and L are each independently -NRC(O)-, -C(O)NR, -OC(O)-, -C(0)0- S-S-, -N(R)C()N(R)-, -OC(0)N(R), -N(R 6 )C(0)0-, -O-N=0-, OR --OC(O)NIH; or L -R 3 and 0-R 4 can be taken together to form an acetal or a ketal; R is fluoro, chioro, bromo, iodo -OR', -N(R)(R) -CN, SR' ,S(O)R' 0 , S(0)2R' 15 Ra is H, QC alkyl, T is 1 or Cj-C alkyl; each RW and R 9 are independently I or CC alkyl; Ri is H or Q-Cf, alkyl; 1 i 2,3, 4, 5, or 6; 20 n is 0, 1, 2, 3, 4, 5, or 6; and pharmaceutical acceptable salts thereof in some embodiments, R is H, a lower alkyl, such as methyl, ethyL propyl, or isopropyl, or a substituted alkyl, such as 2-hydroxyethyl. In some embodimentsR, R is H or a lower alkyl, such as methyl, ethyl, propyl, or 25 isopropyl. in some embodiments, RI or R 2 form a quanadine moiety with the nitrogen of formula (X). SR-i and 1!-R or the combination thereof provide at least one moiety that is cleaved in vivo. In some embodiments, both L"-R and 12-R' are biocieavable. For 30 example. both L'R 3 and 12-R. are independently subject to enzymatic cleavage (eg,, by an esterase, amidase, or a disulfide cleaving enzyme), In some embodiments, both 50 L and K are the same chemical moiety such as an ester, amide or disufide. In other instances, L and L2 are different, for example, one of LI or is an ester an the other of U or U is a disulfide. in some embodiments, C-R 3 and U-R together form an aectal or ketal moiety, 5 which is hydrolyzed in vivo. In some embodiments, one of L-R3 or 1-R 4 is subject to enzymatic cleavage. For example, one of '-R3 or L-R is cleaved in vivo, providing a free hydroxyl moiety or free amine on the lipid, which becomes available to chemically react with the remaining L-R 3 or L-R moiety. Exemplary embodiments are provided below:

R

1 X<m ; N .... 0 R ' 'R'x{ XN ORH R3 HH 0 -aC(Oy II - R 4

NH

2 X or NH 0 Y = 0 oNH Y'R r,'x11 V x LTh 0 H, 37 R X' XOH $~~R'N-- 10,4.AN? "___ ~ ~ S - RC(O} -RY X 0 or NH O Y 0 or' NH ha some preferred embodiments, a carbamate or urea 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 15 chemically react with the carbamate or urea moiety. Some preferred combinations of ' and 12 include two aides, two esters, an amide and an ester, two disalfides, an aide and a disulfide, an ester and a disuufide, 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) $1 Wn n R' R R H, R R'"' .~ ?4A N fl..

R ' R- N O R H Me Et, propyl isopropyt or 2-hydroxyethyl and R" = H; I to 6, m= 1-8, n= 140 R H, Me, Et, propyt isopropyl or 2-hydroxyethyl and R" = Me I to 6, = 14, n:z: I0 R' H, Me Et propyl, isopropyl or 2-hydroxyatnyl and R" = Et; I 1 to 6, m = 1-8, n = 1-10 R' M, Et, propyl, isopropyz or 2-hydroxyetnyl and R'= propyl; " to6, m = 1-8, n =1-1G R' H, Me, Et: propyl, isopropyl or 2-hydroxyethyl and R" isopropyl; I 1 to, = 14 = id Amide Ester linkage with Z configuration (three double bonds) N IN'\ 1 R;m R0 R F__ HN HN N ' N R':; H, Me, EtI propyl, isopropyt or 2-hydroxyethyl anid R*= H: 1 z to 6,;m n n 140J R'= H MeEI, propy isopropyl or 2-hydroxyethyl and R*= Me; I 1o 6, m I 4 n I10 R'= H Me, E1 prOpyl isoprpyt or 2-hydroxyethyl and RP= Et I = to a, m = -8 n 1-0 R H, Me E propyl, isopropyl or 2-hydroxyethyl and R*- propy ; i = Ito , m 1-& n = 140 R'= H, Me, E!, propyt: isopropyl or 2-hydroxyethyl and R" = spropyt = 1 to 6 1, n = 110 5 52 Aides and ester linkages with E configuration (two double bonds) RI 6 n R H O { R"' N N m RT -H Me [t xprop sopropyl or 2-hydroxyethyl and R" H: 1= 1 to 6, m = 148 n 1-10 RFT H, Me, El, propyl isopropyi or 2-hydroxyethyl and R"T' Me; I1 to 6, m = 14,. n =1-10 R': Ht Me, Et, propyL lsopropyl or 2-hydroxjethyi and R" Et; 1 to 6, m 1 ii 1-10 R'= H, Me, Et, propy. isopropyi or 2-hydroxyethyi and R" propy:; i i to 6, m =1- , n = 1-10 R = H, Me, Et, propyh Ispropyl or 2-hydroxyethyi and R" = its i = 16 Ito 4 , = 1- n 1-10 53 Amides and ester linkages with E configuration (three double bonds) nei N 0 H 04' R' N N HN H, l Mt Et, propyl, lsopropyl or 2-hydroxyethyl and R'= Me; 1 to 6, m= 1-8, n =-10 R'P= H, Me Et propyl, isopropyl or 24tydroxyethyl and RW E; i to 6 m 14 n =-1 R' H. Me Et propyl, isopropyl or 2tydroxythyl and R" El poy:I= to 6in 1-8 n 1 0 R it-H Me, Et, propytisopropylor2-hytoxycthyl and R' propyl; 'r I toO,mn -i, nn1-10 R' = 11, Me, Et propyl, isopropyl or 2-bydroxyethyl and R" isopropyl; I Ito 6, m= 1-, n I -10 5 Disnide linkages S K R"' R' = H, Me, Ft propyl, isopropy! or 2tydroxyethy and R H; I= I to 6, m 6-28 R' H, Me, Et, propyl' sopropyl or 2-4ydroxyethyl and R Me; I = 1 to 6, in =626 B =, Mo, Et, propy!' sopropyl or 2-inydroxyethyl and R Et I1 to 6, m 6-26 R' = , Ms Et, propyl. isopropyl or 2tydroxyelhyl and R propyl; I 1 to 6, m = 6-2 R' =H, Me, Et, propyt isopropyl or 2-hydroxyethyl and R isopropy; ! = I to 6, m = 6-28 54 Disulfide linkages with unsaturated alkyl chains, E and Z configuration m R' R"' R"' 0\/n R ,M R" N rn R eEl, propy% isopropyl or 2-hydrcxyeth3yi and R"= Me 1= Ito m 1 $,n 1-1 R H, Me, Et, propy , isocpropyl or 2-hydro xyo-thyl and R"= ME 1 to0 6, m = 1-8 n = 1-10 R7( H, Me, Et, propyl, isopropyl or 2-hydrayethyl and R"= zrpl I 1 o 6, m e 1-8, n 010 R H, me, Elt propy , isopropyl or 2-hydroxyeMyi and R" = iopropytl, to 5. m =, 14 r, 11 5 5 Anide and disuifide linkages with saturated and unsaturated alkyl chains R' HN ' S r. R, -S R' H, Me, Et, propy, isopropyl or 2Ahydroxyethyl and R" H; I= I to 6, mn 6-26 R' H, Me, Et propy, isopropyl or 2-hydroxyethyl and R" = Me; 1 to 6, m = 6-28 R' H, Me. Et, propyt, ;sopropyl or 2-hydroxyethy and R" = Et; I = 1 to 6, m = 6-28 R' H, Me, Et. propyl, isopropy or 2-hydroxyethyl and R' = propyi:I= I to 6, n 6-28 R'= H, Me, Et propyl, -sopropy! or 2-hydroxyethyl and R" = isopropyl; 1 1 to 6 m 6-28 R~ NS RH R" s R"' m R 'IN R' HN n Re s R S m RHN m R' HN R"'N s N S s R'= H, Me, Et, propy,, isopropy) or 2--hydroxyethyl and R" = H;R 1 to 6, m 1-8, n 1410 R'= H, Me, Et propyA sopropyi or 2-hydroxyethyl and R" = Mie; i =I to 6, mr = 1-8, n = 1-10 R'= Hz Me, Et, propyl, :Sopropyi or 2-hydroxyethy and R" = Et, I to 6, m =1-8, n = 1-10 R'z 114, Me, Et. propyl, zisopropyl or 2Zhydroxyethyl and R" = propy ; I* to k9, m = 1-5, n = 1 -10 RI = H, Mle, Et, propyl, isopropyl or 2-hyciroxyethy and R"'= iscpropy ; zz = 1 to C3, m = 1-8, n =1-10 56 3ster andl disulfide linkages with saturted and unsaturated alkyl chains R-N S I R"~- -4 m6, m R'= H, Me, Et, propyl isopropyl or 2-hydroxyethyl and R' H; 1 1 to 6, m = 6-28 R' H. Mde Et, propyt ?sopropyl or 2-hydroxyethyI and R?" =Me; 1 1 to 6, m= 6-28 R'= H, Me Et, propyt ?sopropyl or 2-hydroxyethy and R" a Et; I ito 6, m = 6-28 R'= H, Me Et, propyl. "Zopropyl or 2-hydroxyethyl and R" propyl; I to 6, = = 6-28 R H Me, Et, propyL sopropyl or 2-hydroxyethyl and R" isopropyl; I I to 6, mo 6-28 R" SnR"S R' IN' IN , R'N S

N

3

N

4 m. ni M?' ~ R H, Me ER, propyl isopropyi or 2 hydroxyethyl and R" = H; I=1 to 6l m 1-8 n 10V R H, M, Et, propyl, isopropyl or 2-hydroxyethyl and R" = foe; I =I to 6, m = 148 n = 1-10 R , H: Me Et propyl, isopropyl or 2-hydroxyethyl and R4 = Et; I = 1 to 6, m 1-8, q = 1 -10 'R' = H, le, Et propyl, Isoprouyl or 2-hydroxyethyl and4 R" = propyt i= to 6, mr,= 1-8, n = 1-10 R = H, Me; Et; propy! isopropyl or 2-hydroxyethyl ard R"=z isopropy',; I to 6, mn = 1-8, n = 1-10 57 Carbamate or urea and disulfide linkages with alkyl chains H H a NiO Ny O R' m R' HN m R' HN m R ' 'R" R" m R H Me, Et propyl, isopropyl or 2-hydroxyethy! and R" = H; I = 1 to 6 m = 28 R'= H, Me, Etz propyl, isopropyl or 2-hydroxyethyl and R" = Me; I1 to 6, m = 628 R= H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R" = Et; = to &, m = 6-2B FR H, Me, Et, propyt isopropyl or 2-hydroxyethyl and FR" propyli; I= S to 86 m 6 2 R'= H, Me, Et, propyl, isopropyl or 2-hydroxyethyl and R" =isopropyl; I = to 6, m = 6-2 58 Carbamate or urea and disulfide linkages with unsaturated alkyl chains R O 'mR m N AN - S, H.,, H N - N --- R' R"N"-s''D' R"'N H m R N m R 0 N R"' R"N $ m ' 0 hi HN Rl' HN R'N>- wN H RHN ~H R H M HN = 1 r RT In', Me, Et, propyla isopropy or 2-ydroxyethyl and R" = po; 1 t 6, m = 28 F' = Me, Et, propyl, isopropy or 2-hydroxyethyl and R" =H isopropy; I 1 to 6, = 6-2B 59 Carbamatc or urea and disuifide linkages with unsaturated alkyI chains CO O.ZVN / 0 R' HN R4 NW R N S I' R HN min R' HN n -s R" R S N1N R 1 H Fm HN n R" b ~RN' R' H, Me, Et, propyl, sopropyl or 2-hydroxyethyt and R= Me; I to 6, m 2 R' H, Me, Et, propyl, isopropy or 2-hydroxyethyl and R" = Et; I 1 to 6, m 6-28 R' h, Me, Et, propyl, isopropy or 2-hydroxyethyl and R" = propyl;I - 1 to 6, m 6-28 R' H, Me, Et, propyt, isopropyl or 2-flydroxyethyl and F'W isopropyl; i= 1 to 6, m z 6-28 60 Carbamate and urea linkages with unsaturated alkyl chains R rnR HN 'NR" NNN HH m N RH HmHHRH n R".N N N N NrR H thn R = 1 m NA N RI H, Me, Et propyl isopropyl of 2-hydroxyethyl and R =z Me; i = 1 to 6, m 1=10 M. r 110 R' H, Me, Et, propy sopropy or ydroxyethyl and R"= e; ' 1 to 6, m-= 1-10, n =-10 R H, Me, Et propy, isopropyl or 2-hydroxyethy and R" = f; I = I to 6, -, n 1-10 R H Me, Et propy, isopropyl or 2-hydroxyethyl and R" = propy;I= I to 6, mi 1-10, n 1-10 R' Li He. Pt propyl, Uopropyl or 2-hydroxyethyl and R' isopropy.1:1z I toG, rr" =- lkint 1-10 S i some embodiments, the lipid includes an oxime or hydrazone, which ca n undergo acidic cleavage. R3 and R" are gneally long chain hydrophobic e such as alkyl, alkenyl, or alkvnyt In some embodiments, R3 or R' are substituted with a halo moiety, for example, to provide a perfluoroalkyl or perfluoroalkenyl moiety, Each of R 1 and R" are o independent of each other. In some embodiments, both of R- and R are the same. In some embodiments, Re and R are different, In some embodiments R3 and/or RI are alkyl. For example one or both of RI and/or Ri are C 6 to Cio alkyl, e.g, Co to C2 alkyl, C2 to C 2 alkyt, or C, 2 alkyl. In some dmbodiments, R and/or R4 are alkenyl, In some preerred embodiments, It and/or R4 include 2 or 3 double bonds. For example R3 and/or R" includes 2 double bonds or R3 and/or Pt includes 3 double bonds, The double bonds can each independently have a Z or E configuration. Exemplary alkenyl noieties are provided below: 61 ,A~~ -~ -------- -- X~----, wherein x is an integer from I to 8; and y is an integer from 1- 0. In some preferred embodiments, R' and/or R4 are C6 to C30 alkenyl, elg, Ci to Cs alkenyl, Cn! to C2 5 alkenyl, or C alkenyl, for example having two double bonds, such as two double bonds with Z configuration, R 3 and/or R can be the same or different. In seme preferred embodiments, R' and R are the same. In soie embodiments, R. and/or R4 are alkynyl. For example Ce to Cao alkynyl, eg, Cm to C 26 alkynyl, Cir to C0 alkynyl, R and/or Re can have from I to 3 triple to toads, for example, one, two, or three triple bonds, In som embodiments, the compound of formula (X) is in the form of a salt, such as a pharnaceutically acceptable salt. A salt, for example, can be fonned between an anion and a positively charged substituent (e.g anino) on a compound described herein. Suitable anions include fluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate, phosphate, citrate, methanesulfiate, trifluoroacetate, acetate, tumarate, oleate, valerate, maleate, oxalate, isonicotinate, lactate, salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate, succinate, gentisinato, gluconate, glucaronate, saccharate, fornnate, benzoate, glutamate, ethanesulfonate, benzenesulfonate, p toluensulbnate, and painoate. In some preferred embodiments, the compound of 20 formula (X) is a hydrohalide salt, such as a hydrochloride salt. Compounds of fonnula (X) can. also be present in the fIni of hydrates (e.g., (H2O),) and solvates, which are included herewith in the disclosure. PEGipid 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 fornulated into an association so complex with a nucleic acid moiety, such as siRNA and administered to a subject, the 62 lipid provides enhanced delivery of the nucleic acid moiety This enhanced delivery can be determined, for example, by evaluation in a gene silencing assay such as silencing of FVII. In particular, Applicants have discovered the PEG-lipids of formula (XV) can have desirable properties for the delivery of siRNA, including improved bioavailability, 6 diodegradability, and tolerability. In some embodiment, the PEG is attached via a linker moiety to a structure inciding two hydrophobic moieties, such as a long ehanin alkyl moiety, Examplary PEG-lipids are provided above, for example, those encompassed by fimmda (XV), (XV'), and (XVI), In some preferred embodiments, the PEG-lipid has the structure 10 below: 0 0 N 0 N 0t N, wherein the preferred stereochemistry of the chiral center is 'R' and the repeating PEG moiety has a total average molecular weight of about 2000 daltons, Ih some embodiments, a PEG lipid described herein is conjugated to a targeting OH is moiety, e,g., a glycosyl moiety such as a AcHN in some embodiments, the targeting moiety is attached to the PEG lipid through a linker, for example a hinker described herein. Exemplary targeted PEG lipid compounds are compounds of fornnua (XXI), (XrX)(XX II), and (XXII') described herein. Methods of making such lipids are described, for example, in Examples 42 and 43, 20 Igelids of making cationic lipid compounds and cationic lipid containing preparations The compounds described herein can be obtained from commercial sources (e,g.. Asinex, Moscow, Russia; Bionet, Camelford, England; ChemlDiv, SanDiego, CA; Coigenex, Budapest, Hungary; Enamine, Kiev, Ukraine; IF Lab, Ukraine; 25 Interbioscreen, Moscow, Russia; Maybridge, Tintagel, UK; Specs, The Netherlands: Timtec, Newark, DE; Vitas-M Lab, Moscow, Russia) or synthesized by conventional methods as shown below using commercially available starting materials and reagents. 63 thods of making polyanine lipids In soni embodiments, a compound of formula (I) can be made by reacting a polyamnie of formula (Ill) as provided below H2NIX4 X'NH2 2:H 5 ' formula (III) wherein X', X. and n are defined as above with a 1,4 conjugated system of formula (IV) 0 1 formula (IV) wherein Y and R1 are defined as above to provide a compound of formula (I). In some embodiments, the compounds of formula (Ill) and (IV) are reacted together neat (i.e., free of solvent). For example, the compounds of formula (i1) and 15 () are acted together neat at elevated temperature (e.g., at least about 60 *C, at least about 65 'C, at least about 70 "C at least about 75 *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 fomula (Ill) and (IV) are reacted together with a solvent (e.g., a polar aprotic solvent such as acetonitrile or .DMF). For 2o example, the compounds of formula (I1) and (IV) are Teacted together in solvent at an elevated temperature from about 50 *C to about 120 *C. In some embodiments, the compounds of formula (Ill) and (IV) are reacted together in the presence of a radical quencher or scavenger (eg, hydroquinone). 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. The reaction can be 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 "radical quencher" or "radical scavenger" as used herein refers to a chemical moiety that can absorb fee radicals in a reaction mixture. Examples of radical quenchers/scavengers 64 include hydroquinone, ascorbic acid, cresols, thiamine, 3,5-Di-ert-butyl-4 hvdroxytoiuene. tert-Butyl~4~hydroxyanisole and thiol containing moieties, In some enibodinents, the compounds of fornnula (Ill) and (IV) are reacted together in the presence of a reaction promoter (e.g., water or a Michael addition 5 promoter such as acetic acid, boric acid, citric acid, benzoic acid, tosic acid, pentatluorophenol pictic acid aromatic acids, salts such as bicarbonate, bisulphate, mono and dithydrogen phophates, phenols, perhalophenols, nitrophenols, suiphonic acids, PTTS, etc.), preferably boric acid such as a saturated aqueous boric acid, The reaction conditions including a reaction promoter can be neat or in a solvent e.g., a polar to aprotic solvent such as acetonitrile or DIM. The reaction can be at an elevated temperature (eg, 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, accelerates/enhances the rate of reaction, i5 The ratio of compounds of formula (111) to formula (IV) can be varied, providing variability in the substitution on the polyamine of formula (111). 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)/fonula (IV) are selected to provide for products having a relatively high degree of substitution of the 20 fre amine (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 n is 0 in the polyanine of formula (IlI), and the ratio of compounds of formula (IIl) to compounds of fornula (IV) is from 25 about 1:3 to about 1:5, preferable about 1:4. In sone preferred embodiments, n is 2 in the polyamine of formula (Ill), and the ratio of compound of formula (III) to compounds of formula (IV) is from about 1:3 to about 1:6, preferably about 1:5. in some embodiments, the compounds of fbmula (III) and formula (IV) are reacted in a two step process. For example, the first step process includes a reaction 30 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) 65 and the second step process includes addition of about 0.8 to 1.2 nolar equivalent of compound of formula (IN) to the reaction mixture. Upon completion of the reaction, one or more products having formula (I) can be isolated from the reaction mixture. For example, a compound of fomula (I) can be 5 isolated as a single product (e.g., a single structural isomer) or as a mixture of product (e.g., a plurality of striutural 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 o 0 phase HPLC or RPIPLC), or moving bed chromatography, In some embodiments, a reaction product is purified to provide a preparation containing at least about 80% or 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 with an acid such as HO 15 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 colrrsponding free amine product, In some embodiments, a salt product can prevent or reduce the rate of formation of breakdown product such as N-oxide or N-carbonate formation relative to the corresponding free amine. In some embodiments, a salt 20 product can have improved properties for use in a therapeutic fornulation relative to the corresponding free amine. in some embodiments, the reaction mixture is further treated, for example, to purify one or more products or to remove impurities such as unreacted starting materials, In some embodiments the reaction mixture is treated with an immobilized 25 (e.g, polymer bound) thiol moiety, which can trap unreacted 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 unreacted acrylamide compounds. In some embodiments a reaction product can be treated with an immobilized 30 (e.g, polymer bound) isothiocyanate. For example, a reaction product including tirtiary amines can be treated with an immobilized isothiocyanate to remove primary and/or secondary amines from the product 66 In some embodiments, a compound of formula (1) can be made by reacting a polyamine of formula (HII) as provided below X" X H2Nf' NH, H! 5 formula (Ill) wherein X., X", and n are defined as above with a compound of foniula (VI)), 10 formida (VI) wherein Q is Cl, Br, or I, and Y and R' are as defined above. In some embodiments, the compound of formula (1Il) and formula (VI) are reacted together neat. In some embodiments, the compound of formula (IL) and formAa (VI) are reacted together in the presence of one or more solvents, for example a s polar aprotic solvent such as acetonitrile or DMF. In some embodiments, the reactants (Ibrtrnla (HII) and formula (VI)) are reacted together at elevated temperature (e~g., at least about 50 0C, at least about 60 *C, at least about 70 "C, at least about 80 "C, at least about 90 *C, at least about 100 "C). In some embodiments, the reaction mixture also includes a base, for example a 20 carbonate such as K20C3. In some embodiments, the reaction mixture also includes a catalyst. in some embodiments, the compound of ftrnula (VI) is prepared by reacting an amine moiety with an activated acid such as an acid anhydrate or acid halide (e.g, acid chloride) to provide a compound. of formula (VI). 25 The ratio of compounds of formula (111) to formula (VI) can be varied, providing variability in the substitution on the polyamine of fInnula (i), In general, polyamies having at least about 50% of the hydrogen. moieties substituted with a non-tydrogen moiety are preferred, Accordingly, ratios of compounds of formula (I1)/formula (VI) are selected to provide forproducts having a relatively high degree of substitution of the 67 free amino (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 n is 0 in the polyamine of formula (tII), and the ratio of compounds of formula (I1) 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 polyamine of formula (1il), and the ratio of compound of formula (Dl) to compounds of fonala (VI) is from about 1:3 to about 1:6, preferably about 1:5. In some enbodiments, the compounds of fonnula (Ill) and formula (VI) are to reacted in a two step process. For example, the first step process includes a reaction mixture having from about U 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 (VI) and the second step process includes addition of about 0.8 to 1.2 molar equvalent of compound of formula (VI) to the reaction mixture. 15 In some embodiments, one or more amine moieties of formula (Ill) arc selectively protected using a protecting group prior to reacting the polyamine of fonnula (ll) with a compound of formula (IV) or (VI), thereby providing improved saectivity in 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 20 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 (1) can be 5 isolated from the reaction mixture. For example, a compound of formla (I) 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 fomula (I)), In some embodinents, on or more reaction products can be isolated and/or purified using chromatography, such as flash chromatography, gravity chromatography (eg., 3o gravity separation of isomers using silica gel), column chromatography (e.g, nonal phase HP LC 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 68 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 with an acid such as HCI to prove an amine salt of the product (e.g, a hydrochloride salt). i sone embodiments a 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 the rate of formation of breakdown product such as N-oxide or N-carbonate formation relative to the corresponding free amine. In some enbodiments, a salt product can have improved properties for use in a therapeutic formulation relative to the 10 corresponding free amine, in some embodiments, a polyamine cationic lipid can be made in using a regioselective synthesis approach., The regioselective synthetic approach provides a convenient way to make site specific alkylation on nitrogen(s) of the polyarine backbone hait leads to synthesis of specific alkylated derivatives of interest. in general 5 a compound of formula (1) is initially reacted with a reagent that selectively reacts with primary anines 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 a target compound. After blocking terminal amines of a compound of ormula (1) one or more of the secondary anines could be selectively blocked with an orthogonal amine protecting groups by using appropriate molar ratios of the reagnt aid reaction conditions, Selective alkylations, followed by selective deprotection of the blocked anines and further alkylation of regenerated amiies and appropriate repetition of the sequence of reactions described provides specific compound of interest. For example, terminal amines of triethylenetetramine (1) is 25 selectively blocked with primary amine specific protecting groups (e.g., trifle oroacetamide) under appropriate reaction conditions and subsequently reacted with excess of orthogonal amine protecting reagent [(Boc) 2 0, for e.g.)J in the presence or a base (for e,g, diisopropylethylanii) to block all internal aines (e.g, Boc), Selective removal of the terminal protecting group and subsequent alkylation of the terminal amines, for instance with an acrylamide provides a filly tenninai amine alkyliated derivative of compound 1. Deblocking of the internal amine protection and subsequent alkylation with calculated amount of an acrylamide for instance yields a partially 69 aikylated product 7. Another approach to make compound 7 is to react terminally protected compound I with calculated amount of an orthogonal amine protecting reagent [(Boc) 2 , for e~g.)] to obtain a partially protected derivatives of compound 1. Removal of the terminal amine protecting groups of partially and selectively protected I 5 and subsequent alkylation of all unprotected amines with an acrylamide, for instance, yields compound 7 of interest. Medsho o making lipids having a bioclcavable moiety In sone embodiments, a compound of formula (X) can be made by reacting a compound of formula Rygr OH

R

2 OH formula (XI) with a compound of formula (XII) HO R 3 formula (XII) wherein R, R2 and R' are as defined above. In some embodiments, the compounds of formulas (XI) and (XII) are reacted in the presence of a coupling agent such as a carbodiimide (e.g., a water soluble carbodiimide such as EDCI). Other chemical reactions and starting materials can be employed to provide a 20 compound of formula (X) having two linking groups [) and It. For example, the hydroxyl moieties of fonnula (Xl) could be replaced with amine moieties to provide a precursor to aide or urea linking groups. Upon completion of the reaction, one or more products having formula (X) can be isolated from the reaction mixture. For example, a compound of formula (X) can be 2s 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 compouids of formula (X)). 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, nonnial 30 phase HPLC or RPHPLC), or moving bed chromatography. In some embodiments, a 70 reaction product is purified to provide a prepar nation containing at least about 80% of a. single comnund, 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 with an acid such as Bl' 5 to prove an amine salt of the product (e.g, a hydrochloride salt). I some embodiments a salt product provides improved properties, e.g, tor handling and/or storage, relative to the corresponding free amine product. In some embodiments, a salt product can prevent or reduce the rate of formation of breakdown product such as N-oxide or N-carbonate formation relative to the corresponding free amine, In some embodiments, a satt a product can have improved properties fot use in a therapeutic fonnulation relative to the corresponding free amine, Methods of making PEGipids The PEG-ipid compounds can be made, for example, by reacting a glyceride moiety (e.g., a dimyristyl glyceride, dipalmityl glyceride, or distearyl glyceride) with an i activating moiety under appropriate conditions, for example, to provide an activated inteniediate 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-lipid. For example, a dalkyiglyceride (e.g., dimyristyl glyceride) is initially reacted with NN disuccininidyl carbonate in the presence of a base (for e.g., triethylanilne) and 2f subsequent reaction of the intermediate formed with a PEG-amine (e.g,, mPEG2000

NE

2 ) in the presence of base such as pyridine affords a PEG-ipid of interest Under these conditions the PEG component is attached to the lipid moiety via a carbamrate 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, 25 dimyristoyi glyceride, dipalmitoyl gyceride or distearoyl glyceride) with succinic anhydride and subsequent activation of the carboxyl generated followed by reaction of the activated intermediate with a PEG component with an amine or a hydroxyl group, for instance, to obtain a PEG-ipid In one example, dimyristyl g 1 lyceride is reacted with succine anhydride in the presence of a base such as DMAP to obtain a heini-succinate so The free carboxyl moiety of the hemi-succinate thus obtained is activated using standard carboxyl activating agents sueh as HBTU and diisopropylethylamine and subsequent reaction of the activated carboxyl with mPEH2000-NHx for instance, yields a PEGlipid. In this approach the PEG component is linked to the lipid component via a succinare bridge. Association complexes 5 The lipid compoids and lipid preparations described herein can be used as a component in an association complex, for example a liposome or a lipoplex. 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 10 oligonucleotide agent capable of modifying gene expression by targeting and binding to a nucleic acid. An oligonucleotide agent can be single-stranded or doubic-stranded, and can include, e,g,, a dsiRNA, aa pre-mRNA, an mRNA, a nicroRNA (miRNA), a mi RNA precursor (pre-miRNA), plasmid or DNA, or to a protein. An oligonucleotide agent featured in the invention can be, e.g, a dsRNA, a microRNA, antisense KNA, antagomir, decoy RNA, DNA, plasmid and aptamer. Association complexes can include a plurality of components. In some embodiments, an association complex such as a liposomes can include an active ingredient such as a nucleic acid therapeutic (such as an oligonucleotide agent, e.g.. dsRNA), a cationic Iipid such as a lipid described herein. In some embodiments, the 20 association complex can include a plurality of therapeutic agents, for example tvo or three single or double stranded nucleic acid moieties targeting more than one gene or different regions of the same gene. Other components can also be included in an association complex, including a PEG-lipid such as a PEG-ipid described herein, or a structural component, such as cholesterol. In some embodiments the association 25 complex also includes a fusogenic lipid or component and/or a targeting molecuIe in some preferred embodiments, the association complex is a liposone including an oligonucleotde 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-lipd of fonnula (XV), and a structural component such as cholesterol. 30 Sngl Stranded ibonu cleid acid OHigonucleotide agents include ndcroRNAs (miRNAs), MicrolRNAs are small noncoding RNA molecules that arc capable of causing post-transcriptional silencing of 72 specific genes in cells such as by the inhibition of translation or through degradation of the targeted mlRNA, An miRNA can be completely complementary or can have a region ofnoncomplementarity with a target nucleic acid, consequently resulting in a "buige" at the region of non-complementarity. The region of noncomplementarity (the bulg&) can be flanked by regions of sufficient comrplementarity, preferably complete complemetarity to allow duplex formation, Preferably, the regions of complementarity ire a east 8 to 10 nucleotides long (eg., 8, 9, or 10 nucleotides long) A miRNA can inhibit gene expression by repressing translation, such as when the micrORNA is not cnmpletely complementary to the target nucleic acid, or by causing target RNA 0 degradaton, which is believed to occur only when the miRNA binds its target -with perfect complementarity. The invention also can include double-stranded precursors ot miRNAs that may or may not fbrm 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-miRNA, The oligorucleotide agent featured in the e invention can include naturally occurring nucleobases, sugars, and covalent intenuclkoside (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 the endogenous 20 miRNA tuget, and/or increased stability in the presence of nucleases. An oligonucleotide agent. designed to bind to a specific endogenous miRNA has substantial complementanty, e~g., at least 70, 80, 90, or 100% complementary, with at least 10, 20, or 25 or more bases of the target miRNA. A miRNA or pre-miRNA can be 18-100 nucleotides in length, and more 25 preferably fom 18-80 nucleotides in length. Mivature miRNAs can have a length of 19 30 nueleotides, 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 fom pre-miRNAs by enzymes called Dicer and Drosha that specifically process long pre-miRNA into -o functional miRNA. The nijcroRNAs 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 73 characteristic, For examip le, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cel-type, or celi penneability, e.g,, by an endocytosis-dependent or -independent medanism, Modifications can also increase sequence specificity, and consequently decrease off-site a targeting. Methods of synthesis and chemical modifications are described in greater detail below. Given a sense strand sequence (e,g, the sequence of a sense strand of a cDNA molecule). an niiRNA can be designed according to the rules of Watson and Crick base pairing. The miRUNA can be complementary to a portion of an RNA, e g., a miRNA, a pre-rmiRNA, a pre-mRNA or an niRNA, For example, the miRNA can be complementary to the coding region or noncoding region of an mRNA or pre-mRNA, e.g ite region surrounding the translation start site of a pre-mRNA or mRNA, such as the 5' JUR. An miRNA oligonucleotide can be, for example, from about 12 to 30 nucleotides in length, preferably bout 15 to 28 nuel cotides in length (6,I14 17, 18, 15 19,.20 21, 22, 23,24, or 25 nucleotides in length). in particular, an miRNA or a pre-miRNA featured in the invention can have a chemical modification on a nucleotide in an internal (Ix., non-terminal) region having non complementarity with the target nucleic acid. For example, a modified nuIeotide can be incorporated into the region of a mniRNA that orns a bulge. The modification 20 can include a ligand attached to the miRNA, e.g., by a linker (eag, see diagrams OTIl through OT-iV below). The modification can, for example, improve pharmacokinetics or stability of a therapeutic aiRNA, or improve hybridization properties (e.g:, hybridization thennodynamics) of the riRNA to a target nucleic acid. In some embodiments, it is preferred that the orientation of a modification or ligand incorporated 25 into or tethered to the bulge region of a miRNA is oriented to occupy the space in the 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 buge. The intercalator can be an aromatic, e g, polycyctic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking 30 capabilities, and can include systems with 2, 3, or 4 fased 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 onentaton facilitates the improved hybridization properties or an otherwise desired characteristic of the miRNA, i1 one embodiment, an miRNA or a pre-niRNA can include an aminoglycoside 5 ligand, which can cause the miRNA to have improved hybridization propertes or improved sequence specificity. Exemplary aminoglycosides include glycosylated polyysine; galactosylated polylysine; neomycin B; tobramycin; kanamycin A; and acridane conjugates of aminogl ycosides, such as Neo-N-aeridine, NeoS-acridine, Neo C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can S incase sequee s specificity. For example, neomycin B has a high affiity fr RNA as comparedto DNA, but low sequcnce-specificity, An acridine analog, neo-S-acridine has an increased affinity for the H Rev-response element (RRE). In some embodiments the guanidine analog (the guanidinoglycoside) of an arninoglycoside ligand is tethered to an oligonucleotide agent. In a guanidinoglycoside, the amine group 5 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, the cleaving group is tethered to the miRNA in a manner such that it is positioned in the blige 20 region, where it can access and cleave the target RNA. The cleaving group can be, for example, a blcomycin (eg bleamnycin-As, bleomycin-A 2 or bleumycin-BS)1 pyrene, phenanthroline (eOg O-phenanthrolinc), a polyamine, a tripeptide (eg. lys-tyr-Iys tripeptide), or metal ion chelating group. The metal ion chelating group can include. e.g, an iiLu(i) or 'EU(11) macrocyclic complex, a Zn(II) 2,-dimehlphenanthroline as derivative, a Cu(ii) terpyridine, or acridine, which can promote the selecdve cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III) In some embodiments, a peptide ligand can be tethered to a miRNA or a preomiRNA to promote cleavage of the target RNA, e.g., at the bulge region. For example, I ,8dimethyl 1.3 ,8,10,3~hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (eg., by 30 an amino acid derivative) to promote target RNA cleavage. The methods and compositions featured in the invention include niiRNAs that inhibit target gene expression by a cleavage or non-cleavage dependent mechanism, 75 An miRNA or a pre-miRNA can be designed and synthesized to include a region of noncomplementarity (eg. a region that is 3, 4, 5, or 6 nucleotides long) flanked by regions of suftcient complementarity to form a duplex (e.g. regions that are 7, 8 9,10, or 11 n'ucleotides long), For increased nuclease resistance and/or binding affinity to the target, the miRNA sequences can include 2'-O-methy, 2'-fluorine, 2O-rmethoxyethyl, 2'-O aminopropyl, 2T-amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), 2-thiopyrimidines (eg, 2-thio-U), 2-amino-A, G-clamp modifications, and ethylene nucleic acids (ENA), e g.2' 4'-ethylene-bridged nucleic acids, can also increase finding affnity to the target. 'he inclusion of ftranose sugars in the oligonucleotide backbone cai also decrease endonucleolytic cleavage. An miRNA or a pre-miRNA can he further modified by including a 3 cationic group, or by inverting the nu'cleoside at the 3-terminus with a 3I3* linkage In another ahernative, the T-terminus can be blocked with an aminoalkyl group, eg:, a 3' C5-aminoalkyl dT. Other 3' 15 conjugates can inhibit 3' exonucleolytic cleavage, While not being bound by theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3-5exonucleases. 2o The 5 -terminus can be blocked with an aminoalky group) e?4, a 50 alkylamino substituent. Other 5' conjugates can inhibit 5'3'exonucleolytic cleavage. While not being bound by theory, a S' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding, to the ' end of oligonuclotide. Even small alkyl chains, ary] groups, or heterocyclic 25 conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5' exonucleases, In one embodiment, an miRNA or a pre-miRNA includes a modification, that improves tagcting, e.g a targeting modification described herein. Examples of modifications that target miRNA molecules to particular cell types include carbohydrate 30 sugars such as galactose, N-acetylgalactosamine, nannose; vitamins such as folates; other ligands such as RGDs and ROD mimics; and siall molecules including naproxen, ibuprofen or other known protein-binding molecules. 76 An niRNA or a pre-miRNA can be constructed using chemical syrthesis 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 5 stability of the molecules or to increase the 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 a nucleic acid has been subcloned in an antisense orientation (i.e RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Antisense- type 01aonucleotideAgents The single-stranded oligonucleotide agents featured in the invention include antisense nuceic acids. An "antisense" nucleic acid includes a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a gene expression product, e,g, complementary to the coding strand of a double-stranded CDNA molecule or complementary to an RNA sequence, e.g, a pre-mRNA, mRNA, miRNA, or pre 20 miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target. Given a coding strand sequence (eg, 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 complementary to a 25 portion of the coding or noncoding region of an RNA, e~g, a pre-nRNA or. mRNA, For example, the antisense oligonucleotide can be complementary to the region sun-ounding the translation start site of a pre-mRNA or mRNA, e.g., the 5' UTR. An antisense uligonucleotide can be, for example, about 10 to 25 nucleotides in length (e.g., 1, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). An antisense 30 oligonucleotide can also be complementary to a miRNA or pre-miRNA. An antisense nucleic acid can be constructed using chemical synthesis and/or enzymaic ligation reactions using procedures known in the art, For example, an 777.

antisense nucleic acid (eg., an antisense oligonuldeotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and target nucleic acids, e.g., phosphorothioate 5 derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein, Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subeloned in. an antisense orientation (Ace, RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest), 10 An antiseuse agent can include ribonucleotides only, deoxyribonucleotides only (eg., oligodeoxynucleotides), or both deoxyribonucleotides and ibonucleotides. 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 1 deoxyribonucleotides, 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 an enzyme, e. RN Ase H. Degradation of the target RNA prevents translation. The flanking RNA sequences can include 2'O-methylated nucleotides, and phosphoothioate linkages, and 20 the internal DNA sequence can include phosphorothioate internucleotide linkages. The internal DNA sequence is preferably at least five nucleotides in length when targeting by RN Asei activity is desired. For increased nuclease resistance, an antiscise agent can be further modified by inverting the nucleoside at the 3terminus with a 3'-3 linkagein another alternative. us the 3tterninus 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. Dcoytype li&oeleotide Agns 3Q An oligonulclotide agent featured in the invention can be a decoy nuclei 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, 18 For example, a decoy RNA can mimic the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. The natural binding target can be an endogenous nucleic acid, e:g. a pre niRNA, miRNA, prermRNA,. mRNA or DNA, For example, it has been shown that 5 over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently bind H IV tat 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 e, g- a targeting noditication described herein, 10 The chemical modifications described above for miRNAs and antisense RNAs, and described elsewhere herein, ar also appropriate for use in decoy nuclei acids. ApggnatyeQijgonucleotideAgents An oiigonucleotide agent featured in the invention can be an aptamer. An atamter binds to a non-nucleic acid ligand, such as a small organic molecule or protein, 5 e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity. An aptamer can ild into a specific structure that directs the recognition of the targeted. bin ding site on the non-nucleic acid ligand, An aptamer can. contain any of the modifications described herein, in one embodiment, an aptamer includes a modification that improves targeting, 20 e.g. a targeting modification described herein. The chemical modifications described above for miRNAs and antisense RNA;s, 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 the description below, Other features and advantages of 25 the invention will be apparent from the description and drawings, and from the claims. This application incorporates all cited 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 30 modified oligonucleotides that target a microRNA, An antagomir consisting essentially of or comprising at least 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA and 79 more particulady agents that include 12 or more contiguous nucleotides substantially complementary to a target sequence of an miRNA or pre-iniRNA nucleotide sequence, Preferably, an antagomir featured in the invention includes a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 12 to 25 6 nucleotides, preferably about 15 to 23 nucleotides. More preferably, the target sequence differs by no more than 1, 2, 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, eg., a cholesterol moiety. The non rnuceotide moiety can be attached, e.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 the ligonucleotide asaget. Antagomirs are stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nudelotide modification. In another embodiment, the antagomir includes a phosphorothioate at at least the first, second, or third 15 iternucleotide linkage at the 5' or 3' end of the nucleotide sequence. in yet another em nbodinent, the antagoir includes a 2T-modified aucleotide, e.g., a 2%deoxy, 2 deoxy-2 fluoro, TD-methyl, 2-O-methoxyethyl (2-O-MO E 2'4O-aminopropyl (2-0 AP),2 0-diimethylainoethyl (2t-O-DMAGE), 2UQ~dimethylaminopropyl (2'0 DMAP), 2'O-dimethylaminoethyloxyethyl (2O-DMAEOE), or 2'-O-N 20 methylacetamido (2O-NMA). In a particularly preferred embodiment, the antagonir includes at least one 2'O-methyl-modified nucleotide, and in some embodinents, all of the nucleotides of the antagomir include a 2-methyl modification, An antagomir that is substantially conplementary to a nucleotide sequence of an miRNA can be delivered to a cell or a human to inhibit or reduce the activity of an 25 endogenous niRNA, 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 mnit- 122 (sec Table 1), which hybridizes to numerous RNAs, including aldolase A mRNA, N-myc 3o downstran regulated gene (Ndrg3) mRNA, IQ motif containing OTPase activating protein- i (Iqgapi) mRNA, HMG-CoA-reductase (Hmgcr) mRNA, and citrate synthase mRNA and others. In a preferred embodiment, the antagonir that is substantially 80 complementary to miR-122 is antagomir- 122 (Fable 2a-e), Aldolase A deftciencies have been found to be associated with a variety of disorders, including henolytie anemia, arthrogryposis complex congenia, pituitary ectopia, rhabdomyolysis, hyperkalemia. Humans suffering ftom aldolase A deficiencies also experience 5 symptoms that include growth and developmental retardation, midfacial 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. SDouble-strnded-riboueleic acid (JRNA) In one embodiment, the invention provides a double-stranded ribonucleic acid (daRNA) molecule packaged in an association complex, such as a liposame, for inhibiting the expression of a gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of coniplementarity which is complementary to 1a 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 RNA strands that ire sufficiently complementary to hybridize to forn a duplex structure. One strand 20 of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially conplementary, and generally fuly complementary, to a target sequence, derived ftom the sequence of an 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 a conbined 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 conplermentarity 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 19 and so 21 nucleotides in length, The dsRNA of the invention may furth er 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 81 attonted DNA synthesizer, such as are commercially available -from, for example, Bliosearch, 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 p pairs). :n some embodiments, the dsRNAs include at least one strand that is at least 21nt long lI other embodiments, the dsRNAs include at least one strand that is at least 15, 16, 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 10 embodiment, the dsRNA contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 15 nucleotide from citherthe 5' or 3' end of the region of complementarity, in one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally I or 2 nucleotides. Generally, the single stranded overhang is located at the 3'-terminal end of the antisense strand or, alternatively, at the 3,terminal end of the sense strand, The dsRNA may also have a 20 blunt end, generally located at the 5'-end of the antisense strand, Such dsRNAs have improved stabilityand inhibitory activity, thus allowing administration at low dosages, ie, less than 5 mg/kg body weight of the recipient per day. Generally, the antsense strand of the dsRNA. has a nucleotide overhang at the 3-end, and the 5'-end is blunt, in another embodiment, one or more of the nucleotides in the overhang is replaced with a. 25 nucieoside thiophosphate. hi yet another embodiment, a dsRNA packaged in an association complex, such as a liposome, is chemically modified to enhance stability, Such nucleic acids may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, St et at. (Edrs,), 3o John Wiley & 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 82 non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with 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-known 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 fbr packaging in the association complexes described herein. Generally, the chemical groups that can be used to modify the dsRNA include, 10 without limitation, methylene blue; bifunctional groups, generally bis-(2 chloroethyi)amine; N-acetyl-N'-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the ds.RNA are produced by solid phase synthesis and the bexa-ethylene glycol linker is incorporated according to standard methods (e.g, Williams, DJ., and K.B. Hail, i Bfochem, (1996) 35;14665-14670). In a particular embodiment, the 5'-end of the antiscnse strand and the 3-end of the sense strand are chemically linked via a hexacethylene glycol irker. In another embodiment, at least.ne ucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds. 20 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 celluar enzymes against nicleic acids are known in the art including, but not limited to, 2'-amino modifications, 2T-amino sugar 2 - modifications, 2'-F sugar modifications, 2T'F modifications, 2'-alkyl sugar modifications, 2'-O-akoxyalkyl modifications like 2'-O-methoxyethyl, uncharged and charged backbone modifications, morpholino modifications, 21-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:11 16-8), Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA is replaced by a 3o chemical group, generally by a 2-F or a 2-0-methyl group, Also, at least one nucleotide may be modified to forn a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2T-oxygen of ribose with the 4-carbon of 83 ribose. Oligonucleotides containing the locked nucleotide are descnbed in Koshikin, AA, et 0., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al, Tetrahedron Let. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide iaproves the affinity for complementary sequences and increases the melting 5 temperature by several degrees (Braasch , DA. and DR, Corey. Chem. BloL (2001), $: I 7), Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRN.A to facilitate direct 10 permeation of the cell ular membrane and or uptakce across the liver cells. Altemnatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell penneation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are "5 substantially more active compared to their non-conjugated analogs. See IM, Manoharan Antisense & Ncleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include I -pyrene butyric acid, 1.,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor mediated endocytosis is folic acid. Folie acid enters the cell by folate-receptor-nediated 20 endocytosis dRNA compounds bearing folic acid would be efficiently transported into the cell via the foiate-reeptor-mediated endocytosis. Li 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.; Deshmukh, IL M; Huang, L Phar'n. Res. 1998, 15, 1540, Other ligands that have been conjugated to 25 oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, pophyrin conjugates, delivery peptides and lipids such as cholesterol. Other chemical modifications for siRNAs have been described in Manloharar, M. RNA interference and chemically modified small interfering RNAs. Current Opinion in Chemical Biology (2004), 8(6), 570-579, ao In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of eationic ligands are propylammonium and dim ethylpropylammonium. Interestingly, antisense 84 oligonucleotides were reported to retain their high binding affinity to niRNA when the cationie ligand was dispersed throughout the oligonucleotide. See M. Mancharan Antisense & Nucleic Acid Drug Development 21)02, 12, 103 and references therein. The iigand-conjugated dsRNA of the invention may be synthesized by 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 bearing 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 10 conjugated dsRNA by t use of, in some preferred embodiments, nueleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such IigWI&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 1s moiety located on the 5' position of a nucleoside or oligonucleotide. In certain instances, a dsRNA bearing an aralkyl ligand attached to the 3 terminus of the dSRNA is prepared by first covalently attaching a monomer building block to a controlled-pore glass support via a long-chain aninoalkyI group. Then, nueleotides are bonded via standard solid-phasesynthesis techniques to the monomer building-block bound to the 20 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 may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such syntheis is sold by several vendors including, for example, Applied 25 Biosystems (Foster City, CA). Any other means for such synthesis known. inthe art may additionally or alternatively be employed, It is also known to use similar techniques to prepare other oligonueleotides, such as the phosphorothioates and alkylated derivatives. Teachings regarding the synthesis of particular modified oligonuleotides may be found in the following U.S. patents: U.S, Pat. Nos 5,138,045 and 5,218,105, drawn :30 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. ss Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No, 5,386,023;, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive, coupling; U.S. Pat No. 5,457,1 91, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases 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 f$. lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the ao 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 moieies attached at any of a varety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothinate linkages of high chiral purity; U.S. Pat. No, 5,506,351, drawn to processes for the preparation of 2U0-alkyi is guianosin and related compounds; including 2,6~diaminopurine compounds; U.S. Pat No, 5,5871469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat, No. 5,587,470, drawn to oligonuieotides having 3-deazapurines; 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, and 5,610,289, drawn to backbone-modified oligonucleotide 20 analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2'tfluoro-oligonucieotides In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligoiucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside 25 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 nueleotide-conjugate precursors that already bear a Linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the sM ligand mioeeule is then reacted with the linking moiety to form the ligand-conjugated oligonueleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see 86 Mancoharar et al, PCT 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 conjugates in addition to the standard phosphoramidites and non-standard phosphoramsidites that are a commercially available and routinely used in oligonucleotide synthesis. The dsRNAs packaged in the association complexes described herein can include one or more modified nucleosides, e,g., a 2-0-methyl, 2O-ethyl, 2-0-propyl, 2tO-allyt. 2O-aminoalky! or 2'-deoxy-2-fluoro group in the nucleosides. Such modifications confer enhanced hybridization properties to the oligonucieotide. Further, 1s oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, flnctionalized, linked nucleosides can be augmented to include either or both a phosphorothioate backbone or a 2"0-methyl, 2-0-ethyl, 2-0-propyl, 20 aminoalkyl, 2T-0-allyl or 2deoxy-2'-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT 15 Publication WO 200370918, in sonie embodiments, functionalized nucleoside sequences possessing an amino group at the 5teninus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccininide esters, 0 etrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters, The reaction of the amino group and the active ester produces an oligonucleotide in whicli the selected ligand is attached to the 5-position through a linking group. The amino group at the terminus can be prepared utilizing a S-Amino-Modifier C6 reaget in one embodiment, ligand molecules may be conjugated to oligonucleotides at 25 the 5-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5-hydroxy group directly or indirectly via a linker, Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5-tenninus. Examples of modified internucleoside linkages or backbones include, for so example, phosphorothiontes, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyiphosphotri esters, methyl and other alkyl phosphonates including 3alkylene phosphonates and chiral phosphonates, phosphinates, 87 phosphoramnidates including Y-amino phosphoranidate and aminoalkyiphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, tionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 25' linked analogs of these, and those having inverted polarity wherein die adjacent pairs of s nucleoside units are Iinked 3Q5' to 5'3' or 2'5' to 5!2, Various salts, mixed salts and free-acid forms are also included. Representative United States Patents relating to the preparation of the above phosphoms-atom-containing 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; 10 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5;455,233; 5466,677; 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 (i.e., oligoniuceosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar likages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages, These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and R sulRfone backbones; fonnacetyl and thiofonnacetyl backbones; methylene fonmacetyl and thiofomiacetyIl backbones; alkene containing backbones; sulfamate backbones; Inethylenelinino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH component parts. 25 Representative United States patents relating to the preparation of the above eligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,.34,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,164,562; 5,264,564; 5,405,938; 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; so 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 a nonligand group. A number of nonligand molecules have been cnjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligoncleozide, and procedures for 5 performing such conjugations are available in the scientific literature, Such non~ligand moieties have included lipid moieties, such as cholesterol (Letsinger et at, Proc. Nail. Acad. Sci, USA, 1989, 86:6553), cholic acid (Manoharan et aL, Bioorg. Med. Chem. Lett- 1994, 4.:1053), a thioether, e.g., hexyl-S-tritylthiol (Manolaan et at, Ann. NY. Acad. Sei, 1992, 660:306; Mancharan et al., Bioorg. Med. ChemI, Let., 1993, 3:2765), a Wo thiocholesterol (Oberhauser et at, Nucl Acids Res., 1992, 20:533), an aliphatic chain, e g. dodecandiol or undecyl residues (SaisonBehmoaras et al, El130 J3, 1991, 10:111; Kabanov et aL, FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochinie, 1993, 75:49), a phospholipid, e.g., di-hexadecybraclycerol or triethylammoniim 1,2~di-0 hexadecylraglycero-3-phsphonate(Manoharan et at, Tetrahedron Lett, 1995, 5 36:3651; Shea et al, Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al,, Nucleosides & Nucleotides' 1995, 14:969), or adamantane cetic acid (Manoharan et al, Tetrahedron Lett, 1995, 36:3651), a pahnityl mnoi.ety (Mishra et at, Biochim. Biophys. Acta, 1995, 1264,229), or an ociadecylanine or hexy-amino-carbonyl.'xcholsterol moiety (Crooke et at, J. Pharmacol. Bxp, 'Ter., 2o 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugaion protocols involve the synthesis of oligonueleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conIjugaed using appropriate coupling or activating reagents. The conjugation reaction 25 may be perfomd either with the oligonucleotide still bound to the solid support or following cleavage of the 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 oligonueleotide agent as described herein, 30 Fusoenic pids The term "fusogenic" refers to the ability of a lipid or other drug delivery system to fise with membranes of a cell. The membranes can be either the plasma membrane or 89 membranes surrounding organelles, e.g., endosome, nucleus, etc. Examples of suitable fusogenic lipids include, but are not limited to dioleoylphosphatidylethanolainine (DOPE), DODAC, DODMA, DODAP, or DLinDMA. In some embodinents, the associaton complex include a small molecule such as an uidzole mody conjugated to 5 a lipid, fr example, for endosomal release. PEG or PEG-lipids In addition to cationic and fisogenic lipids, the association complexes include a bilayer stabilizing component (BSC) snch as an ATTN-lipid or a PEG-lipid. Examplary 0 lipids are as follows: PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., WIO 05/026372, PEG coupled to diacylglycerol (PEG-DAD) as described in, e.g., U., Patent Publication Nos. 20030077829 and 2005008689), PEG coupled to phosphatidyiethanolamine (PE) (PEG-PE), or PEG conjugated to ceramides, or a mixture thereof (see, U. Pat, No. 5,885,613). In a preferred embodiment, the 5 association includes a PEG-lipid described here, for example a PEG-lipid of formula (XV), (XV') or (XVI). In one preferred embodiment, the BS( is a conjugated lipid that inhibits aggregation of the SPLPs. Suitable conjugated lipids include, but are not limited to PEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (GPLs) or mixtures thereof. in one preferred embodiment, the SPLPs comprise either a 20 PEG-lipid conjugate or an ATTA-ipid conjugate together with 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 2,00 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons, 25 PEGs are commercially available frm Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEG-O H), mnonmethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succiimidyl succinate (MePEG-S-NHS), monomethoxypolyethyiene glycol amine (McPEG-NHsub.2), monomethoxypolyethylene glycol-tresylate (MePEG 30 TRES), and mnomethoxypolyeth(ylene glycol-imidazolylcarbonyi (MePEG-IM). In addition, monomethoxypolyethyleneglycol-acetio acid (MePEG-CH.sub.2COOH), is 90 particular useful for preparing the PEG-lipid conjugates including, e.g.. PEG-DAA 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 dal tons. The PEG can be optionally substituted by an alkyl, alkoxy; 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 10 coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-conaining linker moieties. In a preferred embodiment, the linker moiety is a non-ester containing linker moiety, As used herein, the term -"non-ester containing linker moiety" refers to a linker moiety that does not contain a carboxylic ester bond (--OC(O)-). Suitable non-ester containing linker moieties include, but are 15 not limited to, anido (-C(0)NH-), amino (--NR-), carbonyl (-C(0)~~), earbamate ( NHC(Q)O-) urea (--NHC(O)NH-), disuiphide (--S-S-), ether (--0-), succinyl ( (0)CCH-sub 2C ub;2C(O)-),succinamidyl (--NIHC(O)CH. sub.2CH, sub 2C(0~ )NH ), ether, disuiphide, etc. as well, as combinations 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--), succinoyl, phosphate esters (-O--(OOH-0-), sulfonate esters, and combinations thereof, WJgettq& mgpts n some embodiments, the association complex includes a targeting agent. For example, targeting agent can be included in the surface of the association complex (e.g, liposome) to help direct the association complex to a targeted area of the body. 3 An example of targeting agents galactose, mannose, and folate, Other examples of targetig agents include sinai molecule receptors, peptides and antibodies. In some embodiments, the targeting agent is conjugated to the therapeutic moiety such as 91 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, hi some embodimentst, he targeting agent is 5 unconjugated, for example on the surface of the association complex. Structural components In some embodiments, the association complex includes one or more components that improves the structure of the complex (e.g., liposme)., In some to embodiments, a therapeutic agents such as dsRNA can be attached (e.g., conjugated) to a lipophilic compound such as cholesterol, thereby providing a lipophilic anchor to the sRNA. i1 some embodiments conjugation of dsRNA to a lipophilic moiety such as cholesterol can improve the encapsulation efficiency of the association complex. 15 nation complexes 15 ProjljjQ aCojaf 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, eg, from about 25 to about 400 nm, e.g., from about 25 nm to about 300 rnim, preferably about 120 nm or less. so In some embodiments, the weight ratio of total excipients within the association complex to RNA is less than about 20:L ,for example about 15:1, in some preferred embodiments, the 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 endozonal conditions (e.g., facilitating the 25 rapture 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 VI. Go In vivo Factor VU1 silencing experiments C57BL'6 mice received tail vein injections of saline or various lipid formulations, Lipid-formulated siRNAs are administered at varying doses in an 92 injection volume of 10 pUg animal body weight. Twentyfour hours after administrationserum samples are collected by retmorbital bleed. Serum Factor VII concentrations are determined using a chromogenic diagnostic kit (Coaset Factor VII Assay Kit, DiaPhanna) according to manufacturer protocols. 5 Methods of making association cormplees 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 som embodiments, a plurality of lipids are included in the solvent, for 10 example, one or more of a cationi lipid (e.g, a polyamine containing lipid or a lipid including a biooleavable moiety as described herein), a PEG-lipid, a targeting lipid or a fusogenic lipid. lr, some embodiments, the buffer is of a strength sufficient to protonate substantially all anines of an amine containing lipid such as lipid described herein, e.g., 15 a lipid of fornula (1) or fomnula (X), In some embodiments, the buffer is an acetate buffer, such as sodium acetate (pH of about 5). In some embodiments, the buffer is present in solution at a concentration of from about 100 mM and about 300 mM. In some embodiments. the solvent is ethanol. For example, in some 20 embodiments, the mixture includes at least about 90% ethanol, or 100% ethanol. In some embodiments, the method includes extruding the mixture to provide association complexes having particles of a size with hydrodynai diameter less than about 500 mn (e.g., a size from about 25 mn to about 300 nm, for example in some preferred embodiments the particle sizes ranges from about 40-120 nni) i some 25 embodients, the method does not include extrusion of the mixture, In ore 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 sucose is 30 dissolved. To this mixture is added a solution of siRNA in acetate buffer, vortexing lightly for about 20 minutes. The mixture is then extruded (e.g., at least about 10 times, 93 e g., 11 times orn more) through at least one filter (e.g., two 200 nm filters) at 40 C, and dialyzed against PBS at p1 7A for about 90 minutes at RT. In one embodiment, a liposome is prepared without extruding the liposomes mixture. A lipid described herein is combined with cholesterol, PEG, and siRNA in 5 100% ethanol; water, and an acetate buffer having a concentration from about I100 mM to about 300 mM (pH of about 5), The combination is rapidly mixed in 90% ethanoL Upon completion, the mixture is dialyzed (or treated with ultrafiltration) against an acetate buffer having a concentration from about 100 mM to about 300 mV (pH of about 5) to remove ethanol, and then dialyzed (or treated with ultrafiltration) against 10 PBS to change buflr conditions. Association complexes can,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 therpauctically active single or double stranded nucleic acid moieties to provide a loaded association complex, ie, an association complex that is loaded with the 15 therpaucitcally 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 liposomes above can be used to fran association complexes ftee of a therapeutic agent, such as a nucleic acid, for example a single or double stnmded RNA such as siRNA. Upon formation of the 20 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 lipid such as a lipid described in formula (1), preferably a cationic lipid of the following formula HHH 9H 25 cholesterol, and a PEGiipid, for example a PEG-lipid described herein, such as the PEG ipid below, 94 0 -'^'"'^w*'^'"'^^-'^r 0 '''-'YN'.O N are 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 5 the association complexes, The association complexes are then treated with the therapeutics agent such as siRNA in ethanol (e.g 35% ethanol) to thereby provide a loaded association complex, i some embodiments, the association complex is then treated with a process that removes the ethanol, such as dialysis. to Characteization of association complexes Association complexes prepared by any of the methods above are characterized in a similar manner. Association complexes are first characterized by visual inspection. In general, preferred association complexes are whitish translucent solutions free from aggregates or sediment, Particie size and particle size distribution of lipid-nanoparticles i are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvem, USA). Preferred parties are 20-300 iny, more preferably, 40-100 urn in size. In some preferred embodiments, the particle size distribution is uniniodal, 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 20 binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation dismpting surfactant, 0.5% Triton-X100. The total siRNA in the formulation ts determined by the signal from 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 in the absence of surfactant) from the total siRNA 25 content. Percent entrapned siRNA is typically >85%. Methods of usin association complexes and compositions including the sue Pharnaceutcal compositions Coipprising oligoucleotide agents 30 An oligonucleotide agent assembled in an association complex can be administered, e.g., to a cell or to a human, in a single-stranded or double-strarded 95 configuranon. An oligonueleotide 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, such as a liposome, as described herein, and a pharmaceutically acceptable carrier, The pOharmaceutical composition comprising the packaged oligonucleotide agent is useful for treating a disease or disorder associated with the expression or activity of a target gene, iW 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 are formulated for delivery to a specific organ/tissue, such as the liver, via parenteral delivery. The pharmaceutical compositions featured in the invention are administered in 1s dosages sufficient to inhibit expression of a target gene. In general, a suitable dose of a packaged oligonucleotide agent will be such that the oligonucleotide agent delivered is in the range of 0.01 to 50 milligrams per kilogram body weight of the recipient per day, generally in the range of I microgram to i mg per kilogram body weight per day. The pharmaceutical composition may be 20 administered once daily, or the oligonucleotide agent may be administered as two, three, or more sub-doses at appropriate intervals throughout the 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 25 delivery over several days, e.g., using a conventional sustained release fohrnulation which provides sustained release of the packaged oligonueleotide agent over a several day period, Sustained release formulations are well known in the art, The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the 30 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 96 series of treatments, Estimates of effctive dosages and in vivo half-lives for the individual oligonuceotide agents packaged in the association conplexes can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein. 5 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 determining a therapeutically effective dose. Any method can. be used to administer an oligonucleotide agent packaged in an o 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 intraperitonena 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) 15 An oligonucleotide agent packaged in an association complex can be fcrmulated 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 intraventricular administration, an oligonucleotide agent can 20 be fbnnulated into compositions such 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 an association complex can be formulated in a pharmaceuticaly acceptable carrier or diluent. A "pharmaceutically 2 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 fBor the desired bulk, consistency, and other pertinent transport and chemical properties. Typical 30 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); 97 lubricants (eg., starch, polyethylene glycol, or sodium acetate); disintegrates (eg., starch or sodium starch glycolate); and weting agents (e.g., sodium. lauryl sulfate), EXAMPLES 5 Exampjeh rynhessspandpurification of compounds 3,4 and 4: akyation of ijethylenetetramine under Michael addition condition method 1 (Scheme 1) Scheme l' HH R R H R (i) 90 "C, Neal, 5 days 10 In a 3 50 mL pressure bottle N-dodecylacrylamnide 1 (84 g, 0, 35 miol) [Sleec, Deborah HM; Romnano, Suzanine J'; Yui, Jinghua; Nguyen, Truc N,; John, Judy K, Rahe~ja, Neil K.; Axe, rank U.; Jones, Todd K,,; Ripka, Willi C. Journal of 1Medicmal Cheomistry (2001), 44(13), 2094-2107] was taken and the solid was melhed under argn bygnly heating the vessel. To this melt was added triethylenetetrfaminje 2 (10,2 g, U7 -15 mol) and the mixture was, heated at 90 'C for 5 days, Michael addition of triethylenetetramine 2 to the acrylamnide.1, yielded two five and the sole six alkylated products along with minor amnounts of low alkylated products under neat reaction conditionThe reaction mixture was analyzed byT LC using CH,2Clh:MeO-H:NBt3 (:55) as the chient. The TLC Showed the near completecconsamption of the Starting 2G acrylam"ide L Th'Ie reaction mixture was dissolved in dichloromethane (40 mL), loaded on a pre-packed columnn of silica a gel ana the mixture was separated using ehuent C1C:M.'VeOl-1N~tj (4 8:1;1 to 8:,1: 1). 1n order to a chieve complete separ-ation, multiple columns using the sam-,e conditions were performed and the followig pure products were obtained, The required ive addition Products 3 and 4 were isolated along with the 99 six addition product 5. In this reaction mixture some of the lower addition products were also detected in the TLC and the LC-MS of the crude reaction mixture, N-Dodecy 3-((2-dodeeylearbamoyl-ethyl)-{2-(2-dodecylearbainoyl-ethyl) 24 (2-dodeeylcarbainoyl-ethyl)-2-(2-dodecykarbamoyl-ethylanino)-ethy-iamino a ethyl-amin>)propionamide. One of the two 5-alkylated derivatives, compound 3 (isomer I), was isolated as light yellow fam (12 g, 13%). MS nt672 (M+21/2), 448 (M+3-1/3) 'H NMR CDC i6 0.87 (t J= 6.5Hz, 15H), 1,20-1.39 (m, 9211), 1.46-1,57 (m, 1211), 2.20-2 50 (mi, 161), 2,60-2.78 (m, 10H), 3.10-3,25 (in, 12H), 6.98 (bs, 31), 7.41 (bs, 111), 7,63 (bs, 1), 8.85 (bs, IH). "C NMR CDC- 8 14.33, 22.90, 27.37. o 2,8 22 3 3, 39-74,172,77. (3i(24{2A{2-Bis-(2-dodecyicarbamnoy1-ethy1)-amninob-ethy)-(2 dodecyicarbamoy-ethyl)-amninoiehylamfiO}-ethy)- (24dodeeykearhamoyiethyI) aminojN-dodeeyl-propionanide). Second 5-alkylated derivative, compound 4 (isomer 11) was isolated as a white powder (13. g, 14%), MS mak 672 (M+2H/2), 448 15 (M+31/3). 'H NMR CDCb 5 0.87 (t, J= 6,5Hz, 15H), 1,20~1.39 (n, 92H), 1.44-154 ( , l21f) 2.30-2,45 (n, 9H), 2.46-2,54 (m 8H), 2,55-2.85 (im, 101) 3,15-3.30 (m, 12H), 6.98 (bs, 31), 7.41 (bs, I i) 7.63 (bs, 1H), 8.85 (bs, 111). '3" NMR CD)31 6 1433,22.9,27-28,27.38.29.59, 2969, 29.88, 29.89,29:92,32,13, 3965,39,74,5084, 17263, 17275, 172.81 20 Along with this a pure mixture of compounds 3 and 4 (L116 g, 12%) in 2:3 (3:4) ratio was also isolated, 34{2-2fBis-(2-dodecylearbamoyl-ethyl)-aminoj-ethyl}-(2~ dodecyfearamaoyethy) amioethyW(2dOdcylcramoWyWethy1)-andnflQthyi} (2-ddecykearbanioyethylaminobN-dodecy-propionlamide. The six alkylated 25 product 5 was isolated as a cream powder (163 , 17%). MS m/z 792 (M+211/2), 528 (M+3H/3), 'H NMR DMSO-d 6 0.87 (r, = 7Hz, 18 ), 1,15-1.40 (m, 112H), L45 i.53 (m, 12H),t 2.20-2.35 (mn, 12H), 2.372,50 (mn, 12H), 2:64~2.78 (m,. 1211), 3,10~3.25 (m, 12H), 726 (b 411), 764 (bs, 2H). ) NMR CD)l1 5 14.32, 2289. 734,27.38 29.59,29.69, 29,9 29.92, 32,13, 39.77, 50.85, 172.80. 30 9p Syntheses and verification of compounds 3, 4and 4: alkylation of tricthylen~et ine nader Michael addition condition -- method 2 cheme 21 99 In another experiment, in order to prevent the poltymerization of the starting acrylamide I at high temperature, a radical quencher benzoquinone was added to the reaction mixture. Scheme2 2IH R R RN 3 4 S 5 H (i) 90 C, Catalytic amount (15 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 ISO mL pressure bottle N-dodecylacrylamide 1 (24 g, 100 mrmol) was taken and to this 15 mg of henzoquinone was added and the solid acrylanide was melted under argon by gendy heating the vessel. To this melt was added triethylenetetramine 2 (2,9 g,20 mmol) and the mixture was heated at 90 NC for 5 days. The reaction mixture was analyzed by TLC using CH 2 C1:MeOH:NEt 3 (90:5:5) as the fluent, The TLC showed the near complete consumption of the starting acrylamide 1. The rTeaction mixture was 6s dissolved in dichloromethane (40 ml) and the desired products 3, 4 and 5 were isolated as described in Example L i this case a slight increase in the amount of six addition product was observed. Compound 3: The ive addition product, isomer I, was isolated as light yellow foam (3.4 g, 13%). The analytical and spectral data for this compound was identical to 20 that of 3 obtained by Method 1. Compound 4: The five addition product, isomer iI, was isolated as a white powder (3,9 g, 14%). The 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 (19 g, 7%) was also isolated, 100 Compound 5: The six. addition product was isolated as a cream powder (6,9 g, 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 conmounds 3. 4 and 4: alkylation of 5 triegamjgderighaeladdition condition: -- method 3(Scheme 3) In this method the Nichacl addition was performed in the presence of a promoter like boric acid (Chaudhuri, Mihir K.; Hussain, Sahid; Kantam, M. Lakshmi; Neelina, B3, Tetrahedron Letters (2005), 46(48L 8329-8331 ) in order to enhance the rate of the reaction, 10 Schemen3" 0 N H H N R R R R R H (1) 90 C, aq. boric acid, 2 days In this method a similar reaction to that of Method 1 (Example 1) was perftnned except that, a Michael addition promoter saturated aqueous boric acid was added to the 15 reaction mixture. In a 150'mL pressure bote Akdodecyl-acrylamide 1 (24 g, 100 mmol) was -melted under argon by gently heating the vessel and to this 3 mL of aqueous boric acid was added, To this meh was added triethylenetetramine 2 (2.9 g, 20 mmol) and the mixture was healed at 90 *C for 2 days. The reaction mixture was analyzed by TLC sing CH12,Cl:MeOH:NEt 3 (90:5:5) as the duent, The TLC showed the near complete 20 consumption of the starting acrylamide 1. The 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 rotary evaporator. This crade product was purified by column chromatography (silica gel) using CH 2 Ch :MeOH:NEt 3 (48:1:1 to 8:1:1), l order to achieve complete separation, multiple columns using the 25 same conditions were performed and the foIl owing pure products were obtained. Under 101 this reaction condition an increase in yields of compound 4 (i somer I) and six addition product 5 were achieved, Compound 3: The live addition product 3, isomer 1, was isolated as light yellow foam (31 g, II %). The analytical and spectral data for this compound was identical to 5 that of 3 obtained by Method I, Compound 4: The five addition product 4, isomer 11, was isolated as a white powder (5. g, 20%), The 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 (2.1 g, 7%) was also isolated. 10 Compound 5: The six addition product 5 was isolated as a cream powder (7,6 g, 28%), The analytical and spectral data for this compound was identical to that of 5 obtaffied by Method 1 Exa e4' Synthses ndpgification ofeq adsland 4: acoyldig f triethylenetetramine murder Michael addition condition. - method 4(Scheme41 is In another experiment, in order to minimize the formation of the six addition product 5, use of solvent was attempted. Scheme 4 ' 0H "'' ~ >tN"'N~N~ R 4- 3, and 2- addeon. pmvduct, R R R 3 .4 S(i) 90C acetonitrile or DMF, 5 days 20 in this method a similar reaction to that of Method I (Example 1) and Method 2 (Example 2) was perfbrmed except that, the reactions were perfdrmed in the presence of solvents at 90 C with stirring. In a 150 nL pressure bottle N-dodecyl-aerylamide 1 (10 g, 41,8 mmol) was dissolved in 210 ml, of either acetonitrile or DMF, To this solution was added triethylenetetanine 2 (1 g, 6.8 mmol) and the mixture was heated at 90 *C 25 tor 5 days. The reaction mixture was analyzed by TLC using CH2C1 2 :MeOiH:1N"Et 3 (90:5:5) as the eluent. The TLC showed the formation of only minor amounts of the 102 required five addition product. The major product in this reaction was a mixture of four addition products along with very polar lower addition products. xampe 5 ration of unreacted aervinide from the reaction mixture and/or the isolated products 3, 4 and 5 PFo remove unreacted aerylamide I from the reaction mixture, the reaction mixture is diluted with ethyl acetate or DMF and stirred with polystyrene or polymer bound thiol (or mea-ptan) to capture all the acrylamide. The irmnobilized thiol was added to the solution and gently shaken at ambient temperature and filter off the solid. Michael addition of immobilized thiol to acrylamide capture all unreacted acrylamide o Traces of acrylamide as contaminant after isolation of each desired isomer could also be completely removed under the same condition, The 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 vacuo affords a pure compound 3 (or 4 or 5) free of acrylamide contamination. SExanplel 6: Separation ofpimary and secondary aine contaminant from compound 5 After column ehromatographic separation of compound 5, to remover traces of primary and secondary amine contaminants, the compound is dissolved in ethyl acetate or DMF and stirre with solid bound or immobilized isothiocyanate at ambient 20 temperature ovemight. Filter off the solid and evaporation of the filtrate affords a pure compound 5 fiee of any primary or secondary amine contamination, zggapl7.: Separation of priman amine contaminants from cmpgound 3 and 4 After the completion of the reaction the reaction mixture is treated with tetrachlorophthalic anhydride in the presence of triethiylamine in. dichloromethane at 25 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, 103 Table I Methods of synthesizing products 3 and 4 Method Temperature ProImoter Solvent Radical Remarks Quencher g 90 C None Neat None Fonnation of 3 and 4 in a combined isolated yield of 39%. The six addition product 5 was isolated in 17%. Reaction took six days for completion. 2 90 "C None Neat Benzoquinone Benzoquinone was -used to prevent the im'nerraton of acrylamide L, The combined yield of 3 and 4 was 34%. However 26% of 5 was also isolated, Reactiontime same as Method 1 3 9(} "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. 80- 120 "C None DMF None Reaction very sluggish,. Only lower addition products formed, 104 Examnole 8: Methods of pre ~.parofflthe ydrochoride salts o th products%3 4 ard 5 1i order to improve the ease of handling and increase the stability of the compounds isted above, they were converted into their corresponding hydrochloride S salts 6, 7 and 8. Hydrochlonde of compound 3 (6): The amine 3 (9,4 g) was dissolved in 100 mL of hot anhydrous 1 ,4-dioxane and 100 mL of 4M HC in dioxane was added and the mixture was stirred at room temperature ovemight. Nitrogen was bubbled into the reaction mixture for 1h to remove the excess HC] and the remaining solution was 10 concentrated to -10 in]L, To this heterogeneous mixture 100 mL. of ltOAc:hex anes (1:1) was added and the precipitated product was filtered, washed with ethyl acetate (50 ml), hexanes (100 mL.) and the resulting powder was dried under vacuum to get the pu re product 6 (9.99 g, 96%) as a cream powder. 'H NMR CDC 5 S 0,83 (r, J=6.5Hz, H), 1.20-139 (m, 92H), 2.64-270 (m, 8H), 2.90410 (nm 16H), 325-3,45 (m, 12H), 15 3)46-3,64 (m, 4H) 5.20-6.0 (bs, 2H), 8.05-8.15 (m, 5H), 10. (bs, 3H), C NMR CDC 8 13.83, 22,04,2648 28.69, 289, 28.90, 29;04, 31,26, 3871, 168.38, 168.53, Elemental Analysis; Calcd CH163N 9 0.4HC13H20: C, 63.05; H, 11,30; N, 8.17; Cl, 9.19. Found: C, 63,13; H, I1, 06; N, 8.21; Cl, 9.21 Scheme 5' HN' R4H R N R 20 H (i) 4M HC in 1,4-dioxane, rt., 12h Com-pomd 7 The amine 4 (13.7 g 10.2 mmol) was converted to the corresponding l salt 7 using a similar procedure used above for 3 to obtain 6, The tetrahydrochloride salt 7 was 25 isolated as a white powder (14.6, 96%). H NMR CDC 8 0.82 (t, J= 6,5 Hz, I H), 1,20-1 41 (mm, 9211), 2,52-2.72 (m, 8H), 2.90-3.10 (m, 16H)3 25-3.45 (m, 12H), 3,46 3,64 (m, 41H'), 5,20."6,0 (bst,2H1), 8,05-8315 (m, 5H), 10. (bis, 3H), "C NMNR CDO, 6 8,42,1384 22.04, 2648, 28.69, 2839, 29.00, 31.26, 45,44, 168,53, 168.60. Elemental 105 Analysis: Calid: C H163N 9 0 4HC1 0: C, 63,79; 11, 1 L30; N, 8,17; Cl, 9,34, Found: C, 63.78; H4, 1 -04; N, 8.40; Cl, 9.73, Scheme 63 R R R H R R R S() 4M H IC in 1,4-dioxane, rt, 12h Compound 8 Tie amine 5 (13.? g, 1.2 minol) was converted to the corresponding HCI 8 using a procedure similar to that described above for the salt 6. The tetrahydrocloride salt 8 was isolated as a vihite powder (1.3 g, 96%). 'H NMR DMSO-d4 6 0,87 (4, J=' 7Hz, 10 18H), 1.13-L 30 (mn, 112H), 1.35-1L53 (mn, 12H1) 2.10~2.25 (m,. 121H), 2.34-2.40 (mn, 12H), 2,60-2 76 (m, 12i1), 3,10-3.25 (m, 1211), 7.26 (bs, 4H), 7.64 (bs 21), 10,' (bs, 12').,I0/.' 2 411), Scheme 7 N. 4HCI Step'1: Preparation of compound 2 (20.55 g, 140.52 mamol, purchased. from Sigma-Aldrich) in acetonitrile (500 mL) was cooled over an. ice 20bath under constant stirring, E thyl triflu-roacetate (3 5,20 mL, 295,09 mmnol) was added to the stirring solution anld stirred for 20h. Solvent and volatiles were removed under reduced pressure and dried under high vacuuim to get 9 as whi te solid (44.4 g, 94%), The product thus- obtained could be used fobr thec next reaction without further purification (Wender P. A. et al.- Organic Letters, 2005 7, 4815), 25Crade- compound 9 (23370, '70 mrmol) was dissolve in actonitrile (400 mL) and stirred over- an ice bath -Bez.xyabnyoy succinate (Z-OSu, 431.3g, 175 106 smiol, purchased from Novabiochem) and triethyl amine (23.40 mL, 21 Ommol) wtere added to tIe reaction Tixture and stirred ovemight, Solvents were removed and the residue was extracted into dihloromethane (DCM), washed successively with water (two times) and brine, dried over anhydrous sodium sulfate, Solvent was removed in 5 vacuo and residue thus obtained was purified by silica gel column chromatography (gradient elution, 30-70% EtOAc/Hexanes) to obtain compound 10 as white solid (382g, 89%), 11 NMR (DMSO-d6, 400MHz) &= 9.60-9-50(m, 211), 7.40-7,20(m, 10H), 5.02(s, 4), 3.40-320(n, 12H). MS: Ca41zsFeN 4 06 Cal. 606.19, Found. 607:2(M*). 1o Step 2: Preparation of compound 11: Compound 10 (12,60 g, 20,8 mmoi) was suspended in methanol (MeOH, 150 mL) at ambient temperature and 8M solution of methylanine in ethanol (40 ml) was added to the suspension under constant stirring. All the solids went into solution, after stirring for lb at ambient temperature, the mix ture was warmed to 50'C and stirred for 8h. Reaction was monitored by TLC All the solvents were removed under reduced pressure and the residue was purified by silca gel column chromatography (gradient elution, I10% MeOH/DCM to 10:10:80 MeOH :TEA:DCM) to yield the product 11 (7.80g, 91%) as pale yellow gummy liquid. '14 NMR (DMdSOd6, 400MHz) 5 = 7,80-7 40(m, 10H), 502-4.94(m, 4H), 3.45-305(mr, 8H), 2,70-2.55(m, 41), t20(bs, 4H), MS: C2 0 aN 4

Q

4 Cal. 414.23, Found 41 5.20(f") Step 3: Preparation of compound 13: Compound 12 was prepared from triethvlenetetramine, 100 (1 0.25g, 70.09mnol) as described in step I for the synthesis of compound 9 by reacting with L 1 molar equivalent of ethyl trifluoroacetate (8,80mL, 77.10mmainol), Crude 12 thus obtained was dissolved in anhydrous DCM (400ml) and cooled to 0 *C. (Bo0c)20 (5353 mmol, 245.31 mmol) and triethylamine (48 ml, 25 350mnol) were added and reaction mixture was allowed to stir ovemight 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 the residue was puriied by silica gel chromatography (gradient elution 50%EtOAc/Hexame to EtOAc) to obtain the desired product 13 (34,20g, 92%) as white ao solid. JH NMR (DMSO-d6, 400MHz) S= 9.51-9.38(m, 1H), 682(bs, 1 H), 3.30-3.00(m, 12H), 1,58-1 30(s, 271). IS: C 23 4

H

1

F

3

N

4 0 7 Cal. 542.29, Found 543,4(m"), 107 Step 4: Preparation of 14: A solution of compound 13 (25g, 47.32 mmol) in MeOH (200 il) was stirred with K 2 COQ (50g) in the presence of water (I mL) at 50 *C ovenight. Progress of the reaction was monitored by TLC, Solid K 2 CO3 was filtered off, washed with MeOh, combined washing and solvents were removed hi vacuo. 5 Residue obtained was purified by silica gel column chromatography to yield the desired product 14 (10,2 g, 50%) as white solid. 'H NMR (DMSO-d6, 400M z) = 6,83(bs, I H), 2.95-3.30(m, 12H), 2.62-2.50(m, 2H), 1.25-1,A5(m, 27H1). MS: C 2 H1 4 0NA Cat.. 446_31, Found 447,4(M!), Scheme 8' Cz E MtsNHg/Mef TENCfriCN 0 H H .1 g ty rxa tl '? HH :t F CH-ON, RT NH 2 e q. Ethyl rifti acmte H ' N N 1T HA OER 114 Kg~os so F MeOH, o 0 c "C4 0 10 1 "Selective protection of triethylenetetrarnipe nitrogens Step 5: Preparation of compound 15: Compound 9 (23.0g, 6802 mmol) was dissolved in a mixture of acetonitile/dichloromthae (1:1, 300mL) and cooled to O'C 108 Z-OSu (1700g, 69 mmol) was added to the solution and stirred for 10 minutes. Triethylamine (23,40 mL, 210mmol) was subsequently added to the reaction mixture and allowed to stir overnight, Solvents and triethylamine were removed in vacau and the residue was extracted into DCM, washed with water (two times), brine and dried. After 5 removing solvent, the residue was purified by silica gel column chromatography (eluied initially with 20-60 % EtOAc/Hexane, then with 5% MeHl/DCM) to obtain the desired product 15 (1 3,3g) as white solid along with side product 10 (8.5g), 'H-1 N MR (DMS0 d6, 400MHz) $ 9,60(bs, 1H), 9.30(bs, 1Hl), 7,40-7,28(m, 5H) 5.01(s, 2H), 3-40 3,10(m, 81), 2.70-2.50(, 4H), MS: C 6HFfN40 4 Cal. 472,15,Found 473,i(M) Step 6: Preparation of compound 16: Treatment of compound 15 (13.4g, 28.38 mmol) with methylamine (50 ml, 8M solution in EtOH) as described in step 2 yielded a colorless liquid compound 16 (6.1Og, 79% ).The product thus obtained could be used for next reaction without further purificaton. H NMR (DMS0-d6, 400MHz) 6 7,45-7,20(6nH, 6), 5,07(s, 21), 3.45-2.90m, 8H). 2.60-2.30(, 4F), MS: C 40 CN 5 Cal. 28019 Found 281 2(M). Scheme 9" z i FN N Nx 5 TEACH cN 9 ao ebMethyarmine(MieOH} F "NK .N r>.Nft 2 -- F 7 t " Selective blocking of single secondary nitrogen of tiethylenetetramine anple 10;tSyLhesis of 5-alkxiatesindge isomer 4 MeIodl 20 Step 1: Reaction of 11 with N-dodeeylacrylanMde: Diamine 11 (1.00g, 2,41 mmol) and N-dodcylacrylamide (3,47g, 14.50 mmol) were taken together in a pressure tube and heated at 90T for 5 days. The reaction was monitored by 'LC, Once the reaction is over, the mixture is dissolved in dichioromethane and purified by flash chromatography to get the products 17, 18 and 19. Step 2: Preparation of compound 20: Compound 19 (2.00g, 1.46 mmol) is dissolved in a mixture of ethylacetate and methanol (1:2, 15 ml) to that 2 eq. of acetie acid is added, 'The mixture is hydrogenated under pressure (50 psi) using pal ladium/carbon (0,200g, 10%wt) as a catalyst to get the desired product 20, 109 Step 3: Preparation of single isomer 4: Compound 20 (1L50g, 1,36 mmol) and the acrylamide 1 (0.325 mmol, 1.36 mnol) is dissolved in toluene (4mL) and heated at 90T days to form compound 4, Progress of the reaction is monitored byT'PLC. After conpletion of reaction, the mixture is cooled to room temperature, dissolved in DCM S and purified by flash silica gel column chromatography to obtain the desired product 4, Scheme 10 Hbz cbz N-Ddecybey~rrmdel Neat, 90 <C Y bz H cbz H b R & N * N R ' N N R -N't,. H CA Cb Gu R, + ,,Pd EtOAcflveOH R R NDodecylacrylarnkeI N N N HH H EIapnple 11: Snthesis of al edsigjl isomer 4 Meihod 2 10 Step 1: Preparation of compound 21: Compound 16 (.0g, 3 56mmnol) and N dodecylacrylamidc (6.00g, 7eq) are taken together in a pressure tube and heated to obtain compound 21. Progress of the reaction is monitored byT TLC. After completion of the reaction th mixture is dissolved in DCM and purified by flash silica gel chromatography to afford the desired compound 21. 15 Step 2: Preparation of compound 4 from 21: Compound 21 (2,00g, 1.35 mnmol) is dissolved in a mixture of ethyl acetate and methanol (1;2, 1.5 ml) to that 2 eq. of acetic acid is added. The mixture is hydrogenated under pressure (50 psi) over palladium-carbon (0.200g, 1 0%wt) to afford the desired single isomer 4, 111 Scheme 1I H N. Nei Nea R R H N~5W h$~tR 4NN~~N.r~~ e i y isomer 3 method I Step 1. Preparatin of compound 22:. Compound 14 (5.06g, 11.30 mmol) and 5 Ndodcyacrlamde(2.94g, 12,43 mmtol) were taken in) toienie and heated at 90'C for five days, TLC wNas checked and showed the formation of product: Theo reaction mixture wa~s directly loaded on a pre-packed column of column s>ioa ge.1 and purified by flash chromatography (5% MeOH/DC to afford compound 22 (4,82g, 62%). '1 NMR (DMSO-d6, 400MN.1-z) 6 = 8.17(bs, ';.H), 6,60(bs, 1H1) 3.30-2.95(m, 12H1-), 2,7 0(t, 10J= 8El,2H-),2.60(t, J=6,00Hz, 211) 2, 18(t, J=6,40Hz., 2H), A,35(m,29H-),1L26 I.15(m, 18H)40.83(It, J=6,00Hiz, 3H-), MS- CSaH71Ns50 Cal. 685.54, Found 6865(M*T); Step 2: Preparation of compound 23: Compound 22 (4,75g, 6.92 mmsol) was; dissolved in dichloromethane (I00tnL) and cooled to W'C, Z-Osu (2.39g, L,5eg) was addedtothe solutionandstirred for1r 15 sfihred with trie~thylamiine (2.82 mL, 20,76mmuol) overnight, Solvent and triethylamine were removed in vacuo and the residue was extracted into dichloromethane, washed successively with wat r (two ies) and hie, ratiddrn odutver . hyrecusonmdiu x sulfate After raeovingsolvent the residue was purified by flashnsilica ge flaumn chromatography (5-10% MeO/DOCM) to obtain the desired compound 23 (5,3'3g 20 94%), IH NR (CDC, 400MHz) 8 = 7,49-7 .25(, 5f), 5.11(s, 2 .), 3 -3,0(m, 14H),2,45-45(m, 411), .50-1,35(m, 27H), 124-L20(m, 18H), 087(t. J=6.00Hz, 3H), MIS; C(nNO, Ca.8t. C.819.57, Fo3ud. 820,C7(MN Con), Step 3: Preparation of compound 24: 4M HC1 in dioxane (50 m ) was added into atsolution of compound 23 (5,30g 6;50 mitmol). The reactionmixtr was suseuently 25 mixture was tihenalowed to stir ovelmit Produ t waspreipitated out during the couseO the re acion, Solvent and Hu were removed under vacun to yield a white II1 solid, The residue was taken in MeOHi containing excess triethylamine and the suspension was stirred for It to obtain a homogeneous solution. Solvents were removed in vacauo and the residue was triturated with EtOAc, filtered off the triethlviatnine hvdrochlioride salt. Combined filtrate was evaporated under vacuum to obtain a gummy & liquid 24 (3,30g, 98%); 'H NMR (CDC, 400MHz) = 7:37-28(m, 51), 105(s 2H), '3,60-3.20(m, 41H), 3.10-2,70(m, I1H),2:.40-220(m, 41-), 1A40~1,30(m,/21-), 1,25~ i,17(m, i8H1) 0,8i(t, J=-00Hz, 3H). MS: 0 29

H

5 -N,0 3 Cal 519.41, Found 520.4(M*); Step 4: Preparation of compound 25: Compound 24 ( 100g, l,925 mmol) and N-dodecyiacrylamnide (3,70g, 8eq) are taken together in a pressure tube and heated at 10 elevated temperature to form desired compound 25. Formation of the product 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 (2.00g, 1.35 mmol) is dissolved in a mixture of ethylacetate and methanol (1:2, 15 ml) to that 2 eq, of acetic in acid is added. The mixture is hydrogenated under pressure (50 psi) over palladium carbon (0- 200g, 10%wt) to afford the desired product 3. Scheme 12 Soc Toluene, heat H H 2 oc 144 ZOsu TEA, DCM Cbz 24 H 2 TEA Cb 23 N-dodecylactamie (eOxcess) heat R 5 R C~d~ H Examne 13: Synthesis of 5-alkylatedsngle isomer 3 - Method 2 20 Step 1: Preparation of compound 26: Benzyl bromide (1,25 ml, L 5eq) to a suspension of compound 22 (4.80g, 7.00mmol) and K 2 C0 3 (96g., 1 0eg) in DMF (10) mL) and the mixture was stirred overnight. Progress of the reaction was monitored by 112 TU.L 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 colunm chromatography (50-1 00% EtOAc/Hiexane) to afford the desired compound 26 (330g 61%). 'H NIR (DMSO-d6 400MHz) S7, 77(bs, 21), 7,28 7,23(n, 5H), 6.85-6.0(m, 1H), 3.59(s, 211), 320-2.20(m,8H), L35(s, 27H), 130 l.23(m. 2), 1,20-L15(m, 1811), 681(t, J= 600Hz, 311). MS: C 4 3HnNSO, Cal 77558, Found 776,5(M t ) Step 2: Preparation of compound 27: Compound 26 (3,30g, 4,25 nnnol) in dioxane (50-m!) was stirred with 4M ICI (50 mL) in dioxane ovemight, Formation of white precipitate was seen during the course of the reaction, Solvent and acid were removed under vacuum and white residue thus obtained was redissolved in methanol containing excess triethlaine. The homogeneous solution was then evaporated under reduced pressure to obtain while residue, The residue was triturated with EtOAe and filtered off triethylamine hydrochloride salt, Filtrate was evaporated under vacuum to 15 afford the desired compound 27 (2,36g, 99%) as gummy liquid, 'H NMR (CDCI 3 , 400MHSz) S &05(t, J= 5.5hz, IlH), 7.40-7.20(m, 51), 3,58(s,. 2H), 310-2,30(m, 1811), A40-L30(mi, 2H) , 25-L,15(n , 18H1), 0, 82 (t, J=-6,00Hz, 3HEl), MS: C2, f N O Cal. 475,43, Found, 498,4(M+Na) Step 3: Preparation of compound 28: Neat compound 27 (L 00g, 2,10 memol) 20 and N-odecylacrylamide (4.Og, 8eg) are mixed in a pressure tube and heated to elevated temperature to form compound 28 Formation of 28 is monitored by VTLC and LC-MS. After completion of the reaction the product is isolated by chromatograpic purification to afford pure compound 28. Step 4. Preparation of compound 3 from compound 28: Compound 28 25 (2.00g, 1.40 mmol) is dissolved in a mixture of ethyl acetate and methanol (1,2, 15 ml) to that 6 eq. of acetic acid is added. The mixture is hydrogenated under pressure (50 psi) over palladium-carbon (0.200g, I 0%wt) to obtain compound 3 113 Scheme 13 R NN ~--~ N. NHSoc BFrtK 2 cO 3 RN N NH H 22 oc DMF 26 NH4M Dioxane) N t-dcdecytacytamde(excess) R NNy 28 R E OAc~teOH H H R-< anmpje 14 Convergent synthesis of isomer 3 Method I Step 1: Preparation of compounds 30, 31 and 32: Ethylenediamine 29 5 (0,978ml, 14,63mmol), N-dodecylacrylamnide (7.00g, 29.26rmmol) and boric acid (i00mg) were taken in 5 mL of water and heated at 90'C for four days. Complete disappearance of acrylamidc 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 column 1W chromatography (2:2:96 to 10:1080% MeOH/TEAIDCM) to get compo und:s 30 (1. 86g) H NMR (CDC, 400MH z) 6= 7.05(bs, 2H), 3.21 (q, J6.30 Hz, 411), 2,87(t J= 6,0011z, 4H), 2173(s, 414), 2,34(t, J= 6,0011z, 414);1 57(bs, 211), L,49-L.45(m, 411), 2 1.19(m, 40H), 0.87(, j::: 6.8Hz, 61) MS: C 32 1i6N 4 0 2 Cal, 538.52, Found 53930(M+), 31 (3.50g4) 'I NMR (DMSO-d6, 400NHz) 5= 8.20(bs, 11H), 3,20-2.15(m, 221-), .36 15 I.30(m 6H), 1,25-I.15(m, 30H4), 0,81(t, f= 6,00Hz, 9H) MS: C 4 7

H

9 5

N

5 , CaL 777.4, Found 778,7(M+) and 32 (L75g) 'H NMR (DMSO-d6, 400MHz) 6 3,23-2Jl5(m, Cal. 1016,97, Found 1018.0(M+)t Step 2: Preparation of compound 33: Compound 31 (1.55g, 2mmoil) and 20 14C%3 (2,76g, 20mmol) are taken in DMF To that chloroacetaddehyde dimethyl acetal (0.453 mi, 4.00mmol) is added and stirred for 24h. Reaction is monitored by 'TLC, fitered off K 2

CQ

3 washed with MeOH. Solvents are removed under reduced pressure and the residue is subjected to chromatographic purification to afford compound 33. 114 Step 3: Preparation of compound 34: Compound 33 (200g, 23 1 nmol) is taken in a mixture of MeOH and DCM, to that 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 5 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 (13 1g, 2.43. mol) are taken in DCM; 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 sodiun tracetoxyborohydride 10 (5 eq.) and acetic acid are added and stirred ovemight. Solvents are removed and extracts with DCM, chromatographic separation of the residue affords pare isomer 3. Scheme 8 N R wavvterdboric acid. 31 32 -o-' e X RPTsA R RN R Na(OA.-3H H as 34 R R N Example 15; Converstent synthesis of isomer 3 - Method 2 15 The desired single isomer 3 is also prepared from compound 30 by selective protection cf one of the nitrogen to obtain compound 35. Compomd 35 is subsequently reacted with aldehyde 34 under reductive conditions to obtain compound 36. Acid treatment of 36 affords desired compound 3. 115 Scheme 15 R R' N RRR R- Borr, R NR 30as 8c 36 R R R IN R x aiple j6 C egrgentsynthesis of isomer 3 - Method 3 'he desired single isomer 3 is also prepared from inonobenzyl ethylenediamine 5 37. Alkylation of 37 with 1 affords a mixture of compounds 38, 39 and 40, Compound 40 is reacted with aldehyde 34 under reductive conditions to obtain compound 41. H ydrogenolysis of 41 affords the desired compound 3. Scheme 16 pH Sy< ,, N R 6 ~ ~ ~ R 3,M 34o RN N H Step 1: Preparation of compounds 43: 'in a 150 mL pressure bottleIVAdodecyl acrylamide 1 (16,4 g, 68&8 mmnol) was melted under argon by gently heating the vessel and to this 3 ml. of aqueous boricacid was added. To this melt was added Boo protectd thylenedamine 42 (5 g, 31 m2nmol) and the mixturevwas heated at 90 *C overnight. 15 he reaction mli-xture Was analyzed by TLC using CH2Ch:MeOH:NEt-.9055)a the fluent. The TILC showed the near comple-te consumption of the strigacrylamnide L. The reaction mixture was dissolved i dichlo-romethane (100 ml) and the solution was stirred with solid sodium bicarbonate and the organic layer was filtered, and conc-entrated inl a rotory evaporator. This crude produt was pur ied by colun 20 C.hromlatography (sflica gel) using CH2Cl,:MeNOH:NEt- (480 :1 to 8:1: 1), The majIor 16 product in this reaction is the double addition product 43. Minor amounts of mono adduct was also observed, Step 2: Preparation of compound 44: Compound 43 (2.00g, 3.13 mmol) is taken in dioxane (50 mL) to that H.C1 (20 m l, 4M solution in dioxane) is added and 5 stirred overnight Solvent is removed to get the compound 44. Step 3: Preparation of single isomer 4 from 34 and 44; Compound 34 (2.00g, 2.43 mnol) and 44 (1.1g, 2.43 mmol) are taken in D)CM; to that activated molecular sieves is added and stirred for 3h. The reaction is monitored by 'LC. Once. the reaction is over solvents are removed. The residue is dissolved in THIF and sodium triacetoxy 10 borohydrid (5 eq.) and acetic acid are added and stirred ovemight, Solvents are removed and extracts with DCM, chromatographic separation of the residue affords pure isomer 4. ScheTme 17 t cyid de40? BoM'^"BocH~CN' R f-.' 34Naoca so R 15 Example 18: Addition f-deyacymieto 1,3-diamiinomooaz4n and ecnofrthe am-ide to nine In order to study the effet of number of charges in the cationic lipid the Michael adducts of acryhunide I with 1,3-diaminopropane 45 was investigated. I117I, Scheme j8* AN N.ANAN."NH N- NH 2 H1 45 N NH A R R R R p IA 49, 0and 51 A$ 47 4@ R RR R N +~ Rt'N. R N H R R R 5254 H ()90 "T, aq, boric acid, 16h; (ii) 4M HCI in 1,4-dioxane rt., 12h and (ii) BII Step I: Synthesis of 46, 47 and 48: In a 150 ml pressure bottle N-dodecyf 5 acrylanide 1 (15.4 g, 64 mmol) was melted under argon by gently heating the vessel and to this 3 mL of aqueous boric acid was added. To this melt was added 1,3 diaminopropane 44 (158 g, 21 mmol) and the mixture was heated at 90 *C overnight The reaction mixture was analyzed by TLC using CH2aC:McOH:NEtj (90:5:5) as the etuent. 'The TLC showed the near complete consumption of the starting acrylamide 1 1c The reaction mixture was dissolved in dichloromethane (100 mL) and the solution was stirred wihi 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 CHiChI:MeQH: NEt 3 (481:1 to 8:1:) The major product in this reaction is the triple addition product 46. Minor amounts of tetra adduct 15 47 and his adduct 48 were also isolated. ethylamino)-propyl -aminol-propionamide 46. The three addition product 46 was isolated as a white powder (5.7 g, 35%). MS wvf 793 (MW), ' 1 1 NMR CDC3 5 0,87 (, j=6,6H, 9H), .20-1,30 (in, 60H), L42-1.66 (m, 611), 2,33 (t, J= 6Hz, 4Hf), 2.38-2,46 M8 (, 411), 2.60-2,70 (n, 4H), 2.84 (t, 2H), 3.1 5-328 (m, 6H), 6.65 (bs, 111), 6.99 (bs, 3H). 4-[ {3-[Bis~(2~dodeecykarhamioyl-ethyi)-anmfaol-propyl}-(2 dedecylearbanoyl-ethyl)aminoj-N-dodeeyl-butyranide 47. The ftu addition 5 produce 47 was also isolated in minor amounts. N-Dodeeyl-3-(2-dodecylcarbamnoyl-ethylamrino)-propylandnoj propionaRidde 48. The diadduct 48 was isolated as a cream powder (1 6 g, 10%), MS tn 553 (MH-). 'H NMR CDC 3 8 0.89 (, J 6.6Hz, 6H), 1 10-1 20 (m. 40K), 1,42 L66 (m, 4H), 2.20 (t 2 61:z, 414), 2.55 (t, 4H), 2.60 (t, 41), 3.00 (m, 4H), 8,00 (s, 10 2K), Step 2: Conversion of anrines 4, 35 and 36 to their corresponding hydrochloride salts 49, 50 and 51. The amine 46 (5.5 g) was converted to the corresponding HCI 49 using a procedure similar to the described in Example 8 and the dihydochloride salt 49 was 15 isolated as a white powder (5.73 g, 92%). 'H NMR DMSQ-d 6 6 0.88 (Q, I = 7Hz, 9H), 1,17130 (, 661H), 135-45 A(i, 68), 2 10-2,25(m, 2H) 25-2,70 (m, 6H), 2.95-3.15 (m, 10H), 3.20-3.35 (in, 6H), 8.16 (t, 1 H). 8,24 (t, 1 H), 9 15 (b, IT), 10.65 (bs, 1 H) In a similar procedure to that described in Example 8 the amine 47 is treated with 4N 11C to obtain the dihydrochioride salt 50, 20 in a similar procedure to that described in Example 8 the amine 48 is treated with 4M HC to obtain the dihydrochloride salt 51, Step 3: Reduction of asiddes 46, 47 and 48 to amines 52, 53 and 54: Amine 46 is refluxed in THF with excess of diborane overnight and subsequent treatment wilh 4M HCI affords hydrohilodde salt of polyanine 52 25 A similar treatment of amines 47 and 48 affords the corresponding reduced product 53 and 54 as their respective hydrochloride salt. Exlie19; Reducion ofsolvanindes 3. 4 and 5 to the correspondng Compound 3 is refluxed with large excess of diborane in THF to obtain the 30 corresponding reduced product 55. After completion of the reaction, the reaction mixture is treated with 4M 11CI prior to work-up and the product is isolated as its I119 hydrocloride salt. Hydrochloride salta of 56 and 57 are also obtained from the corresponding precursors 4 and 5 respectively. Scheme 19" RR H ' R R 5 6 57 H IR () BF rTHF, jaimpi .2Q cg~mn ayL jipids reduction of i miie to anies Preparation of polyamines 60 from 32: Compound 32 (1.02g, I mmol) is taken in THF (20 m), to that BH3..THF (60 ml, I M in THF) is added and refluxed for two days. Reaction is monitored by TLC.Removal of TIF gives a white residue, which 0 is treated with I M 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 under similar conditions described for the preparation 60 respectively affords 58 and 59, 15 Scheme 20 H N R H H R NR RR R N R PH 120 Bx2m-e., hesis ofoovaospolvamino a1k0 - alkylation of mis usiijg allkv1halide Step 1: preparation of compound 62: A solution of chloroacetyl chloride (10.31 u.mL 129.37 mmol) in DCM (200 m L) was cooled over an ice bath and to this a ti soltion of dodecylamine (61, 20.0 , 107.81 mmol) in dichloromethane containing TEA (36.70 i, 269.5 Tmdol) was added dropwise over a period of I hr The reaction mixture tuned brownish-black by this time, continued the stirring for another hour at O'C, The reaction mixture was filtered through a sintered funnel, washed with EtOAe, diluted with chloroform, washed successively with water, sodium bicarbonate solution, I MUHCI antd brine. Organic layer was dried over sodium sulfate. Solvents were removed and the residue was purified by silica gel column chromatography (5~50% EOAc/Hexane) to afford compound 62 (26,00g 92%) as brown solid. HI NMR (CDCI, 400N'1z) 8 = 6,59(bs, 1H), 4.03(s, 211), 325(q, J=6.00Hz, 21), 1,54-1A9(m, 211), 1.45-1,15(mn, 184), 0,86(t, 3=6.00Hz, 311). MS: C 14 1H4CINO Cal, 261:19, Found 15 262.20(M

T

). Step 2; Preparation of 63, 64 and 65: Triethylenetetramnine 2 (100g, 6,83 mnol) and chloroacetamide 62 (10.00g, 5.5 eq) are taken together in a mixture of CiH3CN/DMF (1:3), to that K 2 C0 3 (9.43 g, 10 eq) and KI (50 mg)are added and heated at 85 0 C for three days. The reaction mixture is filtered to remove solids, wash with 20 DCM, solvents are removed in vacuo and chromatographic separation of the crude residue affords pure compounds 63, 64 and 65.

Scheme 21 NH2 H 61 COM 62 0 0 Kp;Y~.Cat 1 1N H CH)CN, DMF 2 H N NI N eeii HNN NNXN.,. A N~r N" H 'N{ H +NRN N NN H :0 HH + H rH HNr 65 NH 6e H LaflpC22 Syrntesijs of noyamido-polyunhiajkyls v .tyaiomf amines usinlik halides withbranched anminoaky Step 1: Preparation of 67: Chloroacetyl chloride (4,05mb, 51 nmol)was taken in DCM (100 mL) and cooled down to 0'C. To this a dichloromethane solution ofN,N didodecylamine (66, 15.00g, 42.41 mmol) and TEA (14.43 ml, 2.5 eq) were added dropwise over a period of i hr. The reaction mixture tuned brownish-black by this time, 10 after the addition the reaction mixture was stirred for 24 h at ambinet temperature. The reaction mixture was filtered through a sintered funnel, washed with EtCAc, diluted with chloroform, washed successively with -water, sodium bicarbonate solution, IM H11CI and brine. Orgamic layer was dried over sodium sulfate. Solvents were removed in vacuo and the residue was purified by silica gel column chromatography (5-50% 15 EOAc/IIexane to obtian the required product 67 (12,5g, 69%) as brownish liquid, VI t122 NMR (CDC1,s, 400MT1,z) 6 =- 4,04(s, 2:H), 3,30(m, 4:H), 1.450-1.45(m, 2H), 1.40-120(m, 18H) 0,87(t, 3= 6.00Hz, 311). MS: Cm1t 2 CINO Cal. 430.15, Found 431.2(M). Step 2: Preparation of 68, 69 and 70: Trethylenetetramine 2 (0.500g, 6.83 mmod) and chloroacetamide 67 (8.10g, 5.5 eq) are taken together in a mixture of CHCN/DMF (1:3), to that K 2 C0 3 (4,72g, 10 eq) and KJ (30 mg) are added and heated at 85 'C for three days. The reaction mixture was filtered to remove insoluble solids wash with DCM. solvents are removed and chromatographic separation of the residue afbrds t 68, 69 and 70, 123 Scheme 22 o C \-NN 6 KMCo:3 Cat. K0 -' -- ~N HNN N N '-N N N - NC 0-0 m 69y diaj) gLaid liolvms s In order to study the effect of addin-g more hydrophobic chains to the Qationic ipids, didodecylamine was used as a precursor to the acrylamide, 124 Scheme 23" H N H R, N -.. RR R + R 72 0 73 74 R= Hydwcolorlde sats 76, 76 and 77 (i) Acryloyl chloride, -10-0 C, DIPEA, C1,C, 4h (ii) 90 * Neat, 5 days and (iii) HCI/Dioxane Step 1: Synthesis of N,N-Didodecylacryhamide 71 To a solution of didodecylamnine 66 (25 g, 70.7 mmol) and diisopropyiethyamnine (18 g, 141 mmol) in anhydrous CH2Cb (700 iL) at -10 "C, a solution of acryloyl chloride (7,68 g, 85 mmol) in CH 2 Ci 2 (100 mL) was added to dropwise over a period of 20 min. After the completion of 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 satd, NaHICO 3 solution (200 mt), water (200 mL), brine (100 mL) and dried over NaSO 4 Concentration of the organic layer provided the product 71 (284 g, 100%) which was 15 used as such in the next step, '11 NMR CDC 6 0,94 (t, J= 6.5Hz, 611), 1:05-169 (ma, 401), 3.15-3.60 (dt, 4H), 5.64 (d,11H), 6.36 (d, 1H), 6.63 (m, 114). Step 2 Reaction of triethyeentetramine 2 and 71 The acrylamnide 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, 25 Step 3: Synthesis of hydrochloride salts 75, 76 and 77: Each single compound obtained is taken in dioxane and 4M HCI in dioxane is added to the solution and stirred as described in example 8 to yield the corresponding hydrochloride salt. ExmpML4:Alkenlation of polyamnes using mono unsaturatpj alkyj 5 Ejdighael addition coiiion In order to study the effect of double bond in the alkyl chain oleylamine Was used as a precursor to the acrylamide 79, Scheme 2 4 a 78 H 'N H HNHt4 R H R R R RR 10"(i) Acryloyl chloride, 10-0 "C, DIPEA, CH2aCb, 4h, (Hi) 90 'C, TNe at., 5 days and (iii) H1CI/Dioxane Step 1: Synthesis of compound 79: T'o a schiution of oleylamine 78 (26,75 g 100 mmol) and. triethylamine (20 g, 200 mrmol) in anhydrous C H2Cl2 (200 mL) at -10 "C; a s Otution Of acryloy) Chloride (9,9 g,110 mmol) in C.12C02(100 ml) was added adropwise ove r a period of 20 min. After the completion of the addition te reaction mixture was stirred for 4 h at 0 "C after which the, TLC of the reaction mixture showed thecomletonof he eatio. Te eacionmitur wa wshe wih aid. N11HCOJ sohution (200 mL), water (200 mL), brine (100 ml, and dried over NaSO,, Concentration of the organic hayer provided the product 79 (32 g, " 00%) which,,was 20 used as, suc h in the next step, H N-MR C DC-1 8 0.91 (t, J=6.5Hz7, 3H1'), 1. 05- 13 5(m 126 24H), 142 (t, 2H) 1.96 (m, 41 5.31 (t, I H), 5,33~5.36 (n, 11), 5,54 (dd, IH), 6.02 (dd, 1f), 6.1 S (dd, &H), 8103 (bs, 1 H). Step 2: Reaction of compound 79 with triethylenetetramine The acrylamide 79 is treated with triethylcnetetramine 2 and after usual work-up and column purtincution of the Michael addition products affords pure compounds 80, 81 and 82. Step 3: Synthesis of hydrochloride salts 83, 84 and 85: Each single compound (80, 81 or 82) obtained is taken in dioxane and 4M HCH in dioxane is added to the solution and stirred as described in example 8 to yield the corresponding hydrochloride 10 salt, mipe25 Alkeylation of diamines usinA&mono unsaturated N-alkvl acrykmide under Michael addition condition Scheme 25 a H 79 6 4 R R R .2 H 15 (i) 90 C aq boric acid, 16 and (ii) HCI/Dioxane In a similar procedure to that of Example 24 the acrylanide 79 is treated with the diamine 45 and aler usual workup and column purification the Michael addition products86, 87 and 88 are isolated. Treatment of the free amine thus obtained with HC1 20 indioxane affords the corresponding hydrochloride salts 89, 90 and 91 respectively. Example 26 Akenylation of polamines usngI polv nsagurated N '..alkyl agvtlamie under Michael addition gndiis In order to study the effect of polyunsaturation in the alkyl ebain. linolylamine 92 was used as a precursor to the acrylamide 93. 127 Scheme 26a i-N NH H R R R R R , R R RR R N H0 079 'Ind 996 (i) Acrloyl ch loride -10-0 TC, DIPEA, CH2-Cl, 4h, (ii) 90 *C, Neat, 5 days and 5 Step 1: Compound 93: Linolylamine 92 is treated-'with acrylo yl chaloride in a similar -procedure to that of Examirple 24, step I and the corresponding acrylamide 93 is Step 2: Reaction of compou-nd 93 with triethylenetetramine The acrylamide 93 is treated with triethylenetetramine 2 in, the presence of boric, 10 aid a, decribed in Example 3 and after usual work-up and crdim prification of the Mlichae!l addition produLcts affobrds pure comapounds 94, 95 and 96. Ste~pl3 Synthesis o hydrochloride salts 97, 98 and 99: Each singe c ompound ('94,95 or 96) obtaied is taken in dioxane and 4M HCI. in dioxane is aded to the solution and stirred as described in example 8 to yield the corresponding bydrochloride 15ssalt. E.mpe lkenlation of di nin uip v!igmtid ah ay Smep und Coon9Michiael addition condition 128 Scheme 27a +HN

NH

2 45 93 H + R .R + HtHydrochoride t R 10 101 102 103,004 and 105 H (i) 90 "C, aq. boric acid, 16h and (ii) HCV/Dioxane n a similar procedure to that of Example 3 the acrylamide 93 is treated with the o diamine 45 in the presence of boric acid 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 HCI in dioxane affords the corresponding hydrochloride salts 103, 104 and 105 respectively, ji prie_28: Alkenylation of poaine g lkyl agrylates uder Michael 10 addition Cotnditoin Scheme 28 a NH t N N rH 2 N H W6 PtH Rl R RR R R'R (i) Methanol-water, 40 *C or Methanol, water, boric acid, room temperature Method I: mnodecylacrylate (106) is stirred with triethylenetetramine 2 in 15 methanol-water at 40 * to obtain compounds 107,108 and 109, The products are isolated by chromatographic separation. Method 2: n-Dodecylacrylate (106) is stirred with triethylenetetramine 2 in the presence of boric acid in methanokwater at 40 'C to obtain compounds 107, 108 and 109. The products are isolated by chromatographic separation. 129 xnle29: Akeylation o d egsiis ,yj te ander Michael addition condition Scheme 294 446 106 4 R RR 110 11 S*(i) Methanoh-water. 40 *C or Methanol, water, boric acid, room tempenture Method 1: n-Dodecylacrlate (106) is stared with triethylenetetramine 2 in methanol-water at 40 CT to obtain compounds 110, 111 and 112. The products are isolated by chmatographic separation. Method 2: n-Dodecylacrylate (106) is stirred with iriethylenetetramine 2 in the 10 preseneC of boric acid in methanol-water at 40 CT to obtain compounds 110, 111 and 112. The products arc isolated by chromatographic separation. Example 30;jynthesis of Octadeca-912-dienoic acid 3-imthiamino-2 octadeca-9 12~diehovloxv-propyl ester 3 15 OH 2 : EO, DIPEA. DMF5 To a solution of the linoleic acid (25 g, 89.1 mmol) in anhydrous DMF (60 nL) 20 diisopropyl ethylamine (17 ml, 100 min) was added at room temperature with stirring followed by 3-(dimethylamino)l2-propanediol (48 g, 40.5 mmol) and EDCI (17,25 g, 89.9 mmoi) and the mixture was stirred at room temperature ovcmight. The TLC of the reaction mixture (eluent 20% EtOAc in hexanes) showed the completion of the reaction The reaction mixture was poured into ice water and extracted with ethyl acetate (2 x i30 100 nL), The combined organic layers were washed with water (100 nl), saturated NaHCO (100 mL) and dried over Na2SO4 Concentration of the orgaic layer provided the crude product which was purified by column chromatography (silica gel, eluent 20% EtOAc in hexanes), The fractions containing pure product was pooled and 5 concentrated. The pure ester was isolated as a clear liquid (5.7 g, 22%), MS in/ 645 (M+H), 11 NNR CDC13 ( 0,88 (, J = 6.3Hz, 6H), 1 20-1.39 (m, 28H), 1.61 (t, = 4.9 Hz, 1211), 2,03-2.08 (m,8H), 2.26-22.38 (m, 101), 244-256 (n 2H), 276 (t, J= 63 Hz, 4 H), 4.09 (dd,,J: 6.1 Hz & 1 .9 Hz, 11), 4.36 (dd, J= 3.3 & 11.9 Hz, 111), 5.29-5.34 (m, I 1$534-5A 1 (m, 8H. 'C NMR CDC11 8 14.30, 22.79, 25.08. 2510, 25,83, 0 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. 130.42, 173.39, 173.65, Example: Exemplarprocedure for making a liposome using extrusion Prepare stock solutions of ND98 (120 mg/ml), cholesterol (25 mggrnI), and Cl 6 PEC-Cer-2000 (100 mg/ml) in 100% ethanol. Store at -204C. Warm in 374C water bath 15 prior to preparing formulations (up to 30 ininutes is helpful - it takes a while for the cholesterol to dissolve completely). 2X 2m! Prep To a 15ml Falcon tube, add: 20 1)125til of lipid 2)200ul of cholesterol 3)70ul of PEG 4)5u; of 100% ethanol 5)600u1 of 25 mM sodium acetate pH 5 25 6)Mix gently (setting 3) on a vortex 7)Add 20 mg sucrose 8) Vortex again until sucrose has dissolved 9)Add I ml of a freshly-prepared (in a new Falcon tube) I ng/mi solution of siRNA in 25 mM sodium acetate ( 100 ul of 10 mg/ml siRNA + 900 ul of 25 maM so sodium acetate) 10)Vortex lightly (setting 1, with Falcon tube holder adapter) for 20 minutes 11 )After 13 minutes (5 ininutes remaining), clean extruder 131 12)Extrude I1 times through two 200 un filters at 40 . 13)Dialyze against PBS, pH1 7.4 for 90 minutes at RT in 3,500 MWCO Pierce cassettes 5 Exanp32Exempary procedure for making a liposome without usgin extrusion Prepare stock solutions ofND98 (120 mg/ml), cholesterol (25 mg/nml), and C1 6 PEG-Cer-2000 (100 mg/ml) in 100% ethanol. Store at -20*C. Warm in 37C water bath prior to preparing formulations (up to 30 minutes is helpful it takes a while for the 10 cholesterol to dissolve completely). To a 15ml Falcon tube, add: ) 1'25d of lipid 2)200r1 of cholesterol 15 3)7oul of PEG 4)495u] of 100% ethanol 5)100ul of water 6)dPrepare I ml of I mg/ml siRNA in 100-300 mM sodium acetate, pH -5 7)Rapidly mix lipids in 90% ethanol with siRNA in acetate buffer 20 8)Dialyze (or use ultrafiltration) against 100-300 mIM sodium acetate, pH -5 to remove ethanol 9)Dialyze (or use ultrafiltration) against PBS to change buffer conditions nEx emn 33: Eftinplary protocol for quantification of RNA in a posme 25 sample The procedure below can be used to quantify (1) the proportion of entrapped siRNA and (2) the total amount of siRNA in a liposome, Materials: 30 RiboGrcen (Molecular Probes) 2 % Triton X-100 TIE buffer 132 Protocol (96-well plate format): L. Dilute samples to be tested in TE buffer such that siRNA concentration is ~ 2 5 ug/L (0.4 - 4 ug/mL). Note dilution of samples, 2. Array 50 uL of each sample into 2 wells (e.g. samples arrayed into 2 rows of niroplate) 3. Add 50 uL of TE buffer to one of each of the 2 samples (e.g. top row samples). This sample will be used to determine "free" siRNA, 10 4, Add 50 uL of 2% Triton X-100 to the remaining of the 2 samples (e.g. bottom row samples). This sample will be used to determine "total" siRNA 5, Prepare standard siRNA dilutions by using known amounts of the siRNA 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. 15 6, licubate for 15 mini 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 flaorimeter (Victor2) using FITC settings. Calculations: Final volume in wells will be 200 ul. RiboGreen will be at 1:200 final dilution, Triton X-100 will be at 0,5%. Standards will be dilutions starting from I ug/mL Plot Standard Curve, perform linear fit. Detraine Entrapment % = 00*(1-"ee" signal/ "total" signal) Detemine {sIRNAj: First convert "total" signal to concentration using the 3l standard curve, then multiply by dilution factor. Example 34: Comparison of Lipid moieties as fbrrulated into Liposomes The effectiveness of lipid compositions can be tested by determining the relative ability of a lipid to deliver an siRNA moiety to a target. For example, the silencing of a as target indicates that the siRNA is delivered into the cell, Applicants have compared Iiposomre complexes that include each of the following lipid moieties together with siRNA that is used to silence Factor VIl (FVI). 133 Initially unpurified reaction mixtures were used, Different ND98 reaction mixtures were generated by synthesizing product at different ND:98 monomer ratios: ND:98 1:1, 2:1, 3:1, 4:1, 5 i, and 6:1, ND98 is generated by reacting ND, the structure of which is provided below: 0 N 5 H , with amine 98, the structure of which is provided below H H2N ' ' ' NH2 NH in the ratios provided above (ibe, ND:98= 1:1, 2:1, 3:1,4:1, 5:1 ,and 6:1) Liposomes were formulated at ND98:cholesterol :FED2000-CerCi 6siRNA = 15:0,8.:1 (wt ratios). Liposomes prepared with ND:98 = 1:1 and 2:1 precipitated dring frmtulatio.n and were not characterized further, Table 1, below provides the average particle size and percent entrapment of the liposomes using the various monomer ratios (ie, the number indicating the ratio of ND relative to 98). 15 Table 1: Z-Avg. Particle size (nm) % Entrapmeut D98 356 95 ND98 4 56 >95 ND98581 93 ND98 6,72 74 Figure I provides the results of the FVIl siliencing assay for the various monomer ratios using an experimental dosing of 2 mg/kg 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 20 abundants species on the ND98 6:1 preparation. As described a 5 tail moiety indicates a 134 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 98 have been reacted with an acrylamide moiety ND, Accordingly, the numer of "tails" indicates the number of reacted hydrogens on the 5 starting nine, Example 35: Detenmination of preferred lipid isomer Applicants purified ND98 lipid products. ND98 lipid moieties are the lipid moiceies resulting in the reaction of ND, the structure of which is provided below: 0 10 H , with amine 98,the structure of which is provided below H H2 Nt 4 Ns/s'.NH2 H 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 i of 5tail ND98 (i,ev, Where for of the amine hydrogens have been reacted with tie ND acryiamide above). Examples of the two 5 tail isomers are provided below: R R H i H R' N rt.N. N R and R'N "N'-' N "N R FR FR R FR , Liposomes of the purified ND98 products were formulated with the following components in the following ratios: ND98:cholesterol:PEG2000CerCI6:siRNA 20 15:5:7:1 (wt ratios). Table 2, below provides the average particle size and percent entrapment of the liposomes using the various monomer ratios (i.e, the number idicating the ratio of ND relative to 98). 135 Table 2: i-Avg. Particle size % Entrapment (nml) ND98 1 88 >95 ND982 104 86

ND

98 3 115 86 ND98 4 92 95 For the purposes of table 2 and Figure 2: ND98 5-tailed (isomer 1); ND9S 2 =aled (isomer 1.); ND98 3 = 5-tailed (isomer i.); and ND9 4 = 4-tailed. The liposomes where administered with siRNA at a does of 2,5 mg/kg, and 5 evaluated. for the silencing of FVTL Figure 2 provides the results of the 4 tailed isomer mixture, the single 5 tailed isomers (i.e., isomer I and 11) and the mixture of 5 tailed isomers (i.e, isomer I and I). jamIc 36;1Determination ofieferred ND98 isomer to A purified isomer of 6 tailed ND98 was prepared and purified. ND98 sticture corresponds with those described in examples 34 and 35 above, The 6 tail indicates that all of the hydrogens of amine 98 have been reacted with the ND starting material. With this lipid starting material, liposomes were formulated at the following ratios: ND98:eholesteroi :PEG2000-CerC 6:siRNA =15:5:7:1 (wt ratios). Figure 3 15 demonstrates the effectiveness of the ND98 6 tail isomer in delivery of siRNA, which effectively silenced FVfl. Example 37:losomeparticesize using various ND8 lipid st mate A plurality of lipid starting materials having the ND98 structures (as provided in 20 examples 34 and 35 above) were fomiulated into liposomes, The particle size of the liposomes were evaluated, the results of which are provided in table 3 below; 136 Fornialadon Particle Diameter (om) ND98 3 (Exp 1) 56 ND98 4 (Exp 1) 56 ND98 5 (Exp 1) 81 ND98 6 (Exp 1) 72 ND98 I (Exp 2) 88 ND98 2 (Exp 2) 104 ND98 3 (Exp 2) 115 ND98 4 (Exp 2) 92 6-tailed ND98 (Exp 3) 1.27 Example 38: Extrusion free liposome fornmilation Liposome complexes were prepared using ND98 lipids. The formulations include the following ratios: ND98:ctholesterol:PEG2000-CerC 16 :siRNA=:-r. 15:5:7:1 5 (wt. ratios). The liposomes wero prepared without extrusion, as generally described in Exanple 32 above, Two samples were prepared, a first sample having the Iblowing: 100 mM = siRNA prepared in 100 mM sodium acetate with a first dialysis step in 100 mM acetate; and a second sample having 300 mM = siRNA prepared in 300 mM sodium acetate with a first dialysis step in 300 mM acetate. F Figure 4 shows the results of an FVH silencing assay, demonstrating the comparative activity of the formulations made using the various processes, 137 Example39:eioselective synthesis of cationic lipid 7 -- strategy I Scheme 31' 2.1 eq. Ethyl hifluroacetate 0 H CH 3 CN, 0 0 C-RT F., N NNF Ff HZN N''-' N H ~ H H F H F 1 (80q)20: DPEA

THF/CH

2 0C O Box MeNH 2 iMeOH FAN NNF

H

2 N N N NH 2 H H F 114 c 80 OC H 113 BOG 0 N AOG H orc acd, Water Soo R R 1 HC Dioxane R N' N R R NH...N 1 115 2. NaHCOs 0 H Rt HP R.N^H' N N_ RN .-N .~N'R 4 HC R 7 117 H Regioselective synthesis of cationic lipid 7 - Approach I Step 1, Preparation of compound 9: Triethylenetetramine, 1 (48,83 g, 0334 mol, purchased from Sigma-Aldrich) in anhydrous acetonitrile (500 mL) was cooled 10 over an ice bath under constant stirring, Ethyl trifluroacetate (79,6 mL, 0.668 mol) was added to the solution and after completion of the addition the reaction mixture was allowed to warm to room temperature and stirred for 20h, Solvent and volatiles were removed under reduced pressure and the residue was dissolved in minimum amount of 138 warm dichloromethanc (100 mL) and to it cold hexanes was added with stirring. The preciptated product was cooled in ice and filtered to get a white solid (112.2 g, 99%), Step 2. Synthesis of ((tert-butoxycarbony1-{2-(2,2-trifluoro 5 acetylanw)ethy jbamino})-2-(2,2,2-trifuoro-acetylamino)ethy]-carbmieacid tert butyl ester 113 The trifluroacetamide 9 (11212 g, 0,332 mol) was dissolved in CH2Cb/THIF (600 mL100 ml) and to it diisopropylethylamine (129.25 g, I mol) was added and stirred over an ice bath. Di-terr-butyl dicarbonate (145 g, 0,664 mol, purchased from Sigma n Aldrich) in CH2Cla (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 NaHCO 3 (400 mL) and filtered and washed with hexanes (100 mL) and dried in vacuo at 45 *C ovemight to obtain the pure diboc compound as a white solid (167 g, 94%). 'H1 NMR for 113 (DMSO-d6, 400MHz) =9.60-9.40(m, 211), 3,35-3.15(m; 1211), 1.36(s, 1811) MS: C1tAFbN404 Cal. 438.17. Found 43920(M) NIS: CH3FzR40a Cal. 538.22, Found 539.20(M*). Step 3. Synthesis of (2-amino-ethyl(2%amine-ethyl)ert butoxycarbonyl-aminob-ethyficarbamic acid tert-butyl ester 2o The acetamide 1.13 (167 g, 0.31 mol) was taken in a stainless steel pressure reactor and to it a solution of methylanine (33% by wt) in ethanol (200 ml) was added. The mixture was warmed to 90*C and stirred for 24 h. Reaction was monitored by nass spectra. AIR the solvents were removed under reduced pressure and the residue was subjected to high vacuum at 80 T to yield the product 114 (103 g, 96 %) as gummy 25 liquid and this compound could be used for the next reaction with out further purification, JH NMR (CDCh, 400MHz) 6 = 3.20-3.00(n, 41), 2,62.38 (in, 81), 132(s, 91), MS: C,1 H 26

N

4 0 2 Cal. 246:21, Found 246.20(M'). Step 4. Synthesis of Michael addition product 115 1o The diamine 114 (103 g, 0.297 mmol), N-dodecylacrylamide (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 for 4 days. The reaction was monitored by TLC and Mass 139 spectra. Thetc reaction mixture was extracted into dichiloromethane (DCM), washed successively with NaHCO3 solution and brine, dried over anhydrous sodium sulfate. Solvent was removed in vacio and residue thus obtained was purified by silica gel column chromatography (gradient elution- Ethyl acetate then 3-10% MeOH/DCM) to obtain 115 as a pale yellow solid (228 g, 59%). MS: CrsoNsOg Cal, 1303,16, Found 1304.20(M v). Step 5-Preparation of diamineli6 4M HCI in dioxane (500 mL) was added to a solution of the diboc compound 115 (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. After the complete disappearance of the starting diboc 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 NaHCOs (1L) and extracted with dichloromethane (3 x 600 mL). The combined organic extracts were dried and 15 concentrated to isolate the tetramer as a white solid (164 g, 85%), MS: CEJimNs04 Cal. 1103.05, Found 1104. 10(M). Step 6. Synthesis of 117: Compound 116 (164 g, 149 mmol) , N. dodecylacrylamide (35.6 g, 149 mmol) and saturated sohition of boric acid in water (30 niL) were taken together in a pressure reactor and heated at 90'C for 3 days. Progress of 20 the reaction was monitored by TLC and Mass spectra, The reaction mixture extracted into dichloromethane (DCM), washed successively with NaHCO 3 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/MeOI/DCM) to obtain 117 as a pale yellow solid (83.8 g, 25 42%), MAS: C 76 F 5 oN 5

O

5 CaL 1303.16, Found 1304.20(M). The material was compared with authentic sample TLC (qualitative), HPLC and Mass spectra. MS: CO 1 41 163

N

9 Os Cal. 1342.28, Found 1343.30(M 4 ). Step 7. Synthesis of the hydrochloride salt 7 The amine 117 (54 g, 40 mmol) was dissolved ethanol (100 mL) and to it 200 30 ml of 2M HC in ether was added and the mixture was stirred at room temperature ovemight. Nitrogen was bubbled to the reaction mixture and the outlet was passed through dryrite and to a 10% solution of KOH. After 30 minute, the reaction mixture 140 was concentrated to dryness and the residue was re-dissolved in 500 mL of 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 " overnight. The pure product was isolated as a cream powder (59.5 g, 99%). Re ipid sj§t4gv2 Method I 21 eq. Ethyl trifuroacetate H H H F

CH

2 CN, 0OCART ' N'N HN< N' NH 2 H HF HH 10 1 0 eq (Bac) 2 O, D3EA DCM/THF MeNH 2 /MeOH H p N2 N-' -YN 103 H 90 OC (2deys) H 102 H F F N H O Y H N'-O H HCI D oxane or Ether H N N N H 4 H H H GStep1: Trehlntta Ine (200g g, L37 mol, purchased front Sigmna Aldrich) in acetonitrile (2 L) in a 4 neck 5L flask wvith overhead stirrer was cooled over an ice bath under Constant stirring, Ethy tritluroacetate (388.5 g, 2,74 mol) -was added to thie stirring solution and stirred for 20h. SolveTnt and. volatiles were removed under reduced. pressure; the residuewas triturated with a mixture of DCM/Heqxame and filteredC' 141 to get 101. as white solid (429 g, 93%). The product thus obtained could b.e used for the next reaction without further purification. MS: CwOHIF&N 4 0 2 Cal 338,12, Found 339.0(M). Step2: Crude compound 101 (427g, 1.26 mol) was dissolved in a mixture of 5 solvents (3 L, THF/DCM (1:2)) and stirred over an ice-water bath. Ditert-butyl dicarbonate ((Boc4 2 0, 270 g, 1.26 mo, purchased from Sigma Aldrich) and DIEA (500 mL, 2.86 mo) were added to the reaction mixture and stirred overmight, Solvents were removed and the residue was extracted into dichloromethane (DCM, 1000 mL), washed successively with NaHCO3 solution (500 mL), water (500 ml x2) and brine, dried over 1D anhydrous sodium sulfate. Solvents were removed in vacuo and residue thus obtained was triturated with DCM/Hexane (2:1) and filtered, Solvents were removed and the residue was dried under high vacuum to get the compound 102 as gummy liquid (523g). Part of the compound 102 was purified by silica gel chromatography (gradient eltion, Ethyl acetate, fiolowed by 3~10% MeOHl/DCM) to obtain compound 102 as t gummy liquid (102,00g). H NMR for 102 (DMSO-d6, 400MIz) 6 = 9.60-9.10(m., 3H), 3,35-3.25(m, 4H), 3,25-3,20(2, 2H), 3.20-3.10(m, 21), 2,68-2.58(m, 41), 135(s, 9H). NIS: C tFN 4 0 4 Cal. 438.17, Found 439.20(M), 20 Step 3: Purified compound 102 (102.0g, 233.40 mmol) was dissolved in Ethanot/Methyl amine (400 ml, 33 wt% methylainine solution in EtCH) 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 25 product 103 (58.00 g, 99 %) as gummy liquid and this compound could be used for the next reaction with out further purification. 'H NMR (CDCl 3 , 400MHzS = 3:20 3.00(m, 411), 2.62-2,38 (m, 81), 1.32(s, 9H). MS: C 0

H

24 N0 2 Cal. 246.21, Found 247.20(M*). Step 4: Triamine 103 (56.00 g, 227,64 mmol), N-dodecylacrylamide (327,00 g, 30 1365 mmol) and saturated solution of boric acid in water (50 mL) were taken together in a pressure reactor and heated at 90'C for 6 days. The reaction was monitored by TLC and Mass spectra. The reaction mixture extracted into dichloromethane (DCM\ washed 142 successive with NaHC10 solution (400 iL) and dried over anhydrous sodium sulfate, Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient ehuion- Ethyl acetate then 3-1.0% MeOH/DCM) to obtain 104 as a pale yellow solid (186 g, 57%). i NMR (CCi, 400M1z) 6 5 7,20(bs, II), 7.05(bs, 1H), 6.85(bs, 1H), 674(bs, IH); 3,25-3,03(m, 1211); 2,80-2.60 (In, 8H), 2.55-221(m, 1211) 152-1L45(m, 10H), 1,42(s, 911), 1.34-1,20(m, 1001) 0U87(t, J 6.5Hz1-, 15H). MS: CadIrnN907 Cal. 1442.33, Found 1443.30(M), Step 5: 4M H11 in dioxane (400 mL) was added into a solution of compound 105 (184,00 g, 127.2$ mmol) in dioxane (300 mL, The reaction mixture was then 10 allowed to stir for overnight. The reaction was monitored by Mass spectra. Excess HC10 was removed by passing nitrogen through the solution, Solvents were removed under vacuum and residue was co evaporated three times with ethanol (500 miL X 3) to yield a pale yellow gummy solid 7 (186.00g , 98%) as tetra hydrochloride sal The material was compared with authentic sample 'LC (qualitative), HPLC and Mass spectra, MS: 15 C1 1

.

53

N

9

O

5 Cal. 1342.28, Found 1343,30(M). Method 2 Compound 102,was prepared as described in Method 1: steps I and 2. The crude product obtained from step 2 of Method I was used for the next reaction without fthiher 20 purification. Step 1: Compound 102 (103.45g, 238.90 mmol, crude compound from step 2. Method I was dissolved in Ethanol/Methyl amine (400 nl, 33 wt% methylamine 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 25 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 (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), N'dodecylacrylamide (320.00 g, 30 1338 mmol) nd saturated solution of boric acid in water (50 mL) were taken together in a pressure reactor and heated at 90'C for 6 days as described in step 4, Method 1, The reaction was monitored by TLC and Mass spectra, The reaction mixture extracted into 143 dichloromethane (DCM), washed successively with Na1CO3 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 elation- Ethyl acetate then 3 -0% MeOH/DCM) to obtain 104 as a pale yellow solid (65,2 g, 20%) Step 4: 2M HC in ether (800 mL) was added to compound 105 (65.00 g, 45 nmol). The reaction mixture was then allowed to stir fir overnight. The reaction was monitored by Mass spectra, Excess I 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 10 tetra hydrochloride salt. The material was compared with authentic sample TLC (qualitative), HPLC and Mass spectra. N: Csil-b3NsOs Cal 1342.28, Found .1343.30MI) Method 3 15 Compound 102 was prepared as described in Method 1: steps I and 2. The crude product obtained from step 2 of Method I was used for the next reaction without further purification Step3: Compound 102 (105.20g, 240 mmol, crude compound from method I) was dissolved in Ethanol/Methyl amine (400 ml, 33 wt% miethylamine solution in 20 EtOH) at ambient temperature in a pressure reactor. The mixture was warmed to 90T 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 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 25 Step 4: Triamine 103 (64.70 g, 240 mmol), Ndodecylacrylamide (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 90*C for 6 days. The reaction was monitored by TLC and Mass spectra, The reaction mixture extracted into dichloroniethane (DCM), washed successively with NaHCO 3 solution (400 mL) and dried over anhydrous sodium sulfate, 3o Solvent was removed in vacuo and residue thus obtained was purified by silica gel column chromatography (gradient ehition- Ethyl acetate then 3-10% MeOH/DCM) to obtain 104 as a pale yellow solid (192 g), 144 Step 5: The desired compound 7 was obtained as hydrochloride salt from compound 104 as described in step 5, Method I of Example 40, Compound 7: 194 g (98%) as tetra hydrochloride salt, The material was compared with authentic sample TLC (qalitative), HPLC and Mass spectra. MS: C 8 41 163 N40 5 Cat 1342,28, Found S 1343.30(M*). -formulated. inlto various ! MmfQi 41 _Compwiso of actity of 4 ionulidJnt da acutiiia gT gi iees: The effectiveness of lipid compositions can be tested by deterimnig the relative 10 ability of a lipid to deliver an siRNA moiety to a target. For example, the silencing of a target indicates ihat the siRNA is delivered into the cell Applicants have compared association complexes that include one of 13 different PEGIipid moieties as provided in Figure 5, together wi siRNA that is used to silence Factor VII (FVh),. PEG-lipids 13 were synthesized using the following procedures: Scheme I RI 1a R = 1411ay 13 R O 4 tf DSC, TEA 0 0m CPC-RT 0 .

mPEGCO-NHz Ror.N NN ^O

M

R -+Q)-Ol Py /DCM R'0 000-RT 4a R = C es 2a R= Ca 4b R = G H 2b R= C 15

H

33 4C R = C H 2c R C Scheme 1: mPEG2000-1 ,2 Di.O..alkyl-sn3-carbomoylglyceride Preparation of compound 5: 1,2-Di-0-tetradecyl -sn-glyceride 1 (30 g 61.80 20 uiol) and N-succinimidylcarboante (DSC, 2336 g, 1,5eg) were taken in dichloromethane (DCM, 500 mL) and stirred over an ice water mixture. Triethylamine (2530 mL, 3eq) was added to stirring solution and subsequently the reaction mixture was allowed to stir ovemight at ambient temperature Progress of the reaction was 145 monitored by TLC. The reaction mixture was diluted with DCM (400 ml) and the organic layer was washed with water (2X500 mL), aqueous Na1CO 3 solution (500 mL followed by standard work-up. Residue obtained was dried at ambient temperature under high vacuum overnight, After drying the cude carbonate 3 thus obtained was 5 dissolved in dicWoromethane (500 nL) and stirred over an ice bath, To the stirring solution tnPEG2o5Nl2 (4, 1030 g, 47.20 mmol, purchased from NCF Corporation, Japan) and anhydrous pyridine (80 mL, excess) were added under argon. The reaction mixture was then allowed stir at ambient temperature overnight. Solvents and volatiles were removed under vacum and the residue was dissolved in DCM (200 mL) and to charged on a column of silica gel packed in ethyl acetate. The colunn was initially elated with ethyl acetate and subsequently with gradient of 5-10 % methanol in dichloronmethane to afford the desired PEG-Lipid 5 as a white solid (10530g, 83%). H1 NMR (CDC 3 , 400 MHz) 6 = 5:20-5.12(m, 1H), 4.18-4.01(m, 211), 380-3,70(m, 2H), 3.70-320(m, -O-CH-C-CyO-. PEG-CI-2), 2,10-2,01(m, 2H), 1.70-1.60 (m, 2H), 1.56 is L45(m, 4H), 1.31-1,5(m, 48$), 0,84(t, J=:: 6.5Hz, 6H). MS range found: 2660-2836, Preparation of 4h: 1,2-Di-O-hexadecyl-sn-glyceride lb (1.00 g, 1,848 nnmol) and DSC (0.710 g, I .Seq) were taken together in dichioromethane (20 niL) and cooled down to O*C in ai ice water mixture. Triethylamine (1L00 mL, 3eq) was added to that 20 and stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (2 times), NaHCO 3 solution and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue 2b under high vacuum ovemight. This compound was directly used for the next reaction without further purification. MPEGra-NH 3 (150g, 0.687 mmol, purchased from NOF Corporation, Japan) and 25 compound from previous step 2b (0,702g, 1,5eq) were dissolved in dichloromethane (20 mL) under argom The reaction was cooled to 0*C. Pyridine (1 mL, excess) was added to tiat and stirred ovemight. 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% MeOHiDCM as a gradient fuon) to get the required 30 compound 4b as white solid (1.46 g, 76 %). H NMR (CDC 3 , 400 MHz) 8= 5.17(t, J 5,5Hz, 1), 4.13(dd, J= 4.00Hz, 11.00 Hz, IH), 4.05(dd, J= 5.00Hz, 11.00 Hz, 1H), 3,82-3.75Y(m, 211), 370-3.20(m, -O-CH 2

-CH

2 -0-, PEG-CH 2 ), 2.05-1L90(fn, 211), 180 146 1.70 (m, 21), 1.61-1 45(m, 61H), 135-1.17(m, 561), 0.85(t, J= 6,z, 6H). MS range found: 2716-2892. Preparation of 4e: ,2-Di -0-otad ecyl-sn-glyceride le (4.00 g, 6.70 mmol) and 5 .:DS (2.58 g, L 5eq) were taken together in dichloromethane (60 mL) and cooled down to O*C in an ice water mixture. Triethylamine (2.75 mL, 3eq) was added to that and stirred overnight. The reaction was followed by TLC, diluted with DCN, washed with water (2 times), NaHCO- solution and dried over sodium sulfate. Solvents were removed tnder reduced pressure and the residue under high vacuum ovemight. This 10 compound was directly used for the next reaction with further purification, MPEG7or NH 3 (LSOg, 0.687 mmol, purchased from NOF Corporation, Japan) and compound from previous step 2 (0.760g, 1 5eq) were dissolved in dichloromethane (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 TLO. Solvents and volatiles were 15 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 coTIpound 4 c as white solid (0:92 g, 48 %), 'H NMR (CDC, 400 MHz) $ = 5,22-5.15(m, 11-1), 4.16(dd, J= 4,00Hz, 11,00 Hz, I H), 4-06(dd J; 5.00Hz, 11,00 iz, 1H), 3.81-3,75(m. 2H), 3.70-3,20(m, -O-CHCH 2 -O , PEG-CH 2 ), 1.80-1.70 (m, 2H), 1.60-.48(m, 41), 20 L31-L 15(m6411), 0.85(t, J= 6.5Hz, 61), NS range found: 2774-2948. 147 Scheme 2a R'U 1bR CeHn ic R C Hk7 0 3 HBTU/IEA R OMG RIDMF/DCM R 5 "a ,;S RHC S R 6 R CAa 51R=C~ 6r, R =C~ Sc R = CRCeHx * Scheme 2: mPEG2000-1 ,2-Di-0-alkyl-sn3~succinylglyceride Preparation of compound 6a: l,2-Di-0-etradecyl-sn-glyceride la (.00 g, 2,06 mmol), succinic anhydride (0.416 g, 2 eq) and DMAP (0,628 g 2.5eq) were taken together in dichioromethane (20 mL) and stirred overnight. The reaction was followed 10 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, M lPEG 2 a,-NH 2 3 (1,50g, 0.687 mmol, purchased from NOF Corporation, Japan), compound from previous step Sa (0.66g, 1.12 eq) and HBTU 15 (0.430g, 1 ,13 mmo ) were dissolved in a mixture of dichloromethan/DMF (2:1, 20 mL) under argon. DIEA (0.358 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 overnight and purified by chromatography (first ethyl acetate then 5-10% MeO/DOCM as a gradient 20 lution) to get the required compound 6a as white solid (0822g, 43 %). 'H. NMR. (CDCI, 40() MHz) = 634-6.30(m, 1t), 416(dd, J= 4.00Hz, 11.00 Hz, 1-), 4,08(dd, J= 5,00Hz, 1L,00 Hz, 1H), 3.82-3.78(m, 2H), 370-3,30(m, -0-CC2-2-bO- PEG CH2), 2.64 (t, J= 7,00Hz, 2H) 2.43(t, J 6.80Hz, 2H), L76-1 .72(m, 2H), L56-L48(m, 41), 1L34-1.1 6(m 48H), 0.85(t, J= 6,5Hz, 611), MS range oiund 2644-2804, 14R Preparation of compound 6b: 1,2-i--hexadecyl-sn-giyceride lb (E00 g, L,848 nol), succinie anhydride (0.0,369 g, 2 eq) and DMAP (0.563 g, 2.5e) were taken together in dichloromethane (20 mL) and stirred ovemight. The reaction was S fioflowed by TILC, 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 fr the next reaction with further purification. MPEG oo-NH2 3 (1. 50g, 0.687 mmol, purchased from NOF Corporation, Japan), compound from previous step 5b (0.66g, 1,03 mmol) and t o HBTIU (0.400g, 1,05 mmol) were dissolved in a mixture of dichloromethane/DMF (2; 1, 20 mi) under argon. DIEA (0.358 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 vacuM overnight and purified by chromatography (first ethyl acetate then 5-10% MeCOH/DCM as a 15 gradient ehio to get the required compound 6b as white solid (0.300g, 16 %), H NMR (CDCP, 400 MHz) 6 = 6.33~6,28(m, 1H), 4.8(dd, J= 4,00Hz, 11LOO Hz, 1H), 4:08(dd, J::; 5.00Hz, 1LOO Hz, H), 3.82-3,76(m, 2H,1 3;0-3,30(m, ~O-CH-CI)

PEG-CH

2 ), 2,65 (t, J= 7.08Hz, 21H), 2.44(t, J: 6,83Hz, 2H), L,76-.68 (im, 211) 157 L.48(m, 4H), 132-117(m, 56H), 0.86(t, J 6.6Hz, 6H). MS range found: 2640-2822. 20 Preparation of compound 6c: I2-Di--octadeoy-sn-glyceride Ic (5.00 g, 837 mmol), succinic anhydride (1.70 g, 2 eq) and DMAP (2.55 g, 2,5eq) were taken together in dichloromethane (50 nL) and stirred overight. The reaction was followed by TLC, diluted with :DCM, washed with cold dilute citric acid, water and dried over sodium 25 sulfate, Solvents were removed under reduced pressure and the residue under high vacuum ovemight This compound was directly used for the next reaction with further purification. MPEO 20 wt-NH 2 3 (1 .50g, 0.687 mmol, purchased from NOF Corporation, Japan), compound from previous step 5c (0.718g. 1.03 mmol) and *IIBTU (0.410g, 108 mmol) were dissolved in a mixture of dichloromethaneDMIF (2:1,20 mL) under argon, 3o 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 overnight and purified by 149 chromatography (first ethyl acetate then 5-10% MeO-i/DCM as a gradient elution) to get the required compound 6c as white solid (11 g, 56 %), 'H NMR (CDC.], 400 MHz) S=:z 63 8-633(m, 11$), 4.19(dd, J= 4,00Hz, i.00 :1z, I H),) 4.07(dd, J= 5,00Bz. M LOO Hz, 1), 3.81-334(m, 2H1). 3.70-320(m, -O-CH-rC-., PEG-CH), 2.63 (t, J= 5 7.3Hz, 2H). 243(t J= 6,87Hz, 2H), 1.76-1,68 (m, 2H), 1.57448Qn, 41H), 1,32-1 17(m, 64811, 86( J 6,60Hz, 611), MS range found: 2680-2922 Scheme 3' OH O 0CM R'6 i R (' 'H SeaR C 1L) RP= CadiZ 1c R O11 1 lb R = 6 I R -CIH37 10) " Scheme 3: mPE*G2000-1,2-Di-0-alkylbsn3-succinylglyceride Preparation of compound 8a: 1,2-Di-O-tetradecyl-sn-glyceride lia (0.300 g, 0.618 mmoA), MPEG-Succinate 7 (LO.g, 0.476 nmiol, purchased from NOF 1 Corporation, Japa), DCC (0.127 g, 13eq) and DMAP (0,058 g, 0.476 nmol) were taken in dichloromethane (20 mL) under argon and stirred overnight. Reaction was monitored by T LC, The reaction mixture was cooled to 0C 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 2- EtOAc, followed by 5-10 % DCM/MeOH gradient elution) to get the compound 8a as a white solid (0-590 g, 48%) 1 H NMR (CDC, 400 MHz) 6 4 25-4718(m, 24), 4.08(dd, J= 5,60Hz 1. 50 Hz, 11-1), 3,80-3.73(m, 211), 3:70-3, 3 0(m, -O-CiECHrAt PE CH2), 1.56-L47(m, 4H), l.50-1.5(m, 481-1), 0.85(t, J= 6,60Hz, 6H) MS range found: 2440-2 708 2 5 Preparation of compound 8b: ,2-Di-O-hexadecyi-sn-glyceride lb 0.334 g, 0 618 mmwl), MPEQ-Sucinate 7(l00g, 0,476 mmol, purchased from NOF 150 Corporation, Japan), DCC (0,127 g, L3eg) and DMAP (0,058 g, 0.476 mmol) were taken in dichloromethane (20 miL) under argon and stirred ovemight. Reaction was monitored by TLC, The reaction mixture was cooled to 0oC alter stirring ovemight and filtered off the precipitated solid, Volatiles and solvents were removed under reduced 5 pressure and the resulting residue was purified by chromatography (first elUted with EtOAc, followed by 5-10 % DCM/MeGH gradient elation) to get the compound 8b as a white solid (0,930 g, 74%), H NNMR (CDCh, 400 MHz) 5 = 4.25-4 171(m, 211), 4.09(dd, 5= 550Hz, 1150 Hz, H), 3.81-3.73(m, 2H), 3,70-31.30(m, -O-CirC~rO-, PEG CH2), 1,58-1.47(m, 4H), L30-1.17(m, 56H), 086(t, J= 6,60Hz, 6H). MS range found: 2452-2760, Preparation of compound Sc: l,2-Di-O-octadeeyisn-glyeerid e (0,369 g, 0,618 mmol. MPEG-Succinate 7 (1.0g, 0.476 imol, purchased from NOP Corporation, Japan), DCC (0.127 g, 1 .3eq) and DMAP (0:058 g, 0,476 mmol) were i5 taken in diehioromethane (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. Volatfies and solvents were removed under reduced pressure and the resulting residue was purified by chromatography (first eluted with EtOAc followed by 5-1.0 % DCM/MeOH gradient elation) to get the compound Sc as a 20 white solid (0.960 g, 75%). 'H N.MR (CDC 3 , 400 MHz) S = 4 27-4,20(m 21H), 4.10(dd, J= 5.80Hz, 1150 Hz, Ill), 3.83-3.74(n, 21), 3,70-335(m. -(>C1-CH-O-, PE G-CH4 154-146(m, 4H), L30-117(m, 6411), 0,86(t, f 6.60Hz, 6H1), MS range fiund: 2508-2816. 25 Scheme 4" 151 O AH 0Y 1o -'i~ 0 --. DCM RK R'0 C,10a. R aC~ 10 R = , E, fiio V ' oOH 0R=c H R O 90R =, C Ha I * Scheme 4; iPEG2000-1,2-Di-O-acyl-sn3-succinylglyceride Preparation of compound 10a: 1,2-Dinyristoyh-sn-glycerol 9a (0.317 g, 06 8 5 unol), MPEG-Succinate 7 (1,00g, 0.476 mmol, purchased from 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 04C after stirring ovemight and filtered off the precipitated solid, Volatiles and solvents were removed under reduced pressure and the 10 resulting residue was purified by chromatography (first elied with RtOAc, followed by 5-10 % DCM/MeOH gradient ehrtion) to get the compound 10a as a white solid (0,960 g,78%),H NMR (CDCb, 400 MHz) 8 = 5.26-5.20(m, 1H), 4.30-4.08(m, 6H), 3,81 33(m, 2H). 3.70-3 4 0 6mO -- CIVCH 2 40, PEG-CH 2 ), 2.65-2.60(m, 4-), 2.35-2,28(ni 4H), 1.63-152(n, 4-), i.30-l.1 5(m, 441), 0,86(t, J 6.601z, 6),NI MS range found: 15 2468 -2732. Preparation of compound 10b: 1,2-Dipalmitoyl~sn-glycerol 9b (0.352 g 0.618 mmol), MPEG-Succinate 7 (LOg, 0.476 mmol, purchased from NOV Corporation, Japan). DCC (0,127 g, .3eg) and DMAP (0.058 g, 0.476 mmol) were taken in 20 dichloromethane (20 mL) under argon and stirred overnight. Reaction was monitored by 'PLC. The reactionmixture was cooled to 0C after stirring ovemight 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 elation) to get the compound 10b as a white solid (1.02 g 25 81%), '1 NMR (CDC1, 400 MHz) 6 = 5.26-5,19(in, 1 H), 4.30-4.05(m 6j), 3180 3,40(m-O-CH 2 -C1O, PEG-CH 2 ), 2,65-2.60(m, 4H), 2.33-2.24(m, 41), 1.63-1.50(m. 4H)j, 130-115 (, 52H), 0,85(t, J 6.60Hz, 6H); MS range found: 2524-2792. 152 Preparation of compound 10c: 1 2Diseroy-sn-glycerol 9c (0.387 g, 0.618 mmol), MPEG~Succinate 7 (1.00g, 0.476 mmol, purchased from NOF Corporation, Japan) DCC (0.127 g, 1.3eq) and DMAP (0.058 g, 0,476 nmmol) were taken in S dichioromethane (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 EtC}Ac, followed by 5-10 % DCMYMeOH gradient elution) to get the compound 10c as a white solid 1 (1,04 g, 80%), 'H NMR (CDC 3 , 400 MHz) 6= 5.26-5.19(m, 11), 4.30405(m, 61), 3.80-340(I -O-CN-CHr-O2- PEG-CM 2 ), 2.66-2,59(m, 4H); 2.31-2.26(m, 4H), 1,63 1 52(m, 4-) 1 30-1.15(m, 52H), 0,95(t, J= 6.60Hz, 611). NIS range Found: 2540-2844, 153 Scheme 5" o' S \ /nf HBTUDIEA DMF/DCM L, 0 12 0 Sebheme 5: Choesteryl-mPEG2000 Preparation of compound 13: mPEGew-OH 11 (6,00g, 3 mmol, purchased rm Sigmna-Aldrich), Cholesterol hemisuccinate 12 (1.50 g, 3.08 mmol mmol) and 10 HBTU (1.23g 3.23 mmol) were dissolved in a mixture of dichloromethane/D.MF (2:, 100 1L) under argon. DIFA (L60 mL, 3 eq.) was added to that and stirred overnight. Solvents and. volatiles w vere 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 chition) to get the required compound 13 as white solid 15 (5.05g, 68 %). 'H NMR (CDC%, 400 MIH;) 6 5.35-5.25(m 1H), 4.60-40(m, 1H1), 4.22-418(m, 2H), 3;80 2 76(m, 2H), 3.72-3,40(m, -O-CH~CYO-, PEG-CH), 2,64 2,56(, 4), 2.31 -2.20(m, 3H),2.01-08(m, 4411)AMS range found: 2390-2654, uxamn4e 2: 2ageted PEG-iid: 154 CH ttN NH2N^W^ftN rO Acc HTU, DEA 15 A WNC 17 AO OAC AcHN H ' H NaO H, DCI'MoH HO-- N~t~ ACHN H rsH 19 Preparation of 19 Step 1: Compound 14 (2.00 g 1,01 mmol) and cholesterol chioroformate 15 (0,453 g, 1.01mmol) were take. together in dichloromethaie (20 mL), The mixture was 5 cooled in an ice-water bath, Triethylamine (0.448 ml) was added amd the reaction mixture was stirred overnight. Reaction was monitored by TLC, Solvent was removed and the residue was purified by silica gel chromatography (Ethyl acetate flowed by 5 10% MeOHDCM) to get the desired compound 16 (110g, 45,40 %). 'H NMR (CDCU, 400 MHz) i 5.35(m, 115(m, 11), 3.40-3.85(m, 0-O-1rCH-Q), 3.10-3,25(mL 10H), 0.80-2.,38(m, 44H, Cholesterol). MS range found: 2220-2490. Step 2: Compound 16 (1 Mg, 0,417 mmol), 17 (0.235g, 0542 mmol) and HBTU (0.190g, 0.5 mmol) were taken in a mixture of DCN/DMF (20 ml 2-1 ) To that DIEA was added and stirred overnight. Reaction was monitored by TLC, solvents were 15 removed under reduced pressure and the residue was purified by chromatography (5 10% MeOH/DCM) to get the desired compound 18 (1.02g, 87 %). 'H NMR (DMSO d6, 400 MHz) 6 z 7.52(d, J= 8.06 Hz, 1H-), 7.33(t, J= 7.02 Hz, H), 7,25(t, J= 7,32 Hz. 1H), 5.27(m, 11), 5. 8(d, J= 3.2 Hz, 11-1) 492(dd, J= 3.17, 11.23 Hz, 11), 4.43(m, 11), 360-4.02(m;5H), 3,20-3.55(m, 0-CH-CH 2 -0), 2.90-3,10(m, I OH), 2,05(s, 3H), 1,96(s, 155 3H), l.84(s 3H), L77(s, 3H), 0.80-2 38(m, 44H, Cholesterod). MS range found: 2680 2990; Step 3: Compound 18 (l02g, 0.362 mmol) was dissolved in a mixture of MeOH/DCM (10 mL) to that 0,5 MA solution of NaOMe in methanol (excess) was added and stirred ovmight. Progress of the reaction was monitored by I'LC, The mixture was neutralized with AcdH, Solvents were removed under vacuum and the residue was purified by &hromatography (5-10 % MeOHl /DCM) to get compound 19 (280 mg, 30%). U NMR (CDC, 400 MHz) S = 538(m, IH), 4,02-4.06(, 71-1), 330-3.80(m, 0 10 CrCH 2 O), 3.20+3.29(m 8H), 2.08(s, 3H), 0.80-2.38(m, 44.1, Cholesterol), MS range found: 2600-2900. Exanmle 43; Targetei PEG-lipids 14 DcM, Py o 0 O-RT H 0 AcOA HTU, DAEA AcHN AcO OAC 0 AcHN HH o 22 NaOMe DCMOMeOH HO N o AQHN M " H 23 15 Preparation of 23: Step 1: Compound 14 (2,00 g, 1.01 mmol) and compowd 20 (0.453 g, L Olmmioi) were taken together in dichloromethane (20 mLh). The mixture was cooled in an ice-water bath, Pyridine (1 mL, excess) was added and tie reaction mixture was 156 stirred overnight, Reaction was monitored by TLC. Solvent was removed and the residue was purified by silica gel chromatography (Ethyl acetate followed by 5-10% MeH/DCM) to get the desired compound 21 (400 mg, 15 %) 'H NMR (CDC.h, 400 MHz) 8 = 5.20(m, 11H), 4.05-4.20(n, 2H), 3,20-3.80(n, 0-C HC 0), L70-. 82(m, 4H), 1,50-1 61(m, 2H), L18-L38(m, 60H), 0,87(t, J= 630 Hz, 6H1). MS range found; 2400-2750. Step 2: Componid 21 (0.415 g, 0.-59 mmol), 17 (0.100g, 13 eq) and HBTU (0,90g, 115 eq) were taken in. a mixture of DCM/DMF (20 mL, 2:1). To that D[EA (0,2 10 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.

4 50g, 94%), 'H NMR (CDI, 400 MHz) 8 = 6,21(d, J= 8.70 Hz, 1H), 5,33(d, J= 2.70 Hz, 1H), 515-5.20(m, 2-), 4.55(d, J= 8.15 Hz, 1B), 4.01-4.20(m, 4H), 3,20-3,90(m, O-C8rC 2 O), 234(s, 3H), 15 2,03(s, 3H), 1,99(s, 3H), U93(s, 31), L70-1,82(m, 4f), L50-1.61(m, 4H), L1i7-1.38(m, 60H), 0.86(1, J= 632 Hz, 6 H). 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 20 and stirred overnight. Progress of the reaction was monitored by TLC. The mixture was neutrlized with AeOh. Solvents were removed under vacuum and the residue was purified by chromatography (5-10 % MeOlH/DCM) to get compound 23 (365 mg, 85 %) 'H NMR (CDC 3 , 400 NHz) 3 = 5.18(m, 1), 4.05-4.20(m, 4H), 3.20-3.90(m, 0 C~t-CHrO). 2.05(s, 311), L71-L80(m, 4H), 1Q50-1.6l(m, 4H), 1.17-L38(m, 60H), 25 0,87 (t, J 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 FVIL For example, firmulation 3 provided a relative high degree of silencing of FV11, as did formulation 5, 6, and 12, 30 Examipt~i14;'Toljerablity of IforulationNPOIasdosd_inmice Empty liposones with composition ND98:cholesterol:PEG-C4= 42:48: 10 (molar ratio) were prepared as described in Example 45. Different amounts of siRNA 157 were then added to the pre-tornied, extruded empty liposomes to yield formulations with initial total excipient:siRNA ratios of 30:1, 20:1, 15:1, 10:1, and 5:1 (wtwt). reparation of a formulation at a total excipient:siRNA ratio of 5:1 results in an excess of siRNA in the formulation, saturating the lipid loading capacity. Excess siRNA was 5 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 fornaulations was assessed by measuring the body weight gain of the animals 24 h and 48 h post administration of the formulation, the results of which are provided in. Figure 7, i0 Example 45: Formation of association complexes byfirst fnning uodd complexes and then treating the unloaded compplxe ithiR A administration of association conlexes including two therapeutic agents Association complexes having two different nucleic acid moieties were prepared 5' as follows, Stock solutions of ND98, cholesterol, and PEG-C14 in ethanol were prepared at the following concentrations: 133 mg/mL, 25 mg/mil, and 100 mngmL for ND98, cholesterol, and PEG-Cl4, respectively. The lipid stocks were then mixed to yield ND98:cholesterol:PEG-C14 molar ratios of 42:48:10. This mixture was then added to aqueous buffer resulting in the spontaneous formulation of lipid nanoparticles 20 in 35% ethanol,.100 mM sodium acetate, pH 5. The unloaded lipid nanoparticles were then passed twice through a 0,08 pm membrane (Whatman, Nucleopore) using an extruder (Lipex, Northern Lipids) to yield unimodal vesicles 20-100 nn in size. The appropriate amount of siRNA in 3 5% ethanol was then added to the pre-sized, unloaded vesicles at a total excipient:siRNA ratio of 7.5:1 (wt:wt). The resulting mixture was 25 then incubated at 37 *C for 30 min to allow for loading of siRNA into the lipid nanopartcles. After incubation, ethanol removal and buffer exchange was performed by either dialysis or tangential flow filtration against PBS. The fmal formulation was then sterile fitered through a 0.2 ptm filter. A flow chart demonstrating the order of addition of exhipients and therapeutic agents is provided in Figure 8, 30 A 1:1 mixture of siRNAs targeting ApoB and Factor VIi were fbrnulated as described in Example 44, Separately, the same ApoB- and Factor VII-targeting siRNAs were individually fb-mutated as described in Example 3. LThe three formulations were 158 then administered at varying doses in injection volume of 10 pLig animal. body weight, Forty-eight hours after administration, serum samples were collected by retroorbital bleed, animals were sacrificed, and livers were harvested, Serum Factor V. concentrations were determined using a chromogenic diagnostic kit (Coaset Factor VII 5 Assay Kit, DiaPharma) according to manufacturer protocols. Liver mRNA levels of ApoB and Factor VII were detennined using a branched-DNA (bDNA) assay (Quantigene, Paiornics), the results of which are provided in figure 9. No evidence of inhibition between the two therapeutic agents was observed. Rather, both of the theraceutic agents demonstrated effectiveness when administered. 10 Examaie 46: Methods of making associationgop ingyrfrmed vsiele Lipid Stock Preparation Stock solutions of lipidoid ND984H1CI (MW 1487), cholesterol, and PEG-Cl 4 15 were prepared in ethanol at the following concentrations; 133 mg/mL, 25 mg/mLand 100 mg/mL for ND98, cholesterol, and PEG-Cl4, respectively Stock solutions were warmed at 50'C to assist in bring lipids into solution. Empty Vesiele Preparation 2 The lipid stocks were then mixed according to the volumes listed below to yield ND98tchoiesterol:PEG-C14 molar ratios of 42:48:10. An aqueous mixture was also prepared according to the volumes listed in the table be] ow. Voume Lipid Mixture (mL) ND98 Cholesterol PEG Tota 56,250 90.0 00 159175 Aqueous Mixture (mL) 3M Water NaOAc Ethanol T otal 378,000 27,000 40.327 445,327 25 The ethanolic Lipid Mixture was then added to the Aqueous Mixture while ripidly stirring on a magnetic sin plate, Upon mixing, lipidoid vesieles formed 159 sotaneously. The resulting vesicles were then extruded (2 passes) through a 0.08 membrane (Whatman, Nucleopore) 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 inM sodiun acetate pH 5 at a concentration of 10 mg/mL An appropriate volume of this siRNA stock was mixed with the appropriate volume of ethanol to yield a diluted siRNA solution in 5% (vol) ethanol (see table below). siRNA Dilution Stock sIRNA mg/mL) (50 nM NaOAc) Ethanol Total 10 19923 276.923 277 mL of diluted siRNA solution was added to 623 ml of empty vesicle mixture while rapidly stirring on a magnetic stir plate. The resuming combined mixture 5 was then incubated at 37*C for 30 main to allow for loading of siRNA. Ultrafiltration and Terminal 0.2 s Filtration After incubation, the 900 nL loaded nanoparticle mixture was diluted into LS , of PBS to yield a 2.7 L diluted mixture. This diluted mixture was then concentrated to ~ 20 L L and diafiltered by tangential flow filtration against 10 volumes of PBS using a Satorius TFF system utilizing two stacked 100,000 MW(C) cartridges, No back pressure was applied to the cartridge and the pump speed was set to 300 rpm. After buffer exchange the resulting solution was concentrated to roughly 2 mg/mL siRNA. Terminal filtration was performed by passing the solution through a 0.2 y filter 25 capsule (Whatmrnan, Polycap 36 AS). A flow chart illustrating this process is shown in Figure 10. 160 Example 47: Conmarison of particle size on effeacy Association Complexes were fomied using the procedure generally described in Example 46. However, because the complexes were being evaluated based on size, different extrusion membranes were used to produce particles having the following S diameters: 150 nn, 85nm, 60 anm, and 50 nm. The siRNAs loaded in the complexes targeted factor VIl The particles were evaluated in a Factor VII silencing assay, demonstrating that the 50 nm patieles were the most efficacious relative to the 1 50 rnm, 85nm, and 60 mn particles. The results of the assay are depicted in Figure I1I o Exanple 48: Conparison of half life of nucleic acid agents unfomilated versus formulated into an association complex The hlf 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 puiposes of comparison unformulated siRNA was also evaluated in vitro in 15 human serum. The percent of full length product detennined by FIPLC was evaluated for both the formulated and unfonmuated siRNA, As demonstrated in Figure 12, the fonmulated siRNA had a significantly improved half life in vitro in human serum. Exanple 49; Comparison of efficacy of association having PEG lipids of vari chain leng th 20 Association complexes were prepared as in Example 46 with variation on the length of the alkyl chain of the PEG 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 in Figure 13, chain lengths of 13,14, and 15 demonstrated the most sileRcing as measured in the 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 the scope of the following claims. 1SF

Claims (208)

1. 2. The preparation of claim 1, wherein when R is not. H, R is 14 25 3, Thew preparation of claim 1, wherein when R.is not H ,R is Rb.
2. 4. The preparation of claim 1, wherein when R is not H, R is :'R 162 5, The preparation of claim 1, wherein when R is not 1, R is Rld
3. 6. The preparation of claim 1 wherein when R is not H R is R, 5 7, The preparation of claim 1, wherein n 2 of the R moieties of formula (1) are not It 8, The preparation of claim 1, wherein n + 3 of the R moieties of formula (1) are not H.
4. 9. The preparation of claim 1, wherein n ± 4 of the k moieties of formula (1) are not H. 10, The preparation of claim 1, wherein n > 0, at least one R of.NR of s formula (1) is I. 11, The preparation of claim 1, wherein at least one R of NR2 of formula (1) is [L 20 12. The preparation of claim 1, wherein at least 80% of the molecules are a single structural isomer,
5. 13. The preparation of claim 12, wherein n + 2 of the Rt moieties of formula (i) are not H. 25
6. 14. The preparation of claim 12, wherein n + 3 of the R moieties of fomida (1) are not H.
7. 15. The preparation of claim 12, wherein n + 4 of the t moieties of fomnnua so (1) are not R. 163
8. 16. The preparation of claim 1, wherein at least n + 2 of the R moieties of formula () in at least about 90% of the compound. of formula (1) are not i.H, 17, The preparation of claim 1, wherein at least n + 2 of the R moieties of 5 formula (I) in at least about 95% of the compound of formula (1) are not [1, 18 The preparation of claim 1, wherein at least n+ 2 of the R moieties of formula (1) in at least about 99% of the compound of fomina (I) are not H1, 10 19. The preparation of claim 1, wherein n is 2,
9. 20. The preparation of claim 1, wherein n is 0, 2 iL The preparation of claim I.wherein X and X are C2 alkylene, 15 22, The preparation of claim 1, wherein n is 0 and X' is ethylene or propylene, 23, The, preparation of claim 1, wherein n >I and Xa varies with at least one 20 occurrence,
10. 24. The preparation of claim 1, wherein when R not H1, R is O Y' 25 25. The preparation of claim 24, wherein Y is 0 or NR 2 , 26, The preparation of claim 24, wherein m is 2.
11. 27. The preparation of claim 24 wherein Y is 0 or NR 2 and m is 2. 30
12. 28. The preparation of claim 24, wherein in is 1. 164
13. 29. The preparation of claim 1, wherein R for at least one occurrence is alkyl 5 30, The preparation of claim I, wherein R' for each occurrence is alkyl 31, The preparation of claim 1, wherein R is alkyl and R2 is -.
14. 32. The preparation of claim 1, wherein R1 and R are alkyi 10
15. 33. The preparation of claim 1, wherein R1 for at least one occurrence is aIkenyl,
16. 34. The preparation of claim I, wherein R for at least one occurrence is 15 alkenyt 35, The preparation of claim 1, wherein when R is not H, R is R,, and wherein Y is O or NH,
17. 36. The preparation of claim 35, wherein Y is 0,
18. 37. The preparation of claim 35, wherein Y is NH. 38, The preparation of claim 35, wherein R is alkyl. 25 39, The preparation of claim 38, wherein R! is Cwei adkyl.
19. 40. The preparation of claim 39,. wherein R1 is C1 alkyl, 30 41. The preparation of claim 35, wherein n is 2, 165 42, The preparation of claim 41, wherein X, for each occurrence is 2 alkylene and X4 is C2 alkylene,
20. 43. The preparation of claim 35, wherein in is 2, 5 44, The preparation of claim 1, wherein n is 2 and R, when R is not i, is R,
21. 45. The preparation of claim 44, wherein 'R is alkyl. 10 46. The preparation of claim 45, wherein RW is Cjo- alkyl 47, The preparation Of claim 46, wherein R is C 2 alkyl 48 The preparation of claini 44, wherein Y is O. 15
22. 49. The preparation of claim 44, wherein Y is NIL
23. 50. The preparation of claim 44, wherein X, for each occurrence is C2 alkylene and Xb is C2 alkylene. 20
24. 51. The preparation of claim 44, wherein m is 2.
25. 52. The preparation of claim 1, wherein at least I R. of NR is : and R, when not 1H is R. and wherein Y is ) or NHI 25
26. 53. The preparation of claim $2, wherein Y is QO 54, The preparation of claim 52, wherein Y is NH. so 55. The preparation of claim 52. wherein R' is alkyL
27. 56. The preparation of claim 55, wherein RI is Cm 3 o alkyl, 166 57, The preparation of clain 56, v/herein R is C, alkyl. 58 The preparation of claim 52, wherein n is 2. 59, The preparadion of claim 58, wherein X", for each occurrence is C2 alkylene and xb is C 2 alkylenc.
28. 60. The preparation of claim 52, wherein n is 2.
29. 61. The preparation of claim 1, wherein n is 2 and at least 1. R of NR is H and when R. is not H, R is 1, and wherein Y is 0 or NI.
30. 62. The preparation of claim 61, wherein R is aky l. 15 63>, The preparation of claim 62, wherein R is Cass alkyL
31. 64. The preparation of claim 63, wherein R is C 12 alkyl. 20 65. The preparation of claim 61, wherein Y is 0,
32. 66. The preparation of claim 61, wherein Y is NH,
33. 67. The preparation of claim 61, wherein X, for each occurrence is C2 25 alkylene and. X is C? alkylene. 68, The preparation of claim 61, wherein m is 2. so 69. The preparation of claim 1, wherein at least I R of NiR 2 is H and R is R, and wherein Y is O or NIH. 167
34. 70. The preparation of claim 69, wherein Y is 0, 1. The preparation of claim 69, wherein Y is NH. 5 72. The preparation of claim 69, wherein R' is alkyl.
35. 73. The preparation of claim 72, wherein R' is Cao alkyL
36. 74. The preparation of claim 73, wherein. R is C, 2 alkyL 10 75, The preparation of claim 69, wherein n is 2.
37. 76. 'The preparation of claim 69, wherein Xa, for each occurrence is C> alkylene and X is C2 alkylene.
38. 77. The preparation of claim 69, wherein m is 2.
39. 78. The preparation of claim 1, wherein n is 2 and at least I R of NRs is H and R is R, and wherein Y is 0 or NH. 20
40. 79. The preparation of claim 78, wherein R is alkyl. 80, The preparation of claim 79, wherein R !is C, 1 0 .. aalkyL 25 81, The preparation of claim 80, wherein R is Ca alkyl.
41. 82. The preparation of claim 78, wherein Y is 0. 83, The preparation of claim 78, wherein Y is NIL 3M) 84, The preparation of claim 78, wherein X", fbr each occurrence is C2 alkylenc and X is C2 alkylene. 168
42. 85. The preparation of claim 78, wherein m is 2.
43. 86. The preparation of claim 1, wherein n is 0 and X is propylene. 5 87, The preparation of claim 86, wherein I R is H. 88 The preparation of clam 86, wherein when R is not H, R. is R, 10 89, The preparation of claim 86, wherein RI is alkyL 90, The preparation of claim 89, wherein R is C.o alkyl. 91, The preparation of claim 90, wherein RW is C1 alkyl. 925 Thepreparation of claim $6. wherein Y is 0. 93 The preparation of claim 86, wherein Y is NO. 20 94 The preparation of claim 86, wherein m is 2.
44. 95. The preparation of claim 1, wherein n is 2 X', for each occurence is C alkylene and X" is C 2 alkyence; and 25 wherein each R is H or 0 R 1 R, i is. 2; so Y is NH or 0; R' is C, alkyl. 169
45. 96. The preparation of claim 95, wherein at least 80% of the molecules of the compound of formula (1) are a single structural isomer. 97, The preparation of claim 95, wherein Y is NH. 98 The preparation of claim 97, wherein at least 80% of the molecules of the compound of formunla (1) are a single structural isomer. 10 99, The preparation of claim 98, wherein R is R., for 5 occurrences, 1.00. The preparation of claim 95, wherein in at least 80% of the molecules of the compound of fommula (1), R is Ra, for 5 occurrences, 15 10 The preparation of claim 100, wherein Y is NH.
46. 102. The preparation of claim 95, wherein the compound of formuda (1) is an inorganic or organic salt thereof 2o 103, The preparation of claim 102, wherein the compound of formula (1) is a hydrohalide salt thereof
47. 104. The preparation of claim 103, the compound of formula (t) is a hydrochloride salt thereof 25
48. 105. The preparation of claim 1, wherein the hydrochloride salt ranges froin a single equivalent of HCL, to n-+2 equivalents of HCI.
49. 106. The preparation of claim 1, comprising a hydrate of the compound of so formula (1) 170 107, The preparation of claim 1, wherein the compound of fiomula (I) is salt of an organic acid. 108 The preparation of daim 107, wherein the salt is an acetate. 109 The preparation of claim 108, wherein the acetate salt ranges from. a single equivalent of acetate, to n+2 equivalents of acetate.
50. 110. The preparation of claim 107, wherein the salt is an fomate, Io I i. The preparation of claim 108, wherein the formate salt rangres from a single equivalent of acetate, to n+2 equivalents of fornmate, 112, The preparation of claim I, wherein R' comprises an alkenyl moiety. '15
51. 113. The preparation of claim 112, wherein I comprises a cis double bond.
52. 114. The preparation of claim 1, wherein the preparation comprises less than H 2 N XN NH2 1.H 11%, by weight, of 20 formula (HO1), wherein X and a are defined as in fonula (1) of claim 1, 115, The preparation of claim 1, wherein the preparation comprises less than 25 90% by weight of 0 formula (IV) wherein Y and R 1 are defined as in fomiula (1) of claim 1 17 1
53. 116. The. preparation of claim 1, the preparation comprising a plurality of compounds of formula (1).
54. 117. The preparation of claim 116, the preparation comprising a mixture of compounds of the fannulas below; R R H R'N 'N and R formula (F) formula (I) wherein in frommla (I") at least live of the R moieties are R' 10
55. 118. The preparation of claim 117, wherein formula (') and (I") are present in a ratio of from about 1:2 to about 2:1,
56. 119. A method of making a compound of fornala (11), 15 RgN' 'NJ ' NR2 R formula (II) wherein each Xa and Xt for each occurrence, is independently Ci. alkylene; 20 nis 0, 1 ,2, 3, 4, or 5; and wherein each R is independently H or Q RI 25 m is 2; Y is 0, NR' or S; R is alkyl or alkenyl; R 2 is H or C alkyl or alkenyl; 172 the method comprising reacting a compound of formula (Il) X 9 X$NH formula (111) with a compound of fonula (V), 0 5 formula (IV) in the presence of a promoter. 10 120, A method of making a compound of fonnula (II), R 2 NiXNL xNR 2 SR, formula (II) wherein s each X< and X , for each occurrence, is independently C6 alkylene; n is 0, 1 , 3, 4, or 5; and wherein each R is indcpendenty H or 0 Ra m is 2; Y is 0, NR 2 , or S; R is aikyl or alkenyi; R' is H or C alkyl or alkenyl; 25 the method comprising reacting a compound of formula (11) 173 HN N{X'NH, n formula (III) with a compound of fornula (IV), 0 Stbrmula (IV) in the presence of a quencher, 12. A method of making a compound of formula (II), 10 R 2 N {xN X'NR RINR formula (II) wherein each Xa and X, for each occurence, is independently Ca alkylene.; 15 n is, 1t 4, or 5; and wherein each R is independently H or 0 4 j,\ ~.. R! 20 n is 2; Y is 0, Nf 2 , or S; R is alkyl or alkenyL; R 4 is H or alkyl or alkenyl; the method comprising reacting a compound of fomiula (iII) 174 H 2 JN 1 fxjX% ay fomula (Ill) with a compound of fbrm ula (IV), 0 5 formula (IV) wherein the reaction mixture comprises from about 0.8 about 1.2 molar equivalents of a compound of f(mula (II), with from about 3.8 to about 6,5 molar equivalents of a compound of fbmula (IV), 10 122, The method of claim 121, wherein the reaction mixture comprises fom about 0.8 about 1.2 molar equivalents of a compound of formula (111), with from about 5.5 to about 65 molar equivalents of a compound of formula (IV). 15 12. The method of claim 122, wherein the reaction mixture conin ses about I molar equivalents of a compound of formula (M.), with from about 6 molar equivalents of a compound of formula (IV).
57. 124. The method of claim 121, wherein the reaction mixture comprises about 20 1 molar equivalents of a compound of formula (III), with f&om about 5 molar equivalents of a compound of fornula (IV).
58. 125. A method of making a compound of formula (['), r I R 2 N XN} 'XNR? 1R, L Jr frmula (I1) wherein 75 each X" and X', for each occurrence, is independently Cv, alkylene; n is 0, 1, 2 , 3, 4, or 5; and wherein each R is independently H or 0 R;, in is 2; Y is 0. NR 2 , or S; R 1 is alkyl or ulkenyl; o R 2 is Hl or alkyl or ilkenyl; the method comprising a two step process of reacting a compound of fonrula (Ill) H 2 N{X NrX NN 9 aX HN ' 'n'H formula (111) 15 with a compound of fornla (IV), fonnula (IV) in the presence of bodc acid and water wherein, the first step process involving the reaction mixture comprises 20 from about 0.8 about 1.2 molar equivalents of a compound of farnula (Hi), 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 L2 molar equivalent of copn.1'd of formula (IV). 25 126. A method of making a compound of formiia (II), 176 R 2 N xN ANR 2 formula (II) wherein ea&h X* and X", for each occurrence; is independently Ct alkylene; 5 n is 0 12, 3, 4, or 5; and wherein each R is independently H or Q R, m is 2; Y is 0, NR, or S; R' is alkyl or alkenyl; R is 1 or alky or alkenyl.; the method comprising reacting a compound of formula (111) lX_ X H 2 N NHz formula (11) with a compound of fomula (IV), .RI formula (IV) 20 and separating at least one structural isomer of formula (II) from the reaction mixture to provide a substantially purified preparation comprising a structural isomer of formula (II). 25 127. The method of claim 126, wherein the structural isomer of formula (11) is separated from the reaction mixture using chromatographic separation, 177
59. 128. The method of claim 127, wherein the chromatographic separation is using flash silica gel for separation of isomers. 5 129. The method of claim 128, wherein the chromatographic separation is gravity separation of isomers using silica gel,
60. 130. The method of claim 128, wherein the chromatographic separation is using moving bed chromatagraphy for separation of isomers 10 131 The method of claim 128, wherein the chromatographic separation is using liquid chromatagraphy (LC) for separation of isomers 132, The method of claim 131, wherein the chromatographic separation using 15' is normal phase HPLC for separation of isomers,
61. 133. The method of claim 131, wherein the chromatographic separation is using reverse phase HPLC for separation of isomers. 20
62. 134. The method of claim 126, wherein the substantially purified preparation comprises at least about 80% of the structural isomer of formula (I), 135, The method of claim. 134, wherein the substantially purified preparation 25 comprises at least about 90% of the structural isomer of formula (II),
63. 136. The method of claim 135, wherein the substantially purified preparation comprises at least about 95% of the structural isomer of formula (11). 30 137. A method of making a compound of formula (V) or a pharmaceutically acceptable salt thereof, 178 R 2 N X N JX'NR 2 'RI' I .n formula (V) wherein each Xa and X', for each occurrence, is independently Cy6 alkylene; n is 0, 1, 2, 3, 4, or 5; and wherein each R is independently H or p R o m s Y is 0e,NR or S; R is alkyl or alkenyl; R2 is H or alkyl or alkenyl; the method comprising reacting a comprmd of fbnnula (.11) Xa, X H 2 N 'r 'NH 2 L formula (III) with a compound of formula (VI), 0 Y R1 C CI or Br or I 23 fornula (VI) to provide a compound of formula (V) or a pharmaceutically acceptable salt thereof 2138 The method of claim 13v7,wherein the pharmaceutically acceptable salt thereof is a hydrochloride salt of the compound of forniula (V). 179 1 39. A compound of fornvula (X), H2 2 S tonnula (X) wherein R' and R(2 are each independently H-, CrC 6 eay optionally substituted with 1-4 RC, alkenyl, optionally substituted with 1-4 R, or C(NR)(NR)2; R 3 and R 4 are each independently alkyl, alkenyl, alkyoly, each of which is 10 optionally substituted with hinoro, chloro, bromo, or iodo; L, and L2 are each independently -NRvC(O), -C(O)NR-, -OC(0)C-, -(Q)O-, S-S-, N(R 0 )C(O)N(R), -OC(O)N(R)-, -N(R)C(0 -ON=C-, OROC(O)NH N=C-, or -NHC()NH-N=C t-R 3 and L- 4 can be taken together to form an acetal, a ketal, or an orthoester, va wherein R' and R4 are defined as above and can. also be H1 or phenyl; R^ is fluoro, chloro, bromo, iodo, -OR , -N(R)(R%, -CN, SR", S(0) S(0)2R(10 R is ,i, CrC alkyl, R7 is H or C,-C6 alkyl; 20 each R( and R' ae independently H or C,-C3 alkyl; Rt"! is H or Crs-f, alkyl; m is 1, 2, 3, 4, 5, or 6; n is 0, 1, 2, 3, 4, 5, or 6; and pharmaceutically acceptable salts thereof
64. 140. The compound of claim 139, wherein the compound is an inorganic salt thereof, 141L The compound of claim 140, wherein the compound is a hydrohalide salt so thereof, I80
65. 142. The compound of claim 141, wherein the compound is a hydrochlori de salt thereof,
66. 143. The compound of claim 139, wherein the compound is an organic salt thereof
67. 144. The compound of claim 139, wherein R1 and R2 are each independently C Ct aIkyl 10 145, The compound of claim 139, wherein R! is methyl. 146, The compound of claim 139, wherein R2 is methyl. 147, The compound of claim 139, wherein R1 and .R. are both methyl.
68. 148. The compound of claim 139, wherein is H, methyl, ethyl, isopropyl, or 2-hydroxyethyl, 149, The compound of claim 148, wherein R 2 is .H 20
69. 150. The compound of claim 148, wherein R is methyl, 15 L The compound of claim 148, wherein R2 is ethyl. 25 152, The compound of claim 148, wherein R is propyl,
70. 153. The compound of claim 14$, wherein R2 is isopropyl. 154, The compound of claim 139, wherein R2 is H, methyl, ethyl, propyl, or 30 isopropyi8 181 155, The compound of claim 139, wherein R 4 is H, methyl, ethyl, isopropyl, or 2-hydroxyethyl and R2 is H, methyl, ethyL propyl, or isopropyl,
71. 156. The compound of claim 139, wherein n is 1.
72. 157. The compound of claim 139, wherein n is I
73. 158. The compound of claim 139, Wherein bothin and n are 1. 10 159, The compound of claim 139, wherein L is -NRC(O)~, or -C(O)NRt-.
74. 160. The compound of claim 139, wherein L' is -OC(Q) or -C()O)O. 161, The compound of claim 139, wherein L is SS-,
75. 162. The compound of claim 139, wherein L, is -N(R7)()N(R&).
76. 163. The compound of claim 139, wherein I) is -0C(0)N(R)- or N(RQ)C(0)0 20
77. 164. The compound of claim 139, wherein LI is -0-N=.C
78. 165. The compound of claim 139, wherein L -OC(0)NH-NC- or~ NHC(0)NH-N C-1 25
79. 166. The compound of claim 139, wherein 1! is --NR 6 C(0)-, or -C(QONRt.
80. 167. The compound of claim 139, wherein L2 is -OC(O)- or -C(0)0-,
81. 168. The compound of clam 139, wherein L2 is S-S, 169, The compound of claim 139, wherein L2 is -N(R)C(O)N(R), 382
82. 170. The compound of claim 139, wherein IU is -OC(0)N(R 6 )- or N(R)C())0-. 5 17L The compound of claim 139, wherein L is -O-N=Cs 172, The compound of claim 139, wherein 1 -0C(0)NH-N=C- or NHC(O)NH-N=C-. 10 173. The compound of claim 139, wherein both L) and 11 areNR'C(0)-, or C(O)NRt.
83. 174. The compound of claim 139, wherein both L' and L 2 are -OC(O)- or C(O)O, 15
84. 175. The compound of claim 139, wherein both 12 and 1 are S-S2
85. 176. The compound of claim 139, wherein both and U are N(R6)C(O)N(R) 20 1'77. The compound of claim 139., wherein both 1 and 12 are -OC(O)N(R) or -N(R)C(0)O-. 178, The compound of claim 139, wherein 1 is -NR*C(o)- and V is -S-S-. 256
86. 179. The compound of claim 139, wherein L' is -OC(O)- and I is S~S 180, The compound of claim 139, wherein L' is -OC(O)N(Re) or N(R)C(0)0- and IU is -- S-S 30 18 $, The compound of claim 139, wherein LV is -N(R*)C(0)N(RT)- and L2 is 183
87. 182. The compound of claim 139, wherein I .R 3 and IF-R 4 are taken together to form an aceal, a ketal, or an orthoester.
88. 183. The compound of claim 139, wherein each R" and R 4 are independently alkyL
89. 184. The compound of claim 139wherein both R and Ri are C 6 -Ca alkyl, 10 185, The compound of claim. 184., wherein each IL' and . -are independently S-S, -O'O)N(R) or -N{RI)C(O)O-, 186, The compound of claim 139, wherein R" is alkyl. 15 182. The compound of claim 139, wherein Ri is alkyl. 188, The compound of claim 139, Wherein R 3 is alkenyl,
90. 189. The compound of claim 139, wherein R4 is alkenyL 20
91. 190. The compound of claim 139, wherein each R and R are independently alkenyli 191 The compound of claim 190, wherein each R 3 and Ri an independently 25 CrCae alkenyt
92. 192. The compound of claim 190, wherein each R3 and R4 are the same alkenyl moiety, li 193. The compound of claim 139, wherein each R and Re includes two double abnd noietices 184 194, The compound of claim 193, wherein at least one of the double bonds have a Z configuration, 19$, The compound of claim 193, wherein both of the double bonds have a Z 5 configuration, 196, The com pound of claim 190, wherein at least one of R 3 and R 4 is provided in formula (II) below -10\ 10 femrula (II) wherein x is an integer from 1 to 8; and y is an integer from 1-10, 15 197. The compound of claim 196., wherein both of R and R are of the formula (1I).
93. 198. The compound of claim 190, wherein at least one of the double bonds have an E configuration, 20
94. 199. The compound of claim 198, wherein both of the double bonds have an E confi guraiion. 200, The compound of claim 198, wherein at least one of R and R2 is 25 provided in formula (111) below formula (Il1) wherein x is an integer from 1 to 8; and 30 y is an integer from 1-10, 18-5
95. 201. The compound of claim 200, wherein both of R1 and R 2 are as provided in formula (111).
96. 202. The compound of claim 190, wherein each R and R 2 includes three nidouble bond moieties.
97. 203. The compound of claim 202, wherein at least one of the double bonds have a Z configuration. 1o 204, The compound of claim 203, wherein at least two of the double bonds have a Z configuration. 205, The compound of claim 204, wherein all three of the double bonds have a Z configuration. 15
98. 206. The compound of claim 190, wherein at least one of Ri and Ris provided in formula (IV) below formula (IV) 20 wherein x is an integer from I to 8: and y is an integer from I - 10.
99. 207. The compound of claim 206, wherein both of R' and R2 are as provided 25 in formula (IV). 20& The compound of claim 190, wherein at least one of the double bonds have an E configuration, so 209. The compound of clahn 208, wherein at least two of the double bonds have an E configuration. 186 210, 1.]1e compound of claim 209, wherein all three of the double bonds have an RE configuration,
100. 211. The compound of claim 210, wherein at least one of RW and R 2 is provided in formula (IV) below formula (V) wherein x is an integer from I to 8; and o y is an integer from 1-10. 212, The compound of claim 212, wherein both of R' and R are as provided in formula (V). 15 213 A preparation comprising a compound of formula (X).
101. 214. A method of making a compound of formula (X), R L R3 R 2 LI'R 20 fonnula (X) wherein R1 and R 4 are each independently C 1 Q alkyl, optionally substituted with 1-4 R-'; R is alkyl, alkenyl, alkynyl 25 L Is -OC(O) R- is -OR , -N(I&)(R", -CN, SR , (O)R 0 , S(OR R is H, C-C 6 alkyl, \ RW is H or C-C 6 alkyl; each R and R 9 are independently H or C 3 -C> alkyl; 30R i s H or CC6 alky; 187 i and n are each independently 1, 2, 3, 4, 5, or 6, the method comprising reacting a compound of formula (VI), R OH fibnula (VOI with a compound of fonnula (VII) HO' 'RS foirmula (VII) in the presence of a coupling agent, thereby providing a compound of formula (X), 10 '215. The method of claim 214, wherein the coupling agent is a carbodimide, 216, The method of claim 215, wherein the coupling reagent is EDCL 1s 217, A method of forming an association complex comprising contacting; a lipid preparation of claim I or claim 213 with a therapeutic agent in the presence of a buffer, wherein said buffer: is of sufficient strength that substantially all amines of the molecules fornia I are protonated; 20 is present at between 100 and 300mMI; is present at a concentration that provides significantly more protonation of than does the same buffer at 20 mM,
102. 218. An association complex made by the method of claim 217, 25
103. 219. A method of forming an association complex comprising contacting a lipid preparation of claim I or claim 213 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 than about 200 WM, 30 188
104. 220. The method of claim 219, wherein the particle has a diameter of less than about 50 uM .221 A method of forming an association complex comprisIng contacting a lipid preparation of claim I or claim 213 with a therapeutic agent in the presence of a buffer, wherein said buffer has a concentration from about 100 to about 300mM.
105. 222. An association complex comprising a preparation of claim 1 or clan 213 and a nucleic acid, 10a
106. 223. Thbe association complex of cl aim 222, further comprising a PE3Cylated lipid.
107. 224. The association complex of claim 222, further comprising a structural I I moiety. 225, The association complex of claim 224, wherein the structural moiety is cholesterol. 20 226. The association complex of claim 222, wherein said nucleic acid is an siRNA. 22'7. The association complex of claim 226, wherein said nucleic acid is an siRNA which has been modified to resist degradation. 25 228, The association complex of claim 226, wherein said nucleic acid is an siRNA which has been modified by modification of the polysaccharide backbone.
108. 229. The association complex of claim 226, wherein the siRNA targets a gene ao or genes of interest 189 230, The association complex of claim 229, wherein the gene or genes of interest is an endogeneously expressed gene in liver; 23 L The association complex of claim 230, wherein the gene of interest is 5 apoB.
109. 232. The association complex of claim 230, wherein the gene of interest is FVIL 233, The association complex of claim 230, wherein the gene of interest is PCSK9,
110. 234. The association coinplex of claim 230, wherein the gene of interest is VEGF, 15 235, The association complex of claim 230, wherein the gene of interest is KSP (eg5). 236 The association complex of claim 230, wherein the gene of interest is 20 hepcidirt 237, The association complex of claim 230, wherein the gene of interest is HCV, 25 238. The association complex of claim 222, wherein said nucleic acid is a single stranded nucleic acid or derivatives thereof,
111. 239. The association complex of claim 238, wherein the nucleic acid is an. antisense nucleic acid. 3O
112. 240. The association complex of claim 238, wherein the nucleic acid is a microRNA. 190
113. 241. The association complex of claim 238 wherein the nucleic acid is an anti sense oligonucleotide ofmicroRNA (antagonir), s 242. The association complex of claim 241. wherein the nucleic acid is against microRNA- 122.
114. 243. The association. complex of claim 241, wherein the nucleic acid is against mi croRNA- 181,
115. 244. The association complex of claim 241, wherein the nucleic acid is against microRNA- 155,
116. 245. The association complex of claim 241. wherein the nuclee acid is against microRNA- 16,
117. 246. The association complex of claim 222, further comprising a stmctural moiety and a PEylated lipid, wherein the ratio, by weight, of preparation of claim I or claim 213, structural moiety, PEGylated lipid, and a nucleic acid, is 8-22:0.4-10:0.4 20 12:0,4-2-2. 247, The association complex of claim 246, wherein the structural moietv is choliesteroi 25 247, The association complexof claim 247,wherein the ratio is 10-20:0.5 8.0:5-10:0,5-2.0.
118. 248. The association complex of claim 248, wherein the ratio is 15:08:7:1 . ;30 249, The association complex of cl aim 246, wherein the average liposome diameter is between 10 nm and 750 nm. 191
119. 250. Thre association complex of claim 249 wherein the average association complex diamee is between 30 and 200 nm. 25 1 The association complex of claim 250, wherein the average association S complex diameter is between 50 and 100 nm. 252, The association complex of claim 222, wherein the preparation is less than IS%, by weight, of unreacted lipid 10253A pharmaceutically acceptable composition comprising the preparation of claim I or claim 213.
120. 254. A pharmaceutically acceptable composition comprising the association cmpilex of claim 222,
121. 255. A method of treating a mammal comprising administering to said maimal a therapeutic amount of an association complex of claim 222,
122. 256. A preparation of claim 1, wherein the preparation comprises one or a 20 mixture of the formula below, wherein R is not H unless specified in the formula below R R H H R'N N N NN R
123. 257. The preparation of claim 1, wherein the preparation consists essentially of one or a mixture of the formula below R R H i H - N N and R' N 25 R R R
124. 258. The preparation of claim 257., wherein each R is 192 RI R2 259, 'The preparation of claim 258 wherein each R is 0 '
125. 260. The preparation of claims 256 or 257, wherein R' isCICa alkyl (e.g, Ca, alky), or C IC alkenyl.
126. 261. The preparation of claim 256 wherein R is 0 N'
127. 262. The preparation of claim 261, wherein R' isCur% a alkyl,
128. 263. The preparation of claim 261, wherein R.' is C a alky and R 2 is H,. 15 264, The preparation of claim 253, wherein fommla (1) is provided below, wherein R is ro H unless specifically recited: R R' N --. N R RR \-'N' Ris R 2 20
129. 265. The preparation of claim 264, wherein R.' is C 12 alkyl and R" is H.
130. 266. The preparation of claim 256, wherein formula (1) is provided below, wherein R is not H unless specifically recited: 193 R H R' N'~_N ",-N R 0 N' RisR2 267, The preparation of claim 266, wherein R1 is C alkyl and R is R 5
131. 268. The preparation of claim 1, wherein formula (1) is provided below, wherein R is not 11 unless specifically recited H R' N. R 10 269, The preparation of claim 268, wherein R is or R N' or R2
132. 270.alkyyo
133. 271. The preparation of claim 268, wherein R is 0
134. 272. The preparation of claim 271, wherein R 1 isCw 1 alkyl,,or CurC 3 0 20 alkenyl and R 2 is H. 27, A method of forrning an association complex comprising a plurality of lipid moieties and a therapeutic agent, the method comprising: 194 mixing a plurality of lipid moieties in ethanol and aqueous NaOAc buffer to provide a particek; and adding the therapeutic agent to the particle, thereby forming the association Complex, 272 ThFe method of claim 271, wherein the lipid moieties are provided in a solution of 100% ethanol.
135. 273. The method of claim 271 wherein the plurality of lipid moieties comprises a cationic lipid, 274, The method of claim 273, wherein the cationic lipid is a lipid of claim I or claim 213.
136. 275. The method of claim 274 wherein the cationic lipid is a lipid of one of the following or a mixture thereof; HH HH ^ H H or H H H c N N N H H
137. 276. The method of claim 271, wherein the plurality of lipid moieties comprises a PEG-lipid.
138. 277. The method of claim 276, wherein the PEG-lipid has the Iblowing structure; 195 R 01' H "m *Q wher n; each I and Ii are independently a bond or C(0); each R and R are independently alky alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; 5 X is-C(O)NH-, C(S)NtH -C(O)CvalkylC(0)NH-; or -C()Cj 3 alkyC(0)0-; m s an integer from. 0-11 and n is ar integer from 1-500. 27$. The method of claim 277, wherein the PEG-lipid is 0
139. 279. The method of claim 271, wherein the plurality of lipid moieties comprises a structural lipid.
140. 280. The method of claim 279, wherein the structural lipid is cholesterol. 2$81 The method of claim 271, further comprising extruding the lipid containing particles.
141. 282. The method of claim 271, wherein the lipid containing particles are extruded prior to addition of the therapeutic agent,
142. 283. The method of claim 271, wherein the therapeutic agents a nuclei acid
143. 284. The method of claim 23, wherein the nucleic acid is an siRNA, 196 284. The method of claim 283, wherein said nucleic acid is an siRNA which has been modified to resist degradation. 285 The method of claim 283 wherein said nucleic acid is an siRNA which 5 has been modified by modification of the polysaccharide backbone,
144. 286. The method of claim 283, siRNA is conjugated to a Lipophilic moiety.
145. 287. Thc method of claim 284, wherein the siRNA targets a gene or genes of 10 interest.
146. 288. The method of claim 287, wherein the gene or genes of is an endogeneously expressed gene in liver. 15 289. The method of claim 288, wherein the gene of interst is apoB.
147. 290. The method of claim 288, wherein the gene of is FVIL 29 L The method of claim 288 wherein the gene of is PCSK9, 20
148. 292. The method of claim 288, wherein the gene of is WEG.
149. 293. The method of claim 288, wherein the gene of is KSP (eg5). 25 294. The method of claim 288, wherein the gene of is hepeidin.
150. 295. The'method of claim 288. wherein the gene of is HCV.
151. 296. The method of claim 283, wherein said nucleic acid is a single stranded an nucleic acid or derivatives thereof, 197
152. 297. The method of claim 296, wherein the nucleic acid is an antisense nuceic acid, 298, The method of claim 296, wherein the nucleic acid is a microRNA. 299, The method of claim 296, wherein the nucleic acid is an antimicroRNA (antagomir).
153. 300. The method of claim 271, wherein the association complex comprises a cationic lipid, a structural lipid, a PEG-lipid and a nucleic acid,
154. 301. The method of claim 300, wherein the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 36-48:42-54:6-14,
155. 302. The method of claim 301, wherein the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 38 -46:44-52:8- 12.
156. 303. The method of claim 302, wherein the molar ratio of the cationic lipid. structural lipid, PEG-lipid and nucleic acid is about 42:48:10.
157. 304. The method of claim 271, wherein the weight ratio of total exipient to nucleic acid is less than about 15:1. 305, The method of claim 304, wherein the weight ratio of total exipient to nucleic acid is about 10:l. 306, The method of claim 305, wherein the weight ratio of total exipient to nucleic acid is about 7.S 1
158. 307. The method of claim 305, wherein the weight ratio of total exipient to nucleic acid is about 51, 198
159. 308. The method of claim 300, wherein the cationic lipid has the following H H N ; ,NN H H H r structure: H the PEli-pid has the following structure: N and the structural lipid is cholesterol.
160. 309. The method of claim 308, wherein the molar ratio of the cationic lipid, structural lipid and PEG-lipid is 38-46:44-52:8-12, 310, The method of claim 309, wherein the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is about 42:48:10. 310, The method of claim 308 wherein the weight ratio of total exipient to nucleic acid is less than about 15:1 31L The method of claim 310, wherein. the weight ratio of total exipient to nucleic acid is about 10:1.
161. 312. The method of claim 310, wherein the weight ratio of total exipient to nucleic acid is about 7.5;1.
162. 313. The method of claim 3 103 wherein the weight ratio of total exipiem to nucleic acid is about 5 1,
163. 314. An association. complex made by a method of any of claims 271 -313. 199
164. 315. An association complex comprising a cationic lipid, a structural lipid, a PEG-lipid and a nucleic acid, wherein the cationic lipid is a lipid of one of the following or a mixture thereof: HHH H or N N. H HOH HH the P EG-pod has the following structure H \ / the structural pid is cholesterol.
165. 316. The association complex of 315) wherein the nucleic acid is an siRNA.
166. 317. The association complex of 315, wherein the cationic lipid has the following formula: H H Y N H
167. 318. The method of claim 315, wherein the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 36-48:42-54:6-14 200 319, The method of claim 318, wherein the molar ratio of the cationic lipid, structural lipid, PEG-lipid and nucleic acid is 38-46:44-52:8-12
168. 320. The method of claim 319, wherein the molar ratio of the cationic lipid, sitmctural lipid, PEG-lipid and nucleic acid is about 42:48:10, 321 The method of clan 315, wherein the weight ratio of total exipient to nucleic acid is less than. about 15:1.
169. 322. he method of claim 321, wherein the weight ratio of total excient to nucleic acid is about 10: 1.
170. 323. The method of claim 321, wherein the weight ratio of total excient to nucleic acid is about 7.5:1
171. 324. The method of claim 321, wherein the weight ratio of total excient to nuceic acid is about 5:1.
172. 325. A compound of fomula (XV) R" L 1 0, ') fornula (XV) wherein; each LI and IY are independently a bond or C(0); each R I and R2 are independently alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; X is -C(0)N C-, -G(S)NH-, -C(O)CakyC(O)NH-; or -C(0)C{ 3 alkylC(0)0-; m is an integer from 0-11 and n is an integer from 1-500. 32. The compound of claim 32$, wherein U and LI are both a bond, 201
173. 327. The compound of claim 325, wherein L and 11 are both C(0), 328, The compound of claim 325, wherein each R' and R 2 are independently 5 alkyL
174. 329. The compound of claim 328, wherein each R' and R2 are independently C6-,2 alkyl, e,g.,CwtoCis, alkyl, e.g,, C14alkyL 330, The compound of claim 325, wherein both R and R are alkyl, e.g., 10 straight chain alkyl having the same length, e.g:, C-2? alkyl, e.g,Cw-Cn alkyl, e.g., C1 alkyl or " afkyl, 33I, The compound of claim 330, wherein both RI and R2 are C alkyl. 15 332. The compound of claim 325, wherein formula XV represents a racemic
175. 333. The compound of claim 325,wherein formula XV represents enantiomerically pure 'R isomer (e.g., a compound having an enantiomeric excess of R 20 isomer, e.g, at least about 95% ee. or greater than 97% ee, e.g, 98%, or 99%).
176. 334. The compound of claim 325, wherein formula XV represents enantiomerically pure 'S' isomer (e,g., a compound having an enantiomeric excess of R. isomer, eg, at least about 95% ce, or greater than 97% ee, e.g, 98%, or 99%).
177. 335. The compound of claim 325; wherein each R( and R(2are independently alkenyl, for example, each RI and R2 are independently C alkenyl or each RI and R are the same alkenyl moiety, 30 336, The compound of claim 335, wherein each R and R2 includes a single double bond, for example a single double bond in the E or Z configuration. 202
178. 337. The compound of claim 335, whereineach R and R2 includes two double bond moieties
179. 338. The compound of claim 325 wherein X is -C(O)NH-,providing a 5 compound of formula (XV) below: 0 V ~~-N9 H. N / R 2 formula (XV)
180. 339. The compound of claim 325, wherein, X is -C(0)C aIkyC(0)OV. to 340, The compound of claim 325, wherein m is an integer from 1-10, fo example an integer from 2-4 or an integer 2. 341, The compound of claim 325, wherein, n is an integer from 1-500, for example an integer from 40-400, from 100-350, from 40-50 or from 42-47, 15 342 The compound of claim 325, wherein the compound is a compound of ior-mula (XV'). 0 fmula (XV'), 20 wherein both L' and are a bond.
181. 343. The b compound of claim 342, wherein each R and R2 are independently alkyl, for example C 6 -C 2 8 alkyl, e.g,CwO-CN alkyl, e.g., C 14 alkyl. 25 344. The compound of claim 343, wherein, both RI and R are alkyl, e.g.. straight chain alkyl having the same length, e.g., C 6 -C alkyl, e.g,Qo-C~ alkyl, e.g., C 1 alkyl or C& alkyl 203
182. 345. The compound of claim 342, wherein m is an integer from 1 -10, for example an integer from 2-4 or an integer 2 5 346. The compound of daim 342 whereinn is an integer from 1-500, for example an integer from 40-400, or from 40-50. 347, The compound of claim 342, wherein, the compound is a compound of formula (XV'), wherein L' and 12 are both bonds, R and R' are both alkyl (e.g., C6-C25 1 alkyl, e g< -C I? alkyl, preferrably Cj alkyl), and n is an integer from about 40-400.
183. 348. The compound of claim 325, wherein, the comound has a formula (XVI) below: formlla (XVI), wherein the repeating PEG moiety has an avenge molecular weight of 2.000 with n value between 42 and 47.
184. 349. The compound of claim 348, wherein the compound of formula XVI is a stereo isomer with preferred absolute configuration 'W (e,g, having an enantiomeric 20. excess of R isomer such as 90%, 95%, 97% 98%, 99%), 204
185. 350. An association complex comprising: a. one or more compounds, each individually having a structure defined by formula (I) or a pharmaceutically acceptable salt thereof R 2 N{X' N' X NR 2 R n formula (I) wherein: each Xa and Xb, for each occurrence, is independently CI- 6 alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H or 0 *rkY' R1 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 0, NR 2 , or S; R' is alkyl or alkenyl; each of which is optionally substituted with one or more substituents; and R2 is H, alkyl or alkenyl; each of which is optionally substituted with one or more substituents; provided that, if n=O, then at least n+3 of the R moieties are not H; b. PEG-lipid having the structure shown in formula (XV) R"'LLO O'O O O R2 formula (XV) wherein: each L' and L 2 are independently a bond or C(O); each R' and R 2 are independently alkyl, alkenyl, or alkynyl; each of which is optionally substituted with one or more substituents; 205 X is -C(O)NH-, -C(S)NH-, -C(O)CI. 3 alkylC(O)NH-; or -C(O)CI- 3 alkyl-C(O)O-; m is an integer from 0-11 and n is an integer from 1-500; c. a steroid; and d. a nucleic acid.
186. 351. The association complex of claim 350, wherein said cationic lipid is one of the following or a mixture thereof: H H N O O N O H N N N'/^'N N N H H O N O H or H N O 0 H H N N -'''N N N H O NO O N H H
187. 352. The association complex of claim 351, wherein said cationic lipid is: H H NO ON O H N k N Ns-- N N H H O N'O H
188. 353. The association complex of claim 351, wherein said cationic lipid is: H N O 0 H H N N NN N N H O N O ON H H 206
189. 354. The association complex of claim 350, wherein said PEG-lipid has the structure 0 R" 1 ' 0 0 O~N 0 0 0 ~n' ' 2m R2 wherein: each L' and L 2 are independently a bond or C(O); each R1 and R 2 are independently alkyl, alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; m is an integer from 0-11 and n is an integer from 1-500.
190. 355. The association complex of claim 354, wherein said PEG-lipid has the structure 0 O O N 0 0 o H0 wherein: n is an integer from 1-500.
191. 356. The association complex of claim 355, wherein said PEG-lipid has an enantiomeric excess of the R isomer.
192. 357. The association complex of claim 350, wherein said steroid is cholesterol.
193. 358. The association complex of claim 350, wherein molar ratio of said cationic lipid, said steroid and said PEG-lipid is 36-48:42-54:6-14.
194. 359. The association complex of claim 358, wherein molar ratio of said cationic lipid, said steroid and said PEG-lipid is 38-48:44-52:8-12. 207
195. 360. The association complex of claim 359, wherein molar ratio of said cationic lipid, said steroid and said PEG-lipid is 42:48:10.
196. 361. The association complex of claim 350, wherein weight ratio of total lipids to nucleic acid is less than about 15:1.
197. 362. The association complex of claim 361, wherein weight ratio of total lipids to nucleic acid is about 10:1.
198. 363. The association complex of claim 361, wherein weight ratio of total lipids to nucleic acid is about 7.5:1.
199. 364. The association complex of claim 361, wherein weight ratio of total lipids to nucleic acid is about 5:1.
200. 365. The association complex of claim 350, wherein said nucleic acid is a siRNA.
201. 366. The association complex of claim 350, wherein said nucleic acid is a single stranded nucleic acid or derivative thereof.
202. 367. The association complex of claim 366, wherein said nucleic acid is an antisense nucleic acid.
203. 368. The association complex of claim 366, wherein said nucleic acid is a microRNA.
204. 369. The association complex of claim 366, wherein said nucleic acid is an antimicroRNA.
205. 370. The association complex of claim 350, wherein: said cationic lipid is 208 H H 0 H 0 N O O NH N N N H H o N O H said steroid is cholesterol; said PEG lipid is 0 0 H O O O O n wherein: n is an integer from 1-500; in a molar ratio of 36-48:42-54:6-14.
206. 371. The association complex of claim 370, wherein molar ratio of said cationic lipid, said steroid and PEG-lipid is 42:48:10.
207. 372. A method of forming an association complex of claim 350, wherein the method comprises: 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.
208. 373. The method of claim 372, further comprising extruding the lipid containing particles. 209