Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`Rational design of novel cationic lipids for use in next-generation siRNA
`delivery systems
`
`
`
`Supplementary Table 1. Linker modifications to DLinDMA
`
`Abbreviated
`Name
`
`Chemical Name
`
`Structure
`
`Mean
`Particle
`Size (nm)a
`
`ED50
`(mg/kg)
`
`O R
`O R
`
`71
`
` 24
`
`~1
`
`DLinDMA
`(Benchmark)
`
`1,2-Dilinoleyloxy-3-
`dimethylaminopropane
`
`DLinDAP
`
`1,2-Dilinoleoyl-3-
`dimethylaminopropane
`
`N
`
`N
`
`65
`
` 21
`
`40-50
`
`79
`
` 26
`
`5-12
`
`73
`
` 25
`
`12-25
`
`69
`
` 25
`
`12-25
`
`67
`
` 22
`
`~0.4
`
`O
`
`O
`
`O
`
`R
`
`O
`
`R
`
`
`
`
`
`
`
`O
`R
`
`O
`R
`
`O
`
`DLin-2-
`DMAP
`
`1-Linoleoyl-2-
`linoeyloxy-3-
`dimethylaminopropane
`
`N
`
`O
`
`DLin-C-DAP
`
`1,2-
`Dilinoleylcarbamoyloxy-
`3-dimethylaminopropane
`
`N
`
`O
`
`DLin-S-DMA
`
`1,2-Dilinoleylthio-3-
`dimethylaminopropane
`
`DLin-K-DMA
`
`2,2-Dilinoleyl-4-
`dimethylaminomethyl-
`[1,3]-dioxolane
`
`N
`
`N
`
`R
`
`O
`H
`N
`
`N
`H
`R
`
`
`
`R
`
`S
`R
`
`O
`
`S
`
`O
`
`O
`
`R
`R
`
`
`
`
`
`R =
`
`
`a Mean particle size of the PFV formulation as tested for FVII activity and containing the indicated
`cationic lipid at 40 mol%.
`
`
`Nature Biotechnology: doi: 10.1038/nbt.1602
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`0001
`
`PROTIVA - EXHIBIT 2022
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
`
`

`

`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`Supplementary Syntheses 1
`
`
`
`Synthesis of 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLinDMA)
`DLinDMA was synthesized according to the method previously published by Heyes et. al15.
`
`
`
`Synthesis of 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP)
`DLinDAP was synthesized according to the method previously published by Bailey and Cullis17.
`
`
`
`1-Linoleoyl-2-linoeyloxy-3-dimethylaminopropane (DLin-2-DMAP)
`
`Synthesis of 1-Triphenylmethyloxy-3-(N,N-dimethylamino)-2-propanol (I)
`
`A mixture of 3-(dimethylamino)-1,2-propanediol (3.0 g, 25 mmol) and triphenylmethyl chloride
`
`(7.75 g, 27.8 mmol) in dry pyridine (100 mL) was refluxed for 30 minutes. Upon cooling, most
`
`of the solvent was evaporated in vacuo, and the resulting residual was re-dissolved in 400 mL of
`
`dichloromethane. The organic phase was washed with water (3 x 200 mL), then brine (150 mL),
`
`and dried over anhydrous Na2SO4. Evaporation of the solvent gave 6.3 g of yellow oil as a crude
`
`product. The crude product was purified by column chromatography on silica gel (230-400
`
`mesh, 500 mL) eluted with 0-10% methanol gradient in dichloromethane. This afforded 4.0 g of
`
`I as yellow oil.
`
`
`
`Synthesis of 1-Triphenylmethyloxy-2-linoleyloxy-3-N,N-dimethylaminopropane (II)
`
`NaH (60%, 2.17 g, 54 mmol) was washed with hexanes (3 x 40 mL) under nitrogen and then
`
`suspended in anhydrous benzene (60 mL). To the suspension was added I (4.0 g, 11 mmol)
`
`dropwise in 20 mL of anhydrous benzene. The resulting mixture was stirred at room temperature
`
`for 20 minutes, then a solution of linoleyl methanesulfonate (4.5 g, 13 mmol) in 40 mL of
`
`anhydrous benzene was added dropwise under nitrogen. The mixture was stirred at room
`
`temperature for 30 minutes and then refluxed overnight. Upon cooling to room temperature,
`
`30 mL of 1:1 (V:V) ethanol-benzene solution were added dropwise under nitrogen followed by
`
`100 mL of benzene and 100 mL of water. Upon shaking, the aqueous phase was separated. The
`
`organic phase was washed with brine (2 x 100 mL) and dried over anhydrous sodium sulfate.
`
`Evaporation of the solvent afforded 6.8 g of yellowish oil. The crude product was
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`chromatographed on a silica gel column (230-400 mesh, 400 mL) eluted with 0-3% methanol
`
`gradient in chloroform. 5.8 g (84%) of II were obtained as yellowish oil.
`
`
`
`Synthesis of 2-Linoleyloxy-3-(N,N-dimethylamino)-1-propanol (III)
`
`II (5.8 g, 9.2 mmoL) was refluxed in 80% HOAc (25 mL) under nitrogen for 10 minutes. Upon
`
`cooling to room temperature, the mixture was diluted with water (100 mL). The resulting
`
`aqueous solution was neutralized to about pH 6 with 0.5% NaOH solution. The aqueous phase
`
`was then extracted with dichloromethane (4 x 100 mL). The combined organic phase was
`
`washed with 0.1% NaOH solution (100 mL), water (100 mL), then brine (100 mL), and dried
`
`over anhydrous sodium sulfate. Evaporation of the solvent gave 5.6 g of a mixture of product and
`
`starting material as yellowish oil. The mixture was chromatographed on a silica gel column (230-
`
`400 mesh, 400 mL) eluted with 0-10% methanol gradient in chloroform. 2.2 g (62%) of III were
`
`afforded as yellowish oil.
`
`
`
`Synthesis of 2-Linoleyoloxyl-3-linoleyloxyl-1-N,N-dimethylaminopropane (DLin-2-DMAP)
`
`To a solution of linoleic acid (2.36 g, 8.4 mmol) in anhydrous benzene (50 mL) was added
`
`dropwise oxalyl chloride (1.45 g, 11.4 mmol) under nitrogen. The resulting mixture was stirred
`
`at room temperature for 4 hours. Solvent and excess of oxalyl chloride was removed in vacuo to
`
`give linoleoyl chloride as light yellowish oil. This was re-dissolved in anhydrous benzene (85
`
`mL). To the resulting solution was added dropwise a solution of III (2.9 g, 7.5 mmol) and dry
`
`pyridine (1 mL) in 15 mL of anhydrous benzene. The mixture was then stirred at room
`
`temperature under nitrogen for 2 days, resulting in a suspension. The mixture was diluted with
`
`benzene (100 mL). The organic phase was washed with a solution of 3:5 (V:V) ethanol-water
`
`(320 mL), then brine (2 x 75 mL), and dried over anhydrous Na2SO4. The solvent was removed
`
`in vacuo affording 5.2 g of oil. The crude product was purified by column chromatography on
`
`silica gel (230-400 mesh, 450 mL) eluted with 0-4% methanol gradient in chloroform. This
`
`afforded 3.9 g (80%) of DLin-2-DMAP as yellowish oil. 1H NMR (400 MHz, CDCl3) δ: 5.25
`
`(8H, m, 4 x CH=CH), 4.17 (1H, dd, J = 11.6 and 4 Hz, OCH), 3.96 (1H, dd, J = 11.6 and 5.2 Hz,
`
`OCH), 3.53-3.64 (1H, m, OCH), 3.35-3.53 (2H, m, OCH2), 2.68 (4H, t, =CH-CH2-CH=), 2.41
`
`(2H, m, CH2), 2.25 (6H, s, 2 x NCH3), 2.21 (2H, m, CH2), 1.96 (8H, q, allylic 4 x CH2), 1.4-1.6
`
`(4H, m, 2 x CH2), 1.21 (30H, s, 15 x CH2), 0.80 (6H, t, 2 x CH3) ppm.
`
`
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`Nature Biotechnology: doi: 10.1038/nbt.1602
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`
`1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP)
`
`Preparation of Linoleyl Phthalimide
`
`A mixture of potassium phthalimide (11.2 g, 59.5 mmol) and linoleyl methanesulfonate (9.3 g,
`
`27 mmol) in 250 mL of anhydrous DMF was stirred at 70ºC under nitrogen overnight. The
`
`resulting suspension was poured into 500 mL of cold water. The aqueous phase was extracted
`
`with EtOAc (3 x 200 mL). The combined extract was washed with water (200 mL), then brine
`
`(200 mL), and dried over anhydrous Na2SO4. Solvent was evaporated to give a mixture of solid
`
`and oily materials. To the mixture was added 300 mL of hexanes. The solid was filtered and
`
`washed with hexanes (2 x 25 mL). The filtrate and washes were combined, and the solvent was
`
`evaporated to result in 11 g of Linoleyl Phthalimide, which was used in the next step without
`
`further purification.
`
`
`
`Preparation of Linoleylamine
`
`The above crude linoleyl phthalimide (11 g, ca. 27 mmol) and hydrazine (10 mL) were refluxed
`
`in 350 mL of ethanol under nitrogen overnight. The resulting white solid was filtered upon
`
`cooling the mixture to about 40 to 50ºC and the solid was washed with warm EtOH (2 x 30 mL).
`
`The filtrate and washes were combined and solvent evaporated. To the residual was added
`
`400 mL of chloroform which resulted in precipitation of white solid. The solid was filtered
`
`again. The organic phase of the resulting filtrate was washed with water (2 x 100 mL), then brine
`
`(100 mL), and dried over anhydrous Na2SO4. Solvent was removed in vacuo to afford 7.3 g of
`
`yellow oil as a crude product. Pure linoleylamine was obtained by column chromatography on
`
`silica gel eluted with 0-20% methanol gradient in chloroform.
`
`
`
`Preparation of Linoleyl Isocyanate
`
`Anhydrous sodium carbonate (11g g) was suspended in a solution of linoleylamine (7.3 g, ca.
`
`27 mmol) in anhydrous CH2Cl2 (200 mL) under good stirring and nitrogen. The suspension was
`
`cooled to 0 to 5ºC with an ice bath. To the suspension was added diphosgene (8.2 g, 41 mmol) in
`
`10 mL of anhydrous CH2Cl2 under vigorous stirring. Upon addition, the resulting suspension was
`
`stirred at 0 to 5ºC under nitrogen for 60 minutes and then at room temperature for 2 hours. Upon
`
`completion of the reaction, 100 mL of water was added to the mixture and the mixture was
`
`stirred at room temperature for 30 minutes. The organic layer was separated, and washed with
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`water (100 mL) then brine (100 mL). After drying with anhydrous Na2SO4, the solvent was
`
`evaporated to give 7.6 g of yellow oil as a crude product, which was used in the following step
`
`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`without further purification.
`
`
`
`1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP)
`
`To a solution of crude linoleyl isocyanate (7.6 g, ca. 25 mmol) in 150 mL of anhydrous benzene
`
`under nitrogen was added dropwise a solution of 3-(dimethylamino)-1,2-propanediol (0.99 g,
`
`8.3 mmol) in 20 mL of anhydrous benzene. The resulting mixture was stirred at room
`
`temperature for 60 minutes and then refluxed for 4 hours, followed by stirring at room
`
`temperature overnight. Upon dilution of the mixture with 150 mL benzene, the organic phase
`
`was washed with water (3 x100 mL), then brine (100 mL), and dried over anhydrous Na2SO4.
`
`Evaporation of the solvent gave 8.4 g of yellow oil. Column purification of the oily material
`
`(500 mL silica gel, 230-400 mesh, eluted with 0-3% methanol gradient in chloroform) afforded
`
`2.2 g (38%) of yellowish oil as the product DLin-C-DAP. 1H NMR (400 MHz, CDCl3) δ: 5.37
`
`(8H, m, 4 x CH=CH), 5.06 (1H, br. CONH), 4.91 (1H, br. CONH), 4.79 (1H, m, OCH), 4.28
`
`(1H, br. d, J = 11 Hz, OCH), 4.16 (1H, dd, J = 12 and 6 Hz, OCH), 3.16 (4H, m, 2 x NCH2), 2.77
`
`(4H, t, J = 6.4 Hz, =CH-CH2-CH=), 2.4-2.7 (2H, m, NCH2), 2.33 (6H, s, 2 x NCH3), 2.05 (8H,
`
`m, allylic 4 x CH2), 1.4-1.55 (4H, m, 2 x CH2), 1.29 (40H, s, 20 x CH2), 0.89 (6H, t, 2 x CH3)
`
`ppm.
`
`
`
`1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA)
`
`Synthesis of Linoleylthio Acetate
`
`First linoleyl mercaptane was synthesized. To a solution of triphenylphosphine (18.0g, 68.2
`
`mmol) in 250 mL of anhydrous THF under nitrogen at 0-5ºC was added dropwise diisopropyl
`
`azodicarboxylate (DIAD, 14.7 mL, 68 mmol). Upon addition, the resulting mixture was stirred at
`
`0 to 5ºC for 45 minutes. A yellow suspension was resulted. A solution of linoleyl alcohol (9.1g,
`
`34 mmol) and thiolacetic acid (5.1 mL, 68 mmol) was then added at 0 to 5ºC dropwise over 30
`
`minutes to the yellow suspension under nitrogen. The resulting mixture was stirred at 0 to 5ºC
`
`for 1 hour and then allowed to warm up to room temperature. After stirring at room temperature
`
`for 60 minutes, a brown solution was obtained. After solvent evaporation the residual was re-
`
`dissolved in 600 mL of ether. The ether phase was washed with water (2 x 250 mL), then brine
`
`(250 mL), and dried over anhydrous Na2SO4. The solvent was evaporated to afford 31g of brown
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
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`oil which partially solidified overnight. This crude mixture was treated with 100 mL of hexanes.
`
`The solid was filtered off and washed with hexanes (2 x 30 mL). The filtrate and washes were
`
`combined and solvent evaporated to give 13 g of brown oil as a crude product. The crude product
`
`was purified by column chromatography twice on silica gel (230-400 mesh, 600 mL) eluted with
`
`0-3% ether gradient in hexanes. This gave 10.0 g (91%) of linoleylthio acetate as yellowish oil.
`
`
`
`Synthesis of Linoleyl Mercaptane
`
`To a suspension of LiAlH4 (4.7g, 124 mmol) in 150 mL of anhydrous ether under nitrogen at
`
`0 to 5ºC was added dropwise a solution (one crystal of iodine in 200 mL of anhydrous ether)
`
`under nitrogen followed by linoleylthio acetate (10.0g, 30.8 mmol) in 100 mL of anhydrous
`
`ether. Upon addition, the suspension was allowed to warm up to room temperature and then
`
`stirred at room temperature for 4 hours. The resulting mixture was cooled to 0 to 5ºC and 10 mL
`
`of NaCl saturated aqueous solution was added very slowly. After stirring at room temperature for
`
`60 minutes, the suspension was filtered through a pad of diatomaceous earth. The solids were
`
`washed with ether (3 x 100 mL). The filtrate and washes were combined and the solvent
`
`evaporated, resulting in 7.2 g (83%) of linoleyl mercaptane as colourless oil.
`
`
`
`Synthesis of 1-Triphenylmethyloxy-2-hydroxy-3-dimethylaminopropane (I)
`
`A mixture of 3-(dimethylamino)-1,2-propanediol (6.3g, 53 mmol) and triphenylmethyl chloride
`
`(15.5g, 55.6 mmol) in anhydrous pyridine (200 mL) was refluxed for 40 minutes. Upon cooling
`
`to room temperature, most of the solvent was removed in vacuo. To the resulting oily residual
`
`was added 400 mL of ethyl acetate. A large amount of solid was formed. The solid was filtered
`
`off and dried in air. The filtrate phase was washed with water (2 x 150 mL), then brine (150 mL)
`
`and dried over anhydrous Na2SO4. Evaporation of the solvent afforded 8.5 g of brown oil as a
`
`crude product (I), which was purified by column chromatography on silica gel (230-400 mesh,
`
`500 mL) eluted with 0-10% methanol gradient in chloroform.
`
`
`
`Synthesis of 1-Triphenylmethyloxy-2-methylsulfonyloxy-3-dimethylaminopropane (II)
`
`To a solution of I (4.2g, 11.7 mmol) and anhydrous triethylamine (2.5 mL, 17.9 mmol) in
`
`150 mL of anhydrous dichloromethane under nitrogen was added dropwise with an ice-water
`
`cooling bath methylsulfonyl chloride (1.0 mL, 13 mmol). Upon addition, the cooling bath was
`
`removed and the mixture stirred at room temperature under nitrogen overnight (20 hours). The
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`resulting mixture was diluted with 100 mL of dichloromethane. The organic phase was washed
`
`with water (2 x 100 mL), then brine (100 mL), and dried over anhydrous Na2SO4. Evaporation of
`
`the solvent gave 4.3 g of crude product (II), which was used in the next step without further
`
`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`purification.
`
`
`
`Synthesis of 1-Triphenylmethyloxy-2-linoleylthio-3-dimethylaminopropane (III)
`
`To a suspension of NaH (2.0 g, 95%, 79 mmol) in 100 mL of anhydrous benzene under nitrogen
`
`was added dropwise a solution of linoleyl mercaptane (3.1 g, 11 mmol) in 30 mL of anhydrous
`
`benzene. The resulting mixture was stirred at room temperature for 20 minutes. A solution of II
`
`(4.5g, 10 mmol) in 30 mL of anhydrous benzene was then added dropwise. After stirring at room
`
`temperature for 15 minutes, the mixture was refluxed gently under nitrogen for 3 days. Upon
`
`cooling, 30 mL of 1:1 (V:V) ethanol-benzene was added slowly to the mixture. The organic
`
`phase was washed once with 1:2 ethanol-water (360 mL) and dried over anhydrous Na2SO4.
`
`Evaporation of the solvent gave 7.1 g of yellowish oil as a crude product (III), which was
`
`purified by column chromatography on silica gel (230-400 mesh, 250 mL) eluted with 0-5%
`
`methanol gradient in chloroform to yield 5.5 g (88%) of III.
`
`
`
`Synthesis of 1-Hydroxy-2-linoleylthio-3-dimethylaminopropane (IV)
`
`III (5.5g, 8.8 mmol) was refluxed in 150 mL of 80% HOAc under nitrogen for 7 hours. Upon
`
`cooling, the solvent was removed to give a pale semi-solid. The material was re-dissolved in
`
`200 mL of ethyl acetate. The organic phase was washed subsequently with 0.5% NaOH aqueous
`
`solution (100 mL), water (100 mL), and brine (100 mL). After drying over anhydrous Na2SO4,
`
`the solvent was evaporated. 5.1 g of a pale solid was resulted. Column chromatography of the
`
`crude product on silica gel (230-400 mesh, 250 mL) eluted with 0-7% methanol gradient in
`
`chloroform afforded 1.3 g (39%) of IV.
`
`
`
`Synthesis of 1-Methylulfonyloxyxy-2-linoleylthio-3-dimethylaminopropane (V)
`
`Methylsulfonyl chloride (0.5g, 4.3 mmol) was added dropwise to a solution of IV (1.3g,
`
`3.2 mmol) and anhydrous triethylamine (0.7 mL, 5 mmol) in 50 mL of anhydrous
`
`dichloromethane under nitrogen. The resulting mixture was stirred at room temperature
`
`overnight (19 hours). The reaction mixture was diluted with 50 mL of dichloromethane. The
`
`organic phase was washed with water (2 x 50 mL), then brine (50 mL), and dried over anhydrous
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
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`Na2SO4. Evaporation of the solvent resulted in 1.4 g of yellowish oil as a crude product (V),
`
`which was used in the following step without further purification.
`
`
`
`Synthesis of 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA)
`
`NaH (0.89g, 60%, 22 mmol) was washed twice with hexanes (2 x 15 mL) under nitrogen and
`
`then suspended in 70 mL of anhydrous benzene. To the suspension was added dropwise a
`
`solution of linoleyl mercaptane (1.1 g, 3.9 mmol) in 15 mL of anhydrous benzene. The resulting
`
`mixture was stirred at room temperature for 20 minutes. A solution of V (1.4g, 3.0 mmol) in
`
`15 mL of anhydrous benzene was then added dropwise. After stirring at room temperature for
`
`20 minutes, the mixture was refluxed gently under nitrogen for 2 days. Upon cooling, 200 mL of
`
`1:1 (V:V) ethanol-benzene was added slowly to the mixture. The organic phase was washed with
`
`water (200 mL) and dried over anhydrous Na2SO4. Evaporation of the solvent gave 2.5 g of
`
`yellowish oil as a crude product. The crude product was purified by repeated column
`
`chromatography on silica gel (230-400 mesh, 250 mL) eluted with 0-3% methanol gradient in
`
`chloroform. This afforded 0.4 g (20%) of DLin-S-DMA as yellowish oil. 1H NMR (400 MHz,
`
`CDCl3) δ: 5.27-5.48 (8H, m, 4 x CH=CH), 2.88-3.0 (1H, m), 2.83 (2H, d, CH2), 2.7 (4H, t, 2 x
`
`C=C-CH2-C=C), 2.63-2.73 (1H, m), 2.58 (4H, double triplet, 2 x SCH2), 2.39-2.49 (1H, m), 2.31
`
`(6H, s, 2 x NCH3), 2.06 (8H, q, 4 x allylic CH2), 1.52-1.65 (4H, m, 2 x CH2), 1.23-1.45 (32H,
`
`m), 0.90 (6H, t, 2 x CH3) ppm.
`
`
`
`2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA)
`
`Synthesis of Linoleyl Bromide
`
`A mixture of linoleyl methane sulfonate (6.2g, 18 mmol) and magnesium bromide etherate (17g,
`
`55 mmol) in anhydrous ether (300 mL) was stirred under argon overnight (21 hours). The
`
`resulting suspension was poured into 300 mL of chilled water. Upon shaking, the organic phase
`
`was separated. The aqueous phase was extracted with ether (2 x 150 mL). The combined ether
`
`phase was washed with water (2 x 150 mL), then brine (150 mL), and dried over anhydrous
`
`Na2SO4. The solvent was evaporated to afford 6.5g of colourless oil. The crude product was
`
`purified by column chromatography on silica gel (230-400 mesh, 300 mL) eluted with hexanes.
`
`This gave 6.2 g (approximately 100%) of linoleyl bromide.
`
`
`
`Synthesis of Dilinoleyl Methanol
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
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`To a suspension of Mg turnings (0.45g, 18.7 mmol) with one crystal of iodine in 200 mL of
`
`anhydrous ether under nitrogen was added a solution of linoleyl bromide in 50 mL of anhydrous
`
`ether at room temperature. The resulting mixture was refluxed under nitrogen overnight. The
`
`mixture was cooled to room temperature. To the cloudy mixture under nitrogen was added
`
`dropwise at room temperature a solution of ethyl formate (0.65g, 18.7 mmol) in 30 mL of
`
`anhydrous ether. Upon addition, the mixture was stirred at room temperature overnight
`
`(20 hours). The ether layer was washed with 10% H2SO4 aqueous solution (100 mL), then water
`
`(2 x 100 mL), then brine (150 mL), and then dried over anhydrous Na2SO4. Evaporation of the
`
`solvent gave 5.0g of pale oil. Column chromatography on silica gel (230-400 mesh, 300 mL)
`
`with 0-7% ether gradient in hexanes as eluent afforded two products, dilinoleyl methanol (2.0g)
`
`and dilinoleylmethyl formate (1.4g). Dilinoleylmethyl formate (1.4g) and KOH (0.2g) were
`
`stirred in 85% EtOH at room temperature under nitrogen overnight. Upon completion of the
`
`reaction, half of the solvent was evaporated. The resulting mixture was poured into 150 mL of
`
`5% HCL solution. The aqueous phase was extracted with ether (3 x 100 mL). The combined
`
`ether extract was washed with water (2 x 100 mL), then brine (100 mL), and dried over
`
`anhydrous Na2SO4. Evaporation of the solvent gave 1.0 g of dilinoleyl methanol as colourless
`
`oil. Overall, 3.0 g (60%) of dilinoleyl methanol were obtained.
`
`
`
`Synthesis of Dilinoleyl Ketone
`
`To a mixture of dilinoleyl methanol (2.0g, 3.8 mmol) and anhydrous sodium carbonate (0.2g) in
`
`100 mL of CH2Cl2 was added pyridinium chlorochromate (PCC, 2.0g, 9.5 mmol). The resulting
`
`suspension was stirred at room temperature for 60 minutes. Ether (300 mL) was then added into
`
`the mixture, and the resulting brown suspension was filtered through a pad of silica gel
`
`(300 mL). The silica gel pad was further washed with ether (3 x 200 mL). The ether filtrate and
`
`washes were combined. Evaporation of the solvent gave 3.0 g of an oily residual as a crude
`
`product. The crude product was purified by column chromatography on silica gel (230-400
`
`mesh, 250 mL) eluted with 0-3% ether in hexanes. This gave 1.8 g (90%) of dilinoleyl ketone.
`
`
`
`Synthesis of 2,2-Dilinoleyl-4-bromomethyl-[1,3]-dioxolane (I)
`
`To make I, a mixture of dilinoleyl methanol (1.3g, 2.5 mmol), 3-bromo-1,2-propanediol (1.5g,
`
`9.7 mmol) and p-toluene sulfonic acid hydrate (0.16g, 0.84 mmol) in 200 mL of toluene was
`
`refluxed under nitrogen for 3 days with a Dean-Stark tube to remove water. The resulting
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`mixture was cooled to room temperature. The organic phase was washed with water (2 x 50 mL),
`
`then brine (50 mL), and dried over anhydrous Na2SO4. Evaporation of the solvent resulted in a
`
`yellowish oily residue. Column chromatography on silica gel (230-400 mesh, 100 mL) with
`
`0-6% ether gradient in hexanes as eluent afforded 0.1 g of pure product and 1.3 g of a mixture of
`
`product and the starting material.
`
`
`
`Synthesis of 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA)
`
`Anhydrous dimethyl amine was bubbled into an anhydrous THF solution (100 mL) containing
`
`1.3 g of a mixture of I and dilinoleyl ketone at 0ºC for 10 minutes. The reaction flask was then
`
`sealed and the mixture stirred at room temperature for 6 days. Evaporation of the solvent left
`
`1.5 g of a residual. The crude product was purified by column chromatography on silica gel
`
`(230-400 mesh, 100 mL) eluted with 0-5% methanol gradient in dichloromethane. This gave
`0.8 g of DLin-K-DMA. 1H NMR (400 MHz, CDCl3) δ: 5.25-5.45 (8, m, 4x CH=CH), 4.28-4.4
`
`(1H, m, OCH), 4.1 (1H, dd, OCH), 3.53 (1H, t OCH), 2.78 (4H, t, 2 x C=C-CH2-C=C), 2.5-2.65
`
`(2H, m, NCH2), 2.41 (6H, s, 2 x NCH3), 2.06 (8H, q, 4 x allylic CH2), 1.56-1.68 (4H, m, 2 x
`
`CH2), 1.22-1.45 (32H, m), 0.90 (6H, t, 2 x CH3) ppm.
`
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`Supplementary Table 2. Headgroup modifications to DLin-K-DMA
`
`Chemical
`Name
`
`2,2-Dilinoleyl-4-
`dimethylamino
`methyl-[1,3]-
`dioxolane
`
`2,2-Dilinoleyl-4-
`N-methyl
`piperazino-[1,3]-
`dioxolane
`
`2,2-Dilinoleyl-4-
`N-morpholino-
`[1,3]-dioxolane
`
`Abbreviated Name
`
`DLin-K-DMA
`(Benchmark)
`
`
`
`DLin-K-MPZ
`
`DLin-K-MA
`
`DLin-K-TMA.Cl
`
`Modification
`
`N
`
`N
`
`N
`
`N
`
`O
`
`C l
`
`N
`
`O
`
`O
`
`R
`
`R
`
`
`
`R
`
`R
`
`
`
`
`
`R
`
`R
`
`R
`
`R
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`Mean
`Particle
`Size (nm)a
`
`ED50
`(mg/kg)
`
`67
`
` 22
`
`~0.4
`
`
`71
`
` 21
`
`~1.5
`
`58
`
` 21
`
`>15
`
`150
`
` 69
`
`>5b
`
`2,2-Dilinoleyl-4-
`trimethylamino-
`[1,3]-dioxolane
`Chloride
`
`2,2-Dilinoleyl-
`4,5-bis
`(dimethylamino
`methyl)-[1,3]-
`dioxolane
`
`2,2-Dilinoleyl
`-4-(2-dimethyl
`aminoethyl)-[1,3]-
`dioxolane
`
`2,2-Dilinoleyl
`-4-(3-dimethyl
`aminopropyl)-
`[1,3]-dioxolane
`
`2,2-Dilinoleyl
`-4-(4-dimethyl
`aminobutyl)-
`[1,3]-dioxolane
`
`R =
`
`DLin-K2-DMA
`
`DLin-KC2-DMA
`
`DLin-KC3-DMA
`
`DLin-KC4-DMA
`
`N
`
`N
`
`O
`
`O
`
`R
`
`R
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`N
`
`N
`
`N
`
`
`
`
`
`R
`
`R
`
`
`
`R
`
`R
`
`
`
`R
`
`R
`
`
`
`68
`
` 32
`
`~0.4
`
`72
`
` 23
`
`~0.1
`
`80
`
` 22
`
`~0.6
`
`64
`
` 15
`
`>3
`
`a
`
`Mean particle size of the PFV formulation as tested for FVII activity and containing the indicated cationic lipid at
`40 mol%.
`
`b No activity observed at 5 mg/kg and lethal at next dose of 15 mg/kg.
`
`
`
`Nature Biotechnology: doi: 10.1038/nbt.1602
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`Novel Cationic Lipids
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`
`Supplementary Syntheses 2
`
`
`
`2,2-Dilinoleyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ)
`
`A mixture of dilinoleyl ketone (1.3 gm, 2.5mmol), 3-bromo1,2-propane diol (1.5 gm, 9.7 mmol)
`
`and PPTS (pyridinium p-toluene sulfonate) (100 mg) in 25 mL of toluene was refluxed under
`
`nitrogen overnight with a Dean-stark tube to remove water. The resulting mixture was cooled to
`
`room temperature. The organic phase was washed with water (2 x 50 mL) and saturated NaHCO3
`
`solution, then dried over anhydrous Na2SO4. Evaporation of solvent resulted in a yellowish oily
`
`residue. Column Chromatography on silica (230-400 mesh) with 0-5% ether as eluent in hexanes
`
`afforded 750 mg of the corresponding ketal. To a mixture of the ketal (250mg, 0.37 mmol) and
`
`K2CO3 (138 mg, 1 mmol) in 5 mL of acetonitrile was added N-methylpiperazine (50 mg, 0.50
`
`mmol). The resulting solution was refluxed under argon overnight. The resulting mixture was
`
`cooled to room temperature, the solvent was evaporated, and the organic phase was washed with
`
`water (2 x 50 mL) and dried over anhydrous Na2SO4. Evaporation of the solvent resulted in a
`
`yellowish oily residue. Column chromatography on silica gel (230-400 mesh, 500 mL) eluted
`
`with 25-50% hexanes and ethyl acetate and then eluted with 0-5% methanol gradient in
`
`dichloromethane gave 225 mg of the desired product DLin-K-MPZ.
`
`
`
`2,2-Dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA)
`
`To a mixture of the above ketal (250mg, 0.37 mmol) and K2CO3(138 mg, 1 mmol) in 5 mL of
`
`acetonitrile was added morpholine (50 mg, 0.57 mmol). The resulting solution was refluxed
`
`under argon overnight. The resulting mixture was cooled to room temperature, the solvent was
`
`evaporated, and the organic phase was washed with water (2 x 50 mL) and dried over anhydrous
`
`Na2SO4. Evaporation of the solvent resulted in a yellowish oily residue. Column chromatography
`
`on silica gel (230-400 mesh, 500 mL) eluted with 25-50% hexanes and ethyl acetate and then
`
`eluted with 0-5% methanol as gradient in dichloromethane gave 225 mg of the desired product
`DLin-K-MA: 1H NMR (300 MHz, CDCl3) δ: 5.27-5.46 (8H, m), 4.21-4.31 (1H, m), 4.06-4.09
`
`(1H, t), 3.71-3.73 (4H, t) 3.49-3.55 (1H, t), 2.78 (4H, t) 2.42-2.62 (6H, m), 2.02-2.09 (8H, m)
`
`1.55-1.65 (4H, m), 1.2-1.47 (32 H, m), 0.87-0.90 (6H, t) ppm.
`
`
`
`2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane Chloride (DLin-K-TMA.Cl)
`
`Nature Biotechnology: doi: 10.1038/nbt.1602
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`0012
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`

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`Semple, S., Akinc, A, Chen J, et al.
`
`Synthesis of 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane Iodide (I)
`
`A mixture of DLin-K-DMA (1.5g, 2.4 mmol) and CH3I (4.0 mL, 64 mmol) in 10 mL of
`
`anhydrous CH2Cl2 was stirred under nitrogen at room temperature for 9 days. Evaporation of the
`
`solvent and excess of iodomethane afforded 20 g of yellow syrup as crude I, which was used in
`
`the following step without further purification.
`
`
`
`Preparation of 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane Chloride (DLin-K-TMA.Cl)
`
`I (2.0 g) was dissolved in 100 mL of CH2Cl2 in a separatory funnel. 30 mL of 1N HCl methanol
`
`solution was added, and the resulting solution was shaken well. To the solution was added 50 mL
`
`of brine and the mixture was shaken well. The organic phase was separated. The aqueous phase
`
`was extracted with 10 mL of CH2Cl2. The organic phase and extract were then combined. This
`
`completed the first step of ion exchange. The ion exchange step was repeated four more times.
`
`The final organic phase was washed with brine (2 x 75 mL) and dried over anhydrous Na2SO4.
`
`Evaporation of the solvent gave 2.0 g of yellowish viscous oil. The product was purified by
`
`column chromatography on silica gel (230-400 mesh, 100 mL) eluted with 0-15% methanol
`
`gradient in chloroform. This afforded 1.2 g of DLin-K-TMA.Cl as a pale waxy material.
`1H NMR (300 MHz, CDCl3) δ: 5.25-5.45 (8H, m, 4 x CH=CH), 4.55-4.75 (2H, m, 2 x OCH),
`
`4.26-4.38 (1H, dd, OCH), 3.48-3.57 (1H, dd, NCH), 3.51 (9H, s, 3 x NCH3), 3.11-3.22 (1H, dd,
`
`NCH), 2.77 (4H, t, 2 x C=C-CH2-C=C), 2.05 (8H, q, 4 x allylic CH2), 1.49-1.7 (4H, m, 2 x CH2),
`
`1.2-1.45 (30H, m), 0.89 (6H, t, 2 x CH3) ppm.
`
`
`
`2,2-Dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane (DLin-K2-DMA)
`
`Synthesis of DLin-K-diethyltartarate
`
`A mixture of dilinoleyl ketone (1 g, 1.9 mmol), diethyl-D-tartarate (412 mg, 2 mmol) and
`
`pyridinium p-tolene sulfonate (250 mg, 1 mmol) in 25 mL of toluene was refluxed under
`
`nitrogen for 2 days with a Dean-stark tube to remove water. The resulting mixture was cooled to
`
`room temperature. The organic phase was washed with water NaHCO3 and brine (2 X 50 mL)
`
`and dried over anhydrous Na2SO4. Evaporation of the solvent resulted in a yellowish oily
`
`residue. Column chromatography on silica gel (230-400 mesh, 500 mL) eluted with 0-10% ether
`
`gradients in hexanes as eluent afforded 400 mg of pure DLin-K-diethyltartarate.
`
`
`
`Synthesis of DLin-K-diethyldiol
`
`Nature Biotechnology: doi: 10.1038/nbt.1602
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`0013
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`

`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`To a solution of lithium aluminum hydride (32 mg, 1 mmol) in dry THF was added a solution of
`
`DLin-K-diethyltartarate (600mg, 0.85 m mol) in dry THF at 0ºC under argon atmosphere; the
`
`reaction was stirred for 4 hours at room temperature. The reaction mixture was quenched with
`
`ice-cold water and then filtered through celite. The evaporation of solvent gave crude reduced
`
`alcohol. Column chromatography on silica gel (230-400 mesh, 500 mL) eluted with 10-40%
`
`ethyl acetate gradients in hexanes as eluent afforded 350 mg of pure DLin-K-diethyldiol.
`
`
`
`Synthesis of DLin-K-diethyldimesylate
`
`To a mixture of DLin-K-diethyldiol (570 mg, 0.95 mmol) in dry dichloromethane was added
`
`pyridine (275 mg, 3.85 mmol) and 4-(dimethylamino)pyridine (122 mg, 1 mmol) under argon,
`
`followed by a solution of methane sulfonyl chloride (500 mg, 2.5 mmol). The resulting mixture
`
`was stirred overnight. The organic phase was washed with water and brine (2 x 50 mL), then the
`
`solvent was evaporated to give a yellowish oil residue. Column chromatography on silica gel
`
`(230-400 mesh, 500 mL) eluted with10-40% ethyl acetate gradients in hexanes as eluent afforded
`
`300 mg of pure DLin-K-diethyldimesylate.
`
`
`
`2,2-Dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane (DLin-K2-DMA)
`
`Anhydrous dimethyl amine solution in THF was added to the reaction vessel containing 300 mg
`
`of DLin-K-diethyldimesylate at room temperature for 5 minutes. The reaction flask was then
`
`sealed and the mixture stirred at room temperature for 6 days. Evaporation of the solvent left
`
`300 mg of residual. The crude product was purified by column chromatography on silica gel
`
`(230-400 mesh, 500 mL) eluted with 0-10% methanol gradients in chloroform as eluent and
`afforded 50 mg of pure DLin-K2-DMA. 1H NMR (300 MHz, CDCl3) δ: 5.27-5.46 (8H, m), 3.72-
`
`3.80 (2H, t), 2.75 (4H, t), 2.49 (4H, d), 2.30 (12H, s), 2.02-2.09 (8H, m) 1.62-1.72 (4H, m), 1.2-
`
`1.47 (32 H, m), 0.87-0.90 (6H, t) ppm.
`
`
`
`2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA
`
`Synthesis of 2,2-Dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxolane (I)
`
`A mixture of dilinoleyl ketone (527 mg, 1.0 mmol); 1,2,4-butanetriol (technical grade, ca. 90%,
`
`236 mg, 2 mmol); and pyridinium p-toluenesulfonate (50 mg, 0.2 mmol) in 50 mL of toluene
`
`was refluxed under nitrogen overnight with a Dean-Stark tube to remove water. The resulting
`
`mixture was cooled to room temperature. The organic phase was washed with water (2 x 30 mL),
`
`Nature Biotechnology: doi: 10.1038/nbt.1602
`
`0014
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`Novel Cationic Lipids
`Semple, S., Akinc, A, Chen J, et al.
`
`