Patent Application
20250213680
This application claims the benefit of Korean Patent Application No. 10-2023-0195342 filed on Dec. 28, 2023, in the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference for all purposes.
The present disclosure relates to a lipid nanoparticle composition for stable delivery of nucleic acid drugs and immunity enhancement and, more specifically, to a lipid nanoparticle composition including a gallic acid derivative lipid and a use thereof.
An mRNA-based vaccine prevents a disease or alleviates the severity thereof by injecting mRNA encoding a specific antigen of the disease into the body and utilizing the immune response to an antigen expressed in vivo. The mRNA-based vaccine can be rapidly mass-produced in a short period of time if the antigen for the disease is clearly identified, which led to emergency approval by the FDA during the COVID-19 pandemic to have mRNA-based vaccines used worldwide.
Although the mRNA-based vaccine has the advantage of being able to be developed quickly, it is rapidly degraded by RNase in vivo, such that a delivery platform is necessarily required to allow it to be stably delivered into cells. mRNA-based vaccines from Pfizer and Moderna used in the COVID-19 pandemic employed lipid nanoparticles as a delivery platform with proven efficiency which had already been utilized in 2018 for delivery of other RNA-based therapeutics. Lipid nanoparticles are mainly manufactured by mixing ionizable lipids, phospholipids, cholesterol, and PEG-lipids in an optimal ratio, and they stabilized the mRNA and increased the efficiency of delivery into the cell compared to injection of the mRNA itself.
However, the ionizable lipid, which plays a role in increasing the embedding efficiency of mRNA carriers, is often cytotoxic as it is, which may cause side effects when excessive doses are applied. In addition, the lipid nanoparticles currently developed have a problem in that the efficiency of producing neutralizing antibodies which help defend cells in the body when infected is still not high.
An object of the present disclosure is to provide a lipid nanoparticle capable of reducing side effects caused by an ionizable lipid and increasing a delivery efficiency and a neutralizing antibody production efficiency by using a bio-friendly material.
Another object of the present disclosure is to provide a drug delivery and immune enhancement use of the lipid nanoparticle.
To achieve the above objects, the present disclosure provides a lipid nanoparticle composition including a compound represented by the following Chemical Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
wherein, in the Chemical Formula 1, R may be a linear, saturated or unsaturated hydrocarbon with 3 to 22 carbon atoms.
The present disclosure provides a lipid nanoparticle composition including the compound represented by Chemical Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof; an ionizable lipid; a helper lipid; and a polyethylene glycol (PEG)-modified lipid.
The present disclosure provides a composition for drug delivery, including the lipid nanoparticle composition; and a therapeutic or prophylactic agent.
In addition, the present disclosure provides an immunoenhancing composition, including the lipid nanoparticle composition.
A lipid nanoparticle according to the present disclosure includes a bio-friendly gallic acid derivative and thus may improve the immune effect by replacing a part of an ionizable lipid, a helper lipid, and a structure-maintaining lipid and, in particular, it is expected that, when a gallic acid derivative replaces a certain ratio of ionizable lipid in a molar component ratio, the toxicity caused by the ionizable lipid may be mitigated.
In addition, when mRNA was delivered using the lipid nanoparticle containing the gallic acid derivative in vivo, the expression efficiency of proteins encoded in the mRNA as well as immunoglobulin G (IgG) were enhanced compared to those not containing the gallic acid derivative, and MCP-1 concentration was lowered, such that it is anticipated to mitigate the toxicity.
Hereinafter, the present disclosure will be described in detail.
The present inventor completed the present disclosure by preparing lipid nanoparticles using gallic acid-based lipid, one of the bio-friendly antioxidants, in an attempt to address issues of conventional lipid nanoparticles, and thus identifying the potential as a therapeutic agent capable of appropriately delivering nucleic acid drugs while reducing side effects caused by immune responses from the prepared lipid nanoparticles.
The present disclosure provides a lipid nanoparticle composition including a gallic acid derivative.
More specifically, the lipid nanoparticle composition according to the present disclosure may include a compound represented by the following Chemical Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
Preferably, the compound may include any one or more selected from the group consisting of compounds represented by the following Chemical Formulas 2 to 6:
In other words, the compound may include propyl gallate, dodecyl gallate, hexadecyl gallate, octadecyl gallate, cis-9-octadecenyl gallate, derivatives thereof, or pharmaceutically acceptable salts thereof.
The compound may be utilized as a helper lipid or structure-maintaining lipid of lipid nanoparticles.
The lipid nanoparticle composition according to the present disclosure may further include any one or more selected from the group consisting of an ionizable lipid, a helper lipid, a structure-maintaining lipid, a polyethylene glycol (PEG)-modified lipid, and an additive, in addition to the compound, a derivative thereof, or a pharmaceutically acceptable salt thereof.
The present disclosure provides a lipid nanoparticle composition including a compound represented by the following Chemical Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof; an ionizable lipid; a helper lipid; and a polyethylene glycol (PEG)-modified lipid:
In the lipid nanoparticle composition according to the present disclosure, the ionizable lipid may be any one or more selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2S)), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), and a pantothenic acid derivative, and preferably, it may be selected from SM-102 or a pantothenic acid derivative, but is not limited thereto.
The pantothenic acid derivative may be a compound selected from a compound represented by the following Chemical Formula 7, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:
Specifically, the pantothenic acid derivative may include any one or more selected from the group consisting of compounds represented by the following Chemical formulas 8 to 13:
In the lipid nanoparticle composition according to the present disclosure, the helper lipid may be any one or more selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and trehalose derivatives, and preferably, it may be selected from DOPE, DSPC, or trehalose derivatives, but is not limited thereto.
The trehalose derivative may include a compound represented by the following Chemical Formula 14:
Specifically, the compound represented by Chemical Formula 14 may be a compound represented by the following Chemical Formula 15:
In the lipid nanoparticle composition according to the present disclosure, the polyethylene glycol (PEG)-modified lipid may be selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkyl glycerol, and a mixture thereof, preferably it may be myristoyl diglyceride (DMG)-PEG, but is not limited thereto.
Preferably, the lipid nanoparticle composition according to the present disclosure may include 10 to 50 mol % of the compound, a derivative thereof, or a salt thereof, 20 to 60 mol % of the ionizable lipid, 5 to 20 mol % of the helper lipid, and 1 to 5 mol % of the PEG-modified lipid.
The composition may further include a structure-maintaining lipid, preferably from 1 to 40 mol %, but is not limited thereto.
The structure-maintaining lipid may be any one or more selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and derivatives thereof, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.
The lithocholic acid derivative may be a compound represented by the following Chemical Formula 16:
Specifically, the compound represented by Chemical Formula 16 may be a compound represented by the following Chemical Formula 17, where R29 is hydrogen, and R30 has an alkyl group with 4 carbon atoms:
The present disclosure provides a composition for drug delivery, including the lipid nanoparticle composition; and a therapeutic or prophylactic agent.
The therapeutic or prophylactic agent may be a vaccine or compound capable of inducing an immune response.
Specifically, the therapeutic or prophylactic agent may be selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and a mixture thereof.
The encapsulation efficiency of the therapeutic and/or prophylactic agent may independently be at least 50% or higher.
A wt/wt ratio of components of the lipid nanoparticle composition to the therapeutic or prophylactic agent may be from about 10:1 to about 60:1.
The N:P ratio of the therapeutic or prophylactic agent may be from about 2:1 to about 30:1. The N/P ratio is a value obtained by dividing the number of ionizable nitrogens in the ionizable lipid by the number of phosphate groups in nucleic acid molecules.
The present disclosure provides an immunoenhancing composition including the lipid nanoparticle composition.
Corresponding features may be substituted in the above-described part.
In addition, the present disclosure provides a method of delivering therapeutic and/or prophylactic agents to mammalian cells by the composition for drug delivery.
The method of delivering the therapeutic and/or prophylactic agent to the mammalian cell includes administering the lipid nanoparticle composition to a subject, wherein the administering may be conducted by bringing the cell in contact with the nanoparticle composition to allow the therapeutic and/or prophylactic agent to be delivered to the cell.
The mammalian cell is from mammals.
The mammal may be a human.
The composition for drug delivery may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. About 0.01 mg/kg to about 10 mg/kg doses of the therapeutic and/or prophylactic agent may be administered to mammals.
Alternatively, the present disclosure may provide a drug delivery method that includes administering the composition for drug delivery into cells of animals other than humans.
Corresponding features may be substituted in the above-described part.
In addition, the present disclosure provides a method of producing a polypeptide of interest in mammalian cells.
The method of producing the polypeptide of interest in the mammalian cell includes brining the cell in contact with a composition for drug delivery including a lipid nanoparticle composition and a therapeutic and/or prophylactic agent, wherein the therapeutic and/or prophylactic agent is an mRNA, and the mRNA encodes the polypeptide of interest, such that the mRNA may be translated in cells to produce the polypeptide of interest.
Corresponding features may be substituted in the above-described part.
Hereinafter, to help the understanding of the present disclosure, example embodiments will be described in detail. However, the following example embodiments are merely illustrative of the content of the present disclosure, and the scope of the present disclosure is not limited to the following example embodiments. The example embodiments of the present disclosure are provided to more completely explain the present disclosure to those of ordinary skill in the art.
Gallic acid derivative compounds represented by the following Chemical Formulas 2 to 6 were purchased and prepared from Tokyo Chemical Industry Co., Ltd.
9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) was purchased from Hanmi Fine Chemical Co., Ltd.
A pantothenic acid derivative was prepared by synthesis according to the following method.
Propane-1,2,3-triol (Compound 54; 300 mg, 1 equiv.) and ethanoic anhydride (432 mg, 1.3 equiv.) were dissolved in an anhydrous acetonitrile (20 mL) solvent, then tetra-n-butylammonium acetate (TBAAc; 687 mg, 0.7 equiv.) was added, and the reaction mixture was stirred at 50° C. for 7 hours. After the termination of the reaction, the reaction mixture was loaded by a solid deposition method and purified by flash tube chromatography (SiO2, n-hexane/EtOAc 5:5) to obtain Compound 55 (297 mg, 68%) in colorless oil.
1H NMR (CDCl3, 400 MHz): δ 2.11 (s, 3H), 2.8 (br s, 1H), 3.14 (br s, 1H), 3.59 (dd, 1H, J=6, 11.6 Hz), 3.69 (dd, 1H, J=3.6, 11.6 Hz), 3.93 (pnt, 1H, J=4.8 Hz), 4.22-4.12 (m, 2H); 13C NMR (CDCl3, 100 MHz): δ 20.83, 63.31, 65.29, 70.12, 171.56.
2-Hexyldecanoic acid (956 mg, 2.5 equiv.) was put into a reaction vessel and then dissolved in DCM (40 mL), and EDCI·HCl (580 mg, 2.5 equiv.) and DMAP (55 mg, 0.3 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Afterwards, a solution in which Compound 55 (200 mg, 1 equiv.) is dissolved in DCM (20 mL) was added dropwise to the reaction mixture at 5° C. and stirred at room temperature for 24 hours. After confirming that the reaction is terminated via TLC (SiO2, EtOAc/hexane 4:6), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (30 mL). A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 1:9) to obtain Compound 69 (801 mg, 88%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.82 (t, J=5.2 Hz, 12H), 1.18-1.25 (m, 42H), 1.36-1.41 (m, 5H), 1.49-1.55 (m, 5H), 2.11 (s, 3H), 2.25-2.31 (m, 2H), 3.5 (dd, J=3.6, 3.9 Hz, 1H), 3.62 (dd, J=3.6, 3.9 Hz, 1H), 4.13 (dd, J=3.4, 4.3 Hz, 1H), 4.30 (dd, J=3.4, 4.3 Hz, 1H), 5.12-5.16 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.04, 14.08, 20.83, 22.61, 22.66, 27.31, 27.34, 27.31, 27.42, 27.46, 29.20, 29.23, 29.28, 29.45, 29.53, 29.56, 31.68, 32.31, 32.34, 32.19, 42.32, 45.65, 45.74, 62.09, 70.17, 171.56, 175.46, 175.87.
After glycerol Compound 69 (700 mg, 1 equiv.) was put into a reaction vessel and then clogged with a rubber stopper, nitrogen gas was filled and left until the termination of the reaction. After that, CH3OH (70 mL) was added through a cannula, and then K2CO3 (316 mg, 2 equiv.) was added subsequently, followed by stirring at room temperature for 6 hours. The reaction mixture was distilled under reduced pressure to remove the solvent, then DCM (50 mL) and distilled water (50 mL) were added, and the mixture was transferred to the separatory funnel. The water layer was extracted with DCM (2×20 mL), and organic layers were collected and washed with saturated aqueous NaCl solution (20 mL). A filtrate filtered by drying the separated organic layers with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 2:8->3:6) to obtain Compound 70 (502 mg, 77%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=5.1 Hz, 12H), 1.18-1.24 (m, 42H), 1.36-1.41 (m, 5H), 1.49-1.57 (m, 5H), 2.25-2.31 (m, 2H), 3.56 (dd, J=3.6, 3.9 Hz, 1H), 3.62 (dd, J=3.6, 3.9 Hz, 1H), 4.12 (dd, J=3.4, 4.3 Hz, 1H), 4.30 (dd, J=3.4, 4.3 Hz, 1H), 5.12-5.16 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.04, 14.08, 22.60, 22.66, 27.30, 27.34, 27.37, 27.41, 27.45, 29.19, 29.23, 29.28, 29.44, 29.53, 29.58, 31.68, 32.30, 32.33, 32.18, 42.32, 45.64, 45.73, 62.08, 70.16, 175.46, 175.86.
While stirring D-pantothenic acid hemicalcium salt (Compound 51; 5 g, 1 equiv.) and 2,2-dimethoxypropane (DMP; 60 mL) in a reaction vessel, p-toluenesulfonic acid (PTSA; 3.97 g, 1.1 equiv.) was added at once. The liquid reaction mixture was stirred at room temperature for 20 hours, and the resulting white solids were filtered by washing with acetone. The yellow solid obtained by distilling the filtrate under reduced pressure was washed with warm n-hexane to obtain Compound 52 (5.45 g, 100%) in while solid.
1H NMR (CDCl3, 400 MHz): δ 0.98 (s, 3H), 1.04 (s, 3H), 1.43 (s, 3H), 1.46 (s, 3H), 2.62 (dt, J=6.0, 2.0, 2H), 3.29 (d, J=11.5, 1H), 3.45-3.53 (m, 1H), 3.56-3.64 (m, 1H), 3.70 (d, J=11.5, 1H), 4.11 (s, 1H), 7.05 (app br s, 1H); 13C NMR (CDCl3, 100 MHz): δ 18.7, 18.8, 22.0, 29.4, 30.9, 33.0, 33.9, 34.1, 71.4, 77.1, 99.1, 170.2.
Pantothenic acid 52 (376 mg, 1.5 equiv.) protected with compound acetal was put into a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (225 mg, 1.5 equiv.) and DMAP (24 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Thereafter, a solution, in which Compound 70 (550 mg, 1 equiv.) is dissolved in DCM (10 mL), was added dropwise in the reaction mixture at 5° C. and then stirred at room temperature for 11 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/hexane 2:8), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (30 mL). The filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and then the mixture obtained was removed by column chromatography (SiO2, EtOAc/hexane 3:7) to obtain Compound 71 (440 mg, 77%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.62 (t, J=5.1 Hz, 12H), 0.73 (s, 3H), 0.80 (s, 3H), 1.01-1.07 (m, 41H), 1.18-1.22 (m, 11H), 1.32-1.37 (m, 5H), 2.08-2.11 (m, 2H), 2.32 (t, J=4.1 Hz, 2H), 3.03 (d, J=6.1 Hz, 1H), 3.20-3.42 (m, 2H), 3.44 (d, J=6.1 Hz, 1H), 3.84-3.94 (m, 3H), 4.06-4.31 (m, 2H), 5.04-5.06 (m, 1H), 7.04 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.03, 14.07, 18.66, 22.08, 22.58, 22.64, 27.26, 27.32, 27.37, 27.42, 29.18, 29.21, 29.27, 29.42, 29.54, 29.57, 31.65, 31.84, 32.21, 32.27, 32.95, 33.83, 33.85, 34.09, 32.12, 45.57, 45.63, 61.95, 62.72, 62.79, 68.64, 71.46, 99.00, 169.78, 171.55, 175.49, 175.87.
Compound 71 (420 mg, 1 equiv.) and DTT (160 mg, 2 equiv.) were put into a reaction vessel and dissolved in DCM, and then p-toluenesulfonic acid (45 mg, 0.5 equiv.) was added at room temperature and stirred for 1 hour. Once the reaction was terminated, extraction was followed with EtOAc (30 mL×2), and organic layers were collected and washed with saturated NaCl solution. The filtrate filtered by drying the separated organic layers with anhydrous Na2SO4 was distilled under reduced pressure to remove the solvent, and then the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 3:7->6:4) to obtain Compound 72 (243 mg, 61%) in light yellow oil.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=5.1 Hz, 12H), 0.95 (s, 3H), 1.00 (s, 3H), 1.18-1.24 (m, 45H), 1.38 (m, 5H), 1.48-1.52 (m, 5H), 2.25-2.29 (m, 2H), 2.43-2.47 (m, 2H), 3.31-3.43 (m, 1H), 3.59-3.62 (m, 1H), 4.02-4.06 (m, 3H), 4.15-4.28 (m, 4H), 4.38-4.42 (m, 1H), 5.32-5.34 (m, 1H), 7.02-7.20 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.03, 14.07, 19.91, 19.98, 20.84, 22.59, 22.65, 27.23, 27.28, 27.33, 27.38, 27.44, 29.11, 29.20, 29.27, 29.43, 29.56, 31.62, 31.65, 31.85, 32.21, 32.26, 32.28, 32.32, 32.43, 33.88, 34.41, 34.61, 38.29, 38.52, 45.62, 45.74, 61.95, 62.08, 62.80, 63.03, 68.70, 68.76, 73.27, 73.32, 74.70, 74.96, 169.66, 172.05, 173.54, 173.71.
3-Morpholinopropanoic acid (Compound 75; 45.42 mg, 1.1 equiv.) was put into a reaction vessel and dissolved in DCM (20 mL), and then EDCI·HCl (75 mg, 1.5 equiv.) and DMAP (6.3 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at room temperature for 20 minutes. Compound 72 (200 mg, 1 equiv.) was put into another reaction vessel and dissolved in DCM (10 mL) followed by cooling to 5° C., and then the liquid mixture of Compound 75 was added dropwise at 5° C. for 30 minutes and stirred at room temperature for 10 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/MeOH 1:1 and 9:1, PMA stain), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (20 mL). The filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and then the mixture obtained was purified by flash column chromatography (SiO2, DCM-MeOH 10:0->10:0.2, added with 28% aqueous NH3 solution) to obtain Compound 8 (151 mg, 64%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.79-0.82 (m, 15H), 1.09 (d, J=2.5 Hz, 3H), 1.18-1.24 (m, 43H), 1.34-1.39 (m, 4H), 1.48-1.51 (m, 4H), 2.24-2.28 (m, 2H), 2.41-2.52 (m, 10H), 3.48-3.63 (m, 7H), 3.85 (d, J=6 Hz, 1H), 4.00-4.04 (m, 2H), 4.23-4.37 (m, 4H), 5.23 (m, 1H), 7.19 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.06, 14.10, 19.22, 19.27, 22.21, 22.61, 22.66, 27.34, 27.39, 24.44, 29.19, 29.24, 29.30, 29.45, 29.59, 31.68, 31.87, 32.22, 32.27, 33.93, 34.33, 34.37, 38.57, 38.65, 45.60, 45.66, 53.45, 54.62, 62.03, 62.80, 62.86, 66.22, 68.65, 71.04, 71.10, 74.42, 74.58, 171.64, 171.72, 172.17, 175.66, 175.84, 176.13, 176.08.
2-Hexyl-1-decanoic acid (500 mg, 1 equiv.) was put into a reaction vessel and dissolved in DCM (50 mL), and then glutaric anhydride (Compound 81; 470 mg, 2 equiv.) and DMAP (630 mg, 2.5 equiv.) were added and stirred vigorously at room temperature for 13 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/Hexane 1:1, PMA stain), 2N aqueous HCl solution (10 mL) was added to the liquid reaction mixture, followed by extraction with DCM (3×25 mL). All organic layers were collected and washed with 2N aqueous HCl solution (2×20 mL) to remove the residual bases, and then washing was followed with aqueous saturated NaCl solution (30 mL). The filtrate filtered by drying the separated organic layer with anhydrous Na2SO4 was distilled under reduced pressure to remove the solvent, and then the mixture was purified by flash column chromatography (SiO2, EtOAc/hexane 1:9->2:8) to obtain Compound 82 (603 mg, 82%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=5.1 Hz, 6H), 1.19 (m, 25H), 1.54 (br s, 1H), 1.90 (qnt, J=5.2 Hz, 2H), 2.31-2.38 (m, 4H), 3.92 (d, J=7.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.07, 14.08, 19.86, 22.64, 22.67, 26.69, 29.30, 29.54, 29.60, 29.93, 31.25, 31.80, 31.89, 33.06, 33.25, 37.27, 67.39, 173.10, 179.25.
Acetal-protected pantothenic acid Compound 52 (107 mg, 1.2 equiv.) was put into a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (119 mg, 1.5 equiv.) and DMAP (10 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Afterwards, a solution in which 2-hexyldecan-1-ol (100 mg, 0.41 mmol) was dissolved in DCM (10 mL) was added dropwise at 5° C. and then stirred at room temperature for 8 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/hexane 3:7, PMA stain), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (20 mL). A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 1:9->4:6) to obtain Compound 86 (177 mg, 89%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.89 (t, J=5.2 Hz, 3H), 0.97 (s, 3H), 1.05 (s, 3H), 1.27-1.32 (m, 25H), 1.43 (s, 3H), 1.47 (s, 3H), 1.62 (br s, 1H), 2.55-2.58 (m, 2H), 3.29 (d, J=9.2 Hz, 1H), 3.45-3.63 (m, 2H), 3.69 (d, J=8.1 Hz, 1H), 4.00 (d, J=9.8 Hz, 2H), 4.08 (s, 1H), 6.95-6.96 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.07, 14.09, 18.68, 18.82, 22.09, 22.63, 22.66, 26.64, 26.67, 29.29, 29.46, 29.54, 29.58, 29.92, 31.20, 31.79, 31.87, 32.69, 34.14, 34.24, 37.27, 67.56, 71.49, 77.15, 98.99, 169.69, 172.30.
After dissolving Compound 86 (177 mg, 1 equiv.) and DTT (122 mg, 2 equiv.) in DCM, p-toluenesulfonic acid (64 mg, 0.5 equiv.) was added and stirred at room temperature for 55 minutes. Once the reaction was terminated, extraction was followed with EtOAc (2×30 mL), all organic layers were collected and washed with saturated NaCl solution, and then the filtrate filtered by drying the separated organic layers with anhydrous Na2SO4 was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by flash column chromatography (SiO2, EtOAc/hexane 1:1->7:3) to obtain Compound 87 (105 mg, 65%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.79-0.83 (m, 9H), 0.92 (s, 3H), 1.19-1.23 (m, 25H), 2.49 (t, J=6.2 Hz, 2H), 1.55 (br s, 1H), 3.38-3.54 (m, 4H), 3.91-3.94 (m, 3H), 7.22-7.25 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.05, 14.03, 22.05, 22.64, 22.63, 26.64, 26.63, 29.29, 29.44, 29.55, 29.58, 29.91, 31.20, 31.79, 31.86, 32.69, 34.14, 34.24, 37.27, 67.56, 71.49, 77.15, 98.99, 169.69, 172.30.
3-(Dimethylamino)propanoic acid (Compound 59; 29 mg, 1.1 equiv.) was put into a reaction vessel and dissolved in DCM (20 mL), and EDCI·HCl (69 mg, 1.6 equiv.) and DMAP (5 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at room temperature for 20 minutes. Compound 87 (100 mg, 1 equiv.) was put into another reaction vessel, dissolved in DCM (10 mL), and cooled to 5° C., and then the liquid mixture of compound 59 was added dropwise at 5° C. for 30 minutes, followed by stirring at room temperature for 8 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/MeOH 1:1, PMA stain), the reaction mixture was distilled under reduced pressure to remove the solvent, DCM (50 mL) was added additionally, and then the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (20 mL). A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, DCM/MeOH 9:1/28% aqueous NH3 solution) to obtain Compound 86 (177 mg, 89%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.79-0.82 (m, 9H), 1.12 (s, 3H), 1.19-1.31 (m, 25H), 1.53 (br. s, 1H), 2.16 (s, 6H), 2.38-2.66 (m, 6H), 3.38-3.53 (m, 3H), 3.81 (s, 1H), 3.91 (d, J=6.2 Hz, 2H), 4.25 (d, J=8.2 Hz, 1H), 7.40 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.08, 14.10, 19.31, 22.49, 22.63, 22.66, 26.64, 26.68, 29.29, 29.55, 29.58, 29.93, 31.18, 31.80, 31.88, 33.33, 34.22, 34.56, 37.25, 38.48, 37.25, 38.48, 45.05, 55.75, 67.56, 71.49, 74.47, 172.17, 172.24, 172.62.
Compound 82 (71 mg, 1.3 equiv.) was put into a reaction vessel and dissolved in DCM (30 mL), and EDCI·HCl (45 mg, 1.5 equiv.) and DMAP (19 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Thereafter, a solution in which Compound 88 (84 mg, 1 equiv.) is dissolved in DCM (10 mL) was added dropwise into the reaction mixture at 5° C., followed by stirring at room temperature for 16 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/hexane 5:5, PMA stain), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (20 mL). A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, the remaining mixture was purified by flash column chromatography (SiO2, EtOAc/hexane 5:5->6:4) to obtain Compound 9 (120 mg, 88%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.79-0.82 (m, 12H), 0.98-0.99 (m, 6H), 1.11-1.36 (m, 47H), 1.53 (br. s, 2H), 1.90 (qnt, J=7.3 Hz, 2H), 2.21 (s, 6H), 2.31 (t, J=6.7 Hz, 2H), 2.41-2.65 (m, 8H), 3.27-3.32 (m, 1H), 3.46-3.53 (m, 1H), 3.74 (d, J=9.3 Hz, 1H), 3.38-3.94 (m, 5H), 4.79 (s, 1H), 7.38 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 20.05, 20.27, 22.04, 22.64, 22.66, 26.64, 26.69, 23.30, 29.56, 29.60, 29.94, 31.16, 31.21, 31.81, 31.88, 33.09, 33.24, 33.78, 35.04, 37.20, 37.24, 37.27, 24.21, 55.13, 67.33, 67.57, 69.59, 76.61, 76.73, 77.05, 77.25, 167.84, 171.69, 191.97, 172.43, 173.04.
Acetal-protected pantothenic acid compound 52 (476 mg, 1.2 equiv.) was put into a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (438 mg, 1.5 equiv.) and DMAP (38 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Thereafter, a solution in which 9-fluorenemethanol (300 mg, 1 equiv.) is dissolved in DCM (10 mL) was added dropwise into the reaction mixture at 5° C., followed by stirring at room temperature for 1 hour. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/hexane 1.9, PMA stain), DCM (50 mL) was added additionally, and the mixture was transferred to the separatory funnel, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (20 mL) sequentially. A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 1.9) to obtain Compound 93 (541 mg, 81%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.86 (s, 3H), 0.94 (s, 3H), 1.27 (s, 3H), 1.31 (s, 3H), 2.56 (t, J=8.2 Hz, 2H), 3.16 (d, J=5.1 Hz, 1H), 3.38-3.52 (m, 2H), 3.56 (d, J=5.5 Hz, 1H), 3.98 (s, 1H), 4.09 (t, J=8.3 Hz, 1H), 4.28 (d, J=7.1 Hz, 2H), 6.89 (s, 1H), 7.17 (t, J=5.1 Hz, 2H), 7.24 (t, J=5.5 Hz, 2H), 7.38 (d, J=5.0 Hz, 2H), 7.57 (d, J=4.8 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.23, 18.67, 18.89, 21.03, 22.14, 29.45, 32.79, 32.98, 34.21, 34.25, 46.72, 46.92, 60.36, 66.66, 71.45, 76.87, 77.19, 77.50, 99.02, 119.78, 120.09, 120.35, 124.69, 125.01, 127.16, 127.87, 141.29, 143.64, 169.82, 171.06.
After dissolving Compound 93 (500 mg, 1 equiv.) and DTT (352 mg, 2 equiv.) in DCM, p-toluenesulfonic acid (98 mg, 0.5 equiv.) was added at room temperature and stirred for 15 minutes. Once the reaction was terminated, the liquid reaction mixture was extracted with EtOAc (30 mL×2) and then washed with saturated NaCl solution after collecting all organic layers. A filtrate filtered by drying the separated organic layers with anhydrous Na2SO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 1:1->7:3) to obtain Compound 94 (277 mg, 61%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.90 (s, 3H), 0.97 (s, 3H), 2.63 (t, J=8.1 Hz, 2H), 3.47-3.58 (m, 4H), 4.00 (s, 1H), 4.20 (t, J=5.1 Hz, 1H), 4.41 (d, J=7.1 Hz, 2H), 7.27-7.30 (m, 3H), 7.33 (t, J=5.5 Hz, 2H), 7.41 (d, J=5.0 Hz, 2H), 7.67 (d, J=4.8 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.19, 20.34, 21.05, 21.31, 34.06, 34.63, 39.26, 46.71, 53.47, 60.47, 66.62, 71.15, 76.82, 77.14, 77.45, 77.48, 120.09, 124.95, 127.18, 127.89, 141.29, 143.59, 171.33, 172.17, 173.54.
Compound 82 (164 mg, 2.2 equiv.) was put into in a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (101 mg, 1.5 equiv.) and DMAP (7 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Subsequently, a solution in which Compound 94 (250 mg, 1 equiv.) is dissolved in DCM (10 mL) was added into the reaction mixture dropwise at 5° C., followed by stirring at room temperature for 4 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc/hexane 1:9, PMA stain), DCM (100 mL) was added additionally, followed by washing with saturated NaHCO3 solution (2×200 mL) and saturated NaCl solution (200 mL) sequentially. A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 1:9) to obtain Compound 102 (300 mg, 81%)) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.80 (t, J=8.0 Hz, 12H), 0.95 (d, J=9.2 Hz, 6H), 1.11-1.28 (m, 50H), 1.52 (s, 2H), 1.63 (s, 1H), 2.00 (qnt, J=5.5 Hz, 4H), 2.26-2.33 (m, 6H), 2.38 (t, J=5.7 Hz, 2H), 2.55 (t, J=5.9 Hz, 2H), 3.38-3.51 (m, 2H), 3.76-3.97 (m, 6H), 4.22 (t, J=5.0 Hz, 1H), 4.33 (d, J=9.1 Hz, 2H), 4.88 (s, 1H), 6.61 (m, 1H), 7.24 (t, J=7.1 Hz, 2H), 7.26 (t, J=8.1 Hz, 2H), 7.49 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ 14.08, 14.10, 20.00, 20.13, 20.90, 21.32, 22.64, 22.67, 26.65, 26.69, 29.30, 29.56, 29.60, 29.95, 31.21, 31.24, 31.81, 31.89, 33.05, 33.17, 33.24, 33.37, 33.66, 34.65, 37.29, 37.38, 46.70, 66.71, 67.35, 67.41, 69.23, 120.08, 124.98, 125.00, 127.17, 127.88, 141.30, 143.57, 143.65, 167.95, 171.62, 172.48, 172.62, 173.01, 173.09.
After mixing and stirring piperidine and DMF in a volume ratio of 2:8 in a reaction vessel, the mixture was cooled to 0° C. to prepare 20% piperidine solution. Compound 102 (300 mg) was prepared by dissolving in DMF (20 mL) in another reaction vessel, and then the mixture was stirred for 5 minutes and cooled to 0° C. The 20% piperidine solution (40 mL) previously prepared was added under argon gas and stirred at 0° C. for 5 minutes. Once the reaction was terminated, 3M aqueous HCl solution was added into the reaction mixture, pH was adjusted to 7, EtOAc (200 mL) was added, and the mixture was transferred to the separatory funnel, followed by washing with distilled water (2×200 mL) and saturated NaCl solution (2×200 mL) sequentially. A filtrate filtered by drying a separated organic layer with anhydrous Na2SO4 was distilled under reduced pressure to remove the solvent, and the mixture obtained was purified by flash column chromatography (SiO2, EtOAc/hexane 0:1->4:6, 1% AcOH added) to obtain Compound 103 (226 mg, 91%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=8.1 Hz, 12H), 0.92 (d, J=9.0 Hz, 6H), 1.12-1.21 (m, 51H), 1.52 (s, 2H), 1.62 (s, 1H), 2.30 (m, 6H), 2.39 (t, J=5.8 Hz, 2H), 2.54 (t, J=5.3 Hz, 2H), 3.38-3.52 (m, 2H), 3.77-3.97 (m, 6H), 4.89 (s, 1H), 6.62 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 14.11, 20.02, 20.14, 20.91, 21.31, 22.66, 22.68, 26.61, 26.65, 29.31, 29.56, 29.61, 29.95, 31.22, 31.24, 31.81, 31.89, 33.05, 33.17, 33.24, 33.37, 33.66, 34.65, 37.29, 37.38, 46.70, 66.71, 67.35, 67.41, 69.23, 167.95, 171.62, 172.48, 172.62, 173.01, 173.09.
Compound 103 (100 mg, 1 equiv.) was put into a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (33 mg, 1.5 equiv.) and DMAP (3 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Subsequently, a solution in which Compound 100 (11 mg, 1.1 equiv.) is dissolved in DCM (10 mL) was added to the reaction mixture dropwise at 5° C., followed by stirring at room temperature for 4 hours. After confirming that the reaction was terminated via TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added additionally, followed by washing with saturated NaHCO3 solution (2×200 mL) and saturated NaCl solution (200 mL) sequentially. A filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was distilled under reduced pressure and purified by column chromatography (SiO2, EtOAc/MeOH 20:0->19:1) to obtain Compound 10 (97 mg, 90%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.82 (t, J=7.1 Hz, 12H), 0.97 (d, J=8.1 Hz, 6H), 1.11-1.32 (m, 50H), 1.54 (s, 2H), 1.88 (qnt, J=8.0 Hz, 4H), 2.22 (s, 6H), 2.31 (t, J=7.0 Hz, 6H), 2.39 (t, J=7.9 Hz, 2H), 2.43-2.51 (m, 4H), 3.38-3.51 (m, 2H), 3.37 (d, J=9.6 Hz, 2H), 3.91 (d, J=9.7 Hz, 4H), 3.96 (d, J=9.9 Hz, 2H), 4.10-4.14 (m, 2H), 4.87 (s, 1H), 6.77 (t, J=6.9 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.04, 19.99, 20.09, 20.79, 21.32, 22.60.22.62, 26.61, 26.66, 29.26, 29.52, 29.56, 29.91, 31.20, 31.77, 31.85, 33.02, 33.16, 33.19, 33.33, 33.75, 34.66, 37.26, 37.30, 45.57, 57.68, 62.16, 67.28, 67.34, 69.21, 167.80, 171.59, 172.44, 192.55, 172.95, 173.01.
After dissolving Compound 103 (100 mg, 1.3 equiv.) in DCM (30 mL) in the reaction vessel, EDCI·HCl (33 mg, 1.5 equiv.) and DMAP (3 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. Subsequently, a solution in which Compound 101 (17 mg, 1.1 equiv.) is dissolved in DCM (10 mL) was added in the reaction mixture dropwise at 5° C. and stirred for 6 hours at room temperature. After confirming that the reaction was terminated via TLC (SiO2, EtOAc 100%, PMA stain), DCM (100 mL) was added additionally, followed by washing with saturated NaHCO3 solution (2×200 mL) and saturated NaCl solution (1×200 mL) sequentially. A mixture obtained by distilling under reduced pressure a filtrate filtered by drying a separated organic layer with anhydrous MgSO4 was purified by flash column chromatography (SiO2, EtOAc/hexane 8:3->10:0) to obtain Compound 11 (89 mg, 79%) in clear oil.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=7.6 Hz, 12H), 0.96 (d, J=8.7 Hz, 6H), 1.10-1.31 (m, 47H), 1.54 (s, 2H), 1.85-1.93 (m, 4H), 2.28-2.57 (m, 16H), 2.33-2.50 (m, 2H), 3.65 (m, 4H), 3.78 (d, 1H), 3.89-3.98 (m, 5H), 4.13-1.19 (m, 2H), 4.88 (s, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.06, 20.01, 20.12, 20.89, 21.31, 22.62, 22.64, 26.64, 26.68, 29.28, 29.54, 29.58, 29.93, 31.22, 31.79, 31.87, 33.06, 33.18, 33.23, 33.35, 33.66, 34.68, 37.29, 37.35, 53.76, 56.97, 61.62, 66.72, 67.34, 37.41, 69.21, 167.88, 171.62, 172.38, 172.59, 173.00, 173.05.
1,2-Octanediol (Compound 104; 500 mg, 1 equiv.) and imidazole (279.32, 1.2 equiv.) were put into a reaction vessel and dissolved in DCM (30 mL) followed by stirring for 5 minutes, and then a solution in which tert-butyldiphenylsilyl chloride (TBDPS-Cl; 936.38 mg, 1 equiv.) was dissolved in DCM (20 mL) was added dropwise for 20 minutes. The liquid reaction mixture was stirred vigorously at room temperature for 5 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 19:1), the liquid reaction mixture was concentrated to a level of 35 mL. Afterwards, 70 mL of n-hexane was added to filter out precipitated solids, and the remaining filtrate was concentrated and purified by column chromatography (SiO2; hexane/ethyl acetate, 39:1 to 19:1, v/v) to obtain Compound 105 (1183 mg, 90%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.97 (t, J=6.2 Hz, 3H), 1.14 (s, 9H), 1.32 (s, 7H), 1.44-1.46 (m, 3H), 2.57 (s, 1H), 3.54-3.58 (m, 1H), 3.72-3.79 (m, 2H), 7.43-7.49 (m, 6H), 7.74 (d, J=6.7 Hz, 4H); 13C NMR (CDCl3, 100 MHz): δ 14.15, 19.10, 22.65, 25.54, 26.92, 29.39, 31.82, 32.85, 68.12, 72.02, 127.83, 129.86, 129.86, 133.25, 133.29, 235.59, 135.61.
Compound 104 (100 mg, 1 equiv.) was put into in a reaction vessel and dissolved in DCM (50 mL), and then octanoic acid (62 mg, 1.3 equiv.), EDCI·HCl (46 mg, 1.5 equiv.), and DMAP (4 mg, 0.2 equiv.) were added to be subjected to reflux heating for 18 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 39:1), DCM (50 mL) was added additionally, followed by washing with distilled water (2×50 mL), saturated NaHCO3 solution (2×50 mL), and brine (1×20 mL) sequentially. Organic layers were collected to remove moisture with anhydrous MgSO4, the filtered filtrate was distilled under reduced pressure and purified by column chromatography (SiO2; hexane/ethyl acetate, 39:1 to 19:1, v/v) to obtain Compound 106 (118 mg, 89%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.98 (t, J=6.1 Hz, 6H), 1.14 (s, 9H), 1.22-1.33 (m, 16H), 1.47-1.55 (m, 4H), 2.17-2.22 (m, 2H), 3.55-3.63 (m, 2H), 4.90-4.95 (m, 1H), 7.27-7.36 (m, 6H), 7.57-7.59 (d, J=6.8 Hz, 4H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 19.26, 22.58, 22.63, 25.10, 25.16, 26.76, 29.00, 29.19, 30.56, 31.69, 31.72, 34.64, 65.09, 74.20, 127.67, 129.67, 129.66, 129.69, 133.53, 135.58, 135.65, 173.51.
After removing moisture from the reaction vessel by flame-drying, it was cooled to 20° C. and then filled with nitrogen gas. Thereafter, Compound 106 (200 mg, 1 equiv.) and dry THE (15 mL) were injected with a syringe and stirred for 1 minute. Thereafter, n-Bu4NF solution (1M, THF; 0.5 mL, 1.2 equiv.) was added dropwise with a syringe for 10 minutes, and then the liquid reaction mixture was stirred at 20° C. for 2 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 9:1), distilled water (2×50 mL) was added for dilution, followed by extraction with DCM (3×40 mL). After organic layers were collected and washed with saturated NaCl solution, the moisture was removed with anhydrous Na2SO4, and a mixture obtained by distilling the filtered filtrate under reduced pressure was purified by column chromatography (SiO2; hexane/ethyl acetate, 1:19, v/v) to obtain Compound 107 (96 mg, 90%) in colorless oil.
1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J=6.3 Hz, 6H), 1.20 (m, 16H), 1.47-1.55 (m, 4H), 2.18-2.22 (m, 2H), 3.56-3.64 (m, 2H), 4.91-4.94 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 19.27, 22.54, 22.65, 25.14, 25.17, 26.88, 29.54, 30.56, 31.74, 31.72, 34.77, 65.09, 74.21, 173.56.
Compound 107 was put into a reaction vessel and dissolved by adding DCM (25 mL), and then Compound 81 (84 mg, 2 equiv.) and DMAP (112 mg, 2.5 equiv.) were added and stirred vigorously at room temperature for 10 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 9:1), 1N aqueous HCl solution was added for acidification, followed by extraction with DCM (3×25 mL). After organic layers were collected and washed with saturated NaCl solution (1×30 mL), the moisture was removed with anhydrous Na2SO4, and a mixture obtained by distilling the filtered filtrate under reduced pressure was purified by column chromatography (SiO2; hexane/ethyl acetate, 9:1 to 8:2, v/v) to obtain Compound 108 (99 mg, 70%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.87 (t, J=6.9 Hz, 6H), 1.24-1.28 (m, 18H), 1.55-1.60 (m, 4H), 1.90-1.98 (m, 2H), 2.38 (t, J=6.1 Hz, 2H), 2.37-2.44 (m, 4H), 3.98-4.02 (m, 1H), 4.23 (dd, J=2.2, 9.1 Hz, 1H), 5.05-5.10 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.12, 19.84, 22.48, 22.55, 24.84, 25.02, 28.87, 28.98, 29.01, 30.66, 31.56, 31.62, 32.90, 33.25, 32.90, 33.25, 34.05, 60.41, 64.81, 71.73, 172.37, 173.60, 178.67.
Compound 108 (170.90 mg, 1.2 equiv.) was dissolved in DCM (30 mL) in a reaction vessel, and then EDCI·HCl (113 mg, 1.6 equiv.) and DMAP (9 mg, 0.2 equiv.) were added, followed by stirring in the presence of argon gas at 5° C. for 10 minutes. A solution in which Compound 88 (200 mg, 1 equiv.) is dissolved in DCM (10 mL) was added dropwise at 5° C. and then stirred at room temperature for 20 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 7:3), DCM (50 mL) was added additionally, followed by washing with saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (1×20 mL) sequentially. By removing moisture from an organic layer with anhydrous MgSO4, filtrated filtered was distilled under reduced pressure and purified by column chromatography (SiO2; ethyl acetate/hexane, 2:8 to 5:5, v/v) to obtain Compound 12 (292 mg, 87%) in clear liquid.
1H NMR (CDCl3, 400 MHz): δ 0.87 (t, J=6.5 Hz, 12H), 0.99 (d, J=4.7 Hz, 6H), 1.19 (m, 47H), 1.53 (m, 1H), 1.88-1.92 (m, 2H), 2.21 (s, 6H), 2.30-2.65 (m, 12H), 3.27-3.32 (m, 1H), 3.46-3.53 (m, 1H), 3.74 (d, J=11.2 Hz, 1H), 3.86-3.94 (m, 5H), 4.79 (s, 1H), 5.16 (br s, 1H), 7.35-7.38 (t, J=5.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.09, 20.05, 20.27, 22.04, 22.64, 22.66, 26.64, 26.69, 29.30, 29.56, 29.60, 29.94, 31.16, 31.21, 31.81, 31.88, 33.09, 33.24, 33.78, 35.04, 37.20, 37.24, 37.27, 45.21, 55.13, 67.33, 67.57, 69.59, 74.47, 76.61, 167.84, 171.69, 171.97, 172.43, 173.04, 176.04.
Compound 105 (500 mg, 1 equiv.) was put into a reaction vessel and dissolved in DCM (50 mL), lipoic acid (349 mg, 1.3 equiv.), EDCI·HCl (374 mg, 1.5 equiv.), EDCI·HCl (374 mg, 1.5 equiv.), and DMAP (31 mg, 0.2 equiv.) were added and subjected to reflux heating for 20 hours. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 29:1), DCM (50 mL) was added additionally, and an organic layer was washed sequentially with distilled water (2×100 mL), saturated NaHCO3 solution (2×100 mL), and saturated NaCl solution (1×40 mL). After distilling under reduced pressure, the filtrate filtered by removing moisture from the obtained organic layer with anhydrous MgSO4, the obtained mixture was purified by column chromatography (SiO2; hexane/ethyl acetate, 49:1 to 39:1, v/v) to obtain Compound 109 (603 mg, 81%) in yellow liquid.
1H NMR (CDCl3, 400 MHz): δ 0.87 (t, J=6.1 Hz, 3H), 1.04 (s, 9H), 1.25 (s, 8H), 1.44-1.66 (m, 8H), 1.68-1.89 (s, 1H), 2.26-2.31 (m, 2H), 2.42-2.43 (m, 1H), 3.09-3.16 (m, 2H), 3.51-3.55 (m, 1H), 3.66-3.68 (m, 2H), 5.00 (m, 1H), 7.25-7.42 (m, 6H), 7.65-7.66 (m, 4H); 13C NMR (CDCl3, 100 MHz): δ 14.10, 19.27, 22.59, 24.79, 25.17, 26.78, 28.82, 29.18, 30.55, 31.71, 34.33, 34.33, 34.66, 38.49, 40.21, 56.12. 65.09, 74.38, 127.69, 129.69, 129.73, 133.45, 133.52, 135.57, 135.64, 173.10.
After removing moisture from the reaction vessel by flame-drying, it was cooled to 20° C. and filled with nitrogen gas. Thereafter, Compound 109 (600 mg, 1 equiv.) and dry THE (30 mL) were injected by a syringe and stirred for 1 minute. Subsequently, n-Bu4NF solution (1M, THF; 1.26 mL, 1.2 equiv.) was added dropwise with a syringe for 10 minutes, and then the liquid reaction mixture was stirred at 20° C. for one hour. After confirming that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 8:2), distilled water (2×50 mL) was added for dilution, followed by extraction with DCM (3×40 mL). Organic layers were collected and washed with saturated NaCl solution, and then the mixture obtained by distilling under reduced pressure the filtrate filtered by removing moisture with anhydrous Na2SO4 was purified by column chromatography (SiO2; hexane/ethyl acetate, 9:1, v/v) to obtain Compound 110 (297 mg, 85%) in yellow oil.
1H NMR (CDCl3, 400 MHz): δ 0.82 (t, J=6.7 Hz, 3H), 1.23-1.21 (m, 8H), 1.41-1.71 (m, 8H), 2.02 (m, 1H), 2.43-2.47 (m, 3H), 3.12-3.19 (m, 2H), 3.56 (qnt, J=6.7 Hz, 1H), 4.02-4.03 (m, 1H), 4.24 (dd, J=2.7, 12.0 Hz, 1H), 5.05 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.11, 19.72, 22.73, 31.81, 25.32, 28.04, 29.02, 30.73, 32.71, 33.41, 34.24, 34.63, 38.55, 40.33, 56.34, 65.56, 70.73, 173.14, 173.65, 178.45.
In the reaction vessel, Compound 81 (204 mg, 2 equiv.) and DMAP (274 mg, 2.5 equiv.) were added to a solution, in which Compound 110 (300 mg, 1 equiv.) and DCM (25 mL) were added, followed by stirring at room temperature. After vigorously stirring the reaction mixture at room temperature for 19 hours and then checking that the reaction was terminated via TLC (SiO2; hexane/ethyl acetate, 8:2), acidification was followed with 1M aqueous HCl solution. The obtained liquid mixture was extracted with DCM (3×50 mL) to collect organic layers, followed by washing with aqueous saturated NaCl solution (1×50 mL), and then the mixture obtained by distilling under reduced pressure the filtrate filtered by removing moisture with anhydrous Na2SO4 was purified by column chromatography (SiO2; hexane/ethyl acetate, 9:1 to 8:2, v/v) to obtain Compound 111 (302 mg, 75%) in yellow liquid.
1H NMR (CDCl3, 400 MHz): δ 0.88 (t, J=6.7 Hz, 3H), 1.27-1.29 (m, 8H), 1.45-1.70 (m, 8H), 1.88-2.02 (m, 5H), 2.35 (t, J=8.0 Hz, 2H), 2.41-2.48 (m, 8H), 3.11-3.18 (m, 2H), 3.55-3.58 (qnt, J=6.8 Hz, 1H), 3.98-4.03 (m, 1H), 4.26 (dd, J=2.7, 11.9 Hz, 1H), 5.09 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.10, 19.72, 22.79, 31.79, 29.31, 25.32, 28.01, 29.03, 30.72, 32.75, 33.31, 34.23, 34.62, 38.51, 40.20, 56.33, 65.55, 70.71, 173.11, 173.60, 178.43.
Compound 111 (215 mg, 1.3 equiv.) was put into a reaction vessel and dissolved in DCM (30 mL), and then EDCI·HCl (113 mg, 1.6 equiv.) and DMAP (9 mg, 0.2 equiv.) were added and stirred vigorously in the presence of argon gas at 5° C. for 10 minutes. A solution in which Compound 88 (200 mg, 1 equiv.) is dissolved in DCM (10 mL) was added dropwise at 5° C. and stirred at room temperature for 21 hours. After confirming that the reaction was terminated via TLC (SiO2; ethyl acetate), DCM (50 mL) was added additionally, followed by washing with anhydrous saturated NaHCO3 solution (2×50 mL) and saturated NaCl solution (1×20 mL) sequentially. After removing moisture from an organic layer with anhydrous MgSO4, and then the filtered filtrate was distilled under reduced pressure and purified by column chromatography (SiO2; ethyl acetate/hexane, 5:5 to 7:3, v/v) to obtain Compound 13 (251 mg, 70%) in yellow liquid.
1H NMR (CDCl3, 400 MHz): δ 0.88 (t, J=6.9 Hz, 9H), 1.06 (d, J=6.5 Hz, 6H), 1.26 (m, 33H), 1.45-1.71 (m, 10H), 1.90-1.99 (m, 3H), 2.28 (s, 6H), 2.32 (t, J=7.4 Hz, 2H), 2.40 (t, J=7.2 Hz, 2H), 2.44-2.61 (m, 8H), 2.67-2.72 (m, 1H), 3.08-3.21 (m, 2H), 3.34-3.39 (m, 1H), 3.53-3.60 (m, 2H), 3.81 (d, J=11.2 Hz, 1H), 3.96-4.04 (m, 4H), 4.23-4.26 (m, 1H), 4.85 (s, 1H), 5.06-5.09 (m, 1H), 7.45 (t, J=5.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 14.01, 14.08, 14.09, 20.04, 20.26, 22.02, 22.51, 22.63, 22.65, 24.56, 25.06, 26.63, 26.67, 28.69, 29.00, 29.29, 29.54, 29.58, 29.92, 30.71, 31.15, 31.59, 31.79, 31.87, 33.02, 33.04, 33.29, 33.79, 33.83, 34.57, 35.05, 37.19, 37.23, 38.46, 40.20, 45.19, 55.10, 56.30, 64.94, 67.57, 67.57, 69.57, 71.69, 76.61, 167.81, 171.66, 171.92, 172.35, 172.39, 173.12.
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phophocholine (DSPC) were purchased from Sigma-Aldrich for preparation as helper lipids, and trehalose-based lipids were synthesized by the following methods.
After dissolving trehalose dihydrate (200 mg, 0.53 mmol) in pyridine (5 mL), TBTU (421 mg, 1.3 mmol), DIPEA (320 μL, 1.2 mmol), and oleic acid (370 μL, 1.2 mmol) were added in order, and after filling with argon gas, the mixture was stirred at room temperature for 20 hours. The solvent was removed using a rotary reduced pressure distiller, and then dried after primary purification by column chromatography (SiO2, MeOH/EtOAc 2:98->10:90). After washing the dried solids several times with EtOAc, it was completely dried to obtain 287 mg (62%) of 6,6′-trehalose dioleate.
1H NMR (400 MHz, CD3OD) δ 5.38 (t, J=4.5 Hz, 4H), 5.08 (d, J=3.8 Hz, 2H), 4.39 (dd, J=2.2, 11.9 Hz, 2H), 4.23 (dd, J=5.1, 11.9 Hz, 2H), 4.04 (ddd, J=2.1, 5.3, 10.2 Hz, 2H), 3.81 (dd, J=9.4, 9.6 Hz, 2H), 3.50 (dd, J=3.6, 9.6 Hz, 2H), 3.35 (dd, J=8.8, 10.0 Hz, 2H), 2.04-2.09 (m, 8H), 2.37 (t, J=7.4 Hz, 4H), 1.61-1.67 (m, 4H), 1.29-1.41 (m, 40H), 0.93 (t, J=6.8 Hz, 6H); 13C NMR (100 MHz, CD3OD) δ 174.0, 129.5, 129.4, 93.8, 73.2, 71.8, 70.5, 70.1, 63.0, 33.7, 31.7, 29.5, 29.4, 29.2, 29.1, 29.0, 28.9, 28.8, 28.8, 26.8, 24.7, 22.4, 13.1.
Cholesterol and butyl lithocholate were prepared as the structure-maintaining lipid, lytocholic acid derivative compounds were synthesized by the following methods, and myristoyl diglyceride (DMG)-PEG was purchased and prepared as the PEG-modified lipid.
Lithocholic acid and n-butanol were put into a reaction vessel, and then HCl solution (35˜37% aqueous solution, 1 equiv.) was injected, followed by stirring at room temperature for 24 hours. After distilling the alcohol using a rotary reduced pressure distiller, it was dissolved again in DCM to extract impurities with saturated NaHCO3 aqueous solution and saturated brine. After that, the moisture was removed from the DCM layer using anhydrous Na2SO4 followed by filtration, and the filtered solution was dried using a rotary reduced pressure distiller and purified by column chromatography (SiO2, DCM/MeOH, 19:1˜49:1 volume ratio) to obtain a lithocholic acid derivative compound (92%).
MS (ESI-MS) calcd. for C28H46NO2 [M+H]+ 428.3523, found 428.3525.
Fabricated was an RNA platform that is inserted with a nucleic acid sequence as a target sequence that encodes Renilla Luciferase (R/L) which is known as one of the reporter genes while having internal ribosome entry site (IRES) element derived from encephalomyocarditis virus (EMCV). The template DNA was designed, and single-stranded nucleic acid molecules for the RNA platform were fabricated using in vitro transcription (IVT).
After preparing the lipid nanoparticle with RNA solution (50 mM sodium citrate buffer, 110 mM NaCl, pH=4.0) in a concentration of 0.625 mg/mL and a lipid mixture solution (ethanol) in Table 1 below using laboratory mixer and emulsifier (Namoassemblr Spark, Precision Nanosystems, Inc.), it was provided by undergoing solvent conversion with normal saline or PBS using a filter tube (UFC5010, Amicon) for centrifugation.
Expression was identified by intradermal injection (ID) of mRNA-LNP into the ears of ICR mice at 5 g/20 μL based on RNA.
The experimental process is as follows. Each experimental group was prepared at a dose of 5 g/20 μL based on RNA, and then injected via I.D. into both ears of the mouse, respectively. Afterwards, the expression was checked according to the maximum expression time band of target proteins. In the case of Renilla Luciferase (R/L), which was actually tested, the expression and maintenance were checked according to two time bands which are 6 hours and 24 hours.
The specific experimental process is as follows.
After anesthesia using an individual respiratory anesthesia machine, the drug administered according to the experimental conditions was injected via an insulin syringe. Afterwards, anesthesia was conducted using CO2 at each corresponding time, and then ears were cut off. The cut ears were completely crushed using scissors and a homogenizer while being placed in 300 μL of 1× Renilla Lysis buffer. Afterwards, 20 μL of the liquid mixture was taken and transferred to a white 96-well plate, 100 μL of Promega's Renilla luciferase assay substrate was added, and then luminescence was measured to compare the expression.
Monocyte chemoattractant protein-1 (MCP-1) is a type of chemokine that causes monocytes to migrate toward vascular endothelial cells for attachment and is known to play an important role in the immune response. In order to identify the effect of LNP on the immune response, mRNA encoding R/L was encapsulated into the LNP, then mRNA-LNP was injected via I.D. into the ears of ICR mice at 5 g/20 μL based on RNA, and blood was collected 6 hours later, followed by concentration measurement through ELISA.
The specific experimental process is as follows.
The drug administered according to the experimental conditions was injected through an insulin syringe. After 6 hours, blood was drawn using a respiratory anesthesia machine. After separating the serum from the secured blood, the experiment was carried out. Expression levels were compared using Invitrogen's MCP-1 mouse uncoated ELISA kit.
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As described above, since a specific part of the content of the present disclosure is described in detail, for those of ordinary skill in the art, it is clear that the specific description is only a preferred embodiment, and the scope of the present disclosure is not limited thereby.
Thus, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2023-0195342 | Dec 2023 | KR | national |
