WO2024253473A1 - Single continuous flow synthesis method for synthesizing functional lipid library used in nucleic acid therapeutic agent - Google Patents

Single continuous flow synthesis method for synthesizing functional lipid library used in nucleic acid therapeutic agent Download PDF

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WO2024253473A1
WO2024253473A1 PCT/KR2024/007830 KR2024007830W WO2024253473A1 WO 2024253473 A1 WO2024253473 A1 WO 2024253473A1 KR 2024007830 W KR2024007830 W KR 2024007830W WO 2024253473 A1 WO2024253473 A1 WO 2024253473A1
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cationic lipid
reactor
propane
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methyl sulfate
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김동표
카왈리산켓 아쇽라오
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Definitions

  • the present invention relates to a single continuous flow synthesis method for synthesizing a functional lipid library used in nucleic acid therapeutics, and more particularly, to a single continuous flow synthesis method for synthesizing an ionizable cationic lipid library including an ionizable cationic lipid such as 1,2-dioleoyl-3-dimethylammonium-propane and a cationic lipid library including a cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane, and a method for preparing lipid nanoparticles or lipid nanoliposomes using the method.
  • LNPs Clinically effective lipid nanoparticles
  • cationic lipids play a key role in liposomal transfection of DNA, RNA, and other negatively charged molecules.
  • cationic lipids typically consist of an ionizable or cationic head group attached to a saturated or unsaturated hydrophobic tail via a linker group.
  • Cationic lipids are positively charged at physiological pH, whereas ionizable cationic lipids are neutral at physiological pH but positively charged at pH below their pKa ( ⁇ 7) due to protonation of the head group-free amines responsible for encapsulation and endosomal release of RNA.
  • cationic lipids in which a head group is linked to a hydrophobic tail via an ester bond exhibit more clinical efficacy due to their biodegradability in the human body.
  • 1,2-Dioleoyl-3-dimethylammonium-propane is an ionizable cationic lipid with low cytotoxicity and high transfection efficiency. It is composed of two hydrophobic oleoyl tails and a hydrophilic head group containing a dimethylammonium moiety and a propane group. In contrast, 1,2-dioleoyl-3-trimethylammonium-propane (3) (DOTAP), a derivative of DODAP (2), contains a cationic trimethylammonium hydrophilic head group ( Figure 1). DODAP(2) and its derivatives are widely used to encapsulate bioactive molecules including mRNA, siRNA and plasmid DNA for disease treatment.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DODAP(2) The synthesis of DODAP(2) involves the synthesis of oleoyl chloride from oleic acid followed by esterification with 3-(dimethylamino)propane-1,2-diol.
  • the synthesis and purification in batch reactors are challenging due to the toxicity of essential chlorinating agents such as oxalyl chloride and the toxic HCl and CO gas byproducts generated during the formation of oleoyl chloride.
  • an alternative synthetic method using the coupling reaction of oleic acid and 3-(dimethylamino)propane-1,2-diol is time-consuming, difficult to purify, and relatively expensive.
  • Patent Documents 1 and 2 report two different approaches to prepare cationic lipid series that utilize highly toxic and carcinogenic methylating agents (chloromethane, iodomethane, etc.), which limit their application in large-scale production ( Figure 2 a and b).
  • Patent Document 3 and Non-Patent Document 2 utilized non-toxic trimethylamine to introduce a cationic head group for DOTAP bromide synthesis (Fig. 2c).
  • Fig. 2c the high cost of using bromopropanediol as a starting material has limited its industrial application.
  • An attempt to use glycerol, a cheap starting material, together with the trimethylamine reagent of Patent Document 4 requires a multi-step process that is time-consuming and labor-intensive and affects the quality of the lipid (Fig. 2d).
  • the shortest route involves the Steglich esterification reaction of oleic acid with 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride starting from 3-chloropropane-1,2-diol (Fig. 2e), but it takes 16 h for complete completion, and it is highly desirable to utilize flow chemistry to shorten the reaction time and productivity.
  • Patent Document 3 DE4013632A1
  • Patent Document 4 WO2005049549
  • Non-patent Document 2 Interactions of Cationic Lipid Vesicles with Negatively Charged Phospholipid Vesicles and Biological Membranes, Leonidas Stamatatos, Rania Leventis, Martin J. Zuckermann, John R. Silvius, Biochemistry 1988, 27 , 3917-3925.
  • Non-patent Document 3 Overcoming solid handling issues in continuous flow substitution reactions through ionic liquid formation, Saeed K. Kashani, Ryan J. Sullivan, Mads Andersen, Stephen G. Newman, Green Chem., 2018 , 20 , 1748-1753.
  • the present invention aims to provide a method for producing lipid nanoparticles or lipid nanoliposomes using a single continuous flow synthesis method for the aforementioned functional lipids.
  • a step of separating HCl and CO gases may be additionally included after step (a).
  • the synthetic method can be performed in a microfluidic device for a single continuous flow process
  • the microfluidic device can include: a first micromixer in which a first channel and a second channel are fluidly connected; a first reactor in which the first micromixer is fluidly connected; a first back pressure regulator in which the first reactor is fluidly connected; a T-shaped liquid-gas separator in which the first back pressure regulator is fluidly connected to separate a solution and a gas; a second micromixer in which a third channel is fluidly connected to the T-shaped liquid-gas separator; a second reactor in which the second micromixer is fluidly connected; and a second back pressure regulator in which the second reactor is fluidly connected.
  • the T-shaped liquid-gas separator may include a first supply channel fluidly connected to the first back pressure regulator, a first discharge channel through which separated gas is discharged, and a second supply channel fluidly connected to the second micro-mixer so that the separated solution is supplied to the second micro-mixer.
  • oxalyl chloride can be injected into the second path
  • HCl and CO gas can be separated through the T-shaped liquid-gas separator
  • 3-(dimethylamino)propane-1,2-diol and a basic compound can be injected into the third path
  • an ionizable cationic lipid having a structure of the chemical formula 1 can be generated in the second reactor.
  • the residence time in the first reactor may be 1 to 2 minutes
  • the residence time in the second reactor may be 2 to 10 minutes
  • the reaction temperature in the second reactor may be 20 to 60°C.
  • the basic compound may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine.
  • the present invention provides a method for synthesizing 6-bromohexyl 2-hexyldecanoate, an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminum, which is an ionizable cationic lipid, comprising the following steps (a) and (b):
  • the present invention includes a step of mixing and reacting an ionizable cationic lipid synthesized by the above-described synthetic method with dimethyl sulfate to produce cationic lipid methyl sulfate having a structure of chemical formula 2,
  • the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.
  • an ionizable cationic lipid having a structure of chemical formula 1 can be injected into the first flow path, and dimethyl sulfate can be injected into the second flow path.
  • cationic lipid methyl sulfate having the structure of the chemical formula 2 can be produced.
  • the ionizable cationic lipid having the structure of the chemical formula 1 and the dimethyl sulfate can be injected at a flow rate of 0.4 to 2.0 ml/min, the reaction temperature in the first reactor can be 40 to 70°C, and the residence time in the first reactor can be 3 to 7 minutes.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one of the following (1) to (7):
  • the synthesis method may additionally include solvent evaporation and recrystallization steps.
  • the recrystallization can be performed in an acetone solvent.
  • the present invention provides a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.
  • the reaction temperature in the first reactor may be 60 to 80°C, and the residence time in the first reactor may be 3 to 7 minutes.
  • the synthesis method may additionally include solvent evaporation and recrystallization steps.
  • the recrystallization can be performed in isopropanol and acetone solvents.
  • the present invention provides a method for preparing lipid nanoparticles comprising the following steps (a) and (b):
  • the present invention provides a method for preparing lipid nano liposomes comprising the following steps (a) and (b):
  • the method for synthesizing a functional lipid used in a nucleic acid therapeutic agent according to the present invention is implemented by a continuous flow synthesis method in a microfluidic reactor, which has significantly higher productivity and provides an ionizable cationic lipid or cationic lipid of superior quality compared to a synthesis method in a conventional batch reactor.
  • DODAP and DOTAP synthesized by the method of the present invention can be utilized as lipid nanoparticles and lipid nanoliposomes that encapsulate physiologically active molecules including mRNA, siRNA, and plasmid DNA for disease treatment.
  • Figure 1 shows the structures of common cationic lipids (1), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) (2) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (3), which are used in lipid nanoparticles or liposomes.
  • DODAP 1,2-dioleoyl-3-dimethylammonium-propane
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • Figure 2 shows a conventionally known method for synthesizing DOTAP (3), and (B) shows a continuous flow method for synthesizing DOTAP using two types of counter ions of the present invention.
  • Figure 3 shows the experimental setup for continuous flow synthesis of oleoyl chloride in a capillary reactor including in-line FTIR.
  • Figure 4 is a photograph of the experimental setup of the in-line FTIR flow cell.
  • Figure 5 shows the experimental results of in-line FTIR monitoring.
  • Figure 6(a) is a photograph showing a custom T-junction used as an effective liquid-gas separator
  • b) is a photograph showing that plugging occurs in the R2 reactor due to high concentration of solid pyridinium hydrochloride salt (8 eq. of base is required) if HCl gas is not removed from the reactor
  • c) shows the results of monitoring the conversion of oleoyl chloride to DODAP(2) using in-line FTIR monitoring.
  • Figure 7 shows the single-flow synthesis of the ionizable cationic lipid DODAP(2) and a library of ionizable cationic lipids in a capillary reactor.
  • Figure 8 shows the experimental setup for the single-flow synthesis of ionizable cationic lipids.
  • Figure 9 shows the experimental setup for continuous flow synthesis of DODAP.
  • Figure 10 shows the experimental setup for continuous flow synthesis of cationic lipids DOTAP methyl sulfate (4), DLinTAP methyl sulfate (17) and other analogues (18-22) in a capillary reactor.
  • Figure 11 shows the experimental setup for continuous flow synthesis of DOTAP methyl sulfate.
  • Figure 12 shows the experimental setup for continuous flow synthesis of a cationic lipid library.
  • Figure 13 shows the continuous flow synthesis of DOTAP chloride in a capillary reactor.
  • Figure 14 shows the experimental setup for continuous flow synthesis of DOTAP chloride using a size-up method.
  • Figure 15 shows the preparation and characterization of nanoparticles generated from synthesized DODAP and DOTAP, where A of Figure 15 shows a series of processes for nanoparticle production, dialysis and analysis by DLS, and B to E show the normalized size distributions of the prepared DODAP LNPs (B), DODAP liposomes (C), DOTAP-chloride liposomes (D) and DOTAP methyl sulfate liposomes (E) before and after purification by dialysis process, respectively.
  • Figure 11 shows the production and analysis of lipid-based nanoparticles, where A is a schematic diagram of the overall procedure and B shows the numerical size and PDI of nanoparticles produced before and after dialysis.
  • Figure 16 shows the numerical sizes and PDI values of the generated DODAP LNPs (A), DODAP liposomes (B), DOTAP-chloride liposomes (C), and DOTAP methyl sulfate liposomes (D).
  • the inventors of the present invention sought to solve the above-mentioned problems by developing a novel synthetic route capable of synthesizing a high-quality functional lipid library with high productivity using a single continuous flow process.
  • a facile single-flow synthesis method for a library of ionizable lipids developed in a simple two-step manner was developed, achieving scalable productivity of up to 10 g/hr through a scale-up approach.
  • acyl chloride synthesis for continuous flow esterification was successfully explored, and clogging was prevented by effectively removing toxic HCl and CO gases using a T-junction liquid-gas separator. Rapid optimization of the continuous flow process was achieved using in-line FTIR monitoring.
  • the flow protocol of the present invention enables the synthesis of a wide range of lipids, including both established commercial types and novel ionizable lipids, in a much shorter time (approximately 4.5 min) compared to conventional batch protocols.
  • a library of biodegradable ionizable lipids was designed with ester or disulfide functionalities in the long chain group. These lipids were synthesized in a facile capillary reactor, and were obtained in 88-92% yields. In addition, rapid alkylation was achieved using dimethyl sulfate while maintaining the flow reactor for 5 min in the present invention to convert the ionizable lipids into cationic lipids. In particular, DOTAP methyl sulfate was produced with a productivity of 7 g/h. Furthermore, DOTAP chloride was effectively synthesized with a yield of 88% via the Steglich esterification reaction using EDC.HCl, resulting in a productivity of 10.2 g/h.
  • DODAP was converted into uniformly sized 64 mm LNPs with a PDI of 0.07 and 72 mm liposomes with a PDI of 0.05 using a self-developed micromixer.
  • DOTAP formed 55 mm liposomes with a PDI of 0.08. This novel fluid-assisted platform for lipid libraries holds great potential to advance gene therapy and serve as next-generation nanomedicine.
  • the present invention in its first aspect, provides a method for synthesizing an ionizable cationic lipid comprising the steps of:
  • the unsubstituted C10 to C20 saturated aliphatic hydrocarbon radical may be a linear or branched saturated aliphatic hydrocarbon radical containing 10 to 20 carbon atoms.
  • it may be a linear or branched decenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group or an eicosenyl group, but is not limited thereto.
  • all reactants of the above-mentioned synthetic method can be used in a state dissolved in an organic solvent that does not adversely affect the reactions of steps (a) and (b).
  • organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform steps (a) and (b).
  • oxalyl chloride in step (a) was selected as a chlorinating agent for synthesizing oleoyl chloride in the presence of a dimethylformamide (DMF) catalyst.
  • DMF dimethylformamide
  • CH 2 Cl 2 was considered as a general solvent for flow synthesis considering the solubility of the formed base.HCl complex.
  • the 3-(dimethylamino)propane-1,2-diol may be replaced with another type of diol compound containing a dimethylamino group.
  • the synthesis method can be performed in a microfluidic device (1) for a single continuous flow process, wherein the microfluidic device (1) comprises: a first micro-mixer (100) in which a first flow path (110) and a second flow path (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); a first back pressure regulator (300) fluidly connected with the first reactor (200); a T-shaped liquid-gas separator (400) fluidly connected with the first back pressure regulator (300) to separate a solution and a gas; a second micro-mixer (500) in which the T-shaped liquid-gas separator (400) and a third flow path (510) are fluidly connected; a second reactor (600) fluidly connected with the second micro-mixer (500); and may include a second back pressure regulator (700) fluidly connected to the second reactor (600).
  • a first micro-mixer (100) in which a first flow path (110) and a second flow path (120) are fluidly
  • the T-shaped liquid-gas separator (400) may include a first supply path (410) fluidly connected to the first back pressure regulator (300), a first discharge path (420) through which separated gas is discharged, and a second supply path (430) fluidly connected to the second micro-mixer (500) so that the separated solution is supplied to the second micro-mixer (500).
  • the first flow path (110) and the second flow path (120) of the microfluidic device (1) are flow paths into which the reactants of step (a) are injected, respectively, and can be fluidly connected to the first micro-mixer (100).
  • reactants flowing in from the first path (110) and the second path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • These byproduct gases can be separated and removed through the T-shaped liquid-gas separator (400).
  • the second micro-mixer (500) may be fluidly connected to the third channel (510).
  • 3-(dimethylamino)propane-1,2-diol and a basic compound may be injected into the third channel (510).
  • reactants flowing in from the second supply path (430) and the third path (510) are mixed to produce a mixture, and the produced mixture can be injected into the second reactor (600) that is fluidly connected to the second micro-mixer (500).
  • step (b) occurs in the second reactor (600), and an ionizable cationic lipid having the structure of the chemical formula 1 is produced as a result of the reaction.
  • the ionizable cationic lipid having the structure of the chemical formula 1 as the final product can be discharged after passing through the second back pressure regulator (700) fluidly connected to the second reactor (600).
  • a first spectrometer (800) may be additionally fluidly connected between the first reactor (200) and the first back pressure regulator (300), and a second spectrometer (900) may be additionally fluidly connected between the second reactor (600) and the second back pressure regulator (700).
  • the residence time in the first reactor (200) may be 1 to 2 minutes.
  • reaction temperature in the first reactor (200) may be 20 to 30°C.
  • the residence time in the second reactor (600) may be 2 to 10 minutes.
  • the reaction temperature in the second reactor (600) may be 20 to 60°C.
  • step (b) It is preferable to perform the reaction of step (b) within the range of the residence time (i.e., reaction time) and reaction temperature in the second reactor (600), and if it is outside the range, the yield of the ionizable cationic lipid having the structure of the chemical formula 1 may decrease.
  • the residence time in the second reactor can be achieved by injecting a solution containing 3-(dimethylamino)propane-1,2-diol and a basic compound into the third flow path (510) at a flow rate of 0.4 to 2.0 ml/min into the second micro-mixer.
  • the basic compound of step (b) may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine.
  • the most preferred basic compound may be triethylamine.
  • triethylamine When triethylamine is used as the basic compound, an ionizable cationic lipid having the structure of the chemical formula 1 can be obtained in a maximum yield of 92% through a reaction at 25°C for 3 minutes in the second reactor (600).
  • the ionizable cationic lipid having the structure of the chemical formula 1 may be any one of the following (1) to (11):
  • step (a) to synthesize 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), an ionizable cationic lipid, step (a) was performed as follows.
  • the reaction was optimized at room temperature (25 °C) with various residence times by varying the flow rates of the reagents.
  • the flow apparatus was connected to an FTIR spectrometer using a microfluidic cell (Fig. 4).
  • IR spectroscopy monitored the formation of acid chloride with a characteristic CO stretching band at 1780 cm -1 and the disappearance of the CO stretching at 1708 cm -1 of the acid functional group of oleic acid (Fig. 5).
  • a 100 psi back pressure regulator (BPR) was used at the FTIR flow cell outlet to prevent the outgassing of by-products such as HCl and CO gases. After some optimization, it was confirmed that complete consumption of oleic acid occurred at a reaction temperature of 25 °C and a residence time of 1.5 min for smooth formation of oleoyl chloride. Further increase in the residence time and temperature in reactor R1 did not result in further increase in the intensity of the CO stretching peak of oleoyl chloride.
  • HCl and CO gases that are not dissolved in the solution are successfully separated using a liquid-gas separator (T-junction). If HCl is not separated, an excessive amount of base is required in step (b), and the concentration of insoluble hydrochloric acid salt in the base is high, which may cause clogging in R2.
  • DODAP 1,2-dioleoyl-3-dimethylammonium-propane
  • the reaction in R2 was optimized at various residence times by changing the length of the PTFE tube reactor.
  • the ester bond formation during the DODAP(2) synthesis was monitored using inline FTIR spectroscopy, and a characteristic CO stretching band was formed at 1735 cm -1 for the ester group ( FIG. 7 ).
  • the productivity of the ionizable cationic lipid library was improved by using the first reactor (200) and the second reactor (600) whose inner diameters were increased to 1.6 mm.
  • the tube diameter of the first reactor of the microfluidic device may be 0.8 to 2.0 mm
  • the tube length may be 200 to 300 cm
  • the tube diameter of the second reactor may be 0.8 to 2.0 mm
  • the tube length may be 500 to 900 cm.
  • a second aspect of the present invention relates to a method for synthesizing 6-bromohexyl 2-hexyldecanoate, which is an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium, which is an ionizable cationic lipid, comprising the following steps (a) and (b).
  • the method for synthesizing 6-bromohexyl 2-hexyldecanoate of the present invention can be performed under the same conditions and in the same microfluidic device as the method for synthesizing an ionizable cationic lipid described above, except that 2-hydroxydecanoic acid is used instead of oleic acid and 6-bromohexanol is used instead of trimethylamine.
  • the residence time in the first reactor can be 1 to 3 minutes, and the reaction temperature can be 20 to 30°C, the residence time in the second reactor can be 2 to 7 minutes, and the reaction temperature in the second reactor can be 20 to 60°C.
  • a third aspect of the present invention comprises a step of mixing and reacting an ionizable cationic lipid synthesized by the synthetic method described above with dimethyl sulfate to produce a cationic lipid methyl sulfate having a structure of chemical formula 2.
  • the method for synthesizing the cationic lipid methyl sulfate can be performed in a microfluidic device (2) for a single continuous flow process, and the microfluidic device (2) can include: a first micro-mixer (100) in which a first channel (110) and a second channel (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); and a first back pressure regulator (300) fluidly connected with the first reactor (200).
  • all reactants of the above cationic lipid methyl sulfate synthesis method can be used in a state dissolved in an organic solvent that does not adversely affect the reaction.
  • organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.
  • the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidically connected to the first micro-mixer (100).
  • an ionizable cationic lipid having the structure of the chemical formula 1 can be injected into the first flow path (110), and dimethyl sulfate can be injected into the second flow path (120).
  • reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • the ionizable cationic lipid and dimethyl sulfate react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 as a reaction product.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 as the final product can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).
  • a first spectrometer (800) may additionally be fluidly connected between the first reactor (200) and the first back pressure regulator (300) of the above microfluidic device.
  • the residence time in the first reactor (200) may be 3 to 7 minutes.
  • the time required for the reactants introduced into the first reactor (200), that is, the ionizable cationic lipid having the structure of the chemical formula 1 and dimethyl sulfate, to react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 may be 3 to 7 minutes.
  • reaction temperature in the first reactor (200) may be 40 to 70°C.
  • reaction time i.e., reaction time
  • reaction temperature in the above first reactor (200)
  • yield of the cationic lipid methyl sulfate having the structure of the chemical formula 2 may decrease.
  • the residence time in the first reactor (200) can be achieved by injecting a solution containing an ionizable cationic lipid having the structure of the chemical formula 1 and a solution containing dimethyl sulfate into the first micro-mixer at a flow rate of 0.4 to 2.0 ml/min, respectively, into the first flow path.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one selected from the group consisting of the following (1) to (7):
  • the alkylation of DODAP(2) synthesized using dimethyl sulfate in flow chemistry was selected to synthesize cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • complete alkylation of DODAP (2) was achieved by using 10 equivalents of dimethyl sulfate at 60 °C and a residence time of 5 min in the R1 reactor.
  • the accelerated reaction kinetics due to the inherent superiority in mixing efficiency are highly advantageous compared to the batch process which takes 48 h to complete the alkylation.
  • another ionizable lipid or a fatty acid having an ester or disulfide functional group introduced into a long chain of an ionizable lipid is (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-Bis((E)-3,7-dimethylocta-2
  • the productivity of the cationic lipid methyl sulfate library was improved by using the first reactor (200) with the inner diameter increased to 1.6 mm.
  • a fourth aspect of the present invention relates to a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • DOTAP methyl sulfate (4) can be converted to DOTAP chloride (5) by treatment with Amberlite® IRA-400(Cl ) ion exchange resin, which is labor- and cost-intensive.
  • Direct synthesis of DOTAP chloride (5) without using methyl chloride as an alkylating agent can be accomplished in a batch reactor by esterifying oleoyl chloride with commercially available and readily synthesizable 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) dissolved in CH 2 Cl 2 in the presence of DMAP as a base.
  • diols (23) are insoluble in common organic solvents except dimethyl sulfoxide (DMSO), making flow chemistry difficult to perform, and furthermore, DMSO was not a suitable solvent for esterification with acyl chlorides.
  • DMSO dimethyl sulfoxide
  • DOTAP chloride (5) was obtained in 78% yield via N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl)-mediated Steglic esterification of oleic acid and diol (23) in the presence of DMAP in DMSO in 16 h. Therefore, it is worth exploring the Steglic esterification reaction in a continuous flow reactor for the direct synthesis of DOTAP chloride (5).
  • the experimental setup including a PTFE tube reactor with an inner diameter of 1.0 mm is shown in Figure 14.
  • Diol (23) was initially prepared in batch by refluxing an ethanolic solution of trimethylamine dissolved in 3-chloropropane-1,2-diol and methanol solvent.
  • all reactants of the method for synthesizing the DOTAP chloride (5) can be used in a state dissolved in an organic solvent that does not adversely affect the reaction.
  • an organic solvent that does not adversely affect the reaction.
  • DMSO dimethyl sulfoxide
  • a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.
  • the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidly connected to the first micro-mixer (100).
  • a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride, and DMAP can be injected into the first flow path (110), and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride can be injected into the second flow path (120).
  • reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • the final product, DOTAP chloride (5) can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).
  • the residence time in the first reactor (200) may be 3 to 7 minutes.
  • the time required for the reactants introduced into the first reactor (200), that is, the mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP, and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride, to react to produce DOTAP chloride (5) may be 3 to 7 minutes.
  • reaction temperature in the first reactor (200) may be 60 to 80°C.
  • reaction time i.e., reaction time
  • reaction temperature in the above first reactor (200)
  • yield of DOTAP chloride (5) may decrease.
  • the residence time in the first reactor (200) can be achieved by injecting a solution containing oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP into the first flow path and a solution containing (3-dimethylamino-propyl)-ethyl-carbodiimide chloride into the first micro-mixer at a flow rate of 0.4 to 1.5 ml/min.
  • the method for synthesizing the DOTAP chloride (5) may additionally include solvent evaporation and recrystallization steps. At this time, the recrystallization may be performed in isopropanol and acetone solvents, but may be performed without limitation in a solvent suitable for recrystallization of the DOTAP chloride (5).
  • the reaction in R1 was optimized at various temperatures and residence times by varying the flow rate of the reagents using a syringe pump. Initially, the yield of DOTAP chloride (5) was low due to incomplete de-esterification of the diol (23) with a residence time of 15 min at room temperature. However, when the temperature was raised to 70 °C to further promote the de-esterification, DOTAP chloride (5) was obtained in 88% yield after purification with only 5.0 min of residence time in R1, which was much shorter than the 16 h of the batch process.
  • the productivity of DOTAP chloride (5) was improved by using a first reactor (200) with an inner diameter increased to 1.6 mm.
  • a first reactor (200) with an inner diameter increased to 1.6 mm.
  • a fifth aspect of the present invention relates to a method for producing lipid nanoparticles, comprising the following steps (a) and (b):
  • step (a) In the method for manufacturing lipid nanoparticles of the present invention, the description of step (a) is the same as described above, so its description is omitted.
  • the ionizable cationic lipid used in step (b) may include all types of ionizable cationic lipids synthesized by the above-described method.
  • the helper lipids used in the step (b) are DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPI (1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPI (1,2-distearoyl-sn-glycero-3-phosphoinositol), and It may be at least one selected from the group consisting of DLPC (1,2-dirinoley
  • the polyethylene glycol (PEG) lipid may be at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, but is not limited thereto.
  • the PEG lipid includes a PEG moiety having a size of 100 Da to 20 kDa, and more preferably, it may be at least one selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and Ceramide PEG2000 (N-palmitoyl-sphingosine-1- ⁇ succinyl[methoxy(polyethylene glycol)2000] ⁇ ), but is not limited thereto.
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)
  • the molar ratio of the ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid may be, but is not limited to, 40-60 : 5-15 : 30-45 : 1-3.
  • the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, sodium citrate buffer, phosphate buffered saline (PBS), or citrate buffer.
  • a sixth aspect of the present invention relates to a method for preparing lipid nano liposomes comprising the following steps (a) and (b):
  • step (a) In the method for producing the lipid nano liposome of the present invention, the description of step (a) is the same as described above, so its description is omitted.
  • the ionizable cationic lipid or cationic lipid used in step (b) may include all types of ionizable cationic lipid or cationic lipid synthesized by the above-described method.
  • the molar ratio of the ionizable cationic lipid, cationic lipid or DOTAP:cholesterol may be 25 to 75:25 to 75, but is not limited thereto.
  • the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, Tris buffer, phosphate buffered saline (PBS), or citrate buffer.
  • DODAP and DOTAP chloride are biomimetic molecules that take the form of liposomes or lipid nanoparticles, enabling efficient delivery of genetic and various therapeutics by enhanced endosomal escape.
  • the uniform size of the nanoparticles encapsulating the therapeutics is very important in terms of delivery by interaction with cells.
  • the on-demand manufacturing of nanoparticle carriers is difficult because complex variables such as mixer type, feed rate, and composition are involved in the nanoprecipitation process.
  • empty LNPs were synthesized using four types of components similar to those reported, i.e., ionizable lipid, helper lipid, cholesterol, and PEG-lipid.
  • ionizable lipid i.e., ionizable lipid
  • helper lipid i.e., helper lipid
  • cholesterol i.e., glycerol
  • PEG-lipid lipid-lipid-lipid-lipid-lipid
  • DCM Dichloromethane
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ACN acetonitrile
  • MeOH methanol
  • DMF dimethylformamide
  • EtOH absolute ethanol
  • Oleic acid (99%), linoleic acid (99%), palmitic acid (99%), stearic acid (99%), myristic acid (99%), citronellol, geraniol, nerol, sebic acid, 6-bromohexan-1-ol, 2-hexyldecanoic acid, ( ⁇ )-3-chloro-1,2-propanediol, 3-(dimethylamino)-1,2-propanediol, oxalyl chloride, and triethylamine were purchased from Sigma-Aldrich.
  • 1,2-Distearoyl-sn-glycero-3-phosphocholine DSPC
  • cholesterol Chol
  • 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DMG-PEG 2000 were purchased from Avanti Polar Lipids, Inc.
  • Inline FT-IR spectra were recorded on a routine FT-IR spectrometer (Jasco FT/IR-4600) equipped with a sealed flow cell accessory (Specac®) based on ZnSe windows (pathlength 0.1 mm). The masses of lipids were confirmed using a BRUKER MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer.
  • DODAP(2) was considered as a parent molecule for the synthesis of a single-flow ionizable lipid library. Therefore, it was first decided to synthesize oleoyl chloride in a continuous flow system to achieve high-quality synthetic DODAP(2). The purity of commercially available oleoyl chloride is only 85%.
  • the experimental setup for the optimization of oleoyl chloride synthesis in flow chemistry is shown in Fig. 3.
  • the reaction was optimized in a flow system consisting of a PTFE tube with an inner diameter of 1.0 mm.
  • IR spectroscopy monitored the formation of acid chloride with a characteristic C-O stretching band at 1780 cm -1 and the disappearance of the C-O stretching band of the acid functional group of oleic acid at 1708 cm -1 . After some optimization, complete consumption of oleic acid was observed within a residence time of 1.5 min in reactor R1 at a flow rate of 0.6 ml/min of each reagent to form oleoyl chloride (Fig. 5).
  • An FTIR sample cell was connected to reactor R2 for in-line FTIR monitoring.
  • the residence time in R2 was varied by changing the length of the reactor R2 (254 cm, 570 cm, 760 cm, 1020 cm, 2040 cm, etc.). After reaching steady state, the resulting solution was monitored using inline FTIR monitoring. IR spectroscopy monitored the formation of DODAP (2) with the characteristic CO stretching band of ester at 1735 cm -1 and the disappearance of the CO stretching of the acid chloride functional group of oleoyl chloride at 1780 cm -1 .
  • triethylamine was found to be a suitable base, affording the highest DODAP(2) yield of 92% of the isolated yield with a residence time of 3 min at R2 at 25 °C. In batch process, complete conversion took about 8 h.
  • DODAP(2) The successful synthesis of DODAP(2) was extended to develop various routes for the synthesis of a library of ionizable cationic lipids by modifying the long chain acid (Fig. 8).
  • Lipid library development is an important aspect of pharmaceutical research aimed at identifying lipids that can effectively encapsulate therapeutic agents and deliver them to target sites while minimizing toxicity. Accordingly, we attempted to synthesize a library of ionizable cationic lipids by co-incorporating commercially available fatty acids such as linoleic acid, myristic acid, stearic acid, and palmitic acid instead of oleic acid.
  • the experimental setup for continuous flow synthesis of ionizable cationic lipids is shown in Fig. 8.
  • monoterpenoids which have many health benefits such as anti-inflammatory, antioxidant, anticancer, antidiabetic, and antifungal activities, are used in ionizable lipids such as lipids 11 (derived from ⁇ -citronellol), 12 (derived from geraniol), and 13 (derived from nerol).
  • the synthetic method of compound 10 is as follows. First, 10-(hexyloxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 1.
  • the synthetic method of compound 12 is as follows. First, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 3.
  • the synthetic method of compound 13 is as follows. First, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 4.
  • the synthetic method of compound 14 is as follows. First, 3-(octadecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 5.
  • the synthetic method of compound 15 is as follows. First, 3-(dodecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 6.
  • a separate experiment was performed using a PTFE tubing reactor with a larger inner diameter of 1.6 mm.
  • the experimental setup for continuous flow synthesis of DODAP using a 1.6 mm PTFE tube reactor is shown in Fig. 9.
  • the resulting solution was passed into the reactor R1 for the synthesis of oleoyl chloride accompanied by HCl and CO gaseous byproducts.
  • a 250 psi back pressure regulator was connected to the R1 outlet.
  • the experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 10.
  • the 100 psi back pressure regulator was connected to the R1 outlet.
  • the reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using an HPLC pump. After some optimization, complete conversion of DODAP (2) to DOTAP methyl sulfate (4) was achieved under the following conditions: flow rate of 0.40 ml/min of each reactant, 60°C, and residence time of 5.0 min in R1. After reaching steady state, the resulting solution was collected from the end of reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to give DOTAP methyl sulfate (4), which was purified using crystallization from acetone to give a clear crystalline compound in 90% yield (0.27 g).
  • the experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 11.
  • the resulting solution was then passed into the reactor R1 for the synthesis of DOTAP methyl sulfate.
  • the 250 psi back pressure regulator was connected to the R1 outlet. After reaching steady state, the resulting solution was collected at the end of the reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to obtain DOTAP methyl sulfate (4), which was purified using crystallization from acetone to obtain a transparent crystalline compound in 86% yield (0.67 g).
  • Spectroscopic identification results of the synthesized final products confirmed that a cationic lipid library having structural formulas of compounds 17 to 22 was successfully synthesized.
  • the experimental setup for the flow synthesis of DOTAP chloride (5) is shown in Fig. 13.
  • a 40 psi back pressure regulator was connected to the R1 outlet.
  • the reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using a syringe pump. After some optimization, complete conversion of 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) to DOTAP chloride (5) was observed under the following conditions: 70 °C, residence time of 5 min in R1. After reaching steady state, the resulting solution was collected at the end of reactor R1 for 5 min. The solution was slowly quenched with 1 N HCl (10.0 mL) and the resulting aqueous layer was washed three times with dichloromethane (50.0 mL ⁇ 3).
  • DOTAP chloride (5) was purified using flash silica gel column chromatography containing 30% MeOH in CH 2 Cl 2 solvent system to give DOTAP chloride (5) (0.45 g) in 88% yield.
  • the experimental setup for the flow synthesis of DOTAP chloride (5) using the size-increasing method is shown in Fig. 14.
  • a custom-made micro-mixer was fabricated using 3D printing.
  • the mixer consists of hemispherical baffles to enable rapid mixing of the injected fluids.
  • each inlet and outlet were modified with a 5 mm thread (1/4-28 flat bottom for 1/16") to enable connection with XP-235 (IDEX Health & Science) fittings.
  • the virtual model created through CAD was fabricated using a DLP-based 3D printer (Pico 2 HD, Asiga) with commercial resin (PlasCLEAR), and each layer was laminated through photocuring and the resin was removed using isopropanol. Finally, the cleaned device was post-cured in a UV chamber for complete polymerization.
  • Lipid-based nanoparticles were prepared by rapid mixing of lipid solutions and buffer solutions. Each lipid solution was fixed at a total concentration of 10 mg/mL.
  • DODAP/DSPC/Chol/DMG-PEG 2000 was dissolved in ethanol at a molar ratio of 50/10/38.5/1.5.
  • 3 M sodium acetate solution was diluted with deionized water to achieve a concentration of 25 mM.
  • the pH of the sodium acetate solution was adjusted to 5.5 using 100 mM acetic acid solution.
  • DODAP (DOTAP) and cholesterol were dissolved at the same total concentration and molar ratio of 50:50.
  • lipid solution and buffer were passed through a 0.22 ⁇ m syringe filter.
  • the solutions were mixed via a micromixer using a syringe pump.
  • the LNP and liposome samples were dialyzed overnight against 1,000-fold volume of buffer (10 mM PBS, pH 7.4 for DODAP; 10 mM Tris buffer, pH 7.2 for DOTAP) using a dialysis bag (MWCO 12,000 Da, Sigma-Aldrich) to remove residual ethanol.
  • DLS analysis was performed using a Zetasizer Nano ZS instrument (Malvern Instruments, Ltd.).
  • DODAP LNPs and liposomes had an average size of 64 nm and 72 nm and a polydispersity index (PDI) of 0.07 and 0.05, respectively (Figs. 15B, C and 16A, B).
  • PDI polydispersity index
  • For DOTAP liposomes with different counterions a size of about 55 nm and a PDI of 0.08 were produced in both cases (Figs. 15D, E and 16C, D).
  • the production of highly uniform nanoparticles, especially the same properties of cationic liposomes with different counterions, is partly due to the high purity of each major lipid component.
  • DODAP LNPs and liposomes were reduced in size to 40 nm and 45 nm with PDI of 0.12 and 0.10, respectively, whereas DOTAP liposomes reached a size of about 39 nm with PDI of 0.11 (Figs. 15B to E and 16A to D). In all cases, a decrease in the average particle size and a slight broadening of the particle distribution were observed. It is worth noting that at this point, membrane fusion occurred due to coexistence with ethanol, whereas removal of ethanol enhanced the stability.
  • the flow-synthesized DODAP and DOTAP lipids exhibited a size of about 50 nm and a PDI of about 0.10, demonstrating their potential for effective drug delivery after self-assembly even after purification.
  • nanoparticle carriers of less than 100 nm allow for prolonged blood circulation and effective endosomal escape, while the narrow size distribution prevents accumulation in unwanted organs, facilitating optimal therapeutic delivery.
  • Microfluidic device 100 First micro mixer
  • T-type liquid-gas separator 410 1st supply path
  • Second reactor 700 Second pressure regulator

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Abstract

The present invention relates to a single continuous flow synthesis method for synthesizing a functional lipid library used in a nucleic acid therapeutic agent. More specifically, provided are: an ionizable cationic lipid library including ionizable cationic lipids such as 1,2-dioleoyl-3-dimethylammonium-propane; a single continuous flow synthesis method for a cationic lipid library including cationic lipids such as 1,2-dioleoyl-3-trimethylammonium-propane; and a method for preparing lipid nanoparticles or lipid nanoliposomes using said method.

Description

핵산 치료제에 사용되는 기능성 지질 라이브러리를 합성하기 위한 단일 연속 유동 합성 방법A single continuous flow synthesis method for synthesizing a library of functional lipids for use in nucleic acid therapeutics

본 발명은 핵산 치료제에 사용되는 기능성 지질 라이브러리를 합성하기 위한 단일 연속 유동 합성 방법에 관한 것으로, 보다 상세하게는 1,2-디올레오일-3-디메틸암모늄-프로판과 같은 이온화 가능한 양이온성 지질을 포함하는 이온화 가능한 양이온성 지질 라이브러리 및 1,2-디올레오일-3-트리메틸암모늄-프로판과 같은 양이온성 지질을 포함하는 양이온성 지질 라이브러리의 단일 연속 유동 합성 방법 및 상기 방법을 이용한 지질 나노입자 또는 지질 나노 리포좀의 제조방법을 제공한다.The present invention relates to a single continuous flow synthesis method for synthesizing a functional lipid library used in nucleic acid therapeutics, and more particularly, to a single continuous flow synthesis method for synthesizing an ionizable cationic lipid library including an ionizable cationic lipid such as 1,2-dioleoyl-3-dimethylammonium-propane and a cationic lipid library including a cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane, and a method for preparing lipid nanoparticles or lipid nanoliposomes using the method.

유전자 치료는 일반적으로 원치 않는 면역 반응을 유발하지 않고 표적 세포 내에서 기능하기 위해 DNA 및 RNA와 같은 치료용 뉴클레오티드를 필요로 한다. 그러나, 뉴클레오티드의 큰 크기와 음전하로 인해 세포 침투가 방해되고 뉴클레아제에 취약해진다. 임상적으로 효과적인 지질 나노입자(LNP)는 RNA를 표적 세포에 전달하는 능력을 가지며, 코로나19 백신의 효과로 입증된 바와 같이 생명을 위협하는 일련의 질병을 해결할 수 있는 광범위한 기회를 제공한다. 사용되는 다른 구성 요소 중에서 양이온성 지질은 DNA, RNA 및 기타 음전하를 띤 분자의 리포좀 형질감염에서 중요한 역할을 한다. 구조적으로 양이온성 지질은 일반적으로 링커 그룹을 통해 포화 또는 불포화 소수성 꼬리에 부착된 이온화 또는 양이온 헤드 그룹으로 구성된다. 양이온 지질은 생리학적 pH에서 양전하를 띠는 반면, 이온화 가능한 양이온 지질은 생리학적 pH에서는 중성이지만 RNA의 캡슐화 및 엔도솜 방출을 담당하는 헤드 그룹 없는 아민의 양성자화로 인해 pKa(<7)보다 낮은 pH에서 양전하를 띈다. 그 중에서도 에스테르 결합을 통해 소수성 꼬리에 헤드 그룹이 연결된 양이온성 지질은 인체 내에서 생분해성으로 인해 더 많은 임상적 효능을 나타낸다. 1,2-디올레오일-3-디메틸암모늄-프로판(2)(1,2-dioleoyl-3-dimethylammonium-propane, DODAP)는 세포 독성이 낮고 형질감염 효율이 높은 이온화 가능한 양이온성 지질이다. 이는 두 개의 소수성 올레오일 꼬리와 디메틸암모늄 모이어티와 프로판기를 포함하는 친수성 헤드 그룹으로 구성된다. 반면, DODAP(2)의 유도체인, 1,2-디올레오일-3-트리메틸암모늄-프로판(3)(1,2-dioleoyl-3-trimethylammonium-propane, DOTAP)은 양이온성 트리메틸암모늄 친수성 헤드 그룹을 포함한다(도 1). DODAP(2) 및 그 유도체는 질병 치료를 위해 mRNA, siRNA 및 플라스미드 DNA를 포함한 생리 활성 분자를 캡슐화하는 데 널리 사용된다.Gene therapy generally requires therapeutic nucleotides such as DNA and RNA to function within target cells without triggering unwanted immune responses. However, the large size and negative charge of nucleotides hinder their cellular penetration and make them vulnerable to nucleases. Clinically effective lipid nanoparticles (LNPs) have the ability to deliver RNA to target cells and offer a wide range of opportunities to address a range of life-threatening diseases, as evidenced by the effectiveness of the COVID-19 vaccine. Among other components used, cationic lipids play a key role in liposomal transfection of DNA, RNA, and other negatively charged molecules. Structurally, cationic lipids typically consist of an ionizable or cationic head group attached to a saturated or unsaturated hydrophobic tail via a linker group. Cationic lipids are positively charged at physiological pH, whereas ionizable cationic lipids are neutral at physiological pH but positively charged at pH below their pKa (<7) due to protonation of the head group-free amines responsible for encapsulation and endosomal release of RNA. Among them, cationic lipids in which a head group is linked to a hydrophobic tail via an ester bond exhibit more clinical efficacy due to their biodegradability in the human body. 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP) is an ionizable cationic lipid with low cytotoxicity and high transfection efficiency. It is composed of two hydrophobic oleoyl tails and a hydrophilic head group containing a dimethylammonium moiety and a propane group. In contrast, 1,2-dioleoyl-3-trimethylammonium-propane (3) (DOTAP), a derivative of DODAP (2), contains a cationic trimethylammonium hydrophilic head group (Figure 1). DODAP(2) and its derivatives are widely used to encapsulate bioactive molecules including mRNA, siRNA and plasmid DNA for disease treatment.

DODAP(2)의 합성에는 올레산으로부터 올레오일 클로라이드 합성과 이어서 3-(디메틸아미노)프로판-1,2-디올과의 에스테르화 반응이 포함된다. 그러나, 옥살릴 클로라이드와 같은 필수 염소화제의 독성과 올레오일 클로라이드 형성 중에 생성되는 독성 HCl 및 CO 가스 부산물로 인해 배치 반응기에서의 합성 및 정제는 까다롭다. 비특허문헌 1에 따르면, 올레산과 3-(디메틸아미노)프로판-1,2-디올의 커플링 반응을 사용하는 대체 합성 방법은 시간이 많이 걸리고 정제가 어려우며 비교적 비용이 많이 든다. 다양한 문헌을 통해 양이온성 지질 DOTAP(3)의 배치 합성 방법이 보고되었다. 특허문헌 1 및 2는 매우 독성이 강하고 발암성이 있는 메틸화제(클로로메탄, 요오도메탄 등)를 활용하는 양이온성 지질 계열을 준비하기 위한 두 가지 다른 접근법을 보고했는데, 이로 인해 대규모 생산에 적용이 제한된다(도 2의 a 및 b).The synthesis of DODAP(2) involves the synthesis of oleoyl chloride from oleic acid followed by esterification with 3-(dimethylamino)propane-1,2-diol. However, the synthesis and purification in batch reactors are challenging due to the toxicity of essential chlorinating agents such as oxalyl chloride and the toxic HCl and CO gas byproducts generated during the formation of oleoyl chloride. According to Non-Patent Document 1, an alternative synthetic method using the coupling reaction of oleic acid and 3-(dimethylamino)propane-1,2-diol is time-consuming, difficult to purify, and relatively expensive. Batch synthesis methods for the cationic lipid DOTAP(3) have been reported in various literatures. Patent Documents 1 and 2 report two different approaches to prepare cationic lipid series that utilize highly toxic and carcinogenic methylating agents (chloromethane, iodomethane, etc.), which limit their application in large-scale production (Figure 2 a and b).

이 문제를 극복하기 위해, 특허문헌 3 및 비특허문헌 2는 DOTAP 브로마이드 합성을 위한 양이온성 헤드 그룹을 도입하기 위해 무독성 트리메틸아민을 활용하였다(도 2의 c). 그러나 원료인 브로모프로판디올을 출발물질로 사용하는 데 드는 비용이 높기 때문에 산업적 적용이 제한되었다. 특허문헌 4의 트리메틸아민 시약과 함께 값싼 출발 물질인 글리세롤을 사용하려는 시도는 시간과 노동력이 많이 소모되고 지질의 품질에 영향을 미치는 다단계 공정을 필요로 한다(도 2의 d). 가장 짧은 경로는 3-클로로프로판-1,2-디올에서 시작하여, 올레산과 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드의 스테글리치(Steglich) 에스테르화 반응을 포함하지만(도 2의 e), 완전한 완료를 위해 16시간이 소요되며 반응 시간과 생산성을 단축하기 위해 유동 화학을 활용하는 것이 매우 바람직하다.To overcome this problem, Patent Document 3 and Non-Patent Document 2 utilized non-toxic trimethylamine to introduce a cationic head group for DOTAP bromide synthesis (Fig. 2c). However, the high cost of using bromopropanediol as a starting material has limited its industrial application. An attempt to use glycerol, a cheap starting material, together with the trimethylamine reagent of Patent Document 4 requires a multi-step process that is time-consuming and labor-intensive and affects the quality of the lipid (Fig. 2d). The shortest route involves the Steglich esterification reaction of oleic acid with 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride starting from 3-chloropropane-1,2-diol (Fig. 2e), but it takes 16 h for complete completion, and it is highly desirable to utilize flow chemistry to shorten the reaction time and productivity.

최근에는 유기 합성을 위한 연속 유동 공정의 활용이 배치 공정에 비해 더 짧은 시간에 더 높은 수율을 생산할 수 있고, 더 안전하고, 더 깨끗하며, 더 저렴하게 세팅되고 작동될 수 있다는 등의 많은 이점이 있음이 성공적으로 입증되었다. 이는 주로 높은 표면 대 부피 비율, 향상된 열 및 물질 전달, 온도 및 반응 시간과 같은 반응 매개변수를 정밀하게 제어할 수 있는 능력 때문이다. 따라서, 고품질의 이온화 가능한 양이온성 지질에 대한 환경 친화적이고 경제적인 합성 경로를 개발하기 위한 유동 기술의 활용은 유전자 치료 비용을 최소화하기 위해 매우 바람직하다. 새로운 생분해성 지질 후보의 효율적인 스크리닝은 체내 양이온성 지질의 축적으로 인한 장기적인 독성을 최소화하기 위해 요구된다.Recently, the utilization of continuous flow processes for organic synthesis has been successfully demonstrated to have many advantages over batch processes, such as producing higher yields in a shorter time, being safer, cleaner, and cheaper to set up and operate. This is mainly due to the high surface-to-volume ratio, enhanced heat and mass transfer, and the ability to precisely control reaction parameters such as temperature and reaction time. Therefore, the utilization of flow technology to develop an environmentally friendly and economical synthetic route to high-quality ionizable cationic lipids is highly desirable to minimize the cost of gene therapy. Efficient screening of novel biodegradable lipid candidates is required to minimize the long-term toxicity due to the accumulation of cationic lipids in the body.

본 발명에서는 DODAP(2) 및 에스테르 링커 그룹을 포함하는 이의 유도체에 대한 유동 합성 경로가 지질 화합물 라이브러리를 개발하는 것으로 입증되었다. 여기에는 옥살릴 클로라이드를 사용하여 지방산을 산 클로라이드로 전환한 후 알코올 함유 이온화 가능한 아민 헤드 그룹과의 하나의 유동 커플링 반응이 포함되었다. HCl 가스를 제거하기 위해 필요한 염기로부터 형성된 염기.HCl 복합체의 침전으로 인해 관찰된 고체 취급 문제를 극복하기 위해, 맞춤형 중력 기반 액체 가스 분리기를 활용하여 독성 HCl 및 CO 가스 부산물을 지속적으로 안전하게 제거하였다. 비특허문헌 3에서는, 염기.HCl 복합체의 침전으로 인한 고체 취급 문제는 더 높은 온도를 요구하는 이온성 액체 형성을 통해 극복되었으며, 유동 시스템에서 염기와 용매 선택에 대한 제한이 있었다. 주요 과제 중 하나는 인라인 FTIR 모니터링을 사용하여 효과적으로 달성한 수분에 민감한 독성 아실 클로라이드의 유동 합성과 이의 후속 반응 매개변수를 최적화하여 시간과 노동력을 효과적으로 절약하는 것이었다. DOTAP 설페이트(4)의 합성을 위해, 유동 반응기에서 디메틸 설페이트를 사용하여 합성된 DODAP(2)의 신속한 후속 알킬화가 입증되었다. 다르게는, DOTAP 클로라이드(5)의 직접 유동 합성을 위해 올레산과 2,3-디히드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드의 스테글리치 에스테르화 반응이 최적화되었다.In the present invention, a flow synthesis route for DODAP(2) and its derivatives containing an ester linker group is demonstrated for developing a library of lipid compounds. It involves conversion of fatty acids to acid chlorides using oxalyl chloride followed by one flow coupling reaction with an alcohol-containing ionizable amine head group. To overcome the solid handling issues observed due to precipitation of the base.HCl complex formed from the base required to remove HCl gas, a custom gravity-based liquid-gas separator was utilized to continuously and safely remove the toxic HCl and CO gas byproducts. In non-patent literature 3, the solid handling issues due to precipitation of the base.HCl complex were overcome through the formation of ionic liquids requiring higher temperatures, and there were limitations on the choice of base and solvent in the flow system. One of the key challenges was to optimize the flow synthesis of moisture-sensitive toxic acyl chlorides and its subsequent reaction parameters, which were effectively achieved using in-line FTIR monitoring, thereby saving time and labor. For the synthesis of DOTAP sulfate (4), rapid subsequent alkylation of synthesized DODAP (2) using dimethyl sulfate in a flow reactor was demonstrated. Alternatively, the Steglich esterification reaction of oleic acid with 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride was optimized for the direct flow synthesis of DOTAP chloride (5).

또한, 이러한 이온화 가능한 양이온성 지질의 규모 확대 생산은 초기 최적화 조건에서 유사한 수율로 더 큰 채널 직경을 사용하는 간단한 규모 확대 접근 방식을 통해 달성되었다. 이러한 고효율 지질 라이브러리의 새로운 합성 플랫폼은 독성 및 표적화 효능 평가를 촉진함으로써 특정 환자에게 지질 기반 나노의약품에 적합한 지질 후보를 제공하는 데 매우 바람직할 것이다.Furthermore, scale-up production of these ionizable cationic lipids was achieved through a simple scale-up approach using larger channel diameters with similar yields under initial optimization conditions. This novel synthetic platform for high-throughput lipid libraries would be highly desirable for facilitating toxicity and targeting efficacy assessments, thereby providing suitable lipid candidates for lipid-based nanomedicines to specific patients.

[선행기술문헌][Prior art literature]

[특허문헌][Patent Document]

(특허문헌 1) US5925623A(Patent Document 1) US5925623A

(특허문헌 2) US5264618A(Patent Document 2) US5264618A

(특허문헌 3) DE4013632A1(Patent Document 3) DE4013632A1

(특허문헌 4) WO2005049549(Patent Document 4) WO2005049549

[비특허문헌][Non-patent literature]

(비특허문헌 1) Synthesis and characterization of novel zwitterionic lipids with pH-responsive biophysical properties, Colin L. Walsh, Juliane Nguyenb, Francis C. Szoka, Chem. Commun., 2012, 48, 5575-5577.(Non-patent Document 1) Synthesis and characterization of novel zwitterionic lipids with pH-responsive biophysical properties, Colin L. Walsh, Juliane Nguyenb, Francis C. Szoka, Chem. Commun. , 2012 , 48 , 5575-5577.

(비특허문헌 2) Interactions of Cationic Lipid Vesicles with Negatively Charged Phospholipid Vesicles and Biological Membranes, Leonidas Stamatatos, Rania Leventis, Martin J. Zuckermann, John R. Silvius, Biochemistry 1988, 27, 3917-3925.(Non-patent Document 2) Interactions of Cationic Lipid Vesicles with Negatively Charged Phospholipid Vesicles and Biological Membranes, Leonidas Stamatatos, Rania Leventis, Martin J. Zuckermann, John R. Silvius, Biochemistry 1988, 27 , 3917-3925.

(비특허문헌 3) Overcoming solid handling issues in continuous flow substitution reactions through ionic liquid formation, Saeed K. Kashani, Ryan J. Sullivan, Mads Andersen, Stephen G. Newman, Green Chem., 2018, 20, 1748-1753.(Non-patent Document 3) Overcoming solid handling issues in continuous flow substitution reactions through ionic liquid formation, Saeed K. Kashani, Ryan J. Sullivan, Mads Andersen, Stephen G. Newman, Green Chem., 2018 , 20 , 1748-1753.

따라서, 본 발명은 핵산 치료제에 사용되는 기능성 지질에 대한 단일 연속 유동(one-flow) 합성 방법을 제공하는 것을 목적으로 한다.Accordingly, the present invention aims to provide a single continuous flow synthesis method for functional lipids used in nucleic acid therapeutics.

또한, 본 발명은 전술한 기능성 지질에 대한 단일 연속 유동 합성 방법을 이용한 지질 나노입자 또는 지질 나노 리포좀의 제조방법을 제공하는 것을 목적으로 한다.In addition, the present invention aims to provide a method for producing lipid nanoparticles or lipid nanoliposomes using a single continuous flow synthesis method for the aforementioned functional lipids.

상술한 과제를 해결하기 위해, 본 발명은 다음의 (a) 및 (b)의 단계를 포함하는 이온화 가능한 양이온성 지질의 합성 방법을 제공한다:To solve the above-mentioned problems, the present invention provides a method for synthesizing an ionizable cationic lipid comprising the following steps (a) and (b):

(a) R-(CH2)2C(=O)OH, 디메틸포름아미드 및 옥살릴 클로라이드를 혼합 및 반응시켜 R-C(=O)Cl을 형성하는 단계; 및(a) a step of mixing and reacting R-(CH 2 ) 2 C(=O)OH, dimethylformamide and oxalyl chloride to form RC(=O)Cl; and

(b) R-C(=O)Cl을 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물과 혼합하여 에스테르화 반응을 일으켜 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 생성하는 단계;를 포함하며,(b) a step of mixing R-C(=O)Cl with 3-(dimethylamino)propane-1,2-diol and a basic compound to cause an esterification reaction to produce an ionizable cationic lipid having a structure of chemical formula 1;

[화학식 1][Chemical Formula 1]

Figure PCTKR2024007830-appb-img-000001
Figure PCTKR2024007830-appb-img-000001

상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이다.The above R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical in which -C(=O)O- or -S-S- is introduced into the chain.

본 발명에 있어서, 상기 (a) 단계 후에 HCl 및 CO 가스를 분리하는 단계를 추가로 포함할 수 있다.In the present invention, a step of separating HCl and CO gases may be additionally included after step (a).

본 발명에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행될 수 있고, 상기 미세유체장치는: 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기; 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기; 상기 제1 배압 조절기와 유체적으로 연결되어 용액과 가스를 분리하는 T자형 액체-가스 분리기; 상기 T자형 액체-가스 분리기와 제3 유로가 유체적으로 연결되는 제2 미세 혼합기; 상기 제2 미세 혼합기와 유체적으로 연결되는 제2 반응기; 및 상기 제2 반응기와 유체적으로 연결되는 제2 배압 조절기를 포함할 수 있다.In the present invention, the synthetic method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first channel and a second channel are fluidly connected; a first reactor in which the first micromixer is fluidly connected; a first back pressure regulator in which the first reactor is fluidly connected; a T-shaped liquid-gas separator in which the first back pressure regulator is fluidly connected to separate a solution and a gas; a second micromixer in which a third channel is fluidly connected to the T-shaped liquid-gas separator; a second reactor in which the second micromixer is fluidly connected; and a second back pressure regulator in which the second reactor is fluidly connected.

본 발명에 있어서, 상기 T자형 액체-가스 분리기는 상기 제1 배압 조절기와 유체적으로 연결되는 제1 공급유로, 분리된 가스가 배출되는 제1 배출유로 및 분리된 용액이 상기 제2 미세 혼합기에 공급되도록 상기 제2 미세 혼합기에 유체적으로 연결되는 제2 공급유로를 포함할 수 있다.In the present invention, the T-shaped liquid-gas separator may include a first supply channel fluidly connected to the first back pressure regulator, a first discharge channel through which separated gas is discharged, and a second supply channel fluidly connected to the second micro-mixer so that the separated solution is supplied to the second micro-mixer.

본 발명에 있어서, 상기 제1 유로에 R-(CH2)2C(=O)OH 및 디메틸포름아미드, 상기 제2 유로에 옥살릴 클로라이드가 주입될 수 있고, 상기 제1 반응기에서 R-C(=O)Cl이 생성될 수 있고, 상기 T자형 액체-가스 분리기를 통해 HCl 및 CO 가스가 분리될 수 있고, 상기 제3 유로에 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물이 주입될 수 있고, 상기 제2 반응기에서 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질이 생성될 수 있다.In the present invention, R-(CH 2 ) 2 C(=O)OH and dimethylformamide can be injected into the first path, oxalyl chloride can be injected into the second path, RC(=O)Cl can be generated in the first reactor, HCl and CO gas can be separated through the T-shaped liquid-gas separator, 3-(dimethylamino)propane-1,2-diol and a basic compound can be injected into the third path, and an ionizable cationic lipid having a structure of the chemical formula 1 can be generated in the second reactor.

본 발명에 있어서, 상기 제1 반응기에서의 체류 시간은 1 내지 2분일 수 있고, 상기 제2 반응기에서의 체류 시간은 2 내지 10분일 수 있고, 상기 제2 반응기에서의 반응 온도는 20 내지 60 ℃일 수 있다.In the present invention, the residence time in the first reactor may be 1 to 2 minutes, the residence time in the second reactor may be 2 to 10 minutes, and the reaction temperature in the second reactor may be 20 to 60°C.

본 발명에 있어서, 상기 염기성 화합물은 피리딘, 피리딘과 DMAP의 혼합, DMAP, 디이소프로필에틸아민 및 트리에틸아민으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the present invention, the basic compound may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine.

본 발명에 있어서, 상기 (a) 단계의 R-(CH2)2C(=O)OH는 올레산, 리놀레산, 미리스트산, 스테아르산, 팔미트산, 10-(헥실옥시)-10-옥소데칸산, (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산, (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, 3-(옥타데실디설파닐)프로판산 및 3-(도데실디설파닐)프로판산으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the present invention, R-(CH 2 ) 2 C(=O)OH in step (a) may be any one selected from the group consisting of oleic acid, linoleic acid, myristic acid, stearic acid, palmitic acid, 10-(hexyloxy)-10-oxodecanoic acid, (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, 3-(octadecyldisulfanyl)propanoic acid, and 3-(dodecyldisulfanyl)propanoic acid.

본 발명에 있어서, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질은 다음의 (1) 내지 (11) 중 어느 하나일 수 있다:In the present invention, the ionizable cationic lipid having the structure of the chemical formula 1 may be any one of the following (1) to (11):

(1) 1,2-디올레오일-3-디메틸암모늄-프로판;(1) 1,2-Dioleoyl-3-dimethylammonium-propane;

(2) (9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트);(2) (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate);

(3) 1,2-디미리스토일-3-디메틸암모늄-프로판;(3) 1,2-Dimyristoyl-3-dimethylammonium-propane;

(4) 1,2-디스테아로일-3-디메틸암모늄-프로판;(4) 1,2-distearoyl-3-dimethylammonium-propane;

(5) 1,2-디팔미토일-3-디메틸암모늄-프로판;(5) 1,2-Dipalmitoyl-3-dimethylammonium-propane;

(6) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트);(6) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate);

(7) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트);(7) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate);

(8) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(8) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((E)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate);

(9) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(9) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((Z)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate);

(10) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(옥타데실디설파닐)프로파노에이트); 및(10) 3-(dimethylamino)propane-1,2-diyl bis(3-(octadecyldisulfanyl)propanoate); and

(11) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(도데실디설파닐)프로파노에이트).(11) 3-(Dimethylamino)propane-1,2-diyl bis(3-(dodecyldisulfanyl)propanoate).

또한, 본 발명은 다음의 (a) 및 (b) 단계를 포함하는, 이온화 가능한 양이온성 지질인 6-((2-헥실데카노일)옥시)-N-(6-((2-헥실데카노일)옥시)헥실)-N-(4-하이드록시부틸)헥산-1-아미늄의 중간체인 6-브로모헥실 2-헥실데카노에이트의 합성 방법을 제공한다:Furthermore, the present invention provides a method for synthesizing 6-bromohexyl 2-hexyldecanoate, an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminum, which is an ionizable cationic lipid, comprising the following steps (a) and (b):

(a) 2-하이드록시데칸산, 디메틸포름아미드 및 옥살릴 클로라이드를 혼합 및 반응시켜 2-하이드록실데카노일 클로라이드를 형성하는 단계; 및(a) a step of mixing and reacting 2-hydroxydecanoic acid, dimethylformamide and oxalyl chloride to form 2-hydroxydecanoyl chloride; and

(b) 2-하이드록실데카노일 클로라이드를 3-(디메틸아미노)프로판-1,2-디올 및 6-브로모헥산올과 혼합하여 에스테르화 반응을 일으켜 6-브로모헥실 2-헥실데카노에이트를 생성하는 단계.(b) a step of mixing 2-hydroxydecanoyl chloride with 3-(dimethylamino)propane-1,2-diol and 6-bromohexanol to cause an esterification reaction to produce 6-bromohexyl 2-hexyldecanoate.

또한, 본 발명은 전술한 합성 방법에 의해 합성된 이온화 가능한 양이온성 지질과 디메틸 설페이트를 혼합 및 반응시켜 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트를 생성하는 단계를 포함하며,In addition, the present invention includes a step of mixing and reacting an ionizable cationic lipid synthesized by the above-described synthetic method with dimethyl sulfate to produce cationic lipid methyl sulfate having a structure of chemical formula 2,

[화학식 2][Chemical formula 2]

Figure PCTKR2024007830-appb-img-000002
Figure PCTKR2024007830-appb-img-000002

상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼인, 양이온성 지질 메틸 설페이트의 합성 방법을 제공한다.The present invention provides a method for synthesizing a cationic lipid methyl sulfate, wherein R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical having -C(=O)O- or -S-S- introduced into the chain.

본 발명에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행될 수 있고, 상기 미세유체장치는: 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기; 및 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기를 포함할 수 있다.In the present invention, the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.

본 발명에 있어서, 상기 제1 유로에 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질, 상기 제2 유로에 디메틸 설페이트를 주입할 수 있고,In the present invention, an ionizable cationic lipid having a structure of chemical formula 1 can be injected into the first flow path, and dimethyl sulfate can be injected into the second flow path.

[화학식 1][Chemical Formula 1]

Figure PCTKR2024007830-appb-img-000003
Figure PCTKR2024007830-appb-img-000003

여기서, 상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼일 수 있고,Here, R may be an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical in which -C(=O)O- or -S-S- is introduced into the chain,

상기 제1 반응기에서 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트가 생성될 수 있다.In the first reactor, cationic lipid methyl sulfate having the structure of the chemical formula 2 can be produced.

본 발명에 있어서, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질 및 상기 디메틸 설페이트는 0.4 내지 2.0ml/분의 유속으로 주입될 수 있고, 상기 제1 반응기에서의 반응 온도는 40 내지 70℃일 수 있고, 상기 제1 반응기에서의 체류 시간은 3 내지 7분일 수 있다.In the present invention, the ionizable cationic lipid having the structure of the chemical formula 1 and the dimethyl sulfate can be injected at a flow rate of 0.4 to 2.0 ml/min, the reaction temperature in the first reactor can be 40 to 70°C, and the residence time in the first reactor can be 3 to 7 minutes.

본 발명에 있어서, 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트는 다음의 다음의 (1) 내지 (7) 중 어느 하나일 수 있다:In the present invention, the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one of the following (1) to (7):

(1) 1,2-디올레오일-3-트리메틸암모늄-프로판 메틸 설페이트;(1) 1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate;

(2) N,N,N-트리메틸-2,3-비스((9Z,12Z)-옥타데카-9,12-디에노일옥시)프로판-1-아미늄 메틸 설페이트;(2) N,N,N-Trimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propane-1-aminum methyl sulfate;

(3) 2,3-비스((10-(헥실옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(3) 2,3-Bis((10-(hexyloxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(4) 2,3-비스((10-(((S)-3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(4) 2,3-Bis((10-(((S)-3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(5) 2,3-비스((10-(((E)-3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(5) 2,3-Bis((10-(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(6) 2,3-비스((10-(((Z)-3,7-디메틸옥타-2,6-디엔-1-일)옥소)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(6) 2,3-Bis((10-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)oxo)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(7) N,N,N-트리메틸-2,3-비스((3-(옥타데실디설파닐)프로파노일)옥시)프로판-1-아미늄 메틸 설페이트.(7) N,N,N-Trimethyl-2,3-bis((3-(octadecyldisulfanyl)propanoyl)oxy)propan-1-aminum methyl sulfate.

본 발명에 있어서, 상기 합성 방법은 용매 증발 및 재결정화 단계를 추가로 포함할 수 있다.In the present invention, the synthesis method may additionally include solvent evaporation and recrystallization steps.

본 발명에 있어서, 상기 재결정화는 아세톤 용매에서 수행될 수 있다.In the present invention, the recrystallization can be performed in an acetone solvent.

또한, 본 발명은 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물과 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드를 혼합 및 반응시켜 스테글리치 에스테르화 반응을 일으켜 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드를 생성하는 단계를 포함하는, 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드의 합성 방법을 제공한다.In addition, the present invention provides a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.

본 발명에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행될 수 있고, 상기 미세유체장치는: 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기; 및 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기를 포함할 수 있다.In the present invention, the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.

본 발명에 있어서, 상기 제1 반응기에서의 반응 온도는 60 내지 80℃일 수 있고, 상기 제1 반응기에서의 체류 시간은 3 내지 7분일 수 있다.In the present invention, the reaction temperature in the first reactor may be 60 to 80°C, and the residence time in the first reactor may be 3 to 7 minutes.

본 발명에 있어서, 상기 합성 방법은 용매 증발 및 재결정화 단계를 추가로 포함할 수 있다.In the present invention, the synthesis method may additionally include solvent evaporation and recrystallization steps.

본 발명에 있어서, 상기 재결정화는 이소프로판올 및 아세톤 용매에서 수행될 수 있다.In the present invention, the recrystallization can be performed in isopropanol and acetone solvents.

추가로, 본 발명은 다음의 (a) 및 (b) 단계를 포함하는 지질 나노입자의 제조방법을 제공한다:Additionally, the present invention provides a method for preparing lipid nanoparticles comprising the following steps (a) and (b):

(a) 전술한 합성 방법으로 이온화 가능한 양이온성 지질을 합성하는 단계; 및(a) a step of synthesizing an ionizable cationic lipid by the synthetic method described above; and

(b) 상기 합성된 이온화 가능한 양이온성 지질, 헬퍼(helper) 지질, 콜레스테롤 및 폴리에틸렌 글리콘 지질을 포함하는 용액을 완충액과 혼합하여 지질 나노입자를 형성하는 단계.(b) a step of forming lipid nanoparticles by mixing a solution containing the synthesized ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid with a buffer solution.

나아가, 본 발명은 다음의 (a) 및 (b) 단계를 포함하는 지질 나노 리포좀의 제조방법을 제공한다:Furthermore, the present invention provides a method for preparing lipid nano liposomes comprising the following steps (a) and (b):

(a) 전술한 방법으로 이온화 가능한 양이온성 지질, 양이온성 지질, 또는 DOTAP 클로라이드를 합성하는 단계; 및 (a) synthesizing an ionizable cationic lipid, cationic lipid, or DOTAP chloride by the method described above; and

(b) 상기 합성된 이온화 가능한 양이온성 지질, 양이온성 지질 또는 DOTAP와 콜레스테롤을 포함하는 용액을 완충액과 혼합하여 지질 나노 리포좀을 형성하는 단계.(b) a step of forming lipid nanoliposomes by mixing the solution containing the synthesized ionizable cationic lipid, cationic lipid or DOTAP and cholesterol with a buffer solution.

본 발명에 따른 핵산 치료제에 사용되는 기능성 지질의 합성 방법은 미세유체반응기에서 연속 유동 합성 방법으로 구현되는 바, 기존의 배치 반응기에서의 합성 방법에 비해 생산성이 현저히 높고, 품질이 우수한 이온화 가능한 양이온성 지질 또는 양이온성 지질을 제공한다. 나아가, 본 발명의 방법으로 합성된 DODAP 및 DOTAP는 질병 치료를 위해 mRNA, siRNA 및 플라스미드 DNA를 포함한 생리 활성 분자를 캡슐화하는 지질 나노입자 및 지질 나노 리포좀으로 활용될 수 있다.The method for synthesizing a functional lipid used in a nucleic acid therapeutic agent according to the present invention is implemented by a continuous flow synthesis method in a microfluidic reactor, which has significantly higher productivity and provides an ionizable cationic lipid or cationic lipid of superior quality compared to a synthesis method in a conventional batch reactor. Furthermore, DODAP and DOTAP synthesized by the method of the present invention can be utilized as lipid nanoparticles and lipid nanoliposomes that encapsulate physiologically active molecules including mRNA, siRNA, and plasmid DNA for disease treatment.

도 1은 지질 나노입자 또는 리포좀에 사용되는 일반적인 양이온성 지질(1), 1,2-디올레오일-3-디메틸암모늄-프로판(DODAP)(2) 및 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP)(3)의 구조를 나타낸 것이다.Figure 1 shows the structures of common cationic lipids (1), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) (2) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (3), which are used in lipid nanoparticles or liposomes.

도 2의 A)는 기존에 공지된 DOTAP(3)의 합성 방법, B)는 본 발명의 2가지 종류의 반대 이온을 사용한 DOTAP 합성을 위한 연속 유동 방법을 보여준다.Figure 2 (A) shows a conventionally known method for synthesizing DOTAP (3), and (B) shows a continuous flow method for synthesizing DOTAP using two types of counter ions of the present invention.

도 3은 인-라인(in-line) FTIR를 포함하는 모세관 반응기에서 올레오일 클로라이드의 연속 유동 합성을 위한 실험 셋팅을 보여준다.Figure 3 shows the experimental setup for continuous flow synthesis of oleoyl chloride in a capillary reactor including in-line FTIR.

도 4는 인-라인 FTIR 유동 셀의 실험 셋팅 사진이다.Figure 4 is a photograph of the experimental setup of the in-line FTIR flow cell.

도 5는 인-라인 FTIR 모니터링의 실험 결과이다.Figure 5 shows the experimental results of in-line FTIR monitoring.

도 6의 a)는 효과적인 액체-가스 분리기로 사용되는 맞춤 T자형 접합부를 보여주는 사진이고, b)는 반응기에서 HCl 가스를 제거하지 않으면 고농도의 고체 피리디늄 염산염 염(8 eq.의 염기를 사용해야 함)으로 인해 R2 반응기에서 막힘이 발생한다는 것을 보여주는 사진이고, c)는 인-라인 FTIR 모니터링을 사용하여 올레오일 클로라이드에서 DODAP(2)로의 전환을 모니터링한 결과를 보여준다.Figure 6(a) is a photograph showing a custom T-junction used as an effective liquid-gas separator, b) is a photograph showing that plugging occurs in the R2 reactor due to high concentration of solid pyridinium hydrochloride salt (8 eq. of base is required) if HCl gas is not removed from the reactor, and c) shows the results of monitoring the conversion of oleoyl chloride to DODAP(2) using in-line FTIR monitoring.

도 7은 모세관 반응기에서의 이온화 가능한 양이온성 지질 DODAP(2) 및 이온화 가능한 양이온성 지질 라이브러리의 단일 유동 합성을 보여준다.Figure 7 shows the single-flow synthesis of the ionizable cationic lipid DODAP(2) and a library of ionizable cationic lipids in a capillary reactor.

도 8은 이온화 가능한 양이온성 지질의 단일 유동 합성을 위한 실험 셋팅을 보여준다.Figure 8 shows the experimental setup for the single-flow synthesis of ionizable cationic lipids.

도 9는 DODAP의 연속 유동 합성을 위함 실험 셋팅을 보여준다.Figure 9 shows the experimental setup for continuous flow synthesis of DODAP.

도 10은 모세관 반응기에서 양이온성 지질인 DOTAP 메틸 설페이트(4), DLinTAP 메틸 설페이트(17) 및 다른 유사체들(18-22)의 연속 유동 합성을 위한 실험 셋팅을 보여준다.Figure 10 shows the experimental setup for continuous flow synthesis of cationic lipids DOTAP methyl sulfate (4), DLinTAP methyl sulfate (17) and other analogues (18-22) in a capillary reactor.

도 11은 DOTAP 메틸 설페이트의 연속 유동 합성을 위한 실험 셋팅을 보여준다.Figure 11 shows the experimental setup for continuous flow synthesis of DOTAP methyl sulfate.

도 12는 양이온성 지질 라이브러리의 연속 유동 합성을 위한 실험 셋팅을 보여준다.Figure 12 shows the experimental setup for continuous flow synthesis of a cationic lipid library.

도 13은 모세관 반응기에서의 DOTAP 클로라이드의 연속 유동 합성을 보여준다.Figure 13 shows the continuous flow synthesis of DOTAP chloride in a capillary reactor.

도 14는 크기 증가(size-up) 방법을 사용한 DOTAP 클로라이드의 연속 유동 합성을 위한 실험 셋팅을 보여준다.Figure 14 shows the experimental setup for continuous flow synthesis of DOTAP chloride using a size-up method.

도 15는 합성된 DODAP 및 DOTAP로부터 생성된 나노입자의 제조 및 특성을 나타낸 것으로, 도 15의 A는 DLS에 의한 나노입자 생성, 투석 및 분석에 대한 일련의 공정을 보여주고, B 내지 E는 각각 투석 공정을 통한 정제 전후에, 제조된 DODAP LNP(B), DODAP 리포좀(C), DOTAP-클로라이드 리포좀(D) 및 DOTAP 메틸 설페이트 리포좀(E)의 정규화된 크기 분포를 보여준다. Figure 15 shows the preparation and characterization of nanoparticles generated from synthesized DODAP and DOTAP, where A of Figure 15 shows a series of processes for nanoparticle production, dialysis and analysis by DLS, and B to E show the normalized size distributions of the prepared DODAP LNPs (B), DODAP liposomes (C), DOTAP-chloride liposomes (D) and DOTAP methyl sulfate liposomes (E) before and after purification by dialysis process, respectively.

도 11은 지질 기반 나노입자의 생산 및 분석을 나타낸 것으로, A는 전체 절차의 모식도이고, B는 투석 전후에 생성된 나노입자의 수치적 크기 및 PDI를 보여준다.Figure 11 shows the production and analysis of lipid-based nanoparticles, where A is a schematic diagram of the overall procedure and B shows the numerical size and PDI of nanoparticles produced before and after dialysis.

도 16은 생성된 DODAP LNP(A), DODAP 리포좀(B), DOTAP-클로라이드 리포좀(C) 및 DOTAP 메틸 설페이트 리포좀(D)의 수치적 크기 및 PDI 값을 나타낸 것이다.Figure 16 shows the numerical sizes and PDI values of the generated DODAP LNPs (A), DODAP liposomes (B), DOTAP-chloride liposomes (C), and DOTAP methyl sulfate liposomes (D).

이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention.

상술한 바와 같이 DODAP 및 DOTAP와 같은 기능성 지질의 기존 합성 방법들은 시간과 비용 소모로 인해 비경제적이거나, 정제가 어렵거나, 대규모 생산에 적용이 제한되는 문제점이 있어, 유동 기술을 이용하여 고품질의 이온화 가능한 양이온성 지질을 친환경적이고 경제적으로 합성하는 방법을 개발하기 위한 노력이 계속되고 있다.As described above, existing synthetic methods for functional lipids such as DODAP and DOTAP have problems such as being uneconomical due to time and cost consumption, being difficult to purify, or being limited in application to large-scale production. Therefore, efforts are being made to develop an environmentally friendly and economical method for synthesizing high-quality ionizable cationic lipids using fluid technology.

이에, 본 발명자들은 단일 연속 유동 공정을 이용하여 높은 생산성으로 고품질의 기능성 지질 라이브러리를 합성할 수 있는 신규 합성 경로를 개발함으로써, 상술한 문제의 해결 방안을 모색하였다. Accordingly, the inventors of the present invention sought to solve the above-mentioned problems by developing a novel synthetic route capable of synthesizing a high-quality functional lipid library with high productivity using a single continuous flow process.

구체적으로, 간단한 2단계 방식으로 개발된 이온화 가능한 지질 라이브러리에 대한 손쉬운 단일 유동 합성 방법을 개발하여 크기 증가 접근 방식을 통해 시간당 최대 10g의 확장 가능한 생산성을 달성하였다. 첫 번째 단계에서는 연속 유동 에스테르화를 위한 아실 클로라이드 합성을 성공적으로 탐색했으며, T 접합 액체-가스 분리기를 사용하여 독성 HCl 및 CO 가스를 효과적으로 제거함으로써 막힘을 방지하였다. 인라인 FTIR 모니터링을 사용하여 연속 유동 공정의 신속한 최적화가 달성되었다. 본 발명의 유동 프로토콜을 사용하면 기존 배치 프로토콜에 비해 훨씬 짧은 기간(약 4.5분)에 확립된 상업용 종류와 새로운 이온화 가능 지질을 모두 포함하는 광범위한 지질을 합성할 수 있다. 생분해성 이온화 가능한 지질 라이브러리는 긴 사슬 그룹에 에스테르 또는 이황화물 작용기를 포함하여 설계되었다. 이러한 지질은 손쉬운 모세관 반응기에서 합성되어, 88~92%의 수율로 얻어졌다. 추가로, 이온화 가능한 지질을 양이온성 지질로 전환하기 위해 본 발명에서는 유동 반응기에서 5분 동안 유지하면서 디메틸 설페이트를 사용하여 신속한 알킬화를 달성하였다. 특히, DOTAP 메틸 설페이트는 시간 당 7g의 생산성으로 생산되었다. 나아가, EDC.HCl을 사용한 스테글리치 에스테르화 반응을 통해 88% 수율로 DOTAP 클로라이드를 효과적으로 합성하여, 시간 당 10.2g의 생산성으로 DOTAP 클로라이드를 얻을 수 있었다. 마지막으로, 자체 개발한 미세 혼합기를 사용하여 DODAP를 PDI가 0.07인 64mm의 균일한 크기의 LNP와 PDI가 0.05인 72mm 크기의 리포좀으로 변환하였다. 유사하게, DOTAP는 PDI가 0.08인 55mm 크기의 리포좀을 형성하였다. 지질 라이브러리를 위한 이러한 신규 유동 보조 플랫폼은 유전자 치료를 발전시키고 차세대 나노의학 역할을 할 수 있는 큰 가능성을 가진다.Specifically, a facile single-flow synthesis method for a library of ionizable lipids developed in a simple two-step manner was developed, achieving scalable productivity of up to 10 g/hr through a scale-up approach. In the first step, acyl chloride synthesis for continuous flow esterification was successfully explored, and clogging was prevented by effectively removing toxic HCl and CO gases using a T-junction liquid-gas separator. Rapid optimization of the continuous flow process was achieved using in-line FTIR monitoring. The flow protocol of the present invention enables the synthesis of a wide range of lipids, including both established commercial types and novel ionizable lipids, in a much shorter time (approximately 4.5 min) compared to conventional batch protocols. A library of biodegradable ionizable lipids was designed with ester or disulfide functionalities in the long chain group. These lipids were synthesized in a facile capillary reactor, and were obtained in 88-92% yields. In addition, rapid alkylation was achieved using dimethyl sulfate while maintaining the flow reactor for 5 min in the present invention to convert the ionizable lipids into cationic lipids. In particular, DOTAP methyl sulfate was produced with a productivity of 7 g/h. Furthermore, DOTAP chloride was effectively synthesized with a yield of 88% via the Steglich esterification reaction using EDC.HCl, resulting in a productivity of 10.2 g/h. Finally, DODAP was converted into uniformly sized 64 mm LNPs with a PDI of 0.07 and 72 mm liposomes with a PDI of 0.05 using a self-developed micromixer. Similarly, DOTAP formed 55 mm liposomes with a PDI of 0.08. This novel fluid-assisted platform for lipid libraries holds great potential to advance gene therapy and serve as next-generation nanomedicine.

본 발명은 제1 측면은 다음의 (a) 및 (b)의 단계를 포함하는 이온화 가능한 양이온성 지질의 합성 방법을 제공한다:The present invention, in its first aspect, provides a method for synthesizing an ionizable cationic lipid comprising the steps of:

(a) R-(CH2)2C(=O)OH, 디메틸포름아미드 및 옥살릴 클로라이드 혼합 및 반응시켜 R-C(=O)Cl을 형성하는 단계; 및(a) a step of mixing and reacting R-(CH 2 ) 2 C(=O)OH, dimethylformamide and oxalyl chloride to form RC(=O)Cl; and

(b) R-C(=O)Cl을 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물과 혼합하여 에스테르화 반응을 일으켜 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 생성하는 단계;를 포함하며,(b) a step of mixing R-C(=O)Cl with 3-(dimethylamino)propane-1,2-diol and a basic compound to cause an esterification reaction to produce an ionizable cationic lipid having a structure of chemical formula 1;

[화학식 1][Chemical Formula 1]

Figure PCTKR2024007830-appb-img-000004
Figure PCTKR2024007830-appb-img-000004

상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이다.The above R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical in which -C(=O)O- or -S-S- is introduced into the chain.

본 발명에 있어서, 상기 비치환된 C10 내지 C20의 포화 지방족 탄화수소 라디칼은 10 내지 20개의 탄소 원자를 포함하는 선형 또는 분지형의 포화 지방족 탄화수소 라디칼일 수 있다. 예를 들어, n-데실기, 이소데실기, sec-데실기, tert-데실기, 네오데실기, n-운데실기, 이소운데실기, sec-운데실기, tert-운데실기, 네오운데실기, n-도데실기, 이소도데실기, sec-도데실기, tert-도데실기, n-트리데실기, 이소트리데실기, sec-트리데실기, tert-트리데실기, 네오트리데실기, n-테트라데실기, 이소테트라데실기, sec-테트라데실기, tert-테트라데실기, 네오테트라데실기, n-펜타데실기, 이소펜타데실기, sec-펜타데실기, tert-펜타데실기, 네오펜타데실기, n-헥사데실기, 이소헥사데실기, sec-헥사데실기, tert-헥사데실기, 네오헥사데실기, n-헵타데실기, 이소헵타데실기, sec-헵타데실기, tert-헵타데실기, 네오헵타데실기, n-옥타데실기, 이소옥타데실기, sec-옥타데실기, tert-옥타데실기, 네오옥타데실기, n-노나데실기, 이소노나데실기, sec-노나데실기, tert-노나데실기, 네오노나데실기, n-에이코실기, 이소에이코실기, sec-에이코실기, tert-에이코실기 또는 네오에이코실기일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the unsubstituted C10 to C20 saturated aliphatic hydrocarbon radical may be a linear or branched saturated aliphatic hydrocarbon radical containing 10 to 20 carbon atoms. For example, n-decyl group, isodecyl group, sec-decyl group, tert-decyl group, neodecyl group, n-undecyl group, isoundecyl group, sec-undecyl group, tert-undecyl group, neoundecyl group, n-dodecyl group, isododecyl group, sec-dodecyl group, tert-dodecyl group, n-tridecyl group, isotridecyl group, sec-tridecyl group, tert-tridecyl group, neotridecyl group, n-tetradecyl group, isotetradecyl group, sec-tetradecyl group, tert-tetradecyl group, neotetradecyl group, n-pentadecyl group, isopentadecyl group, sec-pentadecyl group, tert-pentadecyl group, neopentadecyl group, n-hexadecyl group, isohexadecyl group, It can be a sec-hexadecyl group, a tert-hexadecyl group, a neohexadecyl group, a n-heptadecyl group, an isoheptadecyl group, a sec-heptadecyl group, a tert-heptadecyl group, a neoheptadecyl group, a n-octadecyl group, an isooctadecyl group, a sec-octadecyl group, a tert-octadecyl group, a neooctadecyl group, a n-nonadecyl group, an isononadecyl group, a sec-nonadecyl group, a tert-nonadecyl group, a neononadecyl group, a n-eicosyl group, an isoeicosyl group, a sec-eicosyl group, a tert-eicosyl group, or a neoeicosyl group, but is not limited thereto.

본 발명에 있어서, 상기 비치환된 C10 내지 C20의 불포화 지방족 탄화수소 라디칼은 10 내지 20개의 탄소 원자를 포함하는 선형 또는 분지형의 불포화 지방족 탄화수소 라디칼일 수 있고, 하나 이상의 이중 결합(C=C)을 포함할 수 있다. 예를 들어, 선형 또는 분지형 형태의 데세닐기, 도데세닐기, 트리데세닐기, 테트라데세닐기, 펜타데세닐기, 헥사데세닐기, 헵타데세닐기, 옥타데세닐기, 노나데세닐기 또는 에이코세닐기일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the unsubstituted C10 to C20 unsaturated aliphatic hydrocarbon radical may be a linear or branched unsaturated aliphatic hydrocarbon radical containing 10 to 20 carbon atoms and may include one or more double bonds (C=C). For example, it may be a linear or branched decenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group or an eicosenyl group, but is not limited thereto.

본 발명에 있어서, 상기 합성 방법의 모든 반응물은 상기 (a) 및 (b) 단계의 반응에 악영향을 미치지 않는 유기용매에 용해된 상태로 사용될 수 있다. 예를 들어, 디메틸포름아미드(DMF), 테트라하이드로퓨란(THF), 디클로로메탄 등의 유기용매를 사용할 수 있으나, 이에 한정되지 않으며, 통상의 기술자는 적절한 유기용매를 선택하여 상기 (a) 및 (b) 단계를 수행할 수 있다.In the present invention, all reactants of the above-mentioned synthetic method can be used in a state dissolved in an organic solvent that does not adversely affect the reactions of steps (a) and (b). For example, organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform steps (a) and (b).

본 발명에 있어서, 상기 (a) 단계 후에는 HCl 및 CO 가스 부산물과 함께 R-C(=O)Cl(아실 클로라이드)가 생성될 수 있다. 생성된 HCl 및 CO 가스는 (b) 단계에서는 불필요한 부산물이므로, (b) 단계를 수행하기 전에 분리 및 제거될 수 있다.In the present invention, after step (a), R-C(=O)Cl (acyl chloride) can be generated together with HCl and CO gas byproducts. Since the generated HCl and CO gases are unnecessary byproducts in step (b), they can be separated and removed before performing step (b).

본 발명의 합성 방법에 있어서, 상기 (a) 단계의 옥살릴 클로라이드는, 디메틸포름아미드(DMF) 촉매 존재 하에 올레오일 클로라이드를 합성하기 위한 염소화제(chlorinating agent)로 선택되었다. CH2Cl2는 형성된 염기.HCl 복합체(base.HCl complex)의 용해도를 고려하여 유동 합성의 일반적인 용매로 간주되었다.In the synthetic method of the present invention, oxalyl chloride in step (a) was selected as a chlorinating agent for synthesizing oleoyl chloride in the presence of a dimethylformamide (DMF) catalyst. CH 2 Cl 2 was considered as a general solvent for flow synthesis considering the solubility of the formed base.HCl complex.

본 발명의 합성 방법에 있어서, 상기 3-(디메틸아미노)프로판-1,2-디올은 디메틸아미노기를 포함하는 다른 종류의 디올 화합물로 대체될 수 있다.In the synthetic method of the present invention, the 3-(dimethylamino)propane-1,2-diol may be replaced with another type of diol compound containing a dimethylamino group.

본 발명에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치(1)에서 수행될 수 있고, 상기 미세유체장치(1)는: 제1 유로(110)와 제2 유로(120)가 유체적으로 연결되는 제1 미세 혼합기(100); 상기 제1 미세 혼합기(100)와 유체적으로 연결되는 제1 반응기(200); 상기 제1 반응기(200)와 유체적으로 연결되는 제1 배압 조절기(300); 상기 제1 배압 조절기(300)와 유체적으로 연결되어 용액과 가스를 분리하는 T자형 액체-가스 분리기(400); 상기 T자형 액체-가스 분리기(400)와 제3 유로(510)가 유체적으로 연결되는 제2 미세 혼합기(500); 상기 제2 미세 혼합기(500)와 유체적으로 연결되는 제2 반응기(600); 및 상기 제2 반응기(600)와 유체적으로 연결되는 제2 배압 조절기(700)를 포함할 수 있다.In the present invention, the synthesis method can be performed in a microfluidic device (1) for a single continuous flow process, wherein the microfluidic device (1) comprises: a first micro-mixer (100) in which a first flow path (110) and a second flow path (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); a first back pressure regulator (300) fluidly connected with the first reactor (200); a T-shaped liquid-gas separator (400) fluidly connected with the first back pressure regulator (300) to separate a solution and a gas; a second micro-mixer (500) in which the T-shaped liquid-gas separator (400) and a third flow path (510) are fluidly connected; a second reactor (600) fluidly connected with the second micro-mixer (500); and may include a second back pressure regulator (700) fluidly connected to the second reactor (600).

본 발명에 있어서, 상기 T자형 액체-가스 분리기(400)는 상기 제1 배압 조절기(300)와 유체적으로 연결되는 제1 공급유로(410), 분리된 가스가 배출되는 제1 배출유로(420) 및 분리된 용액이 상기 제2 미세 혼합기(500)에 공급되도록 상기 제2 미세 혼합기(500)에 유체적으로 연결되는 제2 공급유로(430)를 포함할 수 있다.In the present invention, the T-shaped liquid-gas separator (400) may include a first supply path (410) fluidly connected to the first back pressure regulator (300), a first discharge path (420) through which separated gas is discharged, and a second supply path (430) fluidly connected to the second micro-mixer (500) so that the separated solution is supplied to the second micro-mixer (500).

도 7 및 8을 참조하면, 상기 미세유체장치(1)의 제1 유로(110)와 제2 유로(120)는 각각 상기 (a) 단계의 반응물이 주입되는 유로로서, 제1 미세 혼합기(100)와 유체적으로 연결될 수 있다. 구체적으로, 상기 제1 유로(110)에 R-(CH2)2C(=O)OH 및 디메틸포름아미드, 상기 제2 유로(120)에 옥살릴 클로라이드가 주입될 수 있다.Referring to FIGS. 7 and 8, the first flow path (110) and the second flow path (120) of the microfluidic device (1) are flow paths into which the reactants of step (a) are injected, respectively, and can be fluidly connected to the first micro-mixer (100). Specifically, R-(CH 2 ) 2 C(=O)OH and dimethylformamide can be injected into the first flow path (110), and oxalyl chloride can be injected into the second flow path (120).

도 7 및 8을 참조하면, 상기 제1 미세 혼합기(100)에서는 제1 유로(110)와 제2 유로(120)로부터 유입된 반응물들이 혼합되어 혼합물이 생성되며, 생성된 혼합물은 제1 미세 혼합기(100)와 유체적으로 연결되는 제1 반응기(200)로 주입될 수 있다.Referring to FIGS. 7 and 8, in the first micro-mixer (100), reactants flowing in from the first path (110) and the second path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).

도 7 및 8을 참조하면, 상기 제1 반응기(200)에서는 (a) 단계의 반응이 일어나며, 반응의 결과물로서 R-C(=O)Cl이 생성되고, 부산물로 HCl 및 CO 가스가 생성된다. 전술한 바와 같이, 생성된 HCl 및 CO 가스는 (b) 단계에서는 불필요한 부산물이므로, (b) 단계를 수행하기 전에 R-C(=O)Cl을 포함하는 용액으로부터 분리 및 제거되는 것이 바람직하다. 이러한 부산물 가스는 상기 T자형 액체-가스 분리기(400)를 통해 분리 및 제거될 수 있다. 구체적으로, R-C(=O)Cl을 포함하는 용액과 HCl 및 CO 가스는 상기 제1 반응기(200)와 유체적으로 연결되는 제1 배압 조절기(300)를 거쳐, 상기 제1 배압 조절기(300)와 유체적으로 연결되는 제1 공급유로(410)를 거쳐 T자형 액체-가스 분리기(400)를 통과하면서 R-C(=O)Cl을 포함하는 용액과 HCl 및 CO 가스로 분리될 수 있다. 이때, 상기 분리된 HCl 및 CO 가스는 제1 배출유로(420)로 배출되어 제거되고, 상기 분리된 R-C(=O)Cl을 포함하는 용액은 제2 공급유로(430)를 거쳐 제2 미세 혼합기(500)에 주입될 수 있다.Referring to FIGS. 7 and 8, in the first reactor (200), the reaction of step (a) occurs, and R-C(=O)Cl is generated as a result of the reaction, and HCl and CO gas are generated as byproducts. As described above, the generated HCl and CO gas are unnecessary byproducts in step (b), and therefore, it is preferable to separate and remove them from the solution containing R-C(=O)Cl before performing step (b). These byproduct gases can be separated and removed through the T-shaped liquid-gas separator (400). Specifically, a solution containing R-C(=O)Cl and HCl and CO gas may be separated into a solution containing R-C(=O)Cl and HCl and CO gas while passing through a first pressure regulator (300) fluidly connected to the first reactor (200), a first supply path (410) fluidly connected to the first pressure regulator (300), and a T-shaped liquid-gas separator (400). At this time, the separated HCl and CO gas may be discharged through a first discharge path (420) and removed, and the separated solution containing R-C(=O)Cl may be injected into a second micro-mixer (500) through a second supply path (430).

도 7 및 8을 참조하면, 상기 제2 미세 혼합기(500)는 제3 유로(510)와 유체적으로 연결될 수 있다. 상기 제3 유로(510)에는 상기 제2 미세 혼합기(500)에 주입된 R-C(=O)Cl과 반응하여 최종적으로 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 생성할 수 있는 추가의 반응물이 주입될 수 있다. 구체적으로, 상기 제3 유로(510)에는 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물이 주입될 수 있다.Referring to FIGS. 7 and 8, the second micro-mixer (500) may be fluidly connected to the third channel (510). An additional reactant that can react with R-C(=O)Cl injected into the second micro-mixer (500) to ultimately produce an ionizable cationic lipid having the structure of the chemical formula 1 may be injected into the third channel (510). Specifically, 3-(dimethylamino)propane-1,2-diol and a basic compound may be injected into the third channel (510).

도 7 및 8을 참조하면, 상기 제2 미세 혼합기(500)에서는 제2 공급유로(430)와 제3 유로(510)로부터 유입된 반응물들이 혼합되어 혼합물이 생성되며, 생성된 혼합물은 제2 미세 혼합기(500)와 유체적으로 연결되는 제2 반응기(600)로 주입될 수 있다.Referring to FIGS. 7 and 8, in the second micro-mixer (500), reactants flowing in from the second supply path (430) and the third path (510) are mixed to produce a mixture, and the produced mixture can be injected into the second reactor (600) that is fluidly connected to the second micro-mixer (500).

도 7 및 8을 참조하면, 상기 제2 반응기(600)에서는 (b) 단계의 반응이 일어나며, 반응의 결과물로서 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질이 생성된다.Referring to FIGS. 7 and 8, the reaction of step (b) occurs in the second reactor (600), and an ionizable cationic lipid having the structure of the chemical formula 1 is produced as a result of the reaction.

도 7 및 8을 참조하면, 최종 생성물인 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질은 상기 제2 반응기(600)와 유체적으로 연결되는 제2 배압 조절기(700)를 통과한 후 배출될 수 있다.Referring to FIGS. 7 and 8, the ionizable cationic lipid having the structure of the chemical formula 1 as the final product can be discharged after passing through the second back pressure regulator (700) fluidly connected to the second reactor (600).

도 7 및 8을 참조하면, 상기 제1 반응기(200)와 제1 배압 조절기(300) 사이에는 제1 분광계(800)가 추가로 유체적으로 연결될 수 있고, 상기 제2 반응기(600)와 제2 배압 조절기(700) 사이에는 제2 분광계(900)가 추가로 유체적으로 연결될 수 있다.Referring to FIGS. 7 and 8, a first spectrometer (800) may be additionally fluidly connected between the first reactor (200) and the first back pressure regulator (300), and a second spectrometer (900) may be additionally fluidly connected between the second reactor (600) and the second back pressure regulator (700).

본 발명에 있어서, 상기 제1 반응기(200)에서의 체류 시간은 1 내지 2분일 수 있다. 구체적으로, 상기 제1 반응기(200)에 유입된 반응물들, 즉 R-(CH2)2C(=O)OH, 디메틸포름아미드 및 옥살릴 클로라이드가 반응을 일으켜 R-C(=O)Cl을 생성하기까지 소요되는 시간은 1 내지 2분일 수 있다. In the present invention, the residence time in the first reactor (200) may be 1 to 2 minutes. Specifically, the time required for the reactants introduced into the first reactor (200), i.e., R-(CH 2 ) 2 C(=O)OH, dimethylformamide, and oxalyl chloride, to react to produce RC(=O)Cl may be 1 to 2 minutes.

본 발명에 있어서, 상기 제1 반응기(200)에서의 반응 온도는 20 내지 30 ℃일 수 있다. In the present invention, the reaction temperature in the first reactor (200) may be 20 to 30°C.

상기 제1 반응기(200)에서의 체류 시간(즉, 반응 시간) 및 반응 온도 범위로 상기 (a) 단계의 반응을 수행하는 것이 바람직하며, 상기 범위를 벗어나는 경우 R-C(=O)Cl의 수율이 감소될 수 있다.It is preferable to perform the reaction of step (a) within the range of the residence time (i.e., reaction time) and reaction temperature in the first reactor (200), and if it is outside the range, the yield of R-C(=O)Cl may decrease.

상기 제1 반응기(200)에서의 체류 시간은 상기 제1 유로에 주입되는 R-(CH2)2C(=O)OH 및 디메틸포름아미드를 포함하는 용액과 옥살릴 클로라이드를 포함하는 용액을 0.4 내지 2.0ml/분의 유속으로 제1 미세 혼합기에 주입함으로써 달성될 수 있다.The residence time in the first reactor (200) can be achieved by injecting a solution containing R-(CH 2 ) 2 C(=O)OH and dimethylformamide and a solution containing oxalyl chloride into the first micro-mixer at a flow rate of 0.4 to 2.0 ml/min.

본 발명에 있어서, 상기 제2 반응기(600)에서의 체류 시간은 2 내지 10분일 수 있다. 구체적으로, 상기 제2 반응기(600)에 유입된 반응물들, 즉 R-C(=O)Cl, 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물이 반응을 일으켜 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 생성하기까지 소요되는 시간은 2 내지 10분일 수 있다.In the present invention, the residence time in the second reactor (600) may be 2 to 10 minutes. Specifically, the time required for the reactants introduced into the second reactor (600), i.e., R-C(=O)Cl, 3-(dimethylamino)propane-1,2-diol, and a basic compound to react and produce an ionizable cationic lipid having a structure of the chemical formula 1, may be 2 to 10 minutes.

본 발명에 있어서, 상기 제2 반응기(600)에서의 반응 온도는 20 내지 60 ℃일 수 있다. In the present invention, the reaction temperature in the second reactor (600) may be 20 to 60°C.

상기 제2 반응기(600)에서의 체류 시간(즉, 반응 시간) 및 반응 온도 범위로 상기 (b) 단계의 반응을 수행하는 것이 바람직하며, 상기 범위를 벗어나는 경우 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질의 수율이 감소될 수 있다.It is preferable to perform the reaction of step (b) within the range of the residence time (i.e., reaction time) and reaction temperature in the second reactor (600), and if it is outside the range, the yield of the ionizable cationic lipid having the structure of the chemical formula 1 may decrease.

상기 제2 반응기에서의 체류 시간은 상기 제3 유로(510)에 주입되는 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물을 포함하는 용액을 각각 0.4 내지 2.0ml/분의 유속으로 제2 미세 혼합기에 주입함으로써 달성될 수 있다.The residence time in the second reactor can be achieved by injecting a solution containing 3-(dimethylamino)propane-1,2-diol and a basic compound into the third flow path (510) at a flow rate of 0.4 to 2.0 ml/min into the second micro-mixer.

본 발명에 있어서, 상기 (b) 단계의 염기성 화합물은 피리딘, 피리딘과 DMAP의 혼합, DMAP, 디이소프로필에틸아민 및 트리에틸아민으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. 가장 바람직한 염기성 화합물은 트리에틸아민일 수 있다. 염기성 화합물로 트리에틸아민을 사용하는 경우 상기 제2 반응기(600)에서 25℃, 3분의 반응으로 최대 92%의 수율로 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 얻을 수 있다.In the present invention, the basic compound of step (b) may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine. The most preferred basic compound may be triethylamine. When triethylamine is used as the basic compound, an ionizable cationic lipid having the structure of the chemical formula 1 can be obtained in a maximum yield of 92% through a reaction at 25°C for 3 minutes in the second reactor (600).

본 발명에 있어서, 상기 (a) 단계의 R-(CH2)2C(=O)OH는 지방산으로서, 예를 들어 올레산, 리놀레산, 미리스트산, 스테아르산, 팔미트산, 10-(헥실옥시)-10-옥소데칸산, (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산, (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, 3-(옥타데실디설파닐)프로판산 및 3-(도데실디설파닐)프로판산으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다. 통상의 기술자는 합성하고자 하는 이온화 가능한 양이온성 지질의 종류에 따라 상기 지방산의 종류를 적절히 선택하여 사용할 수 있다.In the present invention, R-(CH 2 ) 2 C(=O)OH of step (a) may be any one selected from the group consisting of a fatty acid, for example, oleic acid, linoleic acid, myristic acid, stearic acid, palmitic acid, 10-(hexyloxy)-10-oxodecanoic acid, (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, 3-(octadecyldisulfanyl)propanoic acid, and 3-(dodecyldisulfanyl)propanoic acid, but is not limited thereto. No. A person skilled in the art can appropriately select and use the type of fatty acid according to the type of ionizable cationic lipid to be synthesized.

본 발명에 있어서, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질은 다음의 (1) 내지 (11) 중 어느 하나일 수 있다:In the present invention, the ionizable cationic lipid having the structure of the chemical formula 1 may be any one of the following (1) to (11):

(1) 1,2-디올레오일-3-디메틸암모늄-프로판;(1) 1,2-Dioleoyl-3-dimethylammonium-propane;

(2) (9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트);(2) (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate);

(3) 1,2-디미리스토일-3-디메틸암모늄-프로판;(3) 1,2-Dimyristoyl-3-dimethylammonium-propane;

(4) 1,2-디스테아로일-3-디메틸암모늄-프로판;(4) 1,2-distearoyl-3-dimethylammonium-propane;

(5) 1,2-디팔미토일-3-디메틸암모늄-프로판;(5) 1,2-Dipalmitoyl-3-dimethylammonium-propane;

(6) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트);(6) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate);

(7) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트);(7) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate);

(8) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(8) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((E)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate);

(9) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(9) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((Z)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate);

(10) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(옥타데실디설파닐)프로파노에이트); 및(10) 3-(dimethylamino)propane-1,2-diyl bis(3-(octadecyldisulfanyl)propanoate); and

(11) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(도데실디설파닐)프로파노에이트).(11) 3-(Dimethylamino)propane-1,2-diyl bis(3-(dodecyldisulfanyl)propanoate).

본 발명의 구체적인 일 실시예에서는 이온화 가능한 양이온성 지질인 1,2-디올레오일-3-디메틸암모늄-프로판(DODAP)를 합성하기 위해, 상기 (a) 단계를 다음과 같이 수행하였다. CH2Cl2에 용해된 0.35M 옥살릴 클로라이드 용액 및 0.05M DMF과 함께 0.35M 올레산 용액을 불활성 대기 하에서 별도로 제조하고, HPLC 펌프를 사용하여 제1 미세 혼합기(Ø = 1.0mm)를 통해 반응기 R1(L = 225cm, Ø = 1.0mm)에 주입하였다. 반응은 시약의 유속을 변화시켜 실온(25 ℃에서 다양한 체류 시간으로 최적화되었다. 미세유체 셀을 사용하여 유동 장치를 FTIR 분광계에 연결하였다(도 4). 차례로, IR 분광법은 1780 cm-1에서 특징적인 CO 스트레칭 밴드를 갖는 산 클로라이드(acid chloride)의 형성과 올레산의 산 작용기의 1708 cm-1에서 CO 스트레칭의 소멸을 모니터링하였(도 5). HCl 및 CO 가스와 같은 부산물의 가스 방출을 방지하기 위해 100psi 배압 조절기(BPR)를 FTIR 유동 셀 배출구에 사용하였다. 몇 가지 최적화 후에, 올레오일 클로라이드를 원활하게 형성하기 위해 25℃의 반응 온도 및 1.5분의 체류 시간에서 올레산의 완전한 소비가 발생함을 확인하였다. 반응기 R1에서의 체류 시간 및 온도가 더 증가하더라도 올레오일 클로라이드의 CO 스트레칭 피크의 강도가 추가로 증가하지는 않았다.In a specific embodiment of the present invention, to synthesize 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), an ionizable cationic lipid, step (a) was performed as follows. A 0.35 M oxalyl chloride solution dissolved in CH 2 Cl 2 and a 0.35 M oleic acid solution together with 0.05 M DMF were prepared separately under an inert atmosphere, and injected into the reactor R1 (L = 225 cm, Ø = 1.0 mm) through the first micromixer (Ø = 1.0 mm) using an HPLC pump. The reaction was optimized at room temperature (25 °C) with various residence times by varying the flow rates of the reagents. The flow apparatus was connected to an FTIR spectrometer using a microfluidic cell (Fig. 4). In turn, IR spectroscopy monitored the formation of acid chloride with a characteristic CO stretching band at 1780 cm -1 and the disappearance of the CO stretching at 1708 cm -1 of the acid functional group of oleic acid (Fig. 5). A 100 psi back pressure regulator (BPR) was used at the FTIR flow cell outlet to prevent the outgassing of by-products such as HCl and CO gases. After some optimization, it was confirmed that complete consumption of oleic acid occurred at a reaction temperature of 25 °C and a residence time of 1.5 min for smooth formation of oleoyl chloride. Further increase in the residence time and temperature in reactor R1 did not result in further increase in the intensity of the CO stretching peak of oleoyl chloride.

본 발명의 구체적인 일 실시예에서는 R1에서 올레오일 클로라이드를 합성한 후, 용액에 용해되지 않은 독성 HCl과 CO 가스를 액체-가스 분리기(T-접합)를 사용하여 성공적으로 분리하였다. HCl을 분리하지 않는 경우, (b) 단계에서 과량의 염기가 필요하며, 염기에 난용성 염산염의 농도가 높아 R2에서 막힘 현상이 발생할 수 있다.In a specific embodiment of the present invention, after synthesizing oleoyl chloride in R1, toxic HCl and CO gases that are not dissolved in the solution are successfully separated using a liquid-gas separator (T-junction). If HCl is not separated, an excessive amount of base is required in step (b), and the concentration of insoluble hydrochloric acid salt in the base is high, which may cause clogging in R2.

본 발명의 구체적인 일 실시예에서는 이온화 가능한 양이온성 지질인 1,2-디올레오일-3-디메틸암모늄-프로판(DODAP)를 합성하기 위해, 상기 (b) 단계를 다음과 같이 수행하였다. 올레오일 클로라이드를 CH2Cl2에 용해된 3-(디메틸아미노)프로판-1,2-디올(0.15 M) 및 염기인 피리딘(0.4 M)을 포함하는 용액과 혼합기 M2(Ø = 1.0 mm)에서 분당 1.0 mL의 유속으로 혼합하고 반응기 R2(Ø = 1.0 mm)를 통과하여 에스테르화 반응을 발생시켰다. R2에서의 반응은 PTFE 튜브 반응기의 길이를 변경하여 다양한 체류 시간에서 최적화되었다. DODAP(2) 합성 중 에스테르 결합 형성은 인라인 FTIR 분광법을 사용하여 모니터링되었으며, 에스테르기에 대해 1735cm-1에서 특징적인 CO 스트레칭 밴드가 형성되었다(도 7).In a specific embodiment of the present invention, to synthesize 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), an ionizable cationic lipid, step (b) was performed as follows. Oleoyl chloride was mixed with a solution containing 3-(dimethylamino)propane-1,2-diol (0.15 M) dissolved in CH 2 Cl 2 and pyridine (0.4 M), a base, at a flow rate of 1.0 mL/min in a mixer M2 (Ø = 1.0 mm) and passed through a reactor R2 (Ø = 1.0 mm) to cause an esterification reaction. The reaction in R2 was optimized at various residence times by changing the length of the PTFE tube reactor. The ester bond formation during the DODAP(2) synthesis was monitored using inline FTIR spectroscopy, and a characteristic CO stretching band was formed at 1735 cm -1 for the ester group ( FIG. 7 ).

몇 가지 최적화 후에, DODAP(2)의 수율을 최대화하기 위해서는 염기성 화합물로 트리에틸아민을 사용하여, 25℃의 반응 온도 및 3분의 체류 시간이 가장 바람직함을 확인하였다.After several optimizations, it was confirmed that a reaction temperature of 25°C and a residence time of 3 min, using triethylamine as the basic compound, were the most preferable to maximize the yield of DODAP(2).

본 발명의 구체적인 일 실시예에서는 이온화 가능한 양이온성 지질 라이브러리를 합성하기 위해, 올레산 대신 각각 리놀레산, 미리스트산, 스테아르산 및 팔미트산을 사용하여 (9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트), 1,2-디미리스토일-3-디메틸암모늄-프로판, 1,2-디스테아로일-3-디메틸암모늄-프로판 및 1,2-디팔미토일-3-디메틸암모늄-프로판을 합성하였다.In a specific embodiment of the present invention, in order to synthesize an ionizable cationic lipid library, (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate), 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-distearoyl-3-dimethylammonium-propane and 1,2-dipalmitoyl-3-dimethylammonium-propane were synthesized using linoleic acid, myristic acid, stearic acid and palmitic acid, respectively, instead of oleic acid.

본 발명의 구체적인 다른 일 실시예에서는 이온화 가능한 지질의 긴 사슬에 에스테르 또는 이황화물 작용기가 도입된 지방산인 10-(헥실옥시)-10-옥소데칸산, (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산, (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, 3-(옥타데실디설파닐)프로판산 및 3-(도데실디설파닐)프로판산을 각각 사용하여, O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트), 3-(디메틸아미노)프로판-1,2-디일 비스(3-(옥타데실디설파닐)프로파노에이트) 및 3-(디메틸아미노)프로판-1,2-디일 비스(3-(도데실디설파닐)프로파노에이트)를 합성하였다.In another specific embodiment of the present invention, 10-(hexyloxy)-10-oxodecanoic acid, (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, 3-(octadecyldisulfanyl)propanoic acid and 3-(dodecyldisulfanyl)propanoic acid, which are fatty acids having an ester or disulfide functional group introduced into a long chain of ionizable lipids, are used to prepare O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((E)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((Z)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate), 3-(dimethylamino)propane-1,2-diyl bis(3-(octadecyldisulfanyl)propanoate) and 3-(Dimethylamino)propane-1,2-diyl bis(3-(dodecyldisulfanyl)propanoate) was synthesized.

그 결과, 도 7에서 확인되는 바와 같이 상기 10종의 이온화 가능한 양이온성 지질을 88 내지 93% 수율로 얻을 수 있음을 확인하였다.As a result, as confirmed in Fig. 7, it was confirmed that the 10 types of ionizable cationic lipids could be obtained with a yield of 88 to 93%.

본 발명의 구체적인 또 다른 일 실시예에서는 내경을 1.6mm로 증가시킨 제1 반응기(200) 및 제2 반응기(600)를 사용하여 이온화 가능한 양이온성 지질 라이브러리의 생산성을 향상시키고자 하였다. DODAP(2)의 생산성을 향상시키기 위해, 더 높은 농도의 0.5M 올레산 및 옥살릴 클로라이드를 분당 1.5mL의 유속으로 반응기 R1(L = 225cm, tR1 = 1.5분)에 주입하였고, 0.22M의 3-(디메틸아미노)프로판-1,2-디올과 0.6M 트리에틸아민을 분당 1.5 mL의 유속으로 반응기 R2(L = 900 cm, tR2 = 3분)에 주입하였다. R1에서 용액에 HCl 및 CO 가스를 완전히 용해하려면 250psi 배압 조절기가 필요했다(도 8). 최적화된 조건을 따르면, DODAP(2) 합성 5분당 0.9g으로 생산성이 높아져 1시간 작업에 11g 이상의 규모를 달성하였다.In another specific embodiment of the present invention, the productivity of the ionizable cationic lipid library was improved by using the first reactor (200) and the second reactor (600) whose inner diameters were increased to 1.6 mm. In order to improve the productivity of DODAP (2), higher concentrations of 0.5 M oleic acid and oxalyl chloride were injected at a flow rate of 1.5 mL/min into the reactor R1 (L = 225 cm, t R1 = 1.5 min), and 0.22 M 3-(dimethylamino)propane-1,2-diol and 0.6 M triethylamine were injected at a flow rate of 1.5 mL/min into the reactor R2 (L = 900 cm, t R2 = 3 min). A 250 psi back pressure regulator was required to completely dissolve HCl and CO gases in the solution in R1 (FIG. 8). Under the optimized conditions, the productivity of DODAP(2) synthesis increased to 0.9 g per 5 minutes, achieving a scale of more than 11 g per 1 hour of operation.

본 발명에 있어서, 상기 미세유체장치의 제1 반응기의 튜브 직경은 0.8 내지 2.0mm, 튜브 길이는 200 내지 300cm일 수 있고, 제2 반응기의 튜브 직경은 0.8 내지 2.0mm, 튜브 길이는 500 내지 900cm일 수 있다.In the present invention, the tube diameter of the first reactor of the microfluidic device may be 0.8 to 2.0 mm, the tube length may be 200 to 300 cm, and the tube diameter of the second reactor may be 0.8 to 2.0 mm, the tube length may be 500 to 900 cm.

본 발명의 제2 측면은 다음의 (a) 및 (b) 단계를 포함하는, 이온화 가능한 양이온성 지질인 6-((2-헥실데카노일)옥시)-N-(6-((2-헥실데카노일)옥시)헥실)-N-(4-하이드록시부틸)헥산-1-아미늄의 중간체인 6-브로모헥실 2-헥실데카노에이트의 합성 방법에 관한 것이다.A second aspect of the present invention relates to a method for synthesizing 6-bromohexyl 2-hexyldecanoate, which is an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium, which is an ionizable cationic lipid, comprising the following steps (a) and (b).

(a) 2-하이드록시데칸산, 디메틸포름아미드 및 옥살릴 클로라이드를 혼합 및 반응시켜 2-하이드록실데카노일 클로라이드를 형성하는 단계; 및(a) a step of mixing and reacting 2-hydroxydecanoic acid, dimethylformamide and oxalyl chloride to form 2-hydroxydecanoyl chloride; and

(b) 2-하이드록실데카노일 클로라이드를 3-(디메틸아미노)프로판-1,2-디올 및 6-브로모헥산올과 혼합하여 에스테르화 반응을 일으켜 6-브로모헥실 2-헥실데카노에이트를 생성하는 단계.(b) a step of mixing 2-hydroxydecanoyl chloride with 3-(dimethylamino)propane-1,2-diol and 6-bromohexanol to cause an esterification reaction to produce 6-bromohexyl 2-hexyldecanoate.

본 발명의 6-브로모헥실 2-헥실데카노에이트의 합성 방법은 전술한 이온화 가능한 양이온성 지질의 합성 방법에서 올레산 대신 2-하이드록시데칸산을 사용하고, 트리메틸아민 대신 6-브로모헥산올을 사용하는 것 이외에는 이온화 가능한 양이온성 지질의 합성 방법과 동일한 조건 및 동일한 미세유체장치에서 수행될 수 있다. 이때, 제1 반응기에서의 체류 시간은 1 내지 3분, 반응 온도는 20 내지 30 ℃일 수 있고, 제2 반응기에서의 체류 시간은 2 내지 7분일 수 있고, 상기 제2 반응기에서의 반응 온도는 20 내지 60 ℃일 수 있다.The method for synthesizing 6-bromohexyl 2-hexyldecanoate of the present invention can be performed under the same conditions and in the same microfluidic device as the method for synthesizing an ionizable cationic lipid described above, except that 2-hydroxydecanoic acid is used instead of oleic acid and 6-bromohexanol is used instead of trimethylamine. At this time, the residence time in the first reactor can be 1 to 3 minutes, and the reaction temperature can be 20 to 30°C, the residence time in the second reactor can be 2 to 7 minutes, and the reaction temperature in the second reactor can be 20 to 60°C.

본 발명의 제3 측면은 전술한 합성 방법에 의해 합성된 이온화 가능한 양이온성 지질과 디메틸 설페이트를 혼합 및 반응시켜 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트를 생성하는 단계를 포함하며,A third aspect of the present invention comprises a step of mixing and reacting an ionizable cationic lipid synthesized by the synthetic method described above with dimethyl sulfate to produce a cationic lipid methyl sulfate having a structure of chemical formula 2.

[화학식 2][Chemical formula 2]

Figure PCTKR2024007830-appb-img-000005
Figure PCTKR2024007830-appb-img-000005

상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼인, 양이온성 지질 메틸 설페이트의 합성 방법에 관한 것이다.The present invention relates to a method for synthesizing cationic lipid methyl sulfate, wherein R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical having -C(=O)O- or -S-S- introduced into the chain.

본 발명에 있어서, 상기 양이온성 지질 메틸 설페이트의 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치(2)에서 수행될 수 있고, 상기 미세유체장치(2)는: 제1 유로(110)와 제2 유로(120)가 유체적으로 연결되는 제1 미세 혼합기(100); 상기 제1 미세 혼합기(100)와 유체적으로 연결되는 제1 반응기(200); 및 상기 제1 반응기(200)와 유체적으로 연결되는 제1 배압 조절기(300)를 포함할 수 있다.In the present invention, the method for synthesizing the cationic lipid methyl sulfate can be performed in a microfluidic device (2) for a single continuous flow process, and the microfluidic device (2) can include: a first micro-mixer (100) in which a first channel (110) and a second channel (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); and a first back pressure regulator (300) fluidly connected with the first reactor (200).

상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼 및 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼에 대한 설명은 전술한 바와 동일하므로, 그 기재를 생략한다.Since the description of the above R is the same as that described above for an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical and a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical having -C(=O)O- or -S-S- introduced into the chain, its description is omitted.

본 발명에 있어서, 상기 양이온성 지질 메틸 설페이트의 합성 방법의 모든 반응물은 반응에 악영향을 미치지 않는 유기용매에 용해된 상태로 사용될 수 있다. 예를 들어, 디메틸포름아미드(DMF), 테트라하이드로퓨란(THF), 디클로로메탄 등의 유기용매를 사용할 수 있으나, 이에 한정되지 않으며, 통상의 기술자는 적절한 유기용매를 선택하여 상기 반응 단계를 수행할 수 있다.In the present invention, all reactants of the above cationic lipid methyl sulfate synthesis method can be used in a state dissolved in an organic solvent that does not adversely affect the reaction. For example, organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.

도 10 내지 12를 참조하면, 상기 미세유체장치(2)의 제1 유로(110)와 제2 유로(120)는 각각 반응물이 주입되는 유로로서, 제1 미세 혼합기(100)와 유체적으로 연결될 수 있다. 구체적으로, 상기 제1 유로(110)에는 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질, 상기 제2 유로(120)에는 디메틸 설페이트가 주입될 수 있다.Referring to FIGS. 10 to 12, the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidically connected to the first micro-mixer (100). Specifically, an ionizable cationic lipid having the structure of the chemical formula 1 can be injected into the first flow path (110), and dimethyl sulfate can be injected into the second flow path (120).

도 10 내지 12를 참조하면, 상기 제1 미세 혼합기(100)에서는 제1 유로(110)와 제2 유로(120)로부터 유입된 반응물들이 혼합되어 혼합물이 생성되며, 생성된 혼합물은 제1 미세 혼합기(100)와 유체적으로 연결되는 제1 반응기(200)로 주입될 수 있다.Referring to FIGS. 10 to 12, in the first micro-mixer (100), reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).

도 10 내지 12를 참조하면, 상기 제1 반응기(200)에서는 상기 이온화 가능한 양이온성 지질과 디메틸 설페이트가 반응하여 반응 결과물로서 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트가 생성된다.Referring to FIGS. 10 to 12, in the first reactor (200), the ionizable cationic lipid and dimethyl sulfate react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 as a reaction product.

도 10 내지 12를 참조하면, 최종 생성물인 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트는 상기 제1 반응기(200)와 유체적으로 연결되는 제1 배압 조절기(300)를 통과한 후 배출될 수 있다.Referring to FIGS. 10 to 12, the cationic lipid methyl sulfate having the structure of the chemical formula 2 as the final product can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).

상기 미세유체장치의 제1 반응기(200)와 제1 배압 조절기(300) 사이에는 제1 분광계(800)가 추가로 유체적으로 연결될 수 있다.A first spectrometer (800) may additionally be fluidly connected between the first reactor (200) and the first back pressure regulator (300) of the above microfluidic device.

본 발명에 있어서, 상기 제1 반응기(200)에서의 체류 시간은 3 내지 7분일 수 있다. 구체적으로, 상기 제1 반응기(200)에 유입된 반응물들, 즉 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질과 디메틸 설페이트가 반응을 일으켜 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트를 생성하기까지 소요되는 시간은 3 내지 7분일 수 있다. In the present invention, the residence time in the first reactor (200) may be 3 to 7 minutes. Specifically, the time required for the reactants introduced into the first reactor (200), that is, the ionizable cationic lipid having the structure of the chemical formula 1 and dimethyl sulfate, to react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 may be 3 to 7 minutes.

본 발명에 있어서, 상기 제1 반응기(200)에서의 반응 온도는 40 내지 70℃일 수 있다. In the present invention, the reaction temperature in the first reactor (200) may be 40 to 70°C.

상기 제1 반응기(200)에서의 체류 시간(즉, 반응 시간) 및 반응 온도 범위로 상기 단계의 반응을 수행하는 것이 바람직하며, 상기 범위를 벗어나는 경우 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트의 수율이 감소될 수 있다.It is preferable to perform the reaction of the above step within the range of the residence time (i.e., reaction time) and reaction temperature in the above first reactor (200), and if it is outside the range, the yield of the cationic lipid methyl sulfate having the structure of the chemical formula 2 may decrease.

상기 제1 반응기(200)에서의 체류 시간은 상기 제1 유로에 주입되는 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 포함하는 용액과 디메틸 설페이트를 포함하는 용액을 각각 0.4 내지 2.0ml/분의 유속으로 제1 미세 혼합기에 주입함으로써 달성될 수 있다.The residence time in the first reactor (200) can be achieved by injecting a solution containing an ionizable cationic lipid having the structure of the chemical formula 1 and a solution containing dimethyl sulfate into the first micro-mixer at a flow rate of 0.4 to 2.0 ml/min, respectively, into the first flow path.

본 발명에 있어서, 상기 양이온성 지질 메틸 설페이트의 합성 방법은 용매 증발 및 재결정화 단계를 추가로 포함할 수 있다. 이때, 상기 재결정화는 아세톤 용매에서 수행될 수 있으나, 상기 양이온성 지질 메틸 설페이트의 재결정화에 적합한 용매에서 제한 없이 수행될 수 있다.In the present invention, the method for synthesizing the cationic lipid methyl sulfate may additionally include a solvent evaporation and recrystallization step. At this time, the recrystallization may be performed in an acetone solvent, but may be performed without limitation in a solvent suitable for recrystallization of the cationic lipid methyl sulfate.

본 발명에 있어서, 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트는 다음의 (1) 내지 (7)로 이루어진 군으로부터 선택되는 어느 하나일 수 있다:In the present invention, the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one selected from the group consisting of the following (1) to (7):

(1) 1,2-디올레오일-3-트리메틸암모늄-프로판 메틸 설페이트;(1) 1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate;

(2) N,N,N-트리메틸-2,3-비스((9Z,12Z)-옥타데카-9,12-디에노일옥시)프로판-1-아미늄 메틸 설페이트;(2) N,N,N-Trimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propane-1-aminum methyl sulfate;

(3) 2,3-비스((10-(헥실옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(3) 2,3-Bis((10-(hexyloxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(4) 2,3-비스((10-(((S)-3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(4) 2,3-Bis((10-(((S)-3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(5) 2,3-비스((10-(((E)-3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(5) 2,3-Bis((10-(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate;

(6) 2,3-비스((10-(((Z)-3,7-디메틸옥타-2,6-디엔-1-일)옥소)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트; 및 (6) 2,3-bis((10-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)oxo)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate; and

(7) N,N,N-트리메틸-2,3-비스((3-(옥타데실디설파닐)프로파노일)옥시)프로판-1-아미늄 메틸 설페이트.(7) N,N,N-Trimethyl-2,3-bis((3-(octadecyldisulfanyl)propanoyl)oxy)propan-1-aminum methyl sulfate.

다만, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질의 종류에 따라 보다 다양한 양이온성 지질 메틸 설페이트를 합성할 수 있음이 자명하다.However, it is clear that a wider variety of cationic lipid methyl sulfates can be synthesized depending on the type of ionizable cationic lipid having the structure of the above chemical formula 1.

본 발명의 구체적인 일 실시예에서는 양이온성 지질인 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 메틸 설페이트를 합성하기 위해, 유동 화학에서 디메틸 설페이트를 사용하여 합성된 DODAP(2)의 알킬화를 선택하였다. 반응은 HPLC 펌프를 사용하여 시약의 유속을 변경함으로써 R1 반응기에서의 다양한 체류 시간에서 최적화되었다. 초기 실험에서는 신속하고 완전한 알킬화가 일어나기 위해서는 더 높은 당량의 디메틸 설페이트가 필수적이라는 사실이 밝혀졌다. 따라서, 0.2M의 DODAP(2) 용액과 CH2Cl2에 용해된 2.0M 디메틸 설페이트 용액을 별도로 제조하여 T-혼합기 M1(Ø = 1.0mm)를 통해 미세유체 반응기 R1(Ø = 1.0mm, L = 510cm)에 공급하였다. 몇 가지 최적화 후에, DODAP(2)의 완전한 알킬화는 60 ℃에서 10당량의 디메틸 설페이트를 사용하고 R1 반응기에서 5분의 체류 시간을 사용함으로써 달성되었다. 혼합 효율의 본질적인 우수성으로 인한 가속화된 반응 동역학은 알킬화를 완료하는 데 48시간이 걸리는 배치 공정에 비해 매우 유리하다. 최종적으로, 용매 증발 및 아세톤에서의 재결정화 후에 순수한 DOTAP 메틸 설페이트(4)가 90% 수율로 순조롭게 얻어졌다. 특히, 테트라하이드로푸란과 아세토니트릴을 반응 용매로 사용하면 전환율이 좋지 않았다.In a specific embodiment of the present invention, the alkylation of DODAP(2) synthesized using dimethyl sulfate in flow chemistry was selected to synthesize cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate. The reaction was optimized at various residence times in the R1 reactor by varying the flow rates of the reagents using an HPLC pump. Initial experiments revealed that higher equivalents of dimethyl sulfate were essential for rapid and complete alkylation. Therefore, 0.2 M DODAP(2) solution and 2.0 M dimethyl sulfate solution in CH2Cl2 were prepared separately and fed into the microfluidic reactor R1 (Ø = 1.0 mm, L = 510 cm) through a T-mixer M1 (Ø = 1.0 mm). After some optimization, complete alkylation of DODAP (2) was achieved by using 10 equivalents of dimethyl sulfate at 60 °C and a residence time of 5 min in the R1 reactor. The accelerated reaction kinetics due to the inherent superiority in mixing efficiency are highly advantageous compared to the batch process which takes 48 h to complete the alkylation. Finally, pure DOTAP methyl sulfate (4) was smoothly obtained in 90% yield after solvent evaporation and recrystallization from acetone. In particular, the conversion was poor when tetrahydrofuran and acetonitrile were used as reaction solvents.

본 발명의 구체적인 다른 일 실시예에서는 또 다른 이온화 가능한 지질 또는 이온화 가능한 지질의 긴 사슬에 에스테르 또는 이황화물 작용기가 도입된 지방산인 (9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트), O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트) 및 3-(디메틸아미노)프로판-1,2-디일 비스(3-(헵타데실디설파닐)프로파노에이트)를 각가 사용하여, N,N,N-트리메틸-2,3-비스((9Z,12Z)-옥타데카-9,12-디에노일옥시)프로판-1-아미늄 메틸 설페이트(DLinDAP), 2,3-비스((10-(헥실옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트, 2,3-비스((10-(((S)-3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트, 2,3-비스((10-(((Z)-3,7-디메틸옥타-2,6-디엔-1-일)옥소)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트 및 9 N,N,N-트리메틸-2,3-비스((3-(옥타데실디설파닐)프로파노일)옥시)프로판-1-아미늄 메틸 설페이트를 합성하였다.In another specific embodiment of the present invention, another ionizable lipid or a fatty acid having an ester or disulfide functional group introduced into a long chain of an ionizable lipid is (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-Bis((E)-3,7-dimethylocta-2,6-dien-1-yl)bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-Bis((Z)-3,7-dimethylocta-2,6-dien-1-yl)bis(decanedioate) and 3-(dimethylamino)propane-1,2-diyl bis(3-(heptadecyldisulfanyl)propanoate) were used respectively to prepare N,N,N-trimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propane-1-aminum methyl sulfate (DLinDAP). 2,3-Bis((10-(hexyloxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminium methyl sulfate, 2,3-bis((10-(((S)-3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminium methyl sulfate, 2,3-bis((10-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)oxo)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminium methyl sulfate and 9 N,N,N-trimethyl-2,3-bis((3-(octadecyldisulfanyl)propanoyl)oxy)propan-1-aminium Methyl sulfate was synthesized.

그 결과, 도 12에서 확인되는 바와 같이 상기 7종의 양이온성 지질을 88 내지 90% 수율로 얻을 수 있음을 확인하였다.As a result, as confirmed in Fig. 12, it was confirmed that the seven types of cationic lipids could be obtained with a yield of 88 to 90%.

본 발명의 구체적인 또 다른 일 실시예에서는 내경을 1.6mm로 증가시킨 제1 반응기(200)를 사용하여 양이온성 지질 메틸 설페이트 라이브러리의 생산성을 향상시키고자 하였다. DOTAP 메틸 설페이트(4)의 생산성을 향상시키기 위해, 2.0mL/분의 혼합 유속으로 내경 1.6mm PTFE 튜브 반응기 R1(L = 500cm)을 사용한 추가 실험에서 5분당 0.67g의 DOTAP 메틸 설페이트(4)가 생성되어 1시간의 작업 시간 동안 거의 8g 규모를 달성하였다.In another specific embodiment of the present invention, the productivity of the cationic lipid methyl sulfate library was improved by using the first reactor (200) with the inner diameter increased to 1.6 mm. In order to improve the productivity of DOTAP methyl sulfate (4), in an additional experiment using a PTFE tube reactor R1 (L = 500 cm) with an inner diameter of 1.6 mm at a mixing flow rate of 2.0 mL/min, 0.67 g of DOTAP methyl sulfate (4) was produced per 5 min, achieving a scale of nearly 8 g during an operation time of 1 hour.

본 발명의 제4 측면은 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물과 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드를 혼합 및 반응시켜 스테글리치 에스테르화 반응을 일으켜 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드를 생성하는 단계를 포함하는, 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드의 합성 방법에 관한 것이다.A fourth aspect of the present invention relates to a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.

DOTAP 메틸 설페이트(4)는 노동력과 비용을 필요로 하는 Amberlite® IRA-400(Cl) 이온 교환 수지로 처리하여 DOTAP 클로라이드(5)로 전환될 수 있다. 알킬화제로서 메틸 클로라이드를 사용하지 않고 DOTAP 클로라이드(5)를 직접 합성하는 것은 DMAP가 염기로 존재하는 상태에서, CH2Cl2에 용해된, 시판되고 쉽게 합성 가능한 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드(23)를 사용하여 올레오일 클로라이드를 에스테르화함으로써 배치 반응기에서 달성될 수 있다. 그러나, 디올(23)은 디메틸 설폭사이드(DMSO)를 제외한 일반적인 유기용매에 용해되지 않아 유동 화학을 수행하기 어려우며, 더욱이 DMSO는 아실 클로라이드를 이용한 에스테르화 반응에 적합한 용매가 아니었다. DOTAP methyl sulfate (4) can be converted to DOTAP chloride (5) by treatment with Amberlite® IRA-400(Cl ) ion exchange resin, which is labor- and cost-intensive. Direct synthesis of DOTAP chloride (5) without using methyl chloride as an alkylating agent can be accomplished in a batch reactor by esterifying oleoyl chloride with commercially available and readily synthesizable 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) dissolved in CH 2 Cl 2 in the presence of DMAP as a base. However, diols (23) are insoluble in common organic solvents except dimethyl sulfoxide (DMSO), making flow chemistry difficult to perform, and furthermore, DMSO was not a suitable solvent for esterification with acyl chlorides.

대안적으로, DMSO에서 DMAP의 존재 하에 올레산과 디올(23)의 N-(3-디메틸아미노프로필)-N′-에틸카르보디이미드 하이드로클로라이드(EDC.HCl) 매개 스테글리치 에스테르화 반응을 통해 16시간 동안 DOTAP 클로라이드(5)를 78% 수율로 얻었다. 따라서, DOTAP 클로라이드(5)의 직접 합성을 위해 연속 유동 반응기에서 스테글리치 에스테르화 반응을 탐구할 가치가 있다. 내부 직경이 1.0mm인 PTFE 튜브 반응기를 포함하는 실험 셋팅이 도 14에 나타나 있다. 디올(23)은 3-클로로프로판-1,2-디올과 메탄올 용매에 용해된 트리메틸아민의 에탄올 용액을 환류하여 배치에서 처음 제조되었다.Alternatively, DOTAP chloride (5) was obtained in 78% yield via N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl)-mediated Steglic esterification of oleic acid and diol (23) in the presence of DMAP in DMSO in 16 h. Therefore, it is worth exploring the Steglic esterification reaction in a continuous flow reactor for the direct synthesis of DOTAP chloride (5). The experimental setup including a PTFE tube reactor with an inner diameter of 1.0 mm is shown in Figure 14. Diol (23) was initially prepared in batch by refluxing an ethanolic solution of trimethylamine dissolved in 3-chloropropane-1,2-diol and methanol solvent.

본 발명에 있어서, 상기 DOTAP 클로라이드(5)의 합성 방법의 모든 반응물은 반응에 악영향을 미치지 않는 유기용매에 용해된 상태로 사용될 수 있다. 예를 들어, 디메틸 설폭사이드(DMSO)를 사용할 수 있으나, 이에 한정되지 않으며, 통상의 기술자는 적절한 유기용매를 선택하여 상기 반응 단계를 수행할 수 있다.In the present invention, all reactants of the method for synthesizing the DOTAP chloride (5) can be used in a state dissolved in an organic solvent that does not adversely affect the reaction. For example, dimethyl sulfoxide (DMSO) can be used, but is not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.

도 13 및 14를 참조하면, 상기 미세유체장치(2)의 제1 유로(110)와 제2 유로(120)는 각각 반응물이 주입되는 유로로서, 제1 미세 혼합기(100)와 유체적으로 연결될 수 있다. 구체적으로, 상기 제1 유로(110)에는 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물, 상기 제2 유로(120)에는 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드가 주입될 수 있다.Referring to FIGS. 13 and 14, the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidly connected to the first micro-mixer (100). Specifically, a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride, and DMAP can be injected into the first flow path (110), and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride can be injected into the second flow path (120).

도 13 및 14를 참조하면, 상기 제1 미세 혼합기(100)에서는 제1 유로(110)와 제2 유로(120)로부터 유입된 반응물들이 혼합되어 혼합물이 생성되며, 생성된 혼합물은 제1 미세 혼합기(100)와 유체적으로 연결되는 제1 반응기(200)로 주입될 수 있다.Referring to FIGS. 13 and 14, in the first micro-mixer (100), reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).

도 13 및 14를 참조하면, 상기 제1 반응기(200)에서는 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물 및 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드가 반응하여 반응 결과물로서 DOTAP 클로라이드(5)가 생성된다.Referring to FIGS. 13 and 14, in the first reactor (200), a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride, and DMAP and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride react to produce DOTAP chloride (5) as a reaction product.

도 13 및 14를 참조하면, 최종 생성물인 DOTAP 클로라이드(5)는 상기 제1 반응기(200)와 유체적으로 연결되는 제1 배압 조절기(300)를 통과한 후 배출될 수 있다.Referring to FIGS. 13 and 14, the final product, DOTAP chloride (5), can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).

본 발명에 있어서, 상기 제1 반응기(200)에서의 체류 시간은 3 내지 7분일 수 있다. 구체적으로, 상기 제1 반응기(200)에 유입된 반응물들, 즉 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물 및 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드가 반응을 일으켜 DOTAP 클로라이드(5)를 생성하기까지 소요되는 시간은 3 내지 7분일 수 있다.In the present invention, the residence time in the first reactor (200) may be 3 to 7 minutes. Specifically, the time required for the reactants introduced into the first reactor (200), that is, the mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP, and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride, to react to produce DOTAP chloride (5) may be 3 to 7 minutes.

본 발명에 있어서, 상기 제1 반응기(200)에서의 반응 온도는 60 내지 80℃일 수 있다. In the present invention, the reaction temperature in the first reactor (200) may be 60 to 80°C.

상기 제1 반응기(200)에서의 체류 시간(즉, 반응 시간) 및 반응 온도 범위로 상기 단계의 반응을 수행하는 것이 바람직하며, 상기 범위를 벗어나는 경우 DOTAP 클로라이드(5)의 수율이 감소될 수 있다.It is preferable to perform the reaction of the above step within the range of the residence time (i.e., reaction time) and reaction temperature in the above first reactor (200), and if it is outside the range, the yield of DOTAP chloride (5) may decrease.

상기 제1 반응기(200)에서의 체류 시간은 상기 제1 유로에 주입되는 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP를 포함하는 용액과 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드를 포함하는 용액을 0.4 내지 1.5ml/분의 유속으로 제1 미세 혼합기에 주입함으로써 달성될 수 있다.The residence time in the first reactor (200) can be achieved by injecting a solution containing oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP into the first flow path and a solution containing (3-dimethylamino-propyl)-ethyl-carbodiimide chloride into the first micro-mixer at a flow rate of 0.4 to 1.5 ml/min.

본 발명에 있어서, 상기 DOTAP 클로라이드(5)의 합성 방법은 용매 증발 및 재결정화 단계를 추가로 포함할 수 있다. 이때, 상기 재결정화는 이소프로판올 및 아세톤 용매에서 수행될 수 있으나, 상기 DOTAP 클로라이드(5)의 재결정화에 적합한 용매에서 제한 없이 수행될 수 있다.In the present invention, the method for synthesizing the DOTAP chloride (5) may additionally include solvent evaporation and recrystallization steps. At this time, the recrystallization may be performed in isopropanol and acetone solvents, but may be performed without limitation in a solvent suitable for recrystallization of the DOTAP chloride (5).

본 발명의 구체적인 일 실시예에서는 DMSO에 용해된 0.65 M 올레산, 0.3 M 디올(23) 및 0.1 M DMAP의 혼합물을 DMSO에 용해된 0.65 M EDC.HCl 용액과 T-혼합기(Ø = 1.0 mm)를 이용하여 혼합한 후 에스테르화가 일어나도록 반응기 R1(Ø = 1.0 mm, L = 640 cm)을 통과시켰다. R1에서의 반응은 주사기 펌프를 사용하여 시약의 유속을 변경함으로써 다양한 온도와 다양한 체류 시간에서 최적화되었다. 처음에는 실온에서 15분의 체류 시간으로 디올(23)의 불완전한 디-에스테르화로 인해 DOTAP 클로라이드(5)의 수율이 낮았다. 그러나, 디-에스테르화를 더욱 촉진하기 위해 온도를 70 ℃로 올리면, DOTAP 클로라이드(5)는 배치 공정의 16시간 보다 훨씬 짧은 R1에서의 5.0분의 체류 시간만으로 정제 후 88% 수율로 얻어졌다. In a specific embodiment of the present invention, a mixture of 0.65 M oleic acid, 0.3 M diol (23) and 0.1 M DMAP dissolved in DMSO was mixed with 0.65 M EDC.HCl solution dissolved in DMSO using a T-mixer (Ø = 1.0 mm) and then passed through a reactor R1 (Ø = 1.0 mm, L = 640 cm) to cause esterification. The reaction in R1 was optimized at various temperatures and residence times by varying the flow rate of the reagents using a syringe pump. Initially, the yield of DOTAP chloride (5) was low due to incomplete de-esterification of the diol (23) with a residence time of 15 min at room temperature. However, when the temperature was raised to 70 °C to further promote the de-esterification, DOTAP chloride (5) was obtained in 88% yield after purification with only 5.0 min of residence time in R1, which was much shorter than the 16 h of the batch process.

본 발명의 구체적인 또 다른 일 실시예에서는 내경을 1.6mm로 증가시킨 제1 반응기(200)를 사용하여 DOTAP 클로라이드(5)의 생산성을 향상시키고자 하였다. DOTAP 클로라이드(5)의 생산성을 향상시키기 위해, 2.0mL/분의 혼합 유속으로 1.6mm PTFE 튜브 반응기(L = 500cm)를 사용하였다. 유동 반응기에서의 이러한 크기 증가 방법은 5분당 0.84g의 생산성을 제공하여 결국 시간당 거의 10.2g 규모를 나타내었다.In another specific embodiment of the present invention, the productivity of DOTAP chloride (5) was improved by using a first reactor (200) with an inner diameter increased to 1.6 mm. In order to improve the productivity of DOTAP chloride (5), a 1.6 mm PTFE tube reactor (L = 500 cm) was used at a mixing flow rate of 2.0 mL/min. This size increase method in the flow reactor provided a productivity of 0.84 g per 5 min, which ultimately resulted in a scale of nearly 10.2 g per hour.

본 발명의 제5 측면은 다음의 (a) 및 (b) 단계를 포함하는 지질 나노입자의 제조방법에 관한 것이다:A fifth aspect of the present invention relates to a method for producing lipid nanoparticles, comprising the following steps (a) and (b):

(a) 전술한 합성 방법으로 이온화 가능한 양이온성 지질을 합성하는 단계; 및(a) a step of synthesizing an ionizable cationic lipid by the synthetic method described above; and

(b) 상기 합성된 이온화 가능한 양이온성 지질, 헬퍼(helper) 지질, 콜레스테롤 및 폴리에틸렌 글리콘 지질을 포함하는 용액을 완충액과 혼합하여 지질 나노입자를 형성하는 단계.(b) a step of forming lipid nanoparticles by mixing a solution containing the synthesized ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid with a buffer solution.

본 발명의 지질 나노입자의 제조방법에 있어서, 상기 (a) 단계에 대한 설명은 전술한 바와 동일하므로, 그 기재를 생략한다.In the method for manufacturing lipid nanoparticles of the present invention, the description of step (a) is the same as described above, so its description is omitted.

본 발명의 지질 나노입자의 제조방법에 있어서, 상기 (b) 단계에서 사용되는 이온화 가능한 양이온성 지질은 전술한 방법에 의해 합성되는 모든 종류의 이온화 가능한 양이온성 지질을 포함할 수 있다.In the method for producing lipid nanoparticles of the present invention, the ionizable cationic lipid used in step (b) may include all types of ionizable cationic lipids synthesized by the above-described method.

본 발명의 지질 나노입자의 제조방법에 있어서, 상기 (b) 단계에서 사용되는 헬퍼 지질은 DMPC(1,2-디미리스토일-sn-글리세로-3-포스파티딜콜린), DOPC(1,2-디올레오일-sn-글리세로-3-포스포콜린), DOPI(1,2-디올레오일-sn-글리세로-3-포스포-(1'-미오-이노시톨)), DOPE(1,2-디올레오일-sn-글리세로-3-포스포에탄올아민), DPPC(1,2-디팔미토일-sn-글리세로-3-포스포콜린), DSPC(1,2-디스테아로일-sn-글리세로-3-포스포콜린), DSPI(1,2-디스테아로일-sn-글리세로-3-포스포이노시톨), 및 DLPC(1,2-디리노레오일-sn-글리세로-3-포스포콜린)으로 이루어진 군으로부터 선택되는 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.In the method for producing lipid nanoparticles of the present invention, the helper lipids used in the step (b) are DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPI (1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPI (1,2-distearoyl-sn-glycero-3-phosphoinositol), and It may be at least one selected from the group consisting of DLPC (1,2-dirinoleyloyl-sn-glycero-3-phosphocholine), but is not limited thereto.

본 발명에 있어서, 상기 폴리에틸렌 글리콘(PEG) 지질은 PEG-수식 포스파티딜에탄올아민, PEG-수식 포스파티드산, PEG-수식 세라마이드, PEG-수식 디알킬아민, PEG-수식 디아실글리세롤, 및 PEG-수식 디알킬글리세롤로 이루어진 군으로부터 선택되는 1종 이상일 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 PEG 지질은 100 Da 내지 20 kDa의 크기를 가지는 PEG 부분을 포함하는 것이 바람직하고, 더욱 바람 직하게는 DMG-PEG2000(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPE-PEG2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], 및 Ceramide PEG2000(N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]})로 이루어진 군으로부터 선택되는 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the polyethylene glycol (PEG) lipid may be at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, but is not limited thereto. In addition, it is preferable that the PEG lipid includes a PEG moiety having a size of 100 Da to 20 kDa, and more preferably, it may be at least one selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and Ceramide PEG2000 (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}), but is not limited thereto.

본 발명에 있어서, 상기 이온화 가능한 양이온성 지질, 헬퍼(helper) 지질, 콜레스테롤 및 폴리에틸렌 글리콘 지질의 몰비는 40~60 : 5~15 : 30~45 : 1~3일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the molar ratio of the ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid may be, but is not limited to, 40-60 : 5-15 : 30-45 : 1-3.

본 발명의 지질 나노입자의 제조방법에 있어서, 상기 (b) 단계에 사용되는 완충액은 아세트산 나트륨 완충액, 시트르산 나트륨 완충액, 인산완충식염수(PBS) 또는 시트르산(citrate) 완충액일 수 있으나, 이에 제한되는 것은 아니다.In the method for producing lipid nanoparticles of the present invention, the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, sodium citrate buffer, phosphate buffered saline (PBS), or citrate buffer.

본 발명의 제6 측면은 다음의 (a) 및 (b) 단계를 포함하는 지질 나노 리포좀의 제조방법에 관한 것이다:A sixth aspect of the present invention relates to a method for preparing lipid nano liposomes comprising the following steps (a) and (b):

(a) 전술한 방법으로 이온화 가능한 양이온성 지질, 양이온성 지질, 또는 DOTAP 클로라이드를 합성하는 단계; 및 (a) synthesizing an ionizable cationic lipid, cationic lipid, or DOTAP chloride by the method described above; and

(b) 상기 합성된 이온화 가능한 양이온성 지질, 양이온성 지질 또는 DOTAP와 콜레스테롤을 포함하는 용액을 완충액과 혼합하여 지질 나노 리포좀을 형성하는 단계.(b) a step of forming lipid nanoliposomes by mixing the solution containing the synthesized ionizable cationic lipid, cationic lipid or DOTAP and cholesterol with a buffer solution.

본 발명의 지질 나노 리포좀의 제조방법에 있어서, 상기 (a) 단계에 대한 설명은 전술한 바와 동일하므로, 그 기재를 생략한다.In the method for producing the lipid nano liposome of the present invention, the description of step (a) is the same as described above, so its description is omitted.

본 발명의 지질 나노 리포좀의 제조방법에 있어서, 상기 (b) 단계에서 사용되는 이온화 가능한 양이온성 지질 또는 양이온성 지질은 전술한 방법에 의해 합성되는 모든 종류의 이온화 가능한 양이온성 지질 도는 양이온성 지질을 포함할 수 있다.In the method for producing a lipid nano liposome of the present invention, the ionizable cationic lipid or cationic lipid used in step (b) may include all types of ionizable cationic lipid or cationic lipid synthesized by the above-described method.

본 발명에 있어서, 상기 이온화 가능한 양이온성 지질, 양이온성 지질 또는 DOTAP : 콜레스테롤의 몰비는 25 ~ 75 : 25 ~ 75일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the molar ratio of the ionizable cationic lipid, cationic lipid or DOTAP:cholesterol may be 25 to 75:25 to 75, but is not limited thereto.

본 발명의 지질 나노 리포좀의 제조방법에 있어서, 상기 (b) 단계에 사용되는 완충액은 아세트산 나트륨 완충액, Tris 완충액, 인산완충식염수(PBS) 또는 시트르산(citrate) 완충액일 수 있으나, 이에 제한되는 것은 아니다.In the method for producing lipid nano liposomes of the present invention, the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, Tris buffer, phosphate buffered saline (PBS), or citrate buffer.

DODAP 및 DOTAP 클로라이드는 리포좀 또는 지질 나노입자 형태를 취하는 생체모방 분자로, 강화된 엔도솜 탈출에 의해 유전적 및 다양한 치료제의 효율적인 전달을 가능하게 한다. 치료제를 캡슐화하는 나노입자의 균일한 크기는 세포와의 상호작용에 의한 전달 측면에서 매우 중요하다. 나노입자 담체의 요구에 따른 제조는 혼합기 종류, 공급 속도 및 조성과 같은 복잡한 변수가 나노침전 공정에 관련되어 있기 때문에 어렵다.DODAP and DOTAP chloride are biomimetic molecules that take the form of liposomes or lipid nanoparticles, enabling efficient delivery of genetic and various therapeutics by enhanced endosomal escape. The uniform size of the nanoparticles encapsulating the therapeutics is very important in terms of delivery by interaction with cells. The on-demand manufacturing of nanoparticle carriers is difficult because complex variables such as mixer type, feed rate, and composition are involved in the nanoprecipitation process.

본 발명의 구체적인 실시예에서는 보고된 것과 유사한 4가지 유형의 성분, 즉 이온화 가능한 지질, 헬퍼 지질, 콜레스테롤 및 PEG-지질을 사용하여 빈-LNP(empty LNP)를 합성하였다. 리포좀의 경우, 주요 지질에 콜레스테롤만을 첨가하였다. 각각의 최종 지질 용액은 자체 제작 미세 혼합기를 통해 완충액과 빠르게 혼합하고 투석을 포함한 정제 공정 전후에 동적 광산란(DLS)을 사용하여 추가 분석하였다(도 15의 A). 약산성 25mM 아세트산 나트륨 완충액(pH 5.5)은 핵산 캡슐화를 위한 DODAP의 잠재력을 입증하는 데 사용되었으며, 이온 강도가 낮은 10mM Tris 완충액(pH 7.2)은 100nm 미만 크기의 균일한 DOTAP 리포좀을 생성하는 데 중요했다.In a specific embodiment of the present invention, empty LNPs were synthesized using four types of components similar to those reported, i.e., ionizable lipid, helper lipid, cholesterol, and PEG-lipid. For liposomes, only cholesterol was added as the main lipid. Each final lipid solution was rapidly mixed with buffer using a home-made micromixer and further analyzed using dynamic light scattering (DLS) before and after the purification process including dialysis (Fig. 15A). Mildly acidic 25 mM sodium acetate buffer (pH 5.5) was used to demonstrate the potential of DODAP for nucleic acid encapsulation, while low ionic strength 10 mM Tris buffer (pH 7.2) was important for generating uniform DOTAP liposomes with a size of less than 100 nm.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[준비예 1][Preparation Example 1]

시약 준비Reagent preparation

디클로로메탄(DCM), 디메틸 설폭사이드(DMSO), 테트라히드로푸란(THF), 아세토니트릴(ACN), 메탄올(MeOH), 디메틸포름아미드(DMF) 및 무수 에탄올(EtOH)은 Sigma-Aldrich에서 구입하였다. 올레산(99%), 리놀레산(99%), 팔미트산(99%), 스테아르산(99%), 미리스트산(99%), 시트로넬롤, 게라니올, 네롤, 세빅산, 6-브로모헥산-1-올, 2-헥실데칸산, (±)-3-클로로-1,2-프로판디올, 3-(디메틸아미노)-1,2-프로판디올, 옥살릴 클로라이드, 및 트리에틸아민은 Sigma-Aldrich에서 구입하였다. 1,2-디스테아로일-sn-글리세로-3-포스포콜린(DSPC), 콜레스테롤(Chol) 및 1,2-디미리스토일-rac-글리세로-3-메톡시폴리에틸렌 글리콜-2000(DMG-PEG 2000)은 Avanti Polar Lipids, Inc.에서 구입하였다.Dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile (ACN), methanol (MeOH), dimethylformamide (DMF), and absolute ethanol (EtOH) were purchased from Sigma-Aldrich. Oleic acid (99%), linoleic acid (99%), palmitic acid (99%), stearic acid (99%), myristic acid (99%), citronellol, geraniol, nerol, sebic acid, 6-bromohexan-1-ol, 2-hexyldecanoic acid, (±)-3-chloro-1,2-propanediol, 3-(dimethylamino)-1,2-propanediol, oxalyl chloride, and triethylamine were purchased from Sigma-Aldrich. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) were purchased from Avanti Polar Lipids, Inc.

[준비예 2][Preparation Example 2]

분광학적 동정Spectroscopic identification

핵자기공명(NMR) 스펙트럼은 Bruker Avance III(1H NMR의 경우 500MHz, 13C NMR의 경우 125MHz)에서 기록되었다. 1H 및 13C 화학적 이동은 달리 명시되지 않는 한 CDCl3에서 표준으로 Me4Si의 다운필드(ppm)로 기록되었다. 다중도(Multiplicity)는 다음 약어를 사용하여 보고되었다: s = 단일선, d = 2중선, t = 3중선, q = 4중선, quint = 5중선, sext = 6중선, sept = 7중선, m = 다중선, br = 넓은 공명. 별도의 언급이 없는 한, 모든 시판되는 물질은 추가적인 정제 없이 사용하였다. 인라인 FT-IR 스펙트럼은 ZnSe 윈도우(경로 길이 0.1mm)를 기반으로 하는 밀봉된 유동 셀 액세서리(Specac®)가 장착된 일반적인 FT-IR 분광계(Jasco FT/IR-4600)에서 기록되었다. BRUKER MALDI-TOF(matrix assisted laser desorption/ionization time-of-flight) 질량 분석기를 사용하여 지질의 질량을 확인하였다.Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III (500 MHz for 1 H NMR and 125 MHz for 13 C NMR). 1 H and 13 C chemical shifts are reported in ppm downfield of Me 4 Si as standard in CDCl 3 unless otherwise stated. Multiplicities are reported using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, quint = pentet, sext = sextet, sept = septet, m = multiplet, br = broad resonance. Unless otherwise stated, all commercially available materials were used without further purification. Inline FT-IR spectra were recorded on a routine FT-IR spectrometer (Jasco FT/IR-4600) equipped with a sealed flow cell accessory (Specac®) based on ZnSe windows (pathlength 0.1 mm). The masses of lipids were confirmed using a BRUKER MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer.

[준비예 3][Preparation Example 3]

반응기 시스템용 부품Components for reactor systems

T형 미세 혼합기(Ø = 1.0mm)는 Sanko Seiki Co.에서 구입하였다. 고순도 PTFE 튜브(Ø = 0.1 cm, IDEX HEALTH & SCIENCE)와 배압 조절기(40, 100, 250 psi, IDEX HEALTH & SCIENCE)는 미세 튜브 반응기로 적절한 길이로 절단하여 사용하였다.A T-type micro-mixer (Ø = 1.0 mm) was purchased from Sanko Seiki Co. High-purity PTFE tubing (Ø = 0.1 cm, IDEX HEALTH & SCIENCE) and a back pressure regulator (40, 100, 250 psi, IDEX HEALTH & SCIENCE) were cut to an appropriate length for use in the micro-tube reactor.

[실시예 1][Example 1]

DODAP의 단일 유동 합성Single-flow synthesis of DODAP

1-1. 인라인 FTIR 모니터링을 통한 올레오일 클로라이드의 연속 유동 합성1-1. Continuous flow synthesis of oleoyl chloride using inline FTIR monitoring

DODAP(2)는 단일-유동 이온화 가능한 지질 라이브러리 합성을 위한 모분자로 간주되었다. 따라서, 고품질 합성 DODAP(2)를 달성하기 위해 연속 유동 시스템에서 올레오일 클로라이드를 합성하기로 첫 번째로 결정되었다. 상업적으로 이용 가능한 올레오일 클로라이드의 순도는 85%에 불과하다. 유동 화학에서 올레오일 클로라이드 합성 최적화를 위한 실험 셋팅은 도 3에 나타내었다. DODAP(2) was considered as a parent molecule for the synthesis of a single-flow ionizable lipid library. Therefore, it was first decided to synthesize oleoyl chloride in a continuous flow system to achieve high-quality synthetic DODAP(2). The purity of commercially available oleoyl chloride is only 85%. The experimental setup for the optimization of oleoyl chloride synthesis in flow chemistry is shown in Fig. 3.

반응은 내경 1.0mm PTFE 튜브로 구성된 유동 시스템에서 최적화되었다. 구체적으로, 유동 시스템은 T자형 미세 혼합기(M1, Ø = 1.0 mm), 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.0 mm, L = 225 cm) 및 100psi 및 40psi 배압 조절기로 구성된다.The reaction was optimized in a flow system consisting of a PTFE tube with an inner diameter of 1.0 mm. Specifically, the flow system consists of a T-shaped micro-mixer (M1, Ø = 1.0 mm), a micro-tube reactor R1 (PTFE tube, Ø = 1.0 mm, L = 225 cm), and 100 psi and 40 psi back pressure regulators.

CH2Cl2에 용해된 올레산(0.35M) 및 DMF(0.05M) 용액과 CH2Cl2에 용해된 옥살릴 클로라이드(0.35M) 용액을 HPLC 펌프를 사용하여 T자형 믹서 M1(Ø = 1.0mm)을 통해 반응기 R1에 주입하였다. 그런 다음, 용액을 반응기 R1에 연결된 FT-IR 샘플 셀에 통과시켰다. 100psi 배압 조절기는 FTIR 유동 셀의 배출구에 연결되었다. HPLC 펌프를 사용하여 시약의 유속을 변경함으로써 반응기 R1에서의 체류 시간을 변경하였다. 정상 상태(steady state)에 도달한 후, 인라인(in-line) FTIR 모니터링을 사용하여 생성된 용액을 모니터링하였다(도 4). Solutions of oleic acid (0.35 M) and DMF (0.05 M) dissolved in CH 2 Cl 2 and solutions of oxalyl chloride (0.35 M) dissolved in CH 2 Cl 2 were injected into the reactor R1 through a T-mixer M1 (Ø = 1.0 mm) using an HPLC pump. Then, the solutions were passed into the FT-IR sample cell connected to the reactor R1. A 100 psi back pressure regulator was connected to the outlet of the FTIR flow cell. The residence time in the reactor R1 was changed by changing the flow rates of the reagents using an HPLC pump. After reaching steady state, the produced solutions were monitored using in-line FTIR monitoring (Fig. 4).

IR 분광법은 1780 cm-1에서 특징적인 CO 스트레칭 밴드를 갖는 산 클로라이드의 형성과 올레산의 산 작용기의 1708 cm-1에서 CO 스트레칭의 소멸을 모니터링하였다. 몇 가지 최적화 후에, 올레오일 클로라이드를 형성하기 위한 각 시약의 유속이 0.6ml/분인 반응기 R1에서 1.5분의 체류 시간 내에 올레산의 완전한 소비가 관찰되었다(도 5).IR spectroscopy monitored the formation of acid chloride with a characteristic C-O stretching band at 1780 cm -1 and the disappearance of the C-O stretching band of the acid functional group of oleic acid at 1708 cm -1 . After some optimization, complete consumption of oleic acid was observed within a residence time of 1.5 min in reactor R1 at a flow rate of 0.6 ml/min of each reagent to form oleoyl chloride (Fig. 5).

1-2. 모세관 반응기에서의 DODAP(2) 단일 유동 합성1-2. Single-flow synthesis of DODAP(2) in a capillary reactor

유동 시스템은 T자형 미세 혼합기(M1 & M2, Ø = 1.0 mm), 2개의 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.0 mm, L = 195 cm)과 R2(PTFE 튜브, Ø = 1.0mm), 및 100psi 및 40psi 배압 조절기로 구성된다. FTIR 샘플 셀은 인라인 FTIR 모니터링을 위해 반응기 R2에 연결되었다. CH2Cl2에 용해된 올레산(0.35M) 및 DMF(0.05M) 용액과 CH2Cl2에 용해된 옥살릴 클로라이드(0.35M) 용액을 HPLC 펌프를 사용하여 0.5mL/분 유속으로 M1(Ø = 1.0mm)에 주입하여 반응기 R1(Ø = 1.0 mm)에서 1.5분의 체류 시간을 달성하였다. 생성된 용액을, HCl 및 CO 기체 부산물을 동반하는 올레오일 클로라이드의 합성을 위해 반응기 R1을 통과시켰다. 100psi 배압 조절기는 HCl 및 CO 가스의 가스 방출을 방지하기 위해 R1 끝에 연결되었다. 원치 않는 독성 HCl 및 CO 가스는 배압 조절기의 바깥쪽에 연결하여 맞춤형 T 접합(직경 = 1.6mm)을 사용하여 제거되었다(도 6). 그런 다음, 용액을 HPLC 펌프를 사용하여 혼합기 M2(Ø = 1.0mm)에서 CH2Cl2에 용해된 3-(디메틸아미노)프로판-1,2-디올(0.15M) 및 염기(0.4M) 용액과 혼합하고 반응기 R2(Ø = 1.0mm)를 통과한 다음 FT-IR 샘플 셀을 통과하였다. 40psi 배압 조절기는 FTIR 유동 셀의 배출구에 연결되었다. 반응기 R2의 길이(254 cm, 570 cm, 760 cm, 1020 cm, 2040 cm 등)를 변경하여 R2에서의 체류 시간을 변경하였다. 정상 상태에 도달한 후, 인라인 FTIR 모니터링을 사용하여 생성된 용액을 모니터링하였다. IR 분광학은 1735 cm-1에서 에스테르의 특징적인 CO 스트레칭 밴드를 갖는 DODAP(2)의 형성과 올레오일 클로라이드의 산 클로라이드 작용기의 1780 cm-1에서 CO 스트레칭의 소멸을 모니터링하였다.The flow system consisted of a T-shaped micro-mixer (M1 & M2, Ø = 1.0 mm), two micro-tube reactors R1 (PTFE tube, Ø = 1.0 mm, L = 195 cm) and R2 (PTFE tube, Ø = 1.0 mm), and 100 psi and 40 psi back pressure regulators. An FTIR sample cell was connected to reactor R2 for in-line FTIR monitoring. Solutions of oleic acid (0.35 M) and DMF (0.05 M) dissolved in CH2Cl2 and oxalyl chloride (0.35 M) dissolved in CH2Cl2 were injected into M1 (Ø = 1.0 mm) at a flow rate of 0.5 mL/min using an HPLC pump to achieve a residence time of 1.5 min in reactor R1 (Ø = 1.0 mm). The resulting solution was passed through reactor R1 for the synthesis of oleoyl chloride with HCl and CO gaseous byproducts. A 100 psi back pressure regulator was connected to the end of R1 to prevent the outgassing of HCl and CO gases. The unwanted toxic HCl and CO gases were removed using a custom T-junction (diameter = 1.6 mm) connected to the outside of the back pressure regulator (Fig. 6). Then, the solution was mixed with a solution of 3-(dimethylamino)propane-1,2-diol (0.15 M) and base (0.4 M) dissolved in CH 2 Cl 2 using a HPLC pump in mixer M2 (Ø = 1.0 mm) and passed through reactor R2 (Ø = 1.0 mm) and then through the FT-IR sample cell. A 40 psi back pressure regulator was connected to the outlet of the FTIR flow cell. The residence time in R2 was varied by changing the length of the reactor R2 (254 cm, 570 cm, 760 cm, 1020 cm, 2040 cm, etc.). After reaching steady state, the resulting solution was monitored using inline FTIR monitoring. IR spectroscopy monitored the formation of DODAP (2) with the characteristic CO stretching band of ester at 1735 cm -1 and the disappearance of the CO stretching of the acid chloride functional group of oleoyl chloride at 1780 cm -1 .

R1에서 25 ℃의 인-라인 FTIR을 사용하여 반응을 몇 가지 최적화한 후, 1.5분의 체류 시간에 올레산의 완전한 소비가 발생하여 올레오일 클로라이드를 원활하게 형성하였다. R1에서의 체류 시간 및 온도가 더 증가함에 따라, 올레오일 클로라이드의 CO 스트레칭 피크 강도의 추가 증가는 관찰되지 않았다. 배치(batch) 공정에서는 염소화제로 옥살릴 클로라이드를 사용하여 올레산을 올레오일 클로라이드로 완전히 전환하는 데 3 시간의 반응 시간이 소요되었다. After some optimization of the reaction using in-line FTIR at 25 ℃ at R1, complete consumption of oleic acid occurred at a retention time of 1.5 min, resulting in smooth formation of oleoyl chloride. With further increasing retention time and temperature at R1, no further increase in the intensity of the CO stretching peak of oleoyl chloride was observed. In a batch process, complete conversion of oleic acid to oleoyl chloride took a reaction time of 3 h using oxalyl chloride as the chlorinating agent.

R1에서 올레오일 클로라이드를 합성한 후, 용해되지 않은 독성 HCl과 CO 가스를 분리하기로 결정하였다. HCl 가스를 분리하지 않으면 2단계에서 과량의 염기가 필요한데, 이는 염기 내 난용성 염산염의 높은 농도로 인해 R2에서 막힘이 발생하는 것으로 나타났다(도 6의 b)). 용액에서 용해되지 않은 HCl 및 CO 가스를 안전하고 효율적으로 제거하는 것은 맞춤형 가스-액체 분리기(T 접합, 직경 Ø = 1.6mm)를 사용하여 성공적으로 달성되었으며, 이는 T-접합의 배출구에서 포화 NaOH 용액을 사용하여 퀜칭되었다(도 6의 a)). 끝에서 두 번째 단계에서는 반응기 R2에서 고체 형성이 관찰되지 않았으며, 이는 중력 기반 액체-가스 분리기의 T자형을 통해 용해되지 않은 HCl 가스가 성공적으로 제거되었음을 확인시켜 준다.After the synthesis of oleoyl chloride in R1, it was decided to separate the undissolved toxic HCl and CO gases. Failure to separate the HCl gas would require an excess of base in the second step, which was found to cause clogging in R2 due to the high concentration of sparingly soluble hydrochloric acid salts in the base (Fig. 6b)). Safe and efficient removal of the undissolved HCl and CO gases from the solution was successfully achieved using a custom-made gas-liquid separator (T-junction, diameter Ø = 1.6 mm), which was quenched with saturated NaOH solution at the outlet of the T-junction (Fig. 6a)). In the penultimate step, no solid formation was observed in the reactor R2, confirming that the undissolved HCl gas was successfully removed via the T-junction of the gravity-based liquid-gas separator.

다음으로, R1(Ø = 1.0 mm, L = 195 cm, 각 시약 유속 = 0.5 mL/분)에서 연속적으로 형성된 올레오일 클로라이드를 CH2Cl2에 용해된 3-(디메틸아미노)프로판-1,2-디올(0.15 M) 및 염기인 피리딘(0.4 M)을 포함하는 용액과 혼합기 M2(Ø = 1.0 mm)에서 분당 1.0 mL의 유속으로 혼합하고 반응기 R2(Ø = 1.0 mm)를 통과하여 에스테르화 반응을 발생시켰다. R2에서의 반응은 PTFE 튜브 반응기의 길이를 변경하여 다양한 체류 시간에서 최적화되었다. DODAP(2) 합성 중 에스테르 결합 형성은 인라인 FTIR 분광법을 사용하여 모니터링되었으며, 에스테르기에 대해 1735cm-1에서 특징적인 CO 스트레칭 밴드가 형성되었다(도 7).Next, the continuously formed oleoyl chloride in R1 (Ø = 1.0 mm, L = 195 cm, each reagent flow rate = 0.5 mL/min) was mixed with a solution containing 3-(dimethylamino)propane-1,2-diol (0.15 M) dissolved in CH2Cl2 and pyridine (0.4 M), a base, at a flow rate of 1.0 mL/min in mixer M2 (Ø = 1.0 mm) and passed through reactor R2 (Ø = 1.0 mm) to cause the esterification reaction. The reaction in R2 was optimized at various residence times by changing the length of the PTFE tube reactor. The ester bond formation during the DODAP(2) synthesis was monitored using inline FTIR spectroscopy, and a characteristic CO stretching band was formed at 1735 cm -1 for the ester group (Figure 7 ).

처음에 25 ℃에서 피리딘을 염기로 사용하면 R2에서 8분의 체류 시간 후에도 3-(디메틸아미노)프로판-1,2-디올이 DODAP(2)로의 불완전한 전환이 관찰되었으며, 수율은 50%에 불과하였다. 반응기 R2의 온도를 50 ℃로 증가시키면 DODAP(2)의 수율이 크게 향상되지는 않았다. 낮은 수율은 피리딘의 낮은 염기도에 기인한다. 따라서, 다른 염기를 선별하기로 결정하였다(표 1). Initially, when pyridine was used as a base at 25 °C, incomplete conversion of 3-(dimethylamino)propane-1,2-diol to DODAP(2) was observed even after a residence time of 8 min in R2, with only a 50% yield. Increasing the temperature of reactor R2 to 50 °C did not significantly improve the yield of DODAP(2). The low yield was attributed to the low basicity of pyridine. Therefore, it was decided to screen other bases (Table 1).

상이한 염기의 사용에 따른 DODAP(2)의 합성 최적화Optimization of the synthesis of DODAP(2) by using different bases 염기base R2에서의 온도Temperature at R2
(℃)(℃) R2에서의 체류시간 (분)Time in residence at R2 (minutes) 수율 (%)aYield (%)a 피리딘Pyridine 2525 88 5050 5050 88 6161 피리딘 + DMAPPyridine + DMAP 2525 88 5151 5050 88 6767 DMAPDMAP 2525 88 7070 5050 88 7474 디이소피로필에틸아민Diisopyrrole ethylamine 2525 88 6767 5050 88 7171 트리에틸아민Triethylamine 2525 33 94 (92)b94 (92)b 5050 33 9090

a) 수율은 인-라인 RTIR 분석을 이용하여 계산됨.a) Yields are calculated using in-line RTIR analysis.

b) 분리된 수율은 괄호 안에 표시됨. b) Separated yields are shown in parentheses.

몇 가지 최적화 후에, 트리에틸아민이 적합한 염기인 것으로 밝혀졌으며, 25 ℃의 R2에서 3분의 체류 시간으로 분리된 수율의 92%에 달하는 가장 높은 DODAP(2) 수율을 제공하였다. 배치 공정의 경우, 완전한 변환이 발생하는 데 약 8시간이 걸렸다.After some optimization, triethylamine was found to be a suitable base, affording the highest DODAP(2) yield of 92% of the isolated yield with a residence time of 3 min at R2 at 25 °C. In batch process, complete conversion took about 8 h.

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 2의 구조식을 갖는 DODAP가 성공적으로 합성되었음을 확인하였다.The successful synthesis of DODAP having the structural formula of compound 2 was confirmed through spectroscopic identification results of the synthesized final product.

[화합물 2][Compound 2]

Figure PCTKR2024007830-appb-img-000006
Figure PCTKR2024007830-appb-img-000006

수율: 92%, 무색의 액체. Yield : 92%, colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 5.38 - 5.28 (m, 4 H), 5.25 - 5.15 (m, 1 H), 4.33 (dd, J = 3.2, 11.9 Hz, 1 H), 4.05 (dd, J = 6.2, 12.0 Hz, 1 H), 2.61 - 2.52 (m, 1 H), 2.49 - 2.43 (m, 1 H), 2.33 - 2.20 (m, 10 H), 2.04 - 1.95 (m, 8 H), 1.59 (m, 4 H), 1.35 - 1.23 (m, 40 H), 0.87 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.38 - 5.28 (m, 4 H), 5.25 - 5.15 (m, 1 H), 4.33 (dd, J = 3.2, 11.9 Hz, 1 H), 4.05 (dd , J = 6.2, 12.0 Hz, 1 H), 2.61 - 2.52 (m, 1 H), 2.49 - 2.43 (m, 1 H), 2.33 - 2.20 (m, 10 H), 2.04 - 1.95 (m, 8 H), 1.59 (m, 4 H), 1.35 - 1.23 (m, 40 H), 0.87 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.4, 173.1, 130.0, 130.0, 129.7, 129.7, 68.7, 63.8, 59.0, 45.4, 34.3, 34.1, 31.9, 29.8, 29.7, 29.5, 29.3, 29.22, 29.20, 29.18, 29.11, 29.09, 29.07, 27.20, 27.16, 24.9, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.4, 173.1, 130.0, 130.0, 129.7, 129.7, 68.7, 63.8, 59.0, 45.4, 34.3, 34.1, 31.9, 29.8, 29.7, 29.5, 29.3, 29.22, 29.20, 29.18, 29.11, 29.09, 29.07, 27.20, 27.16, 24.9, 22.7, 14.1.

MALDI-MS: 648.75 m/zMALDI-MS: 648.75 m/z

[실시예 2][Example 2]

이온화 가능한 양이온 지질 라이브러리의 단일 유동 합성Single-flow synthesis of a library of ionizable cationic lipids

DODAP(2)의 성공적인 합성을 이용하여 긴 사슬 산(long chain acid)을 변경하여 이온화 가능한 양이온성 지질 라이브러리의 합성을 위한 다양한 경로를 개발하도록 확장되었다(도 8). 지질 라이브러리 개발은 독성을 최소화하면서 치료 성분을 효과적으로 캡슐화하여 표적 부위에 전달할 수 있는 지질을 식별하는 것을 목표로 하는 제약 연구의 중요한 측면이다. 이에 따라, 올레산 대신 리놀레산, 미리스트산, 스테아르산, 팔미트산과 같은 시판되는 지방산을 공합하여 이온화 가능한 양이온 지질 라이브러리를 합성하고자 하였다.The successful synthesis of DODAP(2) was extended to develop various routes for the synthesis of a library of ionizable cationic lipids by modifying the long chain acid (Fig. 8). Lipid library development is an important aspect of pharmaceutical research aimed at identifying lipids that can effectively encapsulate therapeutic agents and deliver them to target sites while minimizing toxicity. Accordingly, we attempted to synthesize a library of ionizable cationic lipids by co-incorporating commercially available fatty acids such as linoleic acid, myristic acid, stearic acid, and palmitic acid instead of oleic acid.

이온화 가능한 양이온성 지질의 연속 유동 합성을 위한 실험 셋팅은 도 8에 나타내었다. 유동 시스템은 2개의 T자형 미세 혼합기(M1 & M2, Ø = 1.0 mm), 2개의 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.0 mm, L = 195 cm)와 R2(PTFE 튜브, Ø = 1.0mm, L = 570cm)로 구성된다. CH2Cl2에 용해된 지방산(0.35M) 및 DMF(0.05M) 용액과 CH2Cl2에 용해된 옥살릴 클로라이드(0.35M) 용액을 HPLC 펌프를 사용하여 0.5mL/분 유속으로 M1(Ø = 1.0mm)에 주입하여 반응기 R1에서 1.5분의 체류 시간을 달성하였다. 생성된 용액을 HCl 및 CO 기체 부산물을 동반하는 아실 클로라이드의 합성을 위해 반응기 R1을 통과시켰다. 100 psi 배압 조절기는 HCl 및 CO 가스의 가스 방출을 방지하기 위해 R1 배출구에 연결되었다. 원치 않는 독성 HCl 및 CO 가스는 배압 조절기의 배출구에 연결하여 맞춤형 T 접합(직경 = 1.6mm)을 사용하여 제거되었다(도 6). 그런 다음, 용액을 HPLC 펌프를 사용하여 혼합기 M2(Ø = 1.0mm)에서 CH2Cl2에 용해된 알코올(0.15M) 및 트리에틸아민(0.4M) 용액과 혼합하고 반응기 R2(Ø = 1.0mm, L = 570cm)를 3분의 체류 시간 동안 통과하였다. 정상 상태에 도달한 후, 생성된 용액을 포화 NaHCO3 수용액으로 퀜칭하면서 반응기 R2의 말단에서 5분 동안 수집하였다. 유기층을 분리하고 수용성층을 CH2Cl2로 두 번 추출했습니다. 합한 유기층을 건조 및 증발시키고, 헥산(hexane)에 용해된 30% 에틸 아세테이트를 포함하는 실리카 겔 컬럼 크로마토그래피를 사용하여 정제하여 순수한 이온화 가능한 양이온성 지질을 얻었다.The experimental setup for continuous flow synthesis of ionizable cationic lipids is shown in Fig. 8. The flow system consisted of two T-shaped micromixers (M1 & M2, Ø = 1.0 mm) and two microtube reactors R1 (PTFE tube, Ø = 1.0 mm, L = 195 cm) and R2 (PTFE tube, Ø = 1.0 mm, L = 570 cm). Solutions of fatty acid (0.35 M) and DMF (0.05 M) dissolved in CH2Cl2 and oxalyl chloride (0.35 M ) dissolved in CH2Cl2 were injected into M1 (Ø = 1.0 mm) using an HPLC pump at a flow rate of 0.5 mL/min to achieve a residence time of 1.5 min in reactor R1. The resulting solution was passed through reactor R1 for the synthesis of acyl chlorides with HCl and CO gaseous byproducts. A 100 psi back pressure regulator was connected to the R1 outlet to prevent the outgassing of HCl and CO gases. The unwanted toxic HCl and CO gases were removed using a custom T-junction (diameter = 1.6 mm) connected to the outlet of the back pressure regulator (Fig. 6). Then, the solution was mixed with a solution of alcohol (0.15 M) and triethylamine (0.4 M) dissolved in CH2Cl2 using an HPLC pump in mixer M2 (Ø = 1.0 mm) and passed through reactor R2 (Ø = 1.0 mm, L = 570 cm) with a residence time of 3 min. After reaching steady state, the resulting solution was collected at the end of reactor R2 for 5 min while quenching with saturated NaHCO3 aqueous solution. The organic layer was separated, and the aqueous layer was extracted twice with CH2Cl2 . The combined organic layers were dried and evaporated, and purified using silica gel column chromatography containing 30% ethyl acetate dissolved in hexane to obtain the pure ionizable cationic lipid.

올레산 대신 리놀레산, 미리스트산, 스테아르산, 팔미트산과 같은 시판되는 지방산을 공급함에 따라 동일한 조건에서 해당 이온화 지질(즉, 화합물 6 내지 9)가 만족스러운 수율로 얻어짐을 확인하였다(도 7). 화합물 10에서 15까지의 이온화 가능한 지질의 긴 사슬에 에스테르 또는 이황화물 작용기를 도입하면 인체에 존재하는 에스테라제 및 글루타티온 효소의 작용으로 인해 지질이 더욱 생분해된다. 특히, 지질 11(β-시트로넬롤에서 유래), 12(제라니올에서 유래) 및 13(네롤에서 유래)과 같은 이온화 가능한 지질에 항염증, 항산화, 항암, 항당뇨병, 항진균 활성 등 많은 건강상의 이점을 갖는 모노테르페노이드를 사용한다는 점은 주목할만한 가치가 있다. 또한, 코로나19 백신에 사용되는 이온화 가능한 지질 ALC-0315의 합성을 위한 핵심 중간체인 6-브로모헥실-2-헥실데카노에이트(6-bromohexyl-2-hexyldecanoate)(16)도 2-헥사데칸산과 6-브로모헥산-1-올을 출발 물질로 사용하여 88% 수율로 합성되었다(tR1 = 2.5분, tR2 = 4분, 50 ℃).By supplying commercially available fatty acids such as linoleic acid, myristic acid, stearic acid, and palmitic acid instead of oleic acid, it was confirmed that the corresponding ionizable lipids (i.e., compounds 6 to 9) were obtained in satisfactory yields under the same conditions (Fig. 7). The introduction of ester or disulfide functional groups into the long chains of ionizable lipids from compounds 10 to 15 makes the lipids more biodegradable due to the action of esterase and glutathione enzymes present in the human body. In particular, it is worth noting that monoterpenoids, which have many health benefits such as anti-inflammatory, antioxidant, anticancer, antidiabetic, and antifungal activities, are used in ionizable lipids such as lipids 11 (derived from β-citronellol), 12 (derived from geraniol), and 13 (derived from nerol). Additionally, 6-bromohexyl-2-hexyldecanoate (16), a key intermediate for the synthesis of the ionizable lipid ALC-0315 used in the COVID-19 vaccine, was also synthesized in 88% yield using 2-hexadecanoic acid and 6-bromohexan-1-ol as starting materials (t R1 = 2.5 min, t R2 = 4 min, 50 °C).

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 6 내지 16의 구조식을 갖는 이온화 가능한 양이온 지질 라이브러리가 성공적으로 합성되었음을 확인하였다. 화합물 10 내지 16의 합성은 하기에 상세하게 나타내었다.The spectroscopic identification results of the synthesized final products confirmed that the ionizable cationic lipid library having the structural formulas of compounds 6 to 16 was successfully synthesized. The synthesis of compounds 10 to 16 is described in detail below.

[화합물 6][Compound 6]

(9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트) (DLinDAP), 리놀레산을 사용하여 합성됨(9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate) (DLinDAP), synthesized using linoleic acid

Figure PCTKR2024007830-appb-img-000007
Figure PCTKR2024007830-appb-img-000007

수율: 90%, 무색 액체. Yield : 90%, colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 5.43 - 5.30 (m, 8 H), 4.35 (dd, J = 3.5, 11.9 Hz, 1 H), 4.08 (dd, J = 6.0, 12.0 Hz, 1 H), 2.77 (t, J = 6.6 Hz, 4 H), 2.68 (br. s., 1 H), 2.60 (br. s., 1 H), 2.37 (s, 6 H), 2.36 - 2.29 (m, 4 H), 2.3 (q, J = 6.9 Hz, 8 H), 1.65 - 1.60 (m, 4 H), 1.39 - 1.25 (m, 28 H), 0.89 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.43 - 5.30 (m, 8 H), 4.35 (dd, J = 3.5, 11.9 Hz, 1 H), 4.08 (dd, J = 6.0, 12.0 Hz, 1 H ), 2.77 (t, J = 6.6 Hz, 4 H), 2.68 (br. s., 1 H), 2.60 (br. s., 1 H), 2.37 (s, 6 H), 2.36 - 2.29 (m, 4 H), 2.3 (q, J = 6.9 Hz, 8 H), 1.65 - 1.60 (m, 4 H), 1.39 - 1.25 (m, 28 H), 0.89 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.3, 173.1, 130.2, 130.02, 129.97, 128.04, 128.00, 127.89, 127.86, 66.3, 63.7, 58.8, 45.2, 34.3, 34.0, 31.5, 29.6, 29.3, 29.19, 29.17, 29.10, 29.07, 29.05, 27.2, 25.6, 24.9, 24.8, 22.5, 14.0. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.3, 173.1, 130.2, 130.02, 129.97, 128.04, 128.00, 127.89, 127.86, 66.3, 63.7, 58.8, 45.2, 34.3, 34.0, 31.5, 29.6, 29.3, 29.19, 29.17, 29.10, 29.07, 29.05, 27.2, 25.6, 24.9, 24.8, 22.5, 14.0.

[화합물 7][Compound 7]

1,2-디미리스토일-3-디메틸암모늄-프로판, 미리스트산을 사용하여 합성됨1,2-Dimyristoyl-3-dimethylammonium-propane, synthesized using myristic acid

Figure PCTKR2024007830-appb-img-000008
Figure PCTKR2024007830-appb-img-000008

수율: 93%, 무색 액체. Yield : 93%, colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 5.20 (dd, J = 3.1, 6.5 Hz, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.09 (dd, J = 6.4, 11.9 Hz, 1 H), 2.51 - 2.40 (m, 2 H), 2.31 (dt, J = 3.4, 7.5 Hz, 4 H), 2.27 (s, 6 H), 1.65 - 1.58 (m, 4 H), 1.26 (s, 40 H), 0.89 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.20 (dd, J = 3.1, 6.5 Hz, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.09 (dd, J = 6.4, 11.9 Hz, 1 H), 2.51 - 2.40 (m, 2 H), 2.31 (dt, J = 3.4, 7.5 Hz, 4 H), 2.27 (s, 6 H), 1.65 - 1.58 (m, 4 H), 1.26 (s, 40 H), 0.89 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.5, 173.2, 69.2, 63.9, 59.4, 46.0, 34.4, 34.2, 31.9, 29.7, 29.64, 29.62, 29.5, 29.35, 29.29, 29.13, 29.09, 25.0, 24.95, 24.91, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.5, 173.2, 69.2, 63.9, 59.4, 46.0, 34.4, 34.2, 31.9, 29.7, 29.64, 29.62, 29.5, 29.35, 29.29, 29.13, 29.09, 25.0, 24.95, 24.91, 22.7, 14.1.

[화합물 8][Compound 8]

1,2-디스테아로일-3-디메틸암모늄-프로판, 스테아르산을 사용하여 합성됨1,2-Distearoyl-3-dimethylammonium-propane, synthesized using stearic acid

Figure PCTKR2024007830-appb-img-000009
Figure PCTKR2024007830-appb-img-000009

수율: 90%, 무색 액체. Yield : 90%, colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 5.20 (m, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.08 (dd, J = 6.5, 12.0 Hz, 1 H), 2.50 - 2.38 (m, 2 H), 2.30 (m, 4 H), 2.26 (s, 6 H), 1.66 - 1.55 (m, 4 H), 1.29 - 1.23 (m, 56 H), 0.88 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.20 (m, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.08 (dd, J = 6.5, 12.0 Hz, 1 H), 2.50 - 2.38 (m, 2 H), 2.30 (m, 4 H), 2.26 (s, 6 H), 1.66 - 1.55 (m, 4 H), 1.29 - 1.23 (m, 56 H), 0.88 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.5, 173.2, 69.1, 63.9, 59.3, 45.9, 34.4, 34.1, 31.9, 29.7, 29.5, 29.35, 29.29, 29.12, 29.08, 24.93, 24.89, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.5, 173.2, 69.1, 63.9, 59.3, 45.9, 34.4, 34.1, 31.9, 29.7, 29.5, 29.35, 29.29, 29.12, 29.08, 24.93, 24.89, 22.7, 14.1.

[화합물 9][Compound 9]

1,2-디팔미토일-3-디메틸암모늄-프로판, 팔미트산을 사용하여 합성됨1,2-Dipalmitoyl-3-dimethylammonium-propane, synthesized using palmitic acid

Figure PCTKR2024007830-appb-img-000010
Figure PCTKR2024007830-appb-img-000010

수율: 90%, 무색 액체. Yield : 90%, colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 5.23 - 5.15 (m, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.08 (dd, J = 6.4, 11.9 Hz, 1 H), 2.51 - 2.36 (m, 2 H), 2.29 (dt, J = 3.4, 7.5 Hz, 4 H), 2.25 (s, 6 H), 1.66 - 1.53 (m, 4 H), 1.31 - 1.18 (m, 44 H), 0.87 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.23 - 5.15 (m, 1 H), 4.36 (dd, J = 3.2, 11.9 Hz, 1 H), 4.08 (dd, J = 6.4, 11.9 Hz, 1 H) ), 2.51 - 2.36 (m, 2 H), 2.29 (dt, J = 3.4, 7.5 Hz, 4 H), 2.25 (s, 6 H), 1.66 - 1.53 (m, 4 H), 1.31 - 1.18 (m, 44 H), 0.87 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.4, 173.1, 69.1, 63.9, 59.3, 45.9, 34.4, 34.1, 31.9, 29.66, 29.63, 29.59, 29.5, 29.33, 29.26, 29.1, 29.0, 24.90, 24.87, 22.6, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.4, 173.1, 69.1, 63.9, 59.3, 45.9, 34.4, 34.1, 31.9, 29.66, 29.63, 29.59, 29.5, 29.33, 29.26, 29.1, 29.0, 24.90, 24.87, 22.6, 14.1.

[화합물 10][Compound 10]

O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트), 10-(헥실옥시)-10-옥소데칸산을 사용하여 합성됨Synthesized using O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate), 10-(hexyloxy)-10-oxodecanoic acid

Figure PCTKR2024007830-appb-img-000011
Figure PCTKR2024007830-appb-img-000011

수율: 90%, 무색 오일 Yield: 90%, colorless oil

1H NMR (500 MHz, CDCl3) δ 5.17 - 5.08 (m, 1H), 4.32-4.02 (m, 1H), 3.99 (t, J = 6.7 Hz, 4H), 2.42 - 2.32 (m, 2H), 2.31 - 2.12 (m, 14H), 1.57 - 1.52 (m, 12H), 1.24 (s, 28H), 0.83 (t, J = 6.9 Hz, 6H). 1 H NMR (500 MHz, CDCl 3 ) δ 5.17 - 5.08 (m, 1H), 4.32-4.02 (m, 1H), 3.99 (t, J = 6.7 Hz, 4H), 2.42 - 2.32 (m, 2H), 2.31 - 2.12 (m, 14H), 1.57 - 1.52 (m, 12H), 1.24 (s, 28H), 0.83 (t, J = 6.9 Hz, 6H).

13C NMR (125 MHz, CDCl3) δ 173.72 (d, J = 3.3 Hz), 173.25, 172.96, 69.19, 64.25, 63.82, 59.31, 45.96, 34.24 (d, J = 2.8 Hz), 34.00, 32.61, 31.34, 30.79, 29.47 - 28.82 (m), 28.54, 26.25, 25.59 - 25.18 (m), 24.78 (dd, J = 15.1, 9.4 Hz), 22.44, 13.89. 13 C NMR (125 MHz, CDCl 3 ) δ 173.72 (d, J = 3.3 Hz), 173.25, 172.96, 69.19, 64.25, 63.82, 59.31, 45.96, 34.24 (d, J = 2.8 Hz), 34.00, 32.61, 31.34, 30.79, 29.47 - 28.82 (m), 28.54, 26.25, 25.59 - 25.18 (m), 24.78 (dd, J = 15.1, 9.4 Hz), 22.44, 13.89.

MALDI-MS: 656.78 m/z MALDI-MS : 656.78 m/z

화합물 10의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 1과 같이 10-(헥실옥시)-10-옥소데칸산이 배치 반응기에서 제조되었다.The synthetic method of compound 10 is as follows. First, 10-(hexyloxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 1.

[반응식 1][Reaction Formula 1]

Figure PCTKR2024007830-appb-img-000012
Figure PCTKR2024007830-appb-img-000012

CH2Cl2에 용해된 세바스산 2.0g(1.0eq.)의 용액에 디사이클로헥실카르보디이미드(DCC) 2.45g(1.2eq.)을 첨가한 다음, DMAP 120.0mg(0.1eq.) 및 n-헥산올 0.9g(0.9eq.)을 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 여과하고, 생성된 여과물을 회전 증발기에서 감압 하에 농축시켰다. 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)를 이용하여 정제하여 10-(헥실옥시)-10-옥소데칸산 1.56g을 55% 수율로 무색의 고체로 얻었다.To a solution of 2.0 g (1.0 eq.) of sebacic acid in CH 2 Cl 2 was added 2.45 g (1.2 eq.) of dicyclohexylcarbodiimide (DCC), followed by 120.0 mg (0.1 eq.) of DMAP and 0.9 g (0.9 eq.) of n-hexanol. The mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the resulting filtrate was concentrated under reduced pressure using a rotary evaporator. The obtained residue was purified using flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to obtain 1.56 g of 10-(hexyloxy)-10-oxodecanoic acid as a colorless solid in 55% yield.

1H NMR (500 MHz, CDCl3) δ = 7.96 (br. s., 1 H), 4.02 - 3.88 (m, 2 H), 2.32 - 2.12 (m, 4 H), 1.53 (br. s., 6 H), 1.22 (br. s., 14 H), 0.80 (br. s., 3 H) 1 H NMR (500 MHz, CDCl 3 ) δ = 7.96 (br. s., 1 H), 4.02 - 3.88 (m, 2 H), 2.32 - 2.12 (m, 4 H), 1.53 (br. s., 6 H), 1.22 (br. s., 14 H), 0.80 (br. s., 3 H)

13C NMR (125 MHz, CDCl3) δ = 178.4, 173.9, 64.2, 64.1, 34.1, 33.6, 31.2, 28.8, 28.81, 28.77, 28.4, 25.4, 24.7, 24.5, 22.3, 13.7. 13 C NMR (125 MHz, CDCl 3 ) δ = 178.4, 173.9, 64.2, 64.1, 34.1, 33.6, 31.2, 28.8, 28.81, 28.77, 28.4, 25.4, 24.7, 24.5, 22.3, 13.7.

[화합물 11][Compound 11]

O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트), (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산을 사용하여 합성됨Synthesized using O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl)bis(decanedioate), (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid

Figure PCTKR2024007830-appb-img-000013
Figure PCTKR2024007830-appb-img-000013

수율: 92%, 무색 고체. Yield : 92%, colorless solid.

1H NMR (500 MHz, CDCl3) δ 5.10 (dd, J = 31.1, 28.5 Hz, 2H), 4.29 (d, J = 11.8 Hz, 1H), 4.08 - 3.95 (m, 4H), 2.43 - 2.30 (m, 2H), 2.27 - 2.08 (m, 12H), 1.85 (dd, J = 51.6, 43.5 Hz, 6H), 1.59 (d, J = 10.5 Hz, 7H), 1.56 - 1.43 (m, 16H), 1.36 (dd, J = 13.0, 6.4 Hz, 2H), 1.23 (s, 19H), 1.12 (dd, J = 16.0, 10.5 Hz, 4H), 0.84 (d, J = 6.1 Hz, 6H). 1H NMR (500 MHz, CDCl 3 ) δ 5.10 (dd, J = 31.1, 28.5 Hz, 2H), 4.29 (d, J = 11.8 Hz, 1H), 4.08 - 3.95 (m, 4H), 2.43 - 2.30 ( m, 2H), 2.27 - 2.08 (m, 12H), 1.85 (dd, J = 51.6, 43.5 Hz, 6H), 1.59 (d, J = 10.5 Hz, 7H), 1.56 - 1.43 (m, 16H), 1.36 (dd, J = 13.0, 6.4 Hz, 2H), 1.23 (s, 19H), 1.12 (dd, J = 16.0, 10.5 Hz, 4H), 0.84 (d, J = 6.1 Hz, 6H).

13C NMR (125 MHz, CDCl3) δ 173.74, 173.28, 173.01, 154.09, 131.14, 124.53, 69.13, 63.84, 62.66, 59.28, 45.91, 36.90, 35.42, 34.28, 34.02, 32.65, 30.81, 29.41, 29.12, 29.03, 28.98, 28.93, 26.29, 25.64, 25.47, 25.32, 24.89, 24.82, 24.78, 24.70, 19.36, 17.57. 13 C NMR (125 MHz, CDCl 3 ) δ 173.74, 173.28, 173.01, 154.09, 131.14, 124.53, 69.13, 63.84, 62.66, 59.28, 45.91, 36.90, 35.42, 34.28, 34.02, 32.65, 30.81, 29.41, 29.12, 29.03, 28.98, 28.93, 26.29, 25.64, 25.47, 25.32, 24.89, 24.82, 24.78, 24.70, 19.36, 17.57.

MALDI-MS: 764.88 m/z MALDI-MS : 764.88 m/z

화합물 11의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 2와 같이 (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산이 배치 반응기에서 제조되었다.The synthetic method of compound 11 is as follows. First, (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 2.

[반응식 2][Reaction Formula 2]

Figure PCTKR2024007830-appb-img-000014
Figure PCTKR2024007830-appb-img-000014

CH2Cl2에 용해된 세바스산 2.0g(1.0eq.)의 용액에 DCC 2.45g(1.2eq.)을 첨가한 다음 DMAP 120.0mg(0.1eq.) 및 시트로넬롤 1.4g(0.9eq.)을 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 여과하고, 생성된 여과물을 회전 증발기에서 감압 하에 농축시켰다. 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)로 정제하여 (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산을 무색 고체로 2.0g(60%) 얻었다.To a solution of 2.0 g (1.0 eq.) of sebacic acid in CH 2 Cl 2 was added 2.45 g (1.2 eq.) of DCC, followed by 120.0 mg (0.1 eq.) of DMAP and 1.4 g (0.9 eq.) of citronellol. The mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the resulting filtrate was concentrated under reduced pressure using a rotary evaporator. The obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to obtain 2.0 g (60%) of (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid as a colorless solid.

1H NMR (500 MHz, CDCl3) δ = 5.11 - 5.02 (m, 1 H), 4.18 - 4.02 (m, 2 H), 2.33 (t, J = 7.6 Hz, 2 H), 2.27 (t, J = 7.5 Hz, 2 H), 2.03 - 1.89 (m, 2 H), 1.67 (d, J = 0.6 Hz, 3 H), 1.65 - 1.56 (m, 8 H), 1.56 - 1.49 (m, 1 H), 1.45 - 1.38 (m, 1 H), 1.34 - 1.26 (m, 9 H), 1.21 - 1.13 (m, 1 H), 0.90 (d, J = 6.7 Hz, 3 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.11 - 5.02 (m, 1 H), 4.18 - 4.02 (m, 2 H), 2.33 (t, J = 7.6 Hz, 2 H), 2.27 (t, J = 7.5 Hz, 2 H), 2.03 - 1.89 (m, 2 H), 1.67 (d, J = 0.6 Hz, 3 H), 1.65 - 1.56 (m, 8 H), 1.56 - 1.49 (m, 1 H), 1.45 - 1.38 (m, 1 H), 1.34 - 1.26 (m, 9 H), 1.21 - 1.13 (m, 1 H), 0.90 (d, J = 6.7 Hz, 3 H).

13C NMR (125 MHz, CDCl3) δ = 180.0, 173.9, 131.2, 124.5, 62.8, 36.9, 35.4, 34.3, 34.0, 29.4, 29.0, 28.9, 25.6, 25.3, 24.9, 24.6, 19.3, 17.6. 13 C NMR (125 MHz, CDCl 3 ) δ = 180.0, 173.9, 131.2, 124.5, 62.8, 36.9, 35.4, 34.3, 34.0, 29.4, 29.0, 28.9, 25.6, 25.3, 24.9, 24.6, 19.3, 17.6.

[화합물 12][Compound 12]

O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트), (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((E)-3,7-dimethylocta-2,6-dien-1-yl)bis(decanedioate), (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid

Figure PCTKR2024007830-appb-img-000015
Figure PCTKR2024007830-appb-img-000015

수율: 92%, 무색 고체 Yield : 92%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.33 (dt, J = 1.1, 7.1 Hz, 2 H), 5.21 - 5.16 (m, 1 H), 5.11 - 5.05 (m, 2 H), 4.58 (d, J = 7.0 Hz, 4 H), 4.35 (dd, J = 3.1, 12.0 Hz, 1 H), 4.08 (dd, J = 6.3, 12.0 Hz, 1 H), 2.48 - 2.38 (m, 2 H), 2.32 - 2.26 (m, 8 H), 2.25 (s, 6 H), 2.13 - 2.01 (m, 8 H), 1.70 (s, 6 H), 1.68 (s, 6 H), 1.60 (s, 14 H), 1.29 (br. s., 16 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.33 (dt, J = 1.1, 7.1 Hz, 2 H), 5.21 - 5.16 (m, 1 H), 5.11 - 5.05 (m, 2 H), 4.58 (d) , J = 7.0 Hz, 4 H), 4.35 (dd, J = 3.1, 12.0 Hz, 1 H), 4.08 (dd, J = 6.3, 12.0 Hz, 1 H), 2.48 - 2.38 (m, 2 H), 2.32 - 2.26 (m, 8 H), 2.25 (s, 6) H), 2.13 - 2.01 (m, 8 H), 1.70 (s, 6 H), 1.68 (s, 6 H), 1.60 (s, 14 H), 1.29 (br. s., 16 H).

13C NMR (125 MHz, CDCl3) δ = 173.8, 173.4, 173.1, 142.0, 131.7, 123.7, 118.4, 69.2, 63.9, 61.1, 59.4, 46.0, 39.5, 34.33, 34.30, 34.1, 33.9, 29.1, 29.0, 28.97, 26.3, 25.6, 24.92, 24.86, 24.81, 17.6, 16.4. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.8, 173.4, 173.1, 142.0, 131.7, 123.7, 118.4, 69.2, 63.9, 61.1, 59.4, 46.0, 39.5, 34.33, 34.30, 34.1, 33.9, 29.1, 29.0, 28.97, 26.3, 25.6, 24.92, 24.86, 24.81, 17.6, 16.4.

MALDI-MS: 760.60 m/z MALDI-MS : 760.60 m/z

화합물 12의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 3과 같이 (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산이 배치 반응기에서 제조되었다.The synthetic method of compound 12 is as follows. First, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 3.

[반응식 3][Reaction Formula 3]

Figure PCTKR2024007830-appb-img-000016
Figure PCTKR2024007830-appb-img-000016

CH2Cl2에 용해된 세바스산 2.0g(1.0eq.)의 용액에 DCC 2.45g(1.2eq.)을 첨가한 다음 DMAP 120.0mg(0.1eq.) 및 기라니올 1.4g(0.9eq.)을 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 여과하고, 생성된 여과물을 회전 증발기에서 감압 하에 농축시켰다. 얻은 잔류물을 플래시 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)로 정제하여 (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산 1.9g(58%)을 무색 고체로 얻었다.To a solution of 2.0 g (1.0 eq.) of sebacic acid in CH 2 Cl 2 was added 2.45 g (1.2 eq.) of DCC, followed by 120.0 mg (0.1 eq.) of DMAP and 1.4 g (0.9 eq.) of gyraniol. The mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the resulting filtrate was concentrated under reduced pressure using a rotary evaporator. The obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to obtain 1.9 g (58%) of (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid as a colorless solid.

1H NMR (500 MHz, CDCl3) δ = 5.34 (dt, J = 1.1, 7.1 Hz, 1 H), 5.12 - 5.03 (m, 1 H), 4.59 (d, J = 7.2 Hz, 2 H), 2.34 (t, J = 7.6 Hz, 2 H), 2.30 (t, J = 7.6 Hz, 2 H), 2.14 - 2.07 (m, 2 H), 2.07 - 2.01 (m, 2 H), 1.70 (s, 3 H), 1.68 (s, 3 H), 1.65 - 1.58 (m, 7 H), 1.30 (br. s., 8 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.34 (dt, J = 1.1, 7.1 Hz, 1 H), 5.12 - 5.03 (m, 1 H), 4.59 (d, J = 7.2 Hz, 2 H), 2.34 (t, J = 7.6 Hz, 2 H), 2.30 (t, J = 7.6 Hz, 2 H), 2.14 - 2.07 (m, 2 H), 2.07 - 2.01 (m, 2 H), 1.70 (s, 3 H), 1.68 (s, 3 H), 1.65 - 1.58 (m, 7 H), 1.30 (br. s., 8 H).

13C NMR (125 MHz, CDCl3) δ = 180.0, 173.9, 142.1, 131.7, 123.7, 118.4, 61.2, 39.5, 34.3, 34.0, 29.0, 28.9, 26.3, 25.6, 24.9, 24.6, 17.6, 16.4. 13 C NMR (125 MHz, CDCl 3 ) δ = 180.0, 173.9, 142.1, 131.7, 123.7, 118.4, 61.2, 39.5, 34.3, 34.0, 29.0, 28.9, 26.3, 25.6, 24.9, 24.6, 17.6, 16.4.

[화합물 13][Compound 13]

O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트), (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산을 사용하여 합성됨Synthesized using O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((Z)-3,7-dimethylocta-2,6-dien-1-yl)bis(decanedioate), (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid

Figure PCTKR2024007830-appb-img-000017
Figure PCTKR2024007830-appb-img-000017

수율: 90%, 무색 고체 Yield: 90%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.34 (t, J = 7.0 Hz, 2 H), 5.18 - 5.16 (m, 1 H), 5.09 - 5.07 (m, 2 H), 4.54 (d, J = 7.2 Hz, 4 H), 4.34 (dd, J = 3.1, 12.0 Hz, 1 H), 4.07 (dd, J = 6.4, 11.9 Hz, 1 H), 2.48 - 2.36 (m, 2 H), 2.31 - 2.26 (m, 8 H), 2.24 (s, 6 H), 2.13 - 2.01 (m, 8 H), 1.75 (s, 6 H), 1.67 (s, 6 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.34 (t, J = 7.0 Hz, 2 H), 5.18 - 5.16 (m, 1 H), 5.09 - 5.07 (m, 2 H), 4.54 (d, J = 7.2 Hz, 4 H), 4.34 (dd, J = 3.1, 12.0 Hz, 1 H), 4.07 (dd, J = 6.4, 11.9 Hz, 1 H), 2.48 - 2.36 (m, 2 H), 2.31 - 2.26 (m, 8 H), 2.24 (s, 6 H), 2.13 - 2.01 (m, 8 H), 1.75 (s, 6 H), 1.67 (s, 6 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H).

13C NMR (125 MHz, CDCl3) δ = 173.7, 154.1, 142.3, 139.7, 132.0, 123.5, 119.2, 60.8, 55.7, 49.6, 35.8, 34.8, 34.3, 33.9, 32.7, 32.1, 30.8, 29.1, 29.0, 26.6, 26.3, 25.6, 25.44, 25.38, 25.33, 25.26, 24.9, 24.65, 24.60, 23.4, 17.6. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.7, 154.1, 142.3, 139.7, 132.0, 123.5, 119.2, 60.8, 55.7, 49.6, 35.8, 34.8, 34.3, 33.9, 32.7, 32.1, 30.8, 29.1, 29.0, 26.6, 26.3, 25.6, 25.44, 25.38, 25.33, 25.26, 24.9, 24.65, 24.60, 23.4, 17.6.

MALDI-MS: 760.62 m/z MALDI-MS : 760.62 m/z

화합물 13의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 4와 같이 (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산이 배치 반응기에서 제조되었다.The synthetic method of compound 13 is as follows. First, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 4.

[반응식 4][Reaction Formula 4]

Figure PCTKR2024007830-appb-img-000018
Figure PCTKR2024007830-appb-img-000018

CH2Cl2에 용해된 세바스산 2.0g(1.0eq.)의 용액에 DCC 2.45g(1.2eq.)을 첨가한 다음 DMAP 120.0mg(0.1eq.) 및 네롤 1.4g(0.9eq.)을 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 여과하고, 생성된 여과물을 회전 증발기에서 감압 하에 농축시켰다. 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)로 정제하여 (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산 1.84g(55%)을 무색 고체로 얻었다.To a solution of 2.0 g (1.0 eq.) of sebacic acid in CH 2 Cl 2 was added 2.45 g (1.2 eq.) of DCC, followed by 120.0 mg (0.1 eq.) of DMAP and 1.4 g (0.9 eq.) of nerol. The mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the resulting filtrate was concentrated under reduced pressure using a rotary evaporator. The obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to give 1.84 g (55%) of (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid as a colorless solid.

1H NMR (500 MHz, CDCl3) δ = 5.33 (t, J = 7.2 Hz, 1 H), 5.12 - 5.03 (m, 1 H), 4.54 (d, J = 7.2 Hz, 2 H), 2.32 (t, J = 7.5 Hz, 2 H), 2.27 (t, J = 7.6 Hz, 2 H), 2.11 - 2.03 (m, 4 H), 1.74 (s, 3 H), 1.66 (s, 3 H), 1.62 - 1.57 (m, 7 H), 1.29 (br. s., 8 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.33 (t, J = 7.2 Hz, 1 H), 5.12 - 5.03 (m, 1 H), 4.54 (d, J = 7.2 Hz, 2 H), 2.32 ( t, J = 7.5 Hz, 2 H), 2.27 (t, J = 7.6 Hz, 2 H), 2.11 - 2.03 (m, 4 H), 1.74 (s, 3 H), 1.66 (s, 3 H), 1.62 - 1.57 (m, 7 H), 1.29 (br. s., 8H).

13C NMR (125 MHz, CDCl3) δ = 179.8, 173.8, 142.4, 132.0, 123.5, 119.2, 60.9, 34.3, 34.0, 32.1, 28.94, 28.89, 26.6, 25.6, 24.8, 24.6, 23.4, 17.5. 13 C NMR (125 MHz, CDCl 3 ) δ = 179.8, 173.8, 142.4, 132.0, 123.5, 119.2, 60.9, 34.3, 34.0, 32.1, 28.94, 28.89, 26.6, 25.6, 24.8, 24.6, 23.4, 17.5.

[화합물 14][Compound 14]

3-(디메틸아미노)프로판-1,2-디일 비스(3-(옥타데실디설파닐)프로파노에이트), 3-(옥타데실디설파닐)프로판산을 사용하여 합성됨 3-(Dimethylamino)propane-1,2-diyl bis(3-(octadecyldisulfanyl)propanoate), synthesized using 3-(octadecyldisulfanyl)propanoic acid

Figure PCTKR2024007830-appb-img-000019
Figure PCTKR2024007830-appb-img-000019

수율: 88%, 무색 고체 Yield : 88%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.22 (br. s., 1 H), 4.43 - 4.41 (m, 1 H), 4.16 - 4.13 (m, 1 H), 2.91 (br. s., 4 H), 2.77 (t, J = 6.7 Hz, 4 H), 2.69 (t, J = 6.9 Hz, 4 H), 2.52 - 2.41 (m, 2 H), 2.26 (s, 6 H), 1.73 - 1.61 (m, 4 H), 1.37 (br. s., 4 H), 1.26 (br. s., 56 H), 0.88 (t, J = 6.2 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.22 (br. s., 1 H), 4.43 - 4.41 (m, 1 H), 4.16 - 4.13 (m, 1 H), 2.91 (br. s., 4 H), 2.77 (t, J = 6.7 Hz, 4 H), 2.69 (t, J = 6.9 Hz, 4 H), 2.52 - 2.41 (m, 2 H), 2.26 (s, 6 H), 1.73 - 1.61 (m, 4 H), 1.37 (br. s., 4 H) ), 1.26 (br. s., 56 H), 0.88 (t, J = 6.2 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 171.5, 171.2, 69.8, 64.2, 59.2, 46.0, 39.05, 39.00, 34.3, 34.0, 33.9, 33.1, 31.9, 29.7, 29.64, 29.59, 29.5, 29.3, 29.23, 29.18, 28.5, 24.9, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 171.5, 171.2, 69.8, 64.2, 59.2, 46.0, 39.05, 39.00, 34.3, 34.0, 33.9, 33.1, 31.9, 29.7, 29.64, 29.59, 29.5, 29.3, 29.23, 29.18, 28.5, 24.9, 22.7, 14.1.

화합물 14의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 5와 같이 3-(옥타데실디설파닐)프로판산이 배치 반응기에서 제조되었다.The synthetic method of compound 14 is as follows. First, 3-(octadecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 5.

[반응식 5][Reaction Formula 5]

Figure PCTKR2024007830-appb-img-000020
Figure PCTKR2024007830-appb-img-000020

메탄올(10 mL)에 용해된 3-메르캅토프로판산(1.5 g, 1 eq.) 용액에 2,2'-디피리딜 디설파이드(6.2 g, 2 eq.)를 첨가하였다. 혼합물을 실온에서 6시간 동안 교반하였다. 반응 혼합물을 회전증발기를 이용하여 농축하고, 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 70:30)로 정제하여 3-(피리딘-2-일디설파닐)프로판산 2.62g(86%)을 흰색 고체로 얻었다.2,2'-Dipyridyl disulfide (6.2 g, 2 eq.) was added to a solution of 3-mercaptopropanoic acid (1.5 g, 1 eq.) in methanol (10 mL). The mixture was stirred at room temperature for 6 h. The reaction mixture was concentrated using a rotary evaporator, and the obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 70:30) to obtain 2.62 g (86%) of 3-(pyridin-2-yldisulfanyl)propanoic acid as a white solid.

1H NMR (500 MHz, CDCl3) δ = 10.53 (s, 1H), 8.50 (s, 1H), 7.70 (s, 2H), 7.16 (s, 1H), 3.07 (s, 2H), 2.82 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 176.00, 159.36, 149.35, 137.47, 121.16, 120.42, 33.95, 33.65. 1H NMR (500 MHz, CDCl 3 ) δ = 10.53 (s, 1H), 8.50 (s, 1H), 7.70 (s, 2H), 7.16 (s, 1H), 3.07 (s, 2H), 2.82 (s) , 2H). 13 C NMR (126 MHz, CDCl 3 ) δ 176.00, 159.36, 149.35, 137.47, 121.16, 120.42, 33.95, 33.65.

CH2Cl2에 용해된 3-(피리딘-2-일디설파닐)프로판산 1.0g(1.0eq.)의 용액에 1-옥타도다칸티올 1.5g(1.1eq.) 및 AcOH(34eq.)를 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 회전증발기를 이용하여 농축하고, 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)로 정제하여 3-(도데실디설파닐)프로판산 1.5g(82%)을 무색 액체로 얻었다.To a solution of 1.0 g (1.0 eq.) of 3-(pyridin-2-yldisulfanyl)propanoic acid in CH 2 Cl 2 were added 1.5 g (1.1 eq.) of 1-octadodacanethiol and AcOH (34 eq.). The mixture was stirred at room temperature overnight. The reaction mixture was concentrated using a rotary evaporator, and the obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to obtain 1.5 g (82%) of 3-(dodecyldisulfanyl)propanoic acid as a colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 2.95 - 2.89 (m, 2 H), 2.84 - 2.79 (m, 2 H), 2.72 -2.69 (m, 2 H), 1.72 - 1.64 (m, 2 H), 1.39 - 1.35 (m, 2 H), 1.27 (s, 28 H), 0.89 (t, J = 6.8 Hz, 3 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 2.95 - 2.89 (m, 2 H), 2.84 - 2.79 (m, 2 H), 2.72 -2.69 (m, 2 H), 1.72 - 1.64 (m, 2 H) ), 1.39 - 1.35 (m, 2 H), 1.27 (s, 28 H), 0.89 (t, J = 6.8 Hz, 3 H).

13C NMR (125 MHz, CDCl3) δ = 177.4, 39.1, 33.9, 32.7, 31.9, 29.69, 29.65, 29.6, 29.5, 29.3, 29.21, 29.18, 28.5, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 177.4, 39.1, 33.9, 32.7, 31.9, 29.69, 29.65, 29.6, 29.5, 29.3, 29.21, 29.18, 28.5, 22.7, 14.1.

[화합물 15][Compound 15]

3-(디메틸아미노)프로판-1,2-디일 비스(3-(도데실디설파닐)프로파노에이트), 3-(도데실디설파닐)프로판산을 사용하여 합성됨3-(Dimethylamino)propane-1,2-diyl bis(3-(dodecyldisulfanyl)propanoate), synthesized using 3-(dodecyldisulfanyl)propanoic acid

Figure PCTKR2024007830-appb-img-000021
Figure PCTKR2024007830-appb-img-000021

수율: 92%, 무색 고체. Yield : 92%, colorless solid.

1H NMR (500 MHz, CDCl3) δ = 5.23 - 5.19 (dd, J = 2.9, 6.4 Hz, 1 H), 4.42 (dd, J = 2.8, 12.0 Hz, 1 H), 4.14 (dd, J = 6.5, 12.0 Hz, 1 H), 2.94 - 2.85 (m, 4 H), 2.76 (t, J = 7.2 Hz, 4 H), 2.68 (t, J = 6.9 Hz, 4 H), 2.50 - 2.39 (m, 2 H), 2.25 (s, 6 H), 1.70 - 1.62 (m, 4 H), 1.40 - 1.35 (m, 4 H), 1.26 (br. s., 32 H), 0.88 (t, J = 6.9 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.23 - 5.19 (dd, J = 2.9, 6.4 Hz, 1 H), 4.42 (dd, J = 2.8, 12.0 Hz, 1 H), 4.14 (dd, J = 6.5, 12.0 Hz, 1 H), 2.94 - 2.85 (m, 4 H), 2.76 (t, J = 7.2 Hz, 4 H), 2.68 (t, J = 6.9 Hz, 4 H), 2.50 - 2.39 (m, 2 H), 2.25 (s, 6 H) ), 1.70 - 1.62 (m, 4 H), 1.40 - 1.35 (m, 4 H), 1.26 (br. s., 32 H), 0.88 (t, J = 6.9 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 171.4, 171.2, 69.7, 64.2, 59.2, 46.0, 39.01, 38.98, 34.2, 34.0, 33.11, 33.00, 31.9, 29.60, 29.58, 29.54, 29.47, 29.3, 29.2, 29.1, 28.5, 22.6, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 171.4, 171.2, 69.7, 64.2, 59.2, 46.0, 39.01, 38.98, 34.2, 34.0, 33.11, 33.00, 31.9, 29.60, 29.58, 29.54, 29.47, 29.3, 29.2, 29.1, 28.5, 22.6, 14.1.

MALDI-MS: 696.61 m/z MALDI-MS : 696.61 m/z

화합물 15의 합성 방법은 다음과 같다. 첫 번째로, 하기 반응식 6과 같이 3-(도데실디설파닐)프로판산이 배치 반응기에서 제조되었다.The synthetic method of compound 15 is as follows. First, 3-(dodecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 6.

[반응식 6][Reaction Formula 6]

Figure PCTKR2024007830-appb-img-000022
Figure PCTKR2024007830-appb-img-000022

CH2Cl2에 용해된 3-(피리딘-2-일디설파닐)프로판산 1.0g(1.0eq.)의 용액에 1-도데칸티올 1.0g(1.1eq.) 및 AcOH(34eq.)를 첨가하였다. 혼합물을 실온에서 밤새 교반하였다. 반응 혼합물을 감압 농축하고, 얻은 잔류물을 플래쉬 실리카겔 컬럼 크로마토그래피(n-헥산:EtOAc = 50:50)로 정제하여 3-(도데실디설파닐)프로판산 1.1g(80%)을 무색 액체로 얻었다.To a solution of 1.0 g (1.0 eq.) of 3-(pyridin-2-yldisulfanyl)propanoic acid in CH 2 Cl 2 were added 1.0 g (1.1 eq.) of 1-dodecanethiol and AcOH (34 eq.). The mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by flash silica gel column chromatography (n-hexane:EtOAc = 50:50) to obtain 1.1 g (80%) of 3-(dodecyldisulfanyl)propanoic acid as a colorless liquid.

1H NMR (500 MHz, CDCl3) δ = 2.93 - 2.89 (m, 2 H), 2.84 - 2.79 (m, 2 H), 2.72 - 2.68 (m, 2 H), 1.71 - 1.63 (m, 2 H), 1.42 - 1.34 (m, 2 H), 1.32 - 1.23 (m, 16 H), 0.89 (m, 3 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 2.93 - 2.89 (m, 2 H), 2.84 - 2.79 (m, 2 H), 2.72 - 2.68 (m, 2 H), 1.71 - 1.63 (m, 2 H) ), 1.42 - 1.34 (m, 2 H), 1.32 - 1.23 (m, 16 H), 0.89 (m, 3 H).

13C NMR (125 MHz, CDCl3) δ = 177.9, 39.1, 33.9, 32.7, 31.9, 29.61, 29.57, 29.5, 29.3, 29.20, 29.16, 28.5, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 177.9, 39.1, 33.9, 32.7, 31.9, 29.61, 29.57, 29.5, 29.3, 29.20, 29.16, 28.5, 22.7, 14.1.

[화합물 16][Compound 16]

6-브로모헥실 2-헥실데카노에이트, 2-하이드록시데칸산 및 6-브로모헥산올을 사용하여 합성됨Synthesized using 6-bromohexyl 2-hexyldecanoate, 2-hydroxydecanoic acid and 6-bromohexanol

Figure PCTKR2024007830-appb-img-000023
Figure PCTKR2024007830-appb-img-000023

수율: 92%, 무색 액체, R1 = 2.5분, 25 ℃, R2 = 4분, 50 ℃ Yield : 92%, colorless liquid, R1 = 2.5 min, 25 ℃, R2 = 4 min, 50 ℃

1H NMR (500 MHz, CDCl3) δ = 4.07 (t, J = 6.6 Hz, 2 H), 3.40 (t, J = 6.8 Hz, 2 H), 2.31 (ddd, J = 3.8, 5.2, 9.0 Hz, 1 H), 1.87 (quin, J = 7.1 Hz, 2 H), 1.68 - 1.53 (m, 4 H), 1.53 - 1.34 (m, 6 H), 1.30 - 1.22 (m, 20 H), 0.88 (t, J = 6.8 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 4.07 (t, J = 6.6 Hz, 2 H), 3.40 (t, J = 6.8 Hz, 2 H), 2.31 (ddd, J = 3.8, 5.2, 9.0 Hz , 1 H), 1.87 (quin, J = 7.1 Hz, 2 H), 1.68 - 1.53 (m, 4 H), 1.53 - 1.34 (m, 6 H), 1.30 - 1.22 (m, 20 H), 0.88 (t, J = 6.8 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 176.7, 63.8, 45.8, 33.6, 32.6, 32.5, 31.8, 31.7, 29.5, 29.4, 29.23, 29.20, 28.5, 27.8, 27.5, 27.4, 25.2, 22.64, 22.57, 14.1, 14.0. 13 C NMR (125 MHz, CDCl 3 ) δ = 176.7, 63.8, 45.8, 33.6, 32.6, 32.5, 31.8, 31.7, 29.5, 29.4, 29.23, 29.20, 28.5, 27.8, 27.5, 27.4, 25.2, 22.64, 22.57, 14.1, 14.0.

[실시예 3][Example 3]

DODAP의 확장 가능한 단일 유동 합성을 위한 크기 증가 방법Size-up method for scalable single-flow synthesis of DODAP

튜빙 크기 증가 방법을 사용하여 생산성 향상을 위해, 내경이 1.6mm로 더 큰 PTFE 튜빙 반응기를 사용하여 별도의 실험을 수행하였다. 1.6mm PTFE 튜브 반응기를 사용하는 DODAP의 연속 유동 합성을 위한 실험 셋팅은 도 9에 나타내었다. 유동 시스템은 2개의 T자형 미세 혼합기(M1 & M2, Ø = 1.0 mm), 2개의 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.6 mm, L = 225 cm)과 R2(PTFE 튜브, Ø = 1.6mm, L = 900cm)로 구성된다.For improved productivity using the tubing size increase method, a separate experiment was performed using a PTFE tubing reactor with a larger inner diameter of 1.6 mm. The experimental setup for continuous flow synthesis of DODAP using a 1.6 mm PTFE tube reactor is shown in Fig. 9. The flow system consists of two T-shaped micro-mixers (M1 & M2, Ø = 1.0 mm), two micro-tube reactors R1 (PTFE tube, Ø = 1.6 mm, L = 225 cm) and R2 (PTFE tube, Ø = 1.6 mm, L = 900 cm).

CH2Cl2에 용해된 올레산(0.5M) 및 DMF(0.05M) 용액과 CH2Cl2에 용해된 옥살릴 클로라이드(0.5M) 용액을 HPLC 펌프를 사용하여 1.5mL/분의 유속으로 M1(Ø = 1.0mm)에 주입하여 반응기 R1에서 1.5분의 체류 시간을 달성하였다. 생성된 용액을, HCl 및 CO 기체 부산물을 동반하는 올레오일 클로라이드의 합성을 위해 반응기 R1에 통과시켰다. 250psi 배압 조절기는 R1 배출구에 연결되었다. HCl 및 CO 가스는 배압 조절기의 바깥쪽에 연결하여 맞춤형 T 접합(직경 Ø = 1.6mm)(도 6의 a))을 사용하여 제거되었다. 이 용액을 CH2Cl2에 용해된 3-(디메틸아미노)프로판-1,2-디올(0.22 M) 및 염기(0.6 M)의 용액을 HPLC 펌프를 사용하여 1.5 mL/분의 유속으로 믹서 M2(Ø = 1.0 mm)에서 혼합하였다. 정상 상태에 도달한 후, 생성된 용액을 포화 NaHCO3 수용액으로 퀜칭하면서 반응기 R2의 말단에서 5분 동안 수집하였다. 유기층을 분리하고 수용성층을 CH2Cl2로 두 번 추출하였다. 합한 유기층을 건조 및 증발시키고, 헥산에 용해된 30% 에틸 아세테이트를 포함하는 실리카겔 컬럼 크로마토그래피를 사용하여 정제하여 순수한 DODAP 2(0.9g)를 85% 수율로 무색 액체로 생성하였다.Oleic acid (0.5 M) and DMF (0.05 M) solutions dissolved in CH2Cl2 and oxalyl chloride (0.5 M) solution dissolved in CH2Cl2 were injected into M1 (Ø = 1.0 mm) using an HPLC pump at a flow rate of 1.5 mL/min to achieve a residence time of 1.5 min in the reactor R1. The resulting solution was passed into the reactor R1 for the synthesis of oleoyl chloride accompanied by HCl and CO gaseous byproducts. A 250 psi back pressure regulator was connected to the R1 outlet. HCl and CO gases were removed using a custom T-junction (diameter Ø = 1.6 mm) (a) of Figure 6) connected to the outside of the back pressure regulator. This solution was mixed with a solution of 3-(dimethylamino)propane-1,2-diol (0.22 M) and base (0.6 M) dissolved in CH 2 Cl 2 at a flow rate of 1.5 mL/min using an HPLC pump in mixer M2 (Ø = 1.0 mm). After reaching steady state, the resulting solution was collected from the end of reactor R2 for 5 min while quenching with saturated NaHCO 3 aqueous solution. The organic layer was separated, and the aqueous layer was extracted twice with CH 2 Cl 2 . The combined organic layers were dried and evaporated, and purified using silica gel column chromatography containing 30% ethyl acetate dissolved in hexane to give pure DODAP 2 (0.9 g) as a colorless liquid in 85% yield.

[실시예 4][Example 4]

DOTAP 메틸 설페이트의 연속 유동 합성Continuous flow synthesis of DOTAP methyl sulfate

DODAP(2)에서 시작하는 DOTAP 메틸 설페이트(4)의 유동 합성을 위한 실험 셋팅은 도 10에 나타내었다. 유동 시스템은 하나의 T자형 미세 혼합기(M1, Ø = 1.0 mm), 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.0mm, L = 510cm) 및 100psi 배압 조절기로 구성된다. CH2Cl2에 용해된 DODAP(0.2M) 및 디메틸 설페이트(2.0M) 용액을 별도로 제조하고 HPLC 펌프를 사용하여 미세 혼합기 M1(Ø = 1.0mm)에 주입하였다. 다음으로, 생성된 용액을 DOTAP 메틸 설페이트의 합성을 위해 반응기 R1에 통과시켰다. 100psi 배압 조절기는 R1 배출구에 연결되었다. HPLC 펌프를 사용하여 반응물의 유속을 변경함으로써 다양한 체류 시간에서 박층 크로마토그래피를 사용하여 R1에서 반응을 모니터링하였다. 몇 가지 최적화 후에, DODAP(2)가 DOTAP 메틸 설페이트(4)로의 완전한 전환이 다음의 조건에서 달성되었다: 각 반응물의 유속 0.40 ml/분, 60℃, R1에서 5.0분의 체류 시간. 정상 상태에 도달한 후, 생성된 용액을 반응기 R1의 끝에서 5분 동안 수집하였다. 유기 용액을 회전 증발기에서 증발시켜 DOTAP 메틸 설페이트(4)를 얻었고, 이를 아세톤에서 결정화를 사용하여 정제하여 투명 결정질 화합물로서 90% 수율(0.27g)로 생성하였다.The experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 10. The flow system consists of a T-shaped micromixer (M1, Ø = 1.0 mm), a microtube reactor R1 (PTFE tubing, Ø = 1.0 mm, L = 510 cm), and a 100 psi back pressure regulator. DODAP (0.2 M) and dimethyl sulfate (2.0 M) solutions in CH 2 Cl 2 were prepared separately and injected into the micromixer M1 (Ø = 1.0 mm) using an HPLC pump. Next, the resulting solution was passed into the reactor R1 for the synthesis of DOTAP methyl sulfate. The 100 psi back pressure regulator was connected to the R1 outlet. The reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using an HPLC pump. After some optimization, complete conversion of DODAP (2) to DOTAP methyl sulfate (4) was achieved under the following conditions: flow rate of 0.40 ml/min of each reactant, 60°C, and residence time of 5.0 min in R1. After reaching steady state, the resulting solution was collected from the end of reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to give DOTAP methyl sulfate (4), which was purified using crystallization from acetone to give a clear crystalline compound in 90% yield (0.27 g).

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 4의 구조식을 갖는 DOTAP 메틸 설페이트가 성공적으로 합성되었음을 확인하였다.The spectroscopic identification results of the synthesized final product confirmed that DOTAP methyl sulfate having the structural formula of compound 4 below was successfully synthesized.

[화합물 4][Compound 4]

Figure PCTKR2024007830-appb-img-000024
Figure PCTKR2024007830-appb-img-000024

1H NMR (500 MHz, CDCl3) δ = 5.59 (br. s., 1 H), 5.41 - 5.29 (m, 4 H), 4.50 (dd, J = 3.5, 12.2 Hz, 1 H), 4.10 - 4.07 (m, 2 H), 3.75 - 3.73 (m, 1 H), 3.71 (s, 3 H), 3.35 (m, 9 H), 2.37 - 2.30 (m, 4 H), 2.06 - 1.96 (m, 8 H), 1.65 - 1.55 (m, 4 H), 1.35 - 1.22 (m, 40 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.59 (br. s., 1 H), 5.41 - 5.29 (m, 4 H), 4.50 (dd, J = 3.5, 12.2 Hz, 1 H), 4.10 - 4.07 (m, 2 H), 3.75 - 3.73 (m, 1 H), 3.71 (s, 3 H), 3.35 (m, 9 H), 2.37 - 2.30 (m, 4 H), 2.06 - 1.96 (m, 8 H), 1.65 - 1.55 (m, 4 H), 1.35 - 1.22 (m, 40 H).

13C NMR (125 MHz, CDCl3) δ = 173.1, 172.7, 130.0, 130.0, 129.6, 129.6, 65.9, 65.8, 63.1, 54.4, 54.1, 34.1, 33.8, 31.9, 29.73, 29.71, 29.69, 29.5, 29.3, 29.22, 29.17, 29.13, 29.09, 29.07, 29.05, 27.20, 27.16, 27.1, 24.7, 24.6, 22.6, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.1, 172.7, 130.0, 130.0, 129.6, 129.6, 65.9, 65.8, 63.1, 54.4, 54.1, 34.1, 33.8, 31.9, 29.73, 29.71, 29.69, 29.5, 29.3, 29.22, 29.17, 29.13, 29.09, 29.07, 29.05, 27.20, 27.16, 27.1, 24.7, 24.6, 22.6, 14.1.

MALDI-MS: 662.77 m/z. MALDI-MS : 662.77 m/z.

[실시예 5][Example 5]

DOTAP 메틸 설페이트의 확장 가능한 단일 유동 합성을 위한 크기 증가 방법Size-up method for scalable single-flow synthesis of DOTAP methyl sulfate

DODAP(2)에서 시작하는 DOTAP 메틸 설페이트(4)의 유동 합성을 위한 실험 셋팅을 도 11에 나타내었다. 유동 시스템은 T자형 미세 혼합기(M1, Ø = 1.0mm) 1개, 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.6mm, L = 500cm) 및 250psi 배압 조절기로 구성된다. CH2Cl2에 용해된 DODAP(0.2M) 및 디메틸 설페이트(2.0M) 용액을 별도로 제조하고, 각각 1.0mL/분의 유속으로 HPLC 펌프를 사용하여 미세 혼합기 M1(Ø = 1.0mm)에 주입하였다. 이어서, 생성된 용액을 DOTAP 메틸 설페이트의 합성을 위해 반응기 R1에 통과시켰다. 250psi 배압 조절기는 R1 배출구에 연결되었다. 정상 상태에 도달한 후, 생성된 용액을 반응기 R1의 끝에서 5분 동안 수집하였다. 유기 용액을 회전 증발기에서 증발시켜 DOTAP 메틸 설페이트(4)를 얻었고, 이를 아세톤에서의 결정화를 사용하여 정제하여 투명 결정질 화합물로서 86% 수율(0.67g)을 얻었다.The experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 11. The flow system consists of one T-shaped micromixer (M1, Ø = 1.0 mm), a microtube reactor R1 (PTFE tube, Ø = 1.6 mm, L = 500 cm), and a 250 psi back pressure regulator. DODAP (0.2 M) and dimethyl sulfate (2.0 M) solutions dissolved in CH 2 Cl 2 were prepared separately and injected into the micromixer M1 (Ø = 1.0 mm) using an HPLC pump at a flow rate of 1.0 mL/min, respectively. The resulting solution was then passed into the reactor R1 for the synthesis of DOTAP methyl sulfate. The 250 psi back pressure regulator was connected to the R1 outlet. After reaching steady state, the resulting solution was collected at the end of the reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to obtain DOTAP methyl sulfate (4), which was purified using crystallization from acetone to obtain a transparent crystalline compound in 86% yield (0.67 g).

[실시예 6][Example 6]

양이온 지질 라이브러리의 연속 유동 합성Continuous flow synthesis of cationic lipid libraries

개발된 유동 경로는 유사한 생산성을 갖는 널리 사용되는 또 다른 양이온 지질인 DLinTAP(17)의 합성을 위해 확장되었다. 또한, 각각 효소적으로 절단 가능한 에스테르 결합과 이황화 결합을 포함하는 추가의 새로운 양이온성 지질(즉, 화합물 18 내지 22)도 개발된 유동 조건을 사용하여 합성하였다(도 12).The developed flow pathway was extended for the synthesis of another widely used cationic lipid, DLinTAP (17), with similar productivities. In addition, additional novel cationic lipids (i.e., compounds 18 to 22), each containing enzymatically cleavable ester and disulfide bonds, were also synthesized using the developed flow conditions (Figure 12).

DOTAP 메틸 설페이트(4)의 합성과 동일한 조건 하에서, 각각의 이온화 가능한 지질로부터 시작하는 양이온성 지질(화합물 17 내지 22)의 라이브러리를 제조하였다.Under conditions identical to those for the synthesis of DOTAP methyl sulfate (4), a library of cationic lipids (compounds 17 to 22) was prepared starting from each ionizable lipid.

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 17 내지 22의 구조식을 갖는 양이온 지질 라이브러리가 성공적으로 합성되었음을 확인하였다.Spectroscopic identification results of the synthesized final products confirmed that a cationic lipid library having structural formulas of compounds 17 to 22 was successfully synthesized.

[화합물 17][Compound 17]

N,N,N-트리메틸-2,3-비스((9Z,12Z)-옥타데카-9,12-디에노일옥시)프로판-1-아미늄 메틸 설페이트(DLinDAP)N,N,N-Trimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propane-1-aminum methyl sulfate (DLinDAP)

Figure PCTKR2024007830-appb-img-000025
Figure PCTKR2024007830-appb-img-000025

수율: 88%, 무색 고체 Yield : 88%, colorless solid

1H NMR (500MHz, CDCl3) δ = 5.58 (br. s., 1 H), 5.43 - 5.27 (m, 8 H), 4.48 (dd, J = 3.3, 12.1 Hz, 1 H), 4.11 - 4.01 (m, 2 H), 3.75 - 3.71 (m, 1 H), 3.70 (s, 3 H), 3.33 (s, 9 H), 2.76 (t, J = 6.6 Hz, 4 H), 2.37 - 2.29 (m, 4 H), 2.04 (q, J = 6.9 Hz, 8 H), 1.66 - 1.55 (m, 4 H), 1.36 - 1.27 (m, 28 H), 0.88 (t, J = 6.8 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.58 (br. s., 1 H), 5.43 - 5.27 (m, 8 H), 4.48 (dd, J = 3.3, 12.1 Hz, 1 H), 4.11 - 4.01 (m, 2 H), 3.75 - 3.71 (m, 1 H), 3.70 (s, 3 H), 3.33 (s, 9 H), 2.76 (t, J = 6.6 Hz, 4 H), 2.37 - 2.29 (m, 4 H), 2.04 (q, J = 6.9) Hz, 8 H), 1.66 - 1.55 (m, 4 H), 1.36 - 1.27 (m, 28 H), 0.88 (t, J = 6.8 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.1, 172.7, 130.2, 129.92, 129.89, 128.1, 128.0, 127.8, 65.9, 65.8, 63.1, 54.5, 54.1, 34.1, 33.8, 31.5, 29.60, 29.58, 29.3, 29.20, 29.16, 29.12, 29.08, 29.06, 29.03, 27.2, 25.6, 24.7, 24.6, 22.5, 14.0. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.1, 172.7, 130.2, 129.92, 129.89, 128.1, 128.0, 127.8, 65.9, 65.8, 63.1, 54.5, 54.1, 34.1, 33.8, 31.5, 29.60, 29.58, 29.3, 29.20, 29.16, 29.12, 29.08, 29.06, 29.03, 27.2, 25.6, 24.7, 24.6, 22.5, 14.0.

MALDI-MS: 658.80 m/z. MALDI-MS : 658.80 m/z.

[화합물 18][Compound 18]

2,3-비스((10-(헥실옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트2,3-Bis((10-(hexyloxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropane-1-aminum methyl sulfate

Figure PCTKR2024007830-appb-img-000026
Figure PCTKR2024007830-appb-img-000026

수율: 88%, 무색 고체 Yield: 88%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.56 (br. s., 1 H), 4.46 (dd, J = 2.7, 12.1 Hz, 1 H), 4.08 - 3.96 (m, 6 H), 3.74 - 3.69 (m, 1 H), 3.67 (s, 3 H), 3.30 (s, 9 H), 2.34 - 2.23 (m, 8 H), 1.58 - 1.1.56 (m, 12 H), 1.33 - 1.24 (m, 28 H), 0.86 (t, J = 6.5 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.56 (br. s., 1 H), 4.46 (dd, J = 2.7, 12.1 Hz, 1 H), 4.08 - 3.96 (m, 6 H), 3.74 - 3.69 (m, 1 H), 3.67 (s, 3 H), 3.30 (s, 9 H), 2.34 - 2.23 (m, 8 H), 1.58 - 1.1.56 (m, 12 H), 1.33 - 1.24 (m, 28 H), 0.86 (t, J = 6.5 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.8, 173.0, 172.6, 65.9, 65.7, 64.3, 63.1, 54.4, 54.0, 34.24, 34.22, 34.0, 33.7, 31.3, 29.0, 28.97, 28.96, 28.94, 28.92, 28.5, 25.5, 24.9, 24.8, 24.6, 24.5, 22.4, 13.9. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.8, 173.0, 172.6, 65.9, 65.7, 64.3, 63.1, 54.4, 54.0, 34.24, 34.22, 34.0, 33.7, 31.3, 29.0, 28.97, 28.96, 28.94, 28.92, 28.5, 25.5, 24.9, 24.8, 24.6, 24.5, 22.4, 13.9.

MALDI-MS: 670.49 m/z. MALDI-MS : 670.49 m/z.

[화합물 19][Compound 19]

2,3-비스((10-(((S)-3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트2,3-Bis((10-(((S)-3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate

Figure PCTKR2024007830-appb-img-000027
Figure PCTKR2024007830-appb-img-000027

수율: 88%, 무색 고체 Yield : 88%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.60 - 5.52 (m, 1 H), 5.07 - 5.04 (m, 2 H), 4.46 (dd, J = 3.4, 12.1 Hz, 1 H), 4.14 - 3.99 (m, 6 H), 3.75 - 3.69 (m, 1 H), 3.67 (s, 3 H), 2.35 - 2.22 (m, 8 H), 2.03 - 1.87 (m, 4 H), 1.65 (s, 6 H), 1.62 - 1.55 (m, 14 H), 1.55 - 1.47 (m, 3 H), 1.44 - 1.37 (m, 2 H), 1.36 - 1.23 (m, 19 H), 1.20 - 1.11 (m, 2 H), 0.89 (s, 3 H), 0.88 (s, 3 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.60 - 5.52 (m, 1 H), 5.07 - 5.04 (m, 2 H), 4.46 (dd, J = 3.4, 12.1 Hz, 1 H), 4.14 - 3.99 (m, 6 H), 3.75 - 3.69 (m, 1 H), 3.67 (s, 3 H), 2.35 - 2.22 (m, 8 H), 2.03 - 1.87 (m, 4 H), 1.65 (s, 6 H), 1.62 - 1.55 (m, 14) H), 1.55 - 1.47 (m, 3 H), 1.44 - 1.37 (m, 2 H), 1.36 - 1.23 (m, 19 H), 1.20 - 1.11 (m, 2 H), 0.89 (s, 3 H), 0.88 (s, 3 H).

13C NMR (125 MHz, CDCl3) δ = 173.8, 173.0, 172.6, 131.2, 124.5, 65.8, 65.7, 63.1, 62.7, 54.4, 54.0, 36.9, 35.4, 34.2, 34.0, 33.7, 29.4, 29.0, 28.98, 28.96, 28.92, 25.6, 25.3, 24.84, 24.83, 24.6, 24.4, 19.3, 17.5. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.8, 173.0, 172.6, 131.2, 124.5, 65.8, 65.7, 63.1, 62.7, 54.4, 54.0, 36.9, 35.4, 34.2, 34.0, 33.7, 29.4, 29.0, 28.98, 28.96, 28.92, 25.6, 25.3, 24.84, 24.83, 24.6, 24.4, 19.3, 17.5.

MALDI-MS: 778.89 m/z. MALDI-MS : 778.89 m/z.

[화합물 20][Compound 20]

2,3-비스((10-(((E)-3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트2,3-Bis((10-(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate

Figure PCTKR2024007830-appb-img-000028
Figure PCTKR2024007830-appb-img-000028

수율: 90%, 무색 고체 Yield: 90%, colorless solid

1H NMR (500 MHz,CDCl3) δ = 5.57 (br. s., 1 H), 5.37 - 5.28 (m, 3 H), 5.07 (t, J = 6.3 Hz, 2 H), 4.57 (d, J = 7.0 Hz, 4 H), 4.48 (dd, J = 3.4, 12.2 Hz, 1 H), 4.10 - 4.00 (m, 2 H), 3.75 - 3.70 (m, 1 H), 3.69 (s, 3 H), 3.32 (s, 9 H), 2.38 - 2.25 (m, 8 H), 2.13 - 1.99 (m, 8 H), 1.69 (s, 6 H), 1.67 (s, 6 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.57 (br. s., 1 H), 5.37 - 5.28 (m, 3 H), 5.07 (t, J = 6.3 Hz, 2 H), 4.57 (d, J = 7.0 Hz, 4 H), 4.48 (dd, J = 3.4, 12.2 Hz, 1 H), 4.10 - 4.00 (m, 2 H), 3.75 - 3.70 (m, 1 H), 3.69 (s, 3 H), 3.32 (s, 9 H), 2.38 - 2.25 (m, 8) H), 2.13 - 1.99 (m, 8 H), 1.69 (s, 6 H), 1.67 (s, 6 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H).

13C NMR (125 MHz, CDCl3) δ = 173.8, 173.1, 172.6, 142.0, 131.7, 123.7, 118.3, 65.9, 65.8, 63.1, 61.1, 54.5, 54.1, 39.5, 34.3, 34.1, 33.7, 29.04, 29.02, 28.98, 28.95, 26.2, 25.6, 24.88, 24.86, 24.6, 24.5, 17.6, 16.4. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.8, 173.1, 172.6, 142.0, 131.7, 123.7, 118.3, 65.9, 65.8, 63.1, 61.1, 54.5, 54.1, 39.5, 34.3, 34.1, 33.7, 29.04, 29.02, 28.98, 28.95, 26.2, 25.6, 24.88, 24.86, 24.6, 24.5, 17.6, 16.4.

MALDI-MS: 774.59 m/z. MALDI-MS : 774.59 m/z.

[화합물 21][Compound 21]

2,3-비스((10-(((Z)-3,7-디메틸옥타-2,6-디엔-1-일)옥소)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트2,3-Bis((10-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)oxo)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate

Figure PCTKR2024007830-appb-img-000029
Figure PCTKR2024007830-appb-img-000029

수율: 90%, 무색 고체 Yield : 90%, colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.63 - 5.51 (m, 1 H), 5.33 (t, J = 7.1 Hz, 2 H), 5.08 (t, J = 6.2 Hz, 2 H), 4.54 (d, J = 7.3 Hz, 4 H), 4.48 (dd, J = 3.5, 12.2 Hz, 1 H), 4.10 - 4.03 (m, 2 H), 3.76 - 3.70 (m, 1 H), 3.69 (s, 3 H), 3.33 (s, 9 H), 2.36 - 2.24 (m, 8 H), 2.15 - 2.02 (m, 8 H), 1.79 (m, 6 H), 1.67 (s, 7 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.63 - 5.51 (m, 1 H), 5.33 (t, J = 7.1 Hz, 2 H), 5.08 (t, J = 6.2 Hz, 2 H), 4.54 ( d, J = 7.3 Hz, 4 H), 4.48 (dd, J = 3.5, 12.2 Hz, 1 H), 4.10 - 4.03 (m, 2 H), 3.76 - 3.70 (m, 1 H), 3.69 (s, 3 H), 3.33 (s, 9 H), 2.36 - 2.24 (m , 8 H), 2.15 - 2.02 (m, 8 H), 1.79 (m, 6 H), 1.67 (s, 7 H), 1.59 (s, 14 H), 1.28 (br. s., 16 H).

13C NMR (125 MHz, CDCl3) δ = 173.72, 173.70, 173.0, 172.6, 142.4, 132.0, 123.5, 119.2, 65.9, 65.8, 63.1, 60.8, 54.4, 54.1, 34.27, 34.25, 34.0, 33.7, 32.1, 29.03, 29.00, 28.97, 28.94, 26.6, 25.6, 24.9, 24.8, 24.6, 24.5, 23.4, 17.6. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.72, 173.70, 173.0, 172.6, 142.4, 132.0, 123.5, 119.2, 65.9, 65.8, 63.1, 60.8, 54.4, 54.1, 34.27, 34.25, 34.0, 33.7, 32.1, 29.03, 29.00, 28.97, 28.94, 26.6, 25.6, 24.9, 24.8, 24.6, 24.5, 23.4, 17.6.

MALDI-MS: 774.79 m/z. MALDI-MS : 774.79 m/z.

[화합물 22][Compound 22]

N,N,N-트리메틸-2,3-비스((3-(옥타데실디설파닐)프로파노일)옥시)프로판-1-아미늄 메틸 설페이트N,N,N-Trimethyl-2,3-bis((3-(octadecyldisulfanyl)propanoyl)oxy)propan-1-aminum methyl sulfate

Figure PCTKR2024007830-appb-img-000030
Figure PCTKR2024007830-appb-img-000030

수율: 90% 무색 고체 Yield : 90% colorless solid

1H NMR (500 MHz, CDCl3) δ = 5.66 - 5.59 (m, 1 H), 4.60 (dd, J = 3.4, 12.3 Hz, 1 H), 4.23 - 4.13 (m, 2 H), 3.81 (dd, J = 9.1, 14.3 Hz, 1 H), 3.72 (s, 3 H), 3.35 (s, 9 H), 2.95 - 2.88 (m, 4 H), 2.88 - 2.79 (m, 4 H), 2.73 - 2.65 (m, 4 H), 1.67 (dd, J = 5.2, 6.9 Hz, 4 H), 1.38 (br. s., 4 H), 1.26 (s, 56 H), 0.88 (t, J = 6.8 Hz, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.66 - 5.59 (m, 1 H), 4.60 (dd, J = 3.4, 12.3 Hz, 1 H), 4.23 - 4.13 (m, 2 H), 3.81 (dd , J = 9.1, 14.3 Hz, 1 H), 3.72 (s, 3 H), 3.35 (s, 9 H), 2.95 - 2.88 (m, 4 H), 2.88 - 2.79 (m, 4 H), 2.73 - 2.65 (m, 4 H), 1.67 (dd, J = 5.2, 6.9 Hz, 4 H), 1.38 (br. s., 4 H), 1.26 (s, 56 H), 0.88 (t, J = 6.8 Hz, 6 H).

13C NMR (125 MHz, CDCl3) δ = 171.3, 171.0, 66.7, 65.6, 63.2, 54.5, 54.4, 38.9, 38.7, 33.9, 33.6, 32.9, 32.4, 31.9, 29.7, 29.6, 29.5, 29.35, 29.26, 29.21, 29.15, 28.58, 28.55, 22.7, 14.1. 13 C NMR (125 MHz, CDCl 3 ) δ = 171.3, 171.0, 66.7, 65.6, 63.2, 54.5, 54.4, 38.9, 38.7, 33.9, 33.6, 32.9, 32.4, 31.9, 29.7, 29.6, 29.5, 29.35, 29.26, 29.21, 29.15, 28.58, 28.55, 22.7, 14.1.

MALDI-MS: 878.92 m/z.MALDI-MS: 878.92 m/z.

[실시예 7][Example 7]

DOTAP 클로라이드의 연속 유동 합성Continuous flow synthesis of DOTAP chloride

7-1. 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드의 배치 합성7-1. Batch synthesis of 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminum chloride

2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드를 하기 반응식 7과 같이 배치에서 합성하였다.2,3-Dihydroxy-N,N,N-trimethylpropan-1-aminum chloride was synthesized in batch according to the following reaction scheme 7.

[반응식 7][Reaction Formula 7]

Figure PCTKR2024007830-appb-img-000031
Figure PCTKR2024007830-appb-img-000031

농축기(condenser), 자석 교반기 및 적하 깔때기(dropping funnel)가 장착된 2구 250ml 둥근 바닥 플라스크에 트리메틸아민(190mmol)의 에탄올 용액과 메탄올 30ml를 첨가하였다. 불활성 가스 환경에서 플라스크를 닫고 얼음 배스(bath)에서 내용물을 0 ℃로 냉각하였다. 반응 플라스크에 3-클로로프로판-1,2-디올(126.5mmol)을 첨가하였다. 반응 혼합물을 65~68 ℃에서 10시간 동안 교반하고 용매를 증발시켰다. 반응이 완료된 후, 유기 용매를 회전 증발기에서 증발시키고, 생성된 미가공 물질(crude)을 디에틸 에테르(50 mL)로 5회, 아세톤(50 mL)으로 7회 세척하고, 생성된 고체를 진공 하에 건조시켜 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드(24)를 92% 수율로 투명한 결정질 고체로 얻었다.A two-necked 250-mL round-bottomed flask equipped with a condenser, a magnetic stirrer and a dropping funnel was charged with an ethanol solution of trimethylamine (190 mmol) and 30 mL of methanol. The flask was closed under an inert gas atmosphere and the contents were cooled to 0 °C in an ice bath. 3-Chloropropane-1,2-diol (126.5 mmol) was added to the reaction flask. The reaction mixture was stirred at 65–68 °C for 10 h and the solvent was evaporated. After the reaction was completed, the organic solvent was evaporated on a rotary evaporator, and the resulting crude was washed five times with diethyl ether (50 mL) and seven times with acetone (50 mL), and the resulting solid was dried under vacuum to obtain 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminum chloride (24) as a transparent crystalline solid in 92% yield.

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 24의 구조식을 갖는 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드가 성공적으로 합성되었음을 확인하였다.The spectroscopic identification results of the synthesized final product confirmed that 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminum chloride having the structural formula of compound 24 below was successfully synthesized.

[화합물 24][Compound 24]

Figure PCTKR2024007830-appb-img-000032
Figure PCTKR2024007830-appb-img-000032

1H NMR (500 MHz, DMSO-d6) δ = 5.71 (d, J = 5.5 Hz, 1 H), 5.16 (t, J = 5.7 Hz, 1 H), 4.04 (br. s., 1 H), 3.47 (d, J = 13.4 Hz, 1 H), 3.43 - 3.39 (m, 1 H), 3.29 (dd, J = 9.6, 13.4 Hz, 1 H), 3.26 - 3.20 (m, 1 H), 3.17 - 3.12 (s, 9 H). 1 H NMR (500 MHz, DMSO-d 6 ) δ = 5.71 (d, J = 5.5 Hz, 1 H), 5.16 (t, J = 5.7 Hz, 1 H), 4.04 (br. s., 1 H) , 3.47 (d, J = 13.4 Hz, 1 H), 3.43 - 3.39 (m, 1 H), 3.29 (dd, J = 9.6, 13.4 Hz, 1 H), 3.26 - 3.20 (m, 1 H), 3.17 - 3.12 (s, 9 H).

7-2. DOTAP 클로라이드의 연속 유동 합성7-2. Continuous flow synthesis of DOTAP chloride

DOTAP 클로라이드(5)의 유동 합성을 위한 실험 셋팅은 도 13에 나타내었다. 유동 시스템은 T자형 미세 혼합기(M1, Ø = 1.0mm), 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.0mm, L = 640cm)로 구성된다. DMSO에 용해된 올레산(0.65M), 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드(23)(0.3M) 및 DMAP(0.3M) 용액을 DMSO에 주사기 펌프를 사용하여 미세 혼합기 M1 (Ø = 1.0 mm)을 통해 DMSO에 용해된 EDC.HCl(0.65 M) 용액과 혼합하고 반응기 R1에 통과시켰다. 40psi 배압 조절기는 R1 배출구에 연결되었다. 주사기 펌프를 사용하여 반응물의 유속을 변경함으로써 다양한 체류 시간에서 박층 크로마토그래피를 사용하여 R1에서 반응을 모니터링하였다. 몇 가지 최적화 후에, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드(23)에서 DOTAP 클로라이드(5)로의 완전한 전환이 다음과 같은 조건에서 관찰됨을 확인하였다: 70℃, R1에서 5분의 체류 시간. 정상 상태에 도달한 후, 생성된 용액을 반응기 R1의 끝에서 5분 동안 수집하였다. 용액을 1 N HCl(10.0 mL)을 사용하여 천천히 퀜칭하였고, 생성된 수용성층을 디클로로메탄(50.0 mL Х 3)을 사용하여 3회 세척하였다. 합한 유기층을 Na2SO4로 건조시키고, 여과한 다음 회전 증발기에서 농축하여 미가공 물질을 얻었고, 이는 -20 ℃의 이소프로판올 및 아세톤 용매 시스템에서 결정화를 사용하여 정제하여 무정형 분말로 DOTAP 클로라이드(5)를 생성하였다. 대안적으로, 미가공 DOTAP 클로라이드(5)를 CH2Cl2 용매 시스템에 용해된 30% MeOH를 포함하는 플래시 실리카겔 컬럼 크로마토그래피를 사용하여 정제하여 DOTAP 클로라이드(5)(0.45g)를 88% 수율로 얻었다.The experimental setup for the flow synthesis of DOTAP chloride (5) is shown in Fig. 13. The flow system consisted of a T-shaped micromixer (M1, Ø = 1.0 mm) and a microtube reactor R1 (PTFE tube, Ø = 1.0 mm, L = 640 cm). A solution of oleic acid (0.65 M), 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) (0.3 M), and DMAP (0.3 M) dissolved in DMSO were mixed with a solution of EDC.HCl (0.65 M) dissolved in DMSO through the micromixer M1 (Ø = 1.0 mm) using a syringe pump and passed into the reactor R1. A 40 psi back pressure regulator was connected to the R1 outlet. The reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using a syringe pump. After some optimization, complete conversion of 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) to DOTAP chloride (5) was observed under the following conditions: 70 °C, residence time of 5 min in R1. After reaching steady state, the resulting solution was collected at the end of reactor R1 for 5 min. The solution was slowly quenched with 1 N HCl (10.0 mL) and the resulting aqueous layer was washed three times with dichloromethane (50.0 mL Х 3). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated on a rotary evaporator to give the crude material, which was purified using crystallization in isopropanol and acetone solvent system at -20 °C to give DOTAP chloride (5) as an amorphous powder. Alternatively, crude DOTAP chloride (5) was purified using flash silica gel column chromatography containing 30% MeOH in CH 2 Cl 2 solvent system to give DOTAP chloride (5) (0.45 g) in 88% yield.

합성된 최종 산물의 분광학적 동정 결과를 통해 하기 화합물 5의 구조식을 갖는 DOTAP 클로라이드가 성공적으로 합성되었음을 확인하였다.The spectroscopic identification results of the synthesized final product confirmed that DOTAP chloride having the structural formula of compound 5 below was successfully synthesized.

[화합물 5][Compound 5]

Figure PCTKR2024007830-appb-img-000033
Figure PCTKR2024007830-appb-img-000033

1H NMR (500 MHz, CDCl3) δ = 5.58 (br. s., 1 H), 5.31 (br. s., 4 H), 4.59 - 4.46 (m, 2 H), 4.11 - 4.02 (m, 1 H), 3.81 - 3.70 (m, 1 H), 3.50 (s, 12 H), 2.35 - 2.22 (m, 4 H), 1.97 (br. s., 8 H), 1.56 (br. s., 4 H), 1.26 - 1.23 (m, 40 H), 0.88 - 0.83 (m, 6 H). 1 H NMR (500 MHz, CDCl 3 ) δ = 5.58 (br. s., 1 H), 5.31 (br. s., 4 H), 4.59 - 4.46 (m, 2 H), 4.11 - 4.02 (m, 1 H), 3.81 - 3.70 (m, 1 H), 3.50 (s, 12 H), 2.35 - 2.22 (m, 4 H), 1.97 (br. s., 8 H), 1.56 (br. s., 4 H), 1.26 - 1.23 (m, 40 H), 0.88 - 0.83 (m, 6 H).

13C NMR (125 MHz, CDCl3) δ = 173.0, 172.6, 130.0, 129.9, 129.6, 129.5, 65.8, 65.8, 63.1, 54.2, 34.1, 33.8, 31.8, 29.7, 29.64, 29.61, 29.4, 29.23, 29.21, 29.14, 29.08, 29.06, 29.00, 28.97, 27.13, 27.09, 27.07, 24.7, 24.5, 22.6, 14.0. 13 C NMR (125 MHz, CDCl 3 ) δ = 173.0, 172.6, 130.0, 129.9, 129.6, 129.5, 65.8, 65.8, 63.1, 54.2, 34.1, 33.8, 31.8, 29.7, 29.64, 29.61, 29.4, 29.23, 29.21, 29.14, 29.08, 29.06, 29.00, 28.97, 27.13, 27.09, 27.07, 24.7, 24.5, 22.6, 14.0.

MALDI-MS: 662.76 m/z MALDI-MS : 662.76 m/z

[실시예 8][Example 8]

DOTAP 클로라이드의 확장 가능한 단일 유동 합성을 위한 크기 증가 방법Size-up method for scalable single-flow synthesis of DOTAP chloride

크기 증가 방법을 이용한 DOTAP 클로라이드(5)의 유동 합성을 위한 실험 셋팅을 도 14에 나타내었다. 유동 시스템은 하나의 T자형 미세 혼합기(M1, Ø = 1.0mm), 미세 튜브 반응기 R1(PTFE 튜브, Ø = 1.6mm, L = 500cm) 및 100psi 배압 조절기로 구성된다. DMSO에 용해된 올레산(0.65M), 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드(23)(0.3M) 및 DMAP(0.3M) 용액을 HPLC 펌프를 사용하여 각 1.0 ml/분의 유속으로 미세 혼합기 M1 (Ø = 1.0 mm)을 사용하여 DMSO에 용해된 EDC.HCl 용액(0.65 M)과 혼합하였다. 100psi 배압 조절기는 R1 배출구에 연결되었다. 정상 상태에 도달한 후, 생성된 용액을 반응기 R1의 말단에서 5분 동안 수집하였다. 용액을 1.0 N HCl(20.0 mL)을 사용하여 천천히 퀜칭하고, 생성된 수용성층을 디클로로메탄(50.0 mL Х 3)을 사용하여 3회 세척하였다. 합한 유기층을 Na2SO4로 건조시키고, 여과한 다음 회전 증발기에서 농축하여 미가공 물질을 얻었고, 이를 CH2Cl2 용매 시스템에 용해된 30% MeOH를 포함하는 플래시 실리카겔 컬럼 크로마토그래피를 사용하여 정제하여 82% 수율로 DOTAP 클로라이드(5)(0.84g)을 얻었다.The experimental setup for the flow synthesis of DOTAP chloride (5) using the size-increasing method is shown in Fig. 14. The flow system consists of a T-shaped micromixer (M1, Ø = 1.0 mm), a microtube reactor R1 (PTFE tubing, Ø = 1.6 mm, L = 500 cm), and a 100 psi back pressure regulator. Solutions of oleic acid (0.65 M), 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) (0.3 M), and DMAP (0.3 M) dissolved in DMSO were mixed with EDC.HCl solution (0.65 M) dissolved in DMSO using an HPLC pump through the micromixer M1 (Ø = 1.0 mm) at a flow rate of 1.0 ml/min each. The 100 psi back pressure regulator was connected to the R1 outlet. After reaching steady state, the resulting solution was collected at the end of reactor R1 for 5 min. The solution was slowly quenched with 1.0 N HCl (20.0 mL), and the resulting aqueous layer was washed three times with dichloromethane (50.0 mL Х 3). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated on a rotary evaporator to obtain the crude material, which was purified using flash silica gel column chromatography containing 30% MeOH dissolved in CH 2 Cl 2 solvent system to give DOTAP chloride (5) (0.84 g) in 82% yield.

[실시예 9][Example 9]

나노입자 담체의 전구체로서 합성된 지질의 평가Evaluation of synthetic lipids as precursors for nanoparticle carriers

9-1. 자체 제작 미세 혼합기의 설계 및 제작9-1. Design and manufacture of self-made micro-mixer

리포좀 또는 지질나노입자의 합성을 위해, 3D 프린팅을 이용하여 자체 제작한 미세 혼합기를 제작하였다. 믹서는 반구형 배플로 구성되어 주입된 유체 간의 빠른 혼합이 가능하다. 누수 없는 정확한 주입을 위해, 각 주입구와 배출구를 5mm 나사산(1/16"의 경우 1/4-28 평평한 바닥)으로 수정하여 XP-235(IDEX Health & Science) 피팅과 연결이 가능하도록 하였다. CAD(Computer Aided Design)를 통해 제작된 가상 모델은 DLP 기반 3D 프린터(Pico 2 HD, Asiga)를 사용하여 상용 레진(PlasCLEAR)으로 제작되었으며, 광경화를 통해 각 층을 적층하고 이소프로판올을 사용하여 레진을 제거하였다. 마지막으로, 세척된 장치는 완전한 중합을 위해 UV 챔버에서 후경화되었다.For the synthesis of liposomes or lipid nanoparticles, a custom-made micro-mixer was fabricated using 3D printing. The mixer consists of hemispherical baffles to enable rapid mixing of the injected fluids. To ensure accurate injection without leakage, each inlet and outlet were modified with a 5 mm thread (1/4-28 flat bottom for 1/16") to enable connection with XP-235 (IDEX Health & Science) fittings. The virtual model created through CAD (Computer Aided Design) was fabricated using a DLP-based 3D printer (Pico 2 HD, Asiga) with commercial resin (PlasCLEAR), and each layer was laminated through photocuring and the resin was removed using isopropanol. Finally, the cleaned device was post-cured in a UV chamber for complete polymerization.

9-2. 지질 기반 나노입자의 제조 및 특성화9-2. Preparation and characterization of lipid-based nanoparticles

지질 기반 나노입자는 지질 용액과 완충액을 빠르게 혼합하여 생성되었다. 각 지질 용액은 총 농도 10mg/mL로 고정되었다. LNP 제제의 경우, DODAP/DSPC/Chol/DMG-PEG 2000을 에탄올에 50/10/38.5/1.5의 몰비로 용해시켰다. 3M 아세트산 나트륨 용액을 탈이온수로 희석하여 25mM의 농도를 달성하였다. 100mM 아세트산 용액을 사용하여 아세트산 나트륨 용액의 pH를 5.5로 조정하였다. 리포좀의 경우, DODAP(DOTAP)와 콜레스테롤을 동일한 총 농도와 50:50의 몰비로 용해시켰다. 25 mM 아세트산 나트륨 완충액(pH 5.5) 및 10 mM Tris 완충액(pH 7.2)을 각각 이온화 가능한 리포좀과 양이온성 리포좀에 대한 반용매로 사용하였다. 주입하기 전에, 지질 용액과 완충액을 0.22μm 주사기 필터에 통과시켰다. 용액을 주사기 펌프를 사용하여 미세 혼합기를 통해 혼합하였다. LNP 및 리포좀 샘플을 투석 주머니(MWCO 12,000 Da, Sigma-Aldrich)를 사용하여 완충액(DODAP의 경우 10mM PBS, pH 7.4; DOTAP의 경우 10mM Tris 완충액, pH 7.2)의 1,000배에 대해 밤새 투석하여 잔류 에탄올을 제거하였다. 합성된 나노입자의 강도에 따른 크기 분포를 확인하기 위해 Zetasizer Nano ZS 장비(Malvern Instruments, Ltd.)를 사용하여 DLS 분석을 수행하였다.Lipid-based nanoparticles were prepared by rapid mixing of lipid solutions and buffer solutions. Each lipid solution was fixed at a total concentration of 10 mg/mL. For LNP formulations, DODAP/DSPC/Chol/DMG-PEG 2000 was dissolved in ethanol at a molar ratio of 50/10/38.5/1.5. 3 M sodium acetate solution was diluted with deionized water to achieve a concentration of 25 mM. The pH of the sodium acetate solution was adjusted to 5.5 using 100 mM acetic acid solution. For liposomes, DODAP (DOTAP) and cholesterol were dissolved at the same total concentration and molar ratio of 50:50. 25 mM sodium acetate buffer (pH 5.5) and 10 mM Tris buffer (pH 7.2) were used as antisolvents for ionizable and cationic liposomes, respectively. Before injection, the lipid solution and buffer were passed through a 0.22 μm syringe filter. The solutions were mixed via a micromixer using a syringe pump. The LNP and liposome samples were dialyzed overnight against 1,000-fold volume of buffer (10 mM PBS, pH 7.4 for DODAP; 10 mM Tris buffer, pH 7.2 for DOTAP) using a dialysis bag (MWCO 12,000 Da, Sigma-Aldrich) to remove residual ethanol. To determine the size distribution according to the intensity of the synthesized nanoparticles, DLS analysis was performed using a Zetasizer Nano ZS instrument (Malvern Instruments, Ltd.).

생산 후, DODAP LNP 및 리포좀은 평균 크기가 64 nm 및 72 nm였으며, 다분산 지수(PDI)는 각각 0.07 및 0.05였다(도 15의 B, C 및 도 16의 A 및 B). 서로 다른 반대 이온을 갖는 DOTAP 리포좀의 경우, 두 경우 모두 약 55nm의 크기와 0.08의 PDI가 생성되었다(도 15의 D, E 및 도 16의 C, D). 매우 균일한 나노입자의 생성, 특히 서로 다른 반대 이온을 갖는 양이온성 리포좀의 동일한 특성은 부분적으로 각 주요 지질 성분의 높은 순도에 기인한다. 투석을 통해 정제한 후, DODAP LNP와 리포좀은 PDI가 각각 0.12와 0.10인 40nm와 45nm로 크기가 감소한 반면, DOTAP 리포좀은 PDI가 0.11인 약 39ㅋnm의 크기에 도달했다(도 15의 B 내지 E 및 도 16의 A 내지 D). 모든 경우에, 평균 입자 크기가 감소하고 입자 분포가 약간 넓어지는 것이 관찰되었다. 이 시점에서, 에탄올과의 공존으로 인해 막 융합이 발생한 반면, 에탄올을 제거하면 안정성이 향상되었다는 점은 주목할 가치가 있다. 유동 합성된 DODAP 및 DOTAP 지질은 약 50nm 정도의 크기와 약 0.10 정도의 PDI를 나타냈다. 이는 정제 후에도 자가 조립 후 효과적인 약물 전달 가능성을 보여준다. 특히, 100 nm 미만의 나노입자 담체는 혈액 순환을 연장하고 효과적인 엔도솜 탈출을 가능하게 하며, 좁은 크기 분포는 원하지 않는 기관에 축적되는 것을 방지하여 최적의 치료 전달을 촉진한다.After production, DODAP LNPs and liposomes had an average size of 64 nm and 72 nm and a polydispersity index (PDI) of 0.07 and 0.05, respectively (Figs. 15B, C and 16A, B). For DOTAP liposomes with different counterions, a size of about 55 nm and a PDI of 0.08 were produced in both cases (Figs. 15D, E and 16C, D). The production of highly uniform nanoparticles, especially the same properties of cationic liposomes with different counterions, is partly due to the high purity of each major lipid component. After purification via dialysis, DODAP LNPs and liposomes were reduced in size to 40 nm and 45 nm with PDI of 0.12 and 0.10, respectively, whereas DOTAP liposomes reached a size of about 39 nm with PDI of 0.11 (Figs. 15B to E and 16A to D). In all cases, a decrease in the average particle size and a slight broadening of the particle distribution were observed. It is worth noting that at this point, membrane fusion occurred due to coexistence with ethanol, whereas removal of ethanol enhanced the stability. The flow-synthesized DODAP and DOTAP lipids exhibited a size of about 50 nm and a PDI of about 0.10, demonstrating their potential for effective drug delivery after self-assembly even after purification. In particular, nanoparticle carriers of less than 100 nm allow for prolonged blood circulation and effective endosomal escape, while the narrow size distribution prevents accumulation in unwanted organs, facilitating optimal therapeutic delivery.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

1, 2: 미세유체장치 100: 제1 미세 혼합기1, 2: Microfluidic device 100: First micro mixer

110: 제1 유로 120: 제2 유로110: 1st Euro 120: 2nd Euro

200: 제1 반응기 300: 제1 배압 조절기200: 1st reactor 300: 1st back pressure regulator

400: T자형 액체-가스 분리기 410: 제1 공급유로400: T-type liquid-gas separator 410: 1st supply path

420: 제1 배출유로 430: 제2 공급유로420: 1st discharge path 430: 2nd supply path

500: 제2 미세 혼합기 510: 제3 유로500: 2nd fine mixer 510: 3rd euro

600: 제2 반응기 700: 제2 배압 조절기600: Second reactor 700: Second pressure regulator

800: 제1 분광계 900: 제2 분광계800: 1st spectrometer 900: 2nd spectrometer

본 특허출원은 2024년도 대한민국 정부(다부처)의 재원으로 한국연구재단의 지원(1711180482, 지능형 미세유체 기반 정밀의약 합성공정 및 응용연구)을 받아 수행된 연구이다.This patent application was conducted with the support of the National Research Foundation of Korea (1711180482, Intelligent Microfluid-based Precision Medicine Synthesis Process and Application Research) through the multi-departmental government funding from the Republic of Korea in 2024.

Claims (24)

(a) R-(CH2)2C(=O)OH, 디메틸포름아미드 및 옥살릴 클로라이드를 혼합 및 반응시켜 R-C(=O)Cl을 형성하는 단계; 및(a) a step of mixing and reacting R-(CH 2 ) 2 C(=O)OH, dimethylformamide and oxalyl chloride to form RC(=O)Cl; and (b) R-C(=O)Cl을 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물과 혼합하여 에스테르화 반응을 일으켜 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질을 생성하는 단계;를 포함하며,(b) a step of mixing R-C(=O)Cl with 3-(dimethylamino)propane-1,2-diol and a basic compound to cause an esterification reaction to produce an ionizable cationic lipid having a structure of chemical formula 1; [화학식 1][Chemical Formula 1]
Figure PCTKR2024007830-appb-img-000034
Figure PCTKR2024007830-appb-img-000034
 상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼인, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, wherein R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical having -C(=O)O- or -S-S- introduced into the chain. 제1항에 있어서, 상기 (a) 단계 후에 HCl 및 CO 가스를 분리하는 단계를 추가로 포함하는, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, comprising, in the first aspect, a step of separating HCl and CO gases after step (a). 제1항에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행되고,In the first aspect, the synthesis method is performed in a microfluidic device for a single continuous flow process, 상기 미세유체장치는 The above microfluidic device 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기;A first micro-mixer in which the first and second euros are fluidly connected; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기;A first reactor fluidly connected to the first micro-mixer; 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기;A first back pressure regulator fluidly connected to the first reactor; 상기 제1 배압 조절기와 유체적으로 연결되어 용액과 가스를 분리하는 T자형 액체-가스 분리기;A T-shaped liquid-gas separator fluidly connected to the first back pressure regulator for separating the solution and gas; 상기 T자형 액체-가스 분리기와 제3 유로가 유체적으로 연결되는 제2 미세 혼합기;A second micro-mixer fluidly connected to the T-shaped liquid-gas separator and the third flow path; 상기 제2 미세 혼합기와 유체적으로 연결되는 제2 반응기; 및a second reactor fluidly connected to the second micro-mixer; and 상기 제2 반응기와 유체적으로 연결되는 제2 배압 조절기를 포함하는, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, comprising a second pressure regulator fluidly connected to the second reactor. 제3항에 있어서, 상기 T자형 액체-가스 분리기는 상기 제1 배압 조절기와 유체적으로 연결되는 제1 공급유로, 분리된 가스가 배출되는 제1 배출유로 및 분리된 용액이 상기 제2 미세 혼합기에 공급되도록 상기 제2 미세 혼합기에 유체적으로 연결되는 제2 공급유로를 포함하는, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, in claim 3, wherein the T-shaped liquid-gas separator comprises a first supply channel fluidly connected to the first back pressure regulator, a first discharge channel through which separated gas is discharged, and a second supply channel fluidly connected to the second micro-mixer so that the separated solution is supplied to the second micro-mixer. 제3항에 있어서, 상기 제1 유로에 R-(CH2)2C(=O)OH 및 디메틸포름아미드, 상기 제2 유로에 옥살릴 클로라이드가 주입되고,In the third paragraph, R-(CH 2 ) 2 C(=O)OH and dimethylformamide are injected into the first flow path, and oxalyl chloride is injected into the second flow path. 상기 제1 반응기에서 R-C(=O)Cl이 생성되고,In the first reactor above, R-C(=O)Cl is produced, 상기 T자형 액체-가스 분리기를 통해 HCl 및 CO 가스가 분리되고,HCl and CO gases are separated through the above T-shaped liquid-gas separator, 상기 제3 유로에 3-(디메틸아미노)프로판-1,2-디올 및 염기성 화합물이 주입되고,3-(dimethylamino)propane-1,2-diol and a basic compound are injected into the third euro above, 상기 제2 반응기에서 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질이 생성되는, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, wherein an ionizable cationic lipid having a structure of the chemical formula 1 is produced in the second reactor. 제3항에 있어서, 상기 제1 반응기에서의 체류 시간은 1 내지 2분이고, 상기 제2 반응기에서의 체류 시간은 2 내지 10분이고, 상기 제2 반응기에서의 반응 온도는 20 내지 60 ℃인, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, wherein in the third paragraph, the residence time in the first reactor is 1 to 2 minutes, the residence time in the second reactor is 2 to 10 minutes, and the reaction temperature in the second reactor is 20 to 60°C. 제1항에 있어서, 상기 염기성 화합물은 피리딘, 피리딘과 DMAP의 혼합, DMAP, 디이소프로필에틸아민 및 트리에틸아민으로 이루어진 군으로부터 선택되는 어느 하나인, 이온화 가능한 양이온성 지질의 합성 방법.A method for synthesizing an ionizable cationic lipid, wherein in claim 1, the basic compound is any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine. 제1항에 있어서, 상기 (a) 단계의 R-(CH2)2C(=O)OH는 올레산, 리놀레산, 미리스트산, 스테아르산, 팔미트산, 10-(헥실옥시)-10-옥소데칸산, (S)-10-((3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데칸산, (E)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, (Z)-10-((3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데칸산, 3-(옥타데실디설파닐)프로판산, 및 3-(도데실디설파닐)프로판산으로 이루어진 군으로부터 선택되는 어느 하나의 지방산인, 이온화 가능한 양이온성 지질의 합성 방법.In the first paragraph, R-(CH 2 ) 2 C(=O)OH of the step (a) is any one fatty acid selected from the group consisting of oleic acid, linoleic acid, myristic acid, stearic acid, palmitic acid, 10-(hexyloxy)-10-oxodecanoic acid, (S)-10-((3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoic acid, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid, 3-(octadecyldisulfanyl)propanoic acid, and 3-(dodecyldisulfanyl)propanoic acid, of the ionizable cationic lipid. Synthetic method. 제1항에 있어서, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질은 다음의 (1) 내지 (11) 중 어느 하나인, 이온화 가능한 양이온성 지질의 합성 방법:In the first paragraph, the ionizable cationic lipid having the structure of the chemical formula 1 is a method for synthesizing an ionizable cationic lipid, wherein the ionizable cationic lipid is any one of the following (1) to (11): (1) 1,2-디올레오일-3-디메틸암모늄-프로판;(1) 1,2-Dioleoyl-3-dimethylammonium-propane; (2) (9Z,9'Z,12Z,12'Z)-3-(디메틸아미노)프로판-1,2-디일비스(옥타데카-9,12-디에노에이트);(2) (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate); (3) 1,2-디미리스토일-3-디메틸암모늄-프로판;(3) 1,2-Dimyristoyl-3-dimethylammonium-propane; (4) 1,2-디스테아로일-3-디메틸암모늄-프로판;(4) 1,2-distearoyl-3-dimethylammonium-propane; (5) 1,2-디팔미토일-3-디메틸암모늄-프로판;(5) 1,2-Dipalmitoyl-3-dimethylammonium-propane; (6) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-디헥실 비스(데칸디오에이트);(6) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate); (7) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((S)-3,7-디메틸옥트-6-엔-1-일) 비스(데칸디오에이트);(7) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate); (8) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((E)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(8) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((E)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate); (9) O'1,O1-(3-(디메틸아미노)프로판-1,2-디일) 10-비스((Z)-3,7-디메틸옥타-2,6-디엔-1-일) 비스(데칸디오에이트);(9) O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((Z)-3,7-dimethylocta-2,6-dien-1-yl) bis(decanedioate); (10) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(옥타데실디설파닐)프로파노에이트); 및(10) 3-(dimethylamino)propane-1,2-diyl bis(3-(octadecyldisulfanyl)propanoate); and (11) 3-(디메틸아미노)프로판-1,2-디일 비스(3-(도데실디설파닐)프로파노에이트).(11) 3-(Dimethylamino)propane-1,2-diyl bis(3-(dodecyldisulfanyl)propanoate). (a) 2-하이드록시데칸산, 디메틸포름아미드 및 옥살릴 클로라이드를 혼합 및 반응시켜 2-하이드록실데카노일 클로라이드를 형성하는 단계; 및(a) a step of mixing and reacting 2-hydroxydecanoic acid, dimethylformamide and oxalyl chloride to form 2-hydroxydecanoyl chloride; and (b) 2-하이드록실데카노일 클로라이드를 3-(디메틸아미노)프로판-1,2-디올 및 6-브로모헥산올과 혼합하여 에스테르화 반응을 일으켜 6-브로모헥실 2-헥실데카노에이트를 생성하는 단계;를 포함하는, 이온화 가능한 양이온성 지질인 6-((2-헥실데카노일)옥시)-N-(6-((2-헥실데카노일)옥시)헥실)-N-(4-하이드록시부틸)헥산-1-아미늄의 중간체인 6-브로모헥실 2-헥실데카노에이트의 합성 방법.(b) a step of mixing 2-hydroxydecanoyl chloride with 3-(dimethylamino)propane-1,2-diol and 6-bromohexanol to cause an esterification reaction to produce 6-bromohexyl 2-hexyldecanoate; A method for synthesizing 6-bromohexyl 2-hexyldecanoate, which is an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminum, which is an ionizable cationic lipid. 제1항의 합성 방법에 의해 합성된 이온화 가능한 양이온성 지질과 디메틸 설페이트를 혼합 및 반응시켜 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트를 생성하는 단계를 포함하며,A step of mixing and reacting an ionizable cationic lipid synthesized by the synthetic method of claim 1 with dimethyl sulfate to produce cationic lipid methyl sulfate having a structure of chemical formula 2, [화학식 2][Chemical formula 2]
Figure PCTKR2024007830-appb-img-000035
Figure PCTKR2024007830-appb-img-000035
 상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼인, 양이온성 지질 메틸 설페이트의 합성 방법.A method for synthesizing cationic lipid methyl sulfate, wherein R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical having -C(=O)O- or -S-S- introduced into the chain. 제11항에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행되고,In the 11th paragraph, the synthesis method is performed in a microfluidic device for a single continuous flow process, 상기 미세유체장치는 The above microfluidic device 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기;A first micro-mixer in which the first and second euros are fluidly connected; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기; 및a first reactor fluidly connected to the first micro-mixer; and 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기를 포함하는, 양이온성 지질 메틸 설페이트의 합성 방법.A method for synthesizing cationic lipid methyl sulfate, comprising a first pressure regulator fluidly connected to the first reactor. 제12항에 있어서, 상기 제1 유로에 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질, 상기 제2 유로에 디메틸 설페이트를 주입하고,In the 12th paragraph, an ionizable cationic lipid having a structure of chemical formula 1 is injected into the first flow path, and dimethyl sulfate is injected into the second flow path. [화학식 1][Chemical Formula 1]
Figure PCTKR2024007830-appb-img-000036
Figure PCTKR2024007830-appb-img-000036
 여기서, 상기 R은 비치환된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이거나, 또는 -C(=O)O- 또는 -S-S-가 사슬에 도입된 C10 내지 C20의 포화 또는 불포화 지방족 탄화수소 라디칼이고,Here, R is an unsubstituted C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical, or a C10 to C20 saturated or unsaturated aliphatic hydrocarbon radical in which -C(=O)O- or -S-S- is introduced into the chain, 상기 제1 반응기에서 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트가 생성되는, 양이온성 지질의 합성 방법.A method for synthesizing a cationic lipid, wherein cationic lipid methyl sulfate having a structure of the chemical formula 2 is produced in the first reactor. 제13항에 있어서, 상기 화학식 1의 구조를 갖는 이온화 가능한 양이온성 지질 및 상기 디메틸 설페이트는 0.4 내지 2.0ml/분의 유속으로 주입되고, 상기 제1 반응기에서의 반응 온도는 40 내지 70℃이고, 상기 제1 반응기에서의 체류 시간은 3 내지 7분인, 양이온성 지질 메틸 설페이트의 합성 방법.A method for synthesizing cationic lipid methyl sulfate in claim 13, wherein the ionizable cationic lipid having the structure of chemical formula 1 and the dimethyl sulfate are injected at a flow rate of 0.4 to 2.0 ml/min, the reaction temperature in the first reactor is 40 to 70°C, and the residence time in the first reactor is 3 to 7 minutes. 제11항에 있어서, 상기 화학식 2의 구조를 갖는 양이온성 지질 메틸 설페이트는 다음의 (1) 내지 (7) 중 선택되는 어느 하나인, 양이온성 지질 메틸 설페이트의 합성 방법:In the 11th paragraph, the cationic lipid methyl sulfate having the structure of the chemical formula 2 is a method for synthesizing cationic lipid methyl sulfate, wherein the cationic lipid methyl sulfate is one selected from the following (1) to (7): (1) 1,2-디올레오일-3-트리메틸암모늄-프로판 메틸 설페이트;(1) 1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate; (2) N,N,N-트리메틸-2,3-비스((9Z,12Z)-옥타데카-9,12-디에노일옥시)프로판-1-아미늄 메틸 설페이트;(2) N,N,N-Trimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dienyloxy)propane-1-aminum methyl sulfate; (3) 2,3-비스((10-(헥실옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(3) 2,3-Bis((10-(hexyloxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate; (4) 2,3-비스((10-(((S)-3,7-디메틸옥트-6-엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(4) 2,3-Bis((10-(((S)-3,7-dimethyloct-6-en-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate; (5) 2,3-비스((10-(((E)-3,7-디메틸옥타-2,6-디엔-1-일)옥시)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트;(5) 2,3-Bis((10-(((E)-3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate; (6) 2,3-비스((10-(((Z)-3,7-디메틸옥타-2,6-디엔-1-일)옥소)-10-옥소데카노일)옥시)-N,N,N-트리메틸프로판-1-아미늄 메틸 설페이트; 및 (6) 2,3-bis((10-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)oxo)-10-oxodecanoyl)oxy)-N,N,N-trimethylpropan-1-aminum methyl sulfate; and (7) N,N,N-트리메틸-2,3-비스((3-(옥타데실디설파닐)프로파노일)옥시)프로판-1-아미늄 메틸 설페이트.(7) N,N,N-Trimethyl-2,3-bis((3-(octadecyldisulfanyl)propanoyl)oxy)propan-1-aminum methyl sulfate. 제11항에 있어서, 용매 증발 및 재결정화 단계를 추가로 포함하는, 양이온성 지질 메틸 설페이트의 합성 방법.A method for synthesizing cationic lipid methyl sulfate, further comprising solvent evaporation and recrystallization steps in claim 11. 제11항에 있어서, 상기 재결정화는 아세톤 용매에서 수행되는, 양이온성 지질 메틸 설페이트의 합성 방법.A method for synthesizing cationic lipid methyl sulfate, wherein the recrystallization is performed in an acetone solvent. 올레산, 2,3-디하이드록시-N,N,N-트리메틸프로판-1-아미늄 클로라이드 및 DMAP의 혼합물과 (3-디메틸아미노-프로필)-에틸-카르보디이미드 클로라이드를 혼합 및 반응시켜 스테글리치(Steglich) 에스테르화 반응을 일으켜 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드를 생성하는 단계를 포함하는, 양이온성 지질 1,2-디올레오일-3-트리메틸암모늄-프로판(DOTAP) 클로라이드의 합성 방법.A method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride. 제18항에 있어서, 상기 합성 방법은 단일 연속 유동 공정을 위한 미세유체장치에서 수행되며,In claim 18, the synthesis method is performed in a microfluidic device for a single continuous flow process, 상기 미세유체장치는 The above microfluidic device 제1 유로와 제2 유로가 유체적으로 연결되는 제1 미세 혼합기;A first micro-mixer in which the first and second euros are fluidly connected; 상기 제1 미세 혼합기와 유체적으로 연결되는 제1 반응기; 및a first reactor fluidly connected to the first micro-mixer; and 상기 제1 반응기와 유체적으로 연결되는 제1 배압 조절기를 포함하는, 양이온성 지질 DOTAP 클로라이드의 합성 방법.A method for synthesizing a cationic lipid DOTAP chloride, comprising a first back pressure regulator fluidly connected to the first reactor. 제19항에 있어서, 상기 제1 반응기에서의 반응 온도는 60 내지 80℃이고, 상기 제1 반응기에서의 체류 시간은 3 내지 7분인, 양이온성 지질 DOTAP 클로라이드의 합성 방법.A method for synthesizing cationic lipid DOTAP chloride in claim 19, wherein the reaction temperature in the first reactor is 60 to 80°C, and the residence time in the first reactor is 3 to 7 minutes. 제18항에 있어서, 용매 증발 및 재결정화 단계를 추가로 포함하는, 양이온성 지질 DOTAP 클로라이드의 합성 방법.A method for synthesizing cationic lipid DOTAP chloride, further comprising solvent evaporation and recrystallization steps in claim 18. 제21항에 있어서, 상기 재결정화는 이소프로판올 및 아세톤 용매에서 수행되는, 양이온성 지질 DOTAP 클로라이드의 합성 방법.A method for synthesizing cationic lipid DOTAP chloride, wherein the recrystallization is performed in isopropanol and acetone solvents. (a) 제1항 내지 제9항 중 어느 한 항에 따른 방법으로 이온화 가능한 양이온성 지질을 합성하는 단계; 및(a) a step of synthesizing an ionizable cationic lipid by a method according to any one of claims 1 to 9; and (b) 상기 합성된 이온화 가능한 양이온성 지질, 헬퍼(helper) 지질, 콜레스테롤 및 폴리에틸렌 글리콘 지질을 포함하는 용액을 완충액과 혼합하여 지질 나노입자를 형성하는 단계;를 포함하는 지질 나노입자의 제조방법.(b) a step of mixing a solution containing the synthesized ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid with a buffer solution to form lipid nanoparticles; a method for producing lipid nanoparticles, comprising: 제1항 내지 제9항 중 어느 한 항에 따른 방법으로 이온화 가능한 양이온성 지질을 합성하거나, 제11항 내지 제17항 중 어느 한 항에 따른 방법으로 양이온성 지질 메틸 설페이트를 합성하거나, 또는 제18항 내지 제22항의 방법으로 DOTAP 클로라이드를 합성하는 단계; 및 A step of synthesizing an ionizable cationic lipid by a method according to any one of claims 1 to 9, synthesizing cationic lipid methyl sulfate by a method according to any one of claims 11 to 17, or synthesizing DOTAP chloride by a method according to any one of claims 18 to 22; and (b) 상기 합성된 이온화 가능한 양이온성 지질, 양이온성 지질 메틸 설페이트 또는 DOTAP 클로라이드와 콜레스테롤을 포함하는 용액을 완충액과 혼합하여 지질 나노 리포좀을 형성하는 단계;를 포함하는 지질 나노 리포좀의 제조방법.(b) a step of mixing a solution containing the synthesized ionizable cationic lipid, cationic lipid methyl sulfate or DOTAP chloride and cholesterol with a buffer solution to form a lipid nano liposome; a method for producing a lipid nano liposome, comprising:
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