Methods of lipid synthesis

Abstract

The present application provides a compound of formula I useful for the synthesis of lipid-soluble compounds for the purpose of targeting and enhancing the activity of therapeutic molecules, including siRNA. This provides a method for synthesizing lipids in TIFF2023532012000034.tif32160.

Description

Technical Field

[0001] The present application provides a method for synthesizing a lipid useful for synthesizing a lipophilic compound for targeting and enhancing the activity of a therapeutic molecule containing siRNA.

Background Art

[0002] There are a number of techniques (such as viral transfection systems and non-viral transfection systems) for delivering a therapeutic agent (e.g., siRNA) to cells. Non-viral transfection systems include, for example, polymers, lipids, liposomes, micelles, dendrimers, and nanomaterials. Examples of polymers that have been studied to date for use in cell transfection include cationic polymers such as poly(L-lysine) (PLL), polyethyleneimine (PEI), chitosan, and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA). Each type of system has its own advantages and disadvantages. For example, viral systems can have high transfection efficiency, but safety such as that of other non-viral systems can be compromised. Furthermore, viral systems can be complex and / or expensive to implement. Non-viral transfection systems (such as cationic polymers and / or lipids) have been reported to transfer plasmid DNA into cells. Cationic lipids have a number of advantages.

[0003] An example of a cationic lipid is ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl) ditetradecanoate (S104), which is disclosed in U.S. Patent No. 8,308,267. Synthetic strategies for producing S104 and similar compounds to date have had problems such as low yields, inconvenient work-up procedures, and the generation of a large amount of by-products.

[0004] There is a need for synthetic methods that increase the yield of the product, reduce the amount of by-products, and produce cationic lipids using simpler methods.

[0005] (Summary of the present invention) In one embodiment, this application provides a method for manufacturing S104.

[0006] In one embodiment, this application relates to formula I [ka] This invention provides a method for synthesizing the compound (wherein n is an integer between 8 and 16). The above method, a) Equation II [ka] Compounds of formula III (wherein R is a protecting group) and [ka] React the compound (where X is a halogen) with the following: Next, treat with methanesulfonic acid, and then use formula IV. [ka] Forms compounds of; b) Compound IV under coupling conditions of formula V [ka] When reacted with a compound of (wherein each Y is independently a halogen), formula VI [ka] Forms compounds of; c) The compound of formula VI is reacted with 2-(dimethylamino)ethanethiol·HCl under coupling conditions, followed by treatment with oxalic acid to obtain formula VII [ka] Forms compounds of; and d) React the compound of formula VII with a base to produce the compound of formula I. It is characterized by steps.

[0007] In one embodiment, the coupling condition in step b) includes reacting the compound of formula IV with a base. In one embodiment, the base is trimethylamine.

[0008] In one embodiment, the coupling condition in step c) includes reacting the compound of formula VI with a base. In one embodiment, the base is trimethylamine.

[0009] In yet another embodiment, n is 12.

[0010] In another embodiment, R is independently selected from the group consisting of carboxybenzyl, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, tosyl, trichloroethyl chloroformate, (4-nitrophenyl)sulfonyl, methyl, ethyl, propyl, n-butyl, t-butyl, succinimide, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol, trimethylsilyl, allyl, 1,1-dimethylallyl, 2,2,2-trifluoroethyl, phenyl, and 4-methoxybenzyl.

[0011] In a further embodiment, R is t-butyloxycarbonyl.

[0012] In another embodiment, each Y is independently selected from Cl, Br, and I. In yet another embodiment, each Y is identical. In a further embodiment, each Y is Cl.

[0013] In another embodiment, X is independently selected from Cl, Br, and I. In a further embodiment, X is Cl.

[0014] In a further embodiment, the compound of formula VI is

Chemical formula

[0015] In a further embodiment, the yield of step a) is at least about 75%.

[0016] In a further embodiment, the compound of formula I is

Chemical formula

[0017] In a further embodiment, the compound of formula I is obtained from the compound of formula II in a yield of at least about 70%.

[0018] In a further embodiment, compound VIII

Chemical formula

[0019] In a further embodiment, compound VIII is present in the product of step d) at a concentration of less than 100 ppm.

[0020] In a further embodiment, the compound of formula VII is isolated as a solid. In a further embodiment, the compound of formula VII is isolated as a crystalline solid.

[0021] In one embodiment, the purity of the obtained compound of formula VII by liquid chromatography area percent (LCAP) is between about 95% and 99.9% without chromatographic purification.

[0022] In a further embodiment, the compound of formula VI is not isolated.

[0023] In another embodiment, the compound of formula I is prepared by the method of steps a) to d). [Modes for carrying out the invention]

[0024] Throughout this specification and these claims, a given chemical formula or chemical name includes all isomers where stereoisomers, optical isomers, and racemates exist. Unless otherwise specified, all chiral (enantiomers and diastereomers) and racemates are included in the scope of this invention. Many geometric isomers may also exist in this invention, such as C=C double bonds, C=N double bonds, and ring systems, and all such stable isomers are included in this invention. Cis- and trans- (or E- and Z-) geometric isomers of the compounds of this invention are described, and they may be isolated as mixtures of isomers or as separated isomers. The compounds of this invention may be isolated as optically active or racemates. Optically active compounds may be produced by re-separation of racemates or by synthesis from optically active starting materials. Any process used to produce the compounds of this invention and their intermediates is considered part of this invention. When enantiomer or diastereomer products are prepared, the products may be separated by conventional methods, such as chromatography or fractional crystallization.

[0025] Depending on the conditions of this process, the final product of the present invention may be obtained in either a (neutral) free form or a salt form. Both the free and salt forms of these final compounds are included within the scope of the present invention. If desired, one form of a compound may be converted to the other form. A free base or free acid may be converted to a salt; the salt may be converted to its free compound or another salt; a mixture of isomer compounds of the present invention may be separated into their respective isomers. Multiple tautomers may exist of the compounds of the present invention, their free forms and salts, where a hydrogen atom is replaced by a molecule in another part, and the chemical bonds between atoms in the molecule are consequently transferred. Insofar as any tautomer exists, tautomers should be understood to be included within the scope of the present invention.

[0026] The term "stereoisomer" refers to isomers of the same composition but with different spatial arrangements of atoms. Enantiomers and diastereomers are examples of stereoisomers. The term "enantiomer" refers to a type of pair of molecules that are mirror images of each other and cannot be superimposed. The term "diastereomer" refers to stereoisomers that are not mirror images of each other. The term "racemate" or "racemic mixture" refers to a composition in which two enantiomers are present in equimolar amounts and do not have optical activity. It is well understood by those skilled in the art that the stereochemistry of the product can be controlled by selecting the stereochemistry of the starting materials, and that the stereochemistry of the product can be changed by changing the stereochemistry of the starting materials. It is also well understood by those skilled in the art that a racemic mixture can be separated such that the stereochemical purity of the product is >99%.

[0027] The symbols "R" and "S" indicate the arrangement of substituents around a chiral carbon atom. The isomer symbols "R" and "S" are used as described herein to indicate the relative arrangement of atoms to the core molecule and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68, 2193-2222 (1996)).

[0028] The term "chiral" refers to a structural characteristic of a molecule that cannot be superimposed on its own enantiomer. The term "homochiraral" refers to the state of a pure enantiomer. The term "optical activity" refers to the degree to which a chiral molecule (that is not a homochiral molecule or a racemic mixture) rotates the plane of polarization.

[0029] The term "LCAP" refers to the liquid chromatography area percentage obtained using Waters Acquity HPLC.

[0030] The abbreviations used herein are defined as follows: "℃" is Celsius temperature, "eq" is equivalent, "g" is gram, "mg" is milligram, "L" is liter, "mL" is milliliter, "μL" is microliter, "N" is normal, "M" is molar, "mmol" is millimoles, "min" is minutes, "h" is hours, "rt" is room temperature, "RT" is retention time, "conc." is concentrated, "sat" is saturated, "MW" is molecular weight, "ee" is enantiomer excess, "MS" or "Mass Spec" is mass spectrometry, "ESI" is electrospray ionization mass spectrometry, "HR" is high resolution, "HRMS" is high resolution mass spectrometer, "LCMS" is liquid chromatography mass spectrometry, "HPLC" is high-performance liquid chromatography, "NMR" is nuclear magnetic resonance spectroscopy, 1 "H" represents a proton, and "D", "L", "α", "β", "R", "S", "E", and "Z" are stereochemical symbols well known to those skilled in the art.

[0031] Step a) In one embodiment, step a) is Equation II [ka] Compounds of formula III (wherein R is a protecting group) and formula III [ka] (In the formula, n is an integer from 8 to 16, and X is a halogen) react with the compound, Next, it is treated with methanesulfonic acid, and formula IV [ka] It is characterized by forming a compound.

[0032] In one embodiment, R is an amine protecting group. In another embodiment, R is selected from the group consisting of carboxybenzyl, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, tosyl, trichloroethyl chloroformate, and (4-nitrophenyl)sulfonyl. In one embodiment, R is t-butyloxycarbonyl.

[0033] In one embodiment, n is an integer between 8 and 16. In another embodiment, n is an integer between 8 and 9, 8 and 10, 8 and 11, 8 and 12, or 8 and 13. In one embodiment, n is an integer between 10 and 14. In another embodiment, n is an integer between 2 and 10, 3 and 10, 4 and 10, 5 and 10, 6 and 10, 7 and 10, 8 and 10, or 9 and 10. In another embodiment, n is an integer between 9 and 13 or 10 and 12. In one embodiment, n is 8. In one embodiment, n is 9. In one embodiment, n is 10. In one embodiment, n is 11. In one embodiment, n is 12. In one embodiment, n is 13. In one embodiment, n is 14.

[0034] In one embodiment, X is a halogen. In another embodiment, X is selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At). In a further embodiment, X is F. In a further embodiment, X is Cl. In a further embodiment, X is Br. In a further embodiment, X is I.

[0035] In one embodiment, step a) is carried out in a solvent. In one embodiment, step a) is carried out in a mixed solvent. In one embodiment, at least one solvent is a nonpolar solvent. In one embodiment, the solvent is selected from pentane, hexane, cyclohexane, benzene, toluene, chloroform, diethyl ether, heptane, MTBE, and cyclopropyl methyl ether. In one embodiment, step b) is carried out in toluene.

[0036] In one embodiment, the compound of formula IV is isolated as a crystalline solid. In one embodiment, step a) does not require chromatography for isolation or purification.

[0037] In one embodiment, the compound of formula IV is isolated as a crystalline solid. In one embodiment, step a) does not require chromatography for isolation or purification. In one embodiment, the liquid chromatography area percentage (LCAP) purity of the obtained compound of formula IV is between approximately 95% and 99.9% without chromatographic purification. In one embodiment, the LCAP purity of the obtained compound of formula IV is at least approximately 95% without chromatographic purification.

[0038] In one embodiment, the crystalline compound of formula IV is obtained from step a) in a yield between about 70% and 95%. In one embodiment, the compound of formula IV is obtained in a yield between about 70% and 85%, or between about 70% and 75%. In another embodiment, the compound of formula IV is obtained in a yield between about 80% and 95%, or between about 90% and 95%. In yet another embodiment, the compound of formula IV is obtained in yields of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In a further embodiment, the compound of formula IV is obtained in a yield of about 80%. In one embodiment, the compound of formula IV is obtained from step a) in a yield of at least about 65%, at least 70%, at least 75%, at least about 80%, or at least about 85%. In another embodiment, formula IV is obtained from step a) in a yield of at least about 80%.

[0039] In one embodiment, step a) is a two-step method. The first reaction is the N-methyl-morpholine-catalyzed dimyristoylation of the compound of formula I. After the reaction is complete, the mixture is washed with a 1N aqueous acetic acid solution to remove unreacted starting materials and salts, and then distilled dry. The second reaction is the N-Boc-deprotection reaction followed by salt formation with methanesulfonic acid (MSA).

[0040] Step B In one embodiment, step b) involves a compound of formula IV and formula V [ka] By reacting compounds of the form (wherein each Y is independently a halogen), we get formula VI [ka] It is characterized by forming a compound.

[0041] In one embodiment, n is as described in step a). In one embodiment, n is 12.

[0042] In one embodiment, step b) is carried out in a solvent. In one embodiment, at least one solvent is a polar aprotic solvent. In one embodiment, the solvent is selected from dichloromethane (DCM), ethyl acetate (ԅ), tetrahydrofuran (THF), acetone, N,N-dimethylformamide (DMF), acetonitrile, and dimethyl sulfoxide (DMSO). In one embodiment, step b) is carried out in ԅ.

[0043] In one embodiment, step c) involves the reaction of a compound of formula V with a base. In one embodiment, the base is a tertiary amine. In one embodiment, the base is selected from trimethylamine, DIPEA, N-methylmorpholine, sodium hydroxide, and potassium hydroxide. In one embodiment, the base is trimethylamine.

[0044] In one embodiment, step b) is performed between approximately 0°C and approximately 30°C. In one embodiment, the above step is performed between approximately 10°C and approximately 30°C, or between approximately 20°C and approximately 30°C. In one embodiment, step b) is performed between approximately 0°C and approximately 25°C, approximately 0°C and approximately 15°C, or approximately 0°C and approximately 5°C. In one embodiment, step b) is performed between approximately 15°C and approximately 20°C. In one embodiment, step b) is performed at approximately 30°C, approximately 25°C, approximately 20°C, approximately 15°C, approximately 10°C, approximately 5°C, or approximately 0°C. In one embodiment, step b) is performed below approximately 30°C. In one embodiment, step b) is performed below approximately 25°C. In one embodiment, step b) is performed at approximately 20°C. In one embodiment, step b) is performed at or near room temperature.

[0045] In one embodiment, step b) is further characterized by washing with two types of aqueous solutions (an aqueous acetic acid solution and KHCO3 / K2CO3 / saline solution, respectively) to remove unreacted reagents and by-product organic salts.

[0046] In one embodiment, the compound of formula VI is not isolated. In one embodiment, step b) is the chloroacetylation of the compound of formula IV.

[0047] Step C In one embodiment, step c) is to react the compound of formula VI with 2-(dimethylamino)ethanethiol·HCl under coupling conditions, followed by treatment with oxalic acid, to obtain formula VII [ka] It is characterized by forming a compound.

[0048] In one embodiment, n is as described in step a). In one embodiment, n is 12.

[0049] In one embodiment, step c) is carried out in a solvent. In one embodiment, the solvent is a mixed solvent. In another embodiment, at least one solvent is a polar protic solvent. In another embodiment, at least one solvent is a polar aprotic solvent. In another embodiment, at least one solvent is a polar protic solvent. In another embodiment, the solvent is selected from the group consisting of dichloromethane, N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, ethanol, methanol, acetic acid, and water. In one embodiment, step c) is carried out in a mixed solvent of acetonitrile and water.

[0050] In one embodiment, the coupling condition includes a base. In one embodiment, the base is a tertiary amine. In one embodiment, the base is selected from trimethylamine, sodium hydroxide, DIPEA, N-methylmorpholine, and potassium hydroxide. In one embodiment, the base is trimethylamine.

[0051] In one embodiment, step c) is performed between approximately 0°C and approximately 30°C. In one embodiment, the above step is performed between approximately 10°C and approximately 30°C, or between approximately 20°C and approximately 30°C. In one embodiment, step c) is performed between approximately 0°C and approximately 25°C, approximately 0°C and approximately 15°C, or approximately 0°C and approximately 5°C. In one embodiment, step c) is performed between approximately 15°C and approximately 25°C. In one embodiment, step c) is performed at approximately 30°C, approximately 25°C, approximately 20°C, approximately 15°C, approximately 10°C, approximately 5°C, or approximately 0°C. In one embodiment, step c) is performed at less than approximately 30°C. In one embodiment, step c) is performed at approximately 25°C.

[0052] In one embodiment, step c) is further characterized by washing with two types of aqueous solutions (acetic acid aqueous solution / saline solution and KHCO3 / K2CO3 / saline solution, respectively), followed by treatment with oxalic acid to remove unreacted reagents and by-product organic salts.

[0053] In one embodiment, the compound of formula VII is isolated as a crystalline solid. In one embodiment, step c) does not require chromatography for isolation or purification. In one embodiment, the LCAP purity of the obtained compound of formula VII is between approximately 95% and 99.9% without chromatographic purification. In one embodiment, the LCAP purity of the obtained compound of formula VII is approximately 99% without chromatographic purification. In one embodiment, the LCAP purity of the obtained compound of formula VII is at least approximately 95% without chromatographic purification.

[0054] In one embodiment, the crystalline compound of formula VII is obtained from step c) in a yield between about 70% and 95%. In another embodiment, the compound of formula VII is obtained in a yield between about 70% and 85%, or between about 70% and 75%. In yet another embodiment, the compound of formula VII is obtained in a yield between about 80% and 95%, or between about 90% and 95%. In yet another embodiment, the compound of formula VII is obtained in a yield of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In a further embodiment, the compound of formula VII is obtained in a yield of about 80%. In one embodiment, the compound of formula VII is obtained from step c) in a yield of at least about 65%, at least 70%, at least 75%, at least about 80%, or at least about 85%. In yet another embodiment, formula VII is obtained from step c) in a yield of at least about 80%.

[0055] In one embodiment, step c) is a coupling reaction. In one embodiment, step c) involves 2-(dimethylamino)ethanethiol·HCl and the compound of formula VI forming a CS bond and subsequently forming an oxalate.

[0056] Step d In one embodiment, step d) is characterized by the reaction of a compound of formula VII with a base to form a compound of formula I.

[0057] In one embodiment, the crystalline compound of formula I is obtained from step d) in a yield between about 70% and 95%. In one embodiment, the compound of formula VII is obtained in a yield between about 70% and 85%, or between about 70% and 75%. In another embodiment, the compound of formula VII is obtained in a yield between about 80% and 95%, or between about 90% and 95%. In yet another embodiment, the compound of formula VII is obtained in a yield of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In a further embodiment, the compound of formula VII is obtained in a yield of about 80%. In one embodiment, the compound of formula VII is obtained from step d) in a yield of at least about 65%, at least 70%, at least 75%, at least about 80%, or at least about 85%. In another embodiment, formula VII is obtained from step d) in a yield of at least about 80%.

[0058] In a further embodiment, compound VIII [ka] However, it is present in the product of step d) at a concentration of less than approximately 500 ppm. This by-product is the cause of the genotoxic impurity (GTI) in the conventional synthesis route.

[0059] In another embodiment, compound VIII is present in the product of step d) at a concentration of less than about 300 ppm. In another embodiment, compound VIII is present in the product of step d) at a concentration of less than about 200 ppm. In another embodiment, compound VIII is present in the product of step d) at a concentration of less than about 100 ppm. In another embodiment, compound VIII is present in the product of step d) at a concentration of less than about 50 ppm. In a further embodiment, compound VIII is not produced in the above step.

[0060] Entire step In one embodiment, the compound of formula I is obtained from the compound of formula II in a final yield between about 40% and about 80%. In one embodiment, the compound of formula I is obtained from the compound of formula III in yields between about 40% and about 70%, about 40% and about 60%, about 40% and about 50%, or about 40% and about 45%. In one embodiment, the compound of formula I is obtained from the compound of formula II in yields between about 50% and about 80%, about 60% and about 80%, about 70% and about 80%, about 75% and about 80%, about 80% and about 80%, or about 85% and about 80%. In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of about 55%. In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of about 60%.

[0061] In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of at least about 40%, at least about 50%, at least about 60%, or at least about 65%. In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of at least about 50%. In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of at least about 60%. In one embodiment, the compound of formula I is obtained from the compound of formula II in a yield of at least about 65%.

[0062] (Examples) Example 1. Synthesis of Azandiylbis(ethane-2,1-diyl)ditetradecanoate methanesulfonate / methanesulfonic acid solvate [ka]

[0063] Toluene (26.1 kg, 30.0 L, 30 L / kg) was added to Reactor 1, followed by N-Boc-diethanolamine (1.00 kg, 0.920 L). Note: The reactor must be cleaned and completely dried before use, as residual water can cause the myristoylation reaction to stall, increasing impurities (myristic acid). The jacket temperature was set to 15-25°C (target 20°C), and N-methylmorpholine (1.68 kg, 1.82 L, 3.4 equivalents) was added. The batch temperature was maintained at <30°C (target 20°C), and myristic chloride (2.65 kg, 2.94 L, 2.2 equivalents) was added to the reactor over a period of 30 minutes or more. The reaction was aged at 15-25°C (target 20°C) for 5 hours or more. 1N acetic acid / semisaturated saline solution (11.0 kg, 10 L, 10 L / kg) was placed in the reactor. The reaction mixture was heated at 25-35°C (target 30°C) while stirring and aged for 30 minutes. Stirring was stopped, and the reaction mixture layers were allowed to stand for at least 30 minutes. The layers separated, and the lower aqueous solution was discarded. This washing process was repeated. The jacket temperature was set to 45°C, and the organic layer was distilled under vacuum (P < 100 mbar) until the final volume was 14 L / kg. Note: Distillation is generally carried out by adding and removing, and it is desirable to add a minimum volume (20 L / kg) of toluene to dry the solution. In subsequent steps, the product may be decomposed by the remaining water. These impurities significantly increase the solubility of the product, resulting in a significant decrease in yield. Furthermore, this change in solubility also affects the sowing point in step 28. The reaction mixture was transferred to Reactor 2 via a polish filter, and the precipitated NaCl was removed. To wash Reactor 1, toluene was added using a spray ball and transferred to Reactor 2 via the polish filter line (3.48 kg, 4.0 L, 4.0 L / kg). The reaction was heated under a nitrogen atmosphere at 30-40°C (target 35°C) with stirring. Maintaining the batch temperature at 30-40°C (target 35°C), methanesulfonic acid (0.936 kg, 0.693 L, 2.0 equivalents) was added over 30 minutes or more. Note: Here, methanesulfonic anhydride (KF < 0.75 wt%) was used because excess water can decompose the product. Anhydrous ethyl acetate (2.70 kg, 3.0 L, 3.0 L / kg) was added to the reaction mixture, and the reaction was aged at 30-40°C (target 35°C) for at least 1 hour, after which it was cooled to 27-31°C (target 29°C). Seed crystals (0.050 kg, 5 wt%) of azandiylbis(ethane-2,1-diyl)ditetradecanoate methanesulfonate / methanesulfonic acid solvate were added to the reaction solution, and then the mixture was cooled to 10-20°C (target 15°C) over a period of 5 hours or more. The cooling process was carried out in stages as follows: i. More than 30% of the total cooling time should be at 29°C to 27.25°C. ii. More than 30% of the total cooling time should be at 27.25°C to 24.5°C. iii. More than 40% of the total cooling time should be between 24.5°C and 15°C. Record: Taking advantage of the fact that temperature affects the solubility of the product in toluene / ethyl acetate, the mixture was cooled in stages to adjust the filtration rate and removal of impurities. The reaction batch was then cooled at 10-20°C (target 15°C) for 1 hour and filtered. Reactor 1 was charged with tert-butyl methyl ether (MTBE, 2.22 kg, 3.0 L, 3.0 L / kg) to flush the crystallizer and cooled to 10-20°C (target 15°C). The product cake in Reactor 1 was then washed with MTBE and the washing solution was filtered. The cake was vacuum dried and exposed to nitrogen, and the jacket temperature was set to 20-30°C (target 25°C) to obtain the crystal: azandiylbis(ethane-2,1-diyl)ditetradecanoate methanesulfonate methanesulfonic acid solvate in 90% yield. Note: At high temperatures, the wet cake may melt / dissolve, and the product may deteriorate significantly.

[0064] Example 2: Synthesis of ((2-chloroacetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate [ka] Azandiylbis(ethane-2,1-diyl)ditetradecanoate methanesulfonate / methanesulfonic acid solvate (1 kg, 1 equivalent) was added to Reactor 3, followed by butyl (10 L, 10.0 L / kg). Then, triethylamine (0.457 kg, 3.25 equivalents) and acetyl chlorochloride (0.213 kg, 1.36 equivalents) were added to Reactor 3. Record: Excitation occurred upon addition. The substance was added slowly, and the batch temperature was maintained below 25°C. The reaction solution was aged at 20°C for at least 1 hour. After the reaction was complete, water (9.68 L, 9.68 L / kg) and then glacial acetic acid (0.29 L, 0.29 L / kg) were added to Reactor 3 and left to stand at 25°C for at least 0.5 hours without stirring. Record: Unreacted reagents and reaction byproducts were removed with an acidic buffer. The mixture was aged at 25°C for at least 0.5 hours, then the layers were separated, and the lower aqueous layer was discarded. The organic layer was stirred, and a pH 9.5 carbonate buffer solution (10 L, 10 L / kg) was added. The reaction mixture was aged at 25°C for at least 0.5 hours. Stirring was stopped, and the reaction mixture was allowed to stand at 25°C for at least 0.5 hours, after which the layers were separated, and the lower aqueous layer was discarded from Reactor 3.

[0065] Example 3: Synthesis of ((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate oxalate [ka] 2-(dimethylamino)ethanethiol·HCl (0.375 kg, 1.90 equivalents), followed by acetonitrile (10 L, 10.0 L / kg), was added to Reactor 4. Water (0.16 L, 0.16 L / kg) was added, and the reaction mixture was aged at 25°C for at least 0.5 hours. Note: It appears that complete dissolution is necessary. Otherwise, a larger-than-usual amount of impurities that cannot be removed will be formed.

[0066] Triethylamine (0.538 kg, 3.82 equivalents) was added to Reactor 3, and the reaction mixture was transferred from Reactor 3 to Reactor 4 over a period of more than 2 hours, followed by a further aging period of more than 3 hours. Record: To a solution of ((2-chloroacetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate, the solution of ((2-chloroacetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate was slowly added to maintain a high relative concentration of 2-(dimethylamino)ethanethiol and minimize the formation of impurities. Water (8.03 L, 8.03 L / kg) was added to Reactor 4, followed by glacial acetic acid (0.29 L, 0.29 L / kg). 25% sodium chloride aqueous solution (1.68 L, 1.68 L / kg) was added, and the reaction mixture was aged at 25°C for at least 0.5 hours. Stirring was stopped, and the reaction mixture was allowed to stand at 25°C for at least 0.5 hours. The layers were separated, and the lower aqueous layer was discarded from Reactor 4. The remaining organic layer was stirred, and a pH 9.5 carbonate buffer solution (10 L, 10 L / kg) was added. The reaction mixture was aged at 25°C for at least 0.5 hours. Stirring was stopped, and the reaction mixture was allowed to stand at 25°C for at least 0.5 hours. The layers were separated, and the lower aqueous layer was discarded from Reactor 4. The organic layer was distilled at a pressure of 150 mbar or less and a maximum batch temperature of 30°C until the batch size reached 5 L (target 5 L / kg). Toluene (10 L, 10.0 L / kg) was added, and the reaction mixture was concentrated by distillation at a pressure of 75 mbar or less and a maximum batch temperature of 30°C until it reached 5 L, and then toluene (2.5 L, 2.5 L / kg) was added. ₹ (12.5 L, 12.5 L / kg) was placed in Reactor 4, and the reaction mixture was heated at 38°C. Then, oxalic acid solution (5 L, 5.0 L / kg, 1.14 equivalents) was added over at least 2 hours. Record: During the addition process, the oxalic acid solution was added at an approximately constant rate. If the addition rate changes significantly (i.e., adding slowly followed by a rapid addition), the batch may become supersaturated, potentially leading to secondary nucleation. The reaction mixture was then cooled to 20°C over 2 hours and aged for 1 hour. The resulting slurry was filtered, and Reactor 4 was then washed with SiO4 (5 L, 5.0 L / kg). The product cake was washed with the washing solution. The product cake was vacuum-dried with a jacket temperature set to ≤40°C to obtain ((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl bis(ethane-2,1-diyl)ditetradecanoate oxalate from azandiylbis(ethane-2,1-diyl)ditetradecanoate methanesulfonate methanesulfonic acid solvate in 83% yield.

[0067] Example 4: Synthesis of ((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate [ka] SiO (20 L), followed by ((2-((2-(dimethylamino)ethyl)thioacetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate oxalate (1 kg) was added to Reactor 1. Next, a pH 9.5 carbonate buffer solution (15 L) was added to Reactor 1, the solution was heated to 25°C, and then aged for at least 0.5 hours. Stirring was stopped, and the mixture was allowed to stand at 20°C for at least 0.5 hours. The layers were separated, and the lower aqueous layer was discarded from Reactor 1. Next, water (8.32 L), followed by a 25% sodium chloride aqueous solution (1.75 L) was added to Reactor 1, and the mixture was aged at 25°C for at least 0.5 hours. Stirring was stopped, and the mixture was allowed to stand at 25°C for at least 0.5 hours. The layers were separated, and the lower aqueous layer was discarded. At a pressure of 150 mbar and a maximum batch temperature of <30°C, the product layer was distilled and concentrated to 10 L (target 10 L / kg). Because the product can degrade at high temperatures in the presence of water, HCl (6 L) was added to Reactor 1 and the mixture was concentrated to 10 L (target 10 L / kg). Distillation was performed at a pressure of 150 mbar and a maximum batch temperature of <30°C. Because the product can degrade at high temperatures in the presence of water, HCl (6 L) was added to Reactor 1 and the mixture was then concentrated to 5 L (target 5 L / kg). Distillation was performed at a pressure of 150 mbar and a maximum batch temperature of <30°C. The product can degrade at high temperatures in the presence of water. Next, the product was polish-filtered from Reactor 1 to Reactor 2. The solvent was replaced with MeCN by distillation. Distillation was performed at a pressure of 150 mbar and a maximum batch temperature of <30°C. MeCN (13 L / kg) was added to Reactor 2 at 25°C, and Reactor 2 was cooled to 23 ± 0.5°C. The product (0.005 kg) was added to Reactor 2 and aged at 23°C for at least 1 hour. Cooling was carried out in stages as follows. Cooling the Reactor 2 to 20°C over 2 hours Cooling Reactor 2 to 10°C over 2.5 hours. Cooling Reactor 2 to 0°C over 1.5 hours. Cooling Reactor 2 to -10°C over 1 hour It is crucial to control the temperature and cool gradually and thoroughly to prevent uncontrolled nucleation at the reactor walls, which is a factor in product loss. The product was aged at -10°C for at least 1 hour. The slurry was then transferred and filtered. The cake was washed by cooling the washing solution of Reactor 2 to -10°C with agitation and then filtering. The cake was vacuum-dried at a temperature of ≤25°C to obtain ((2-((2-(dimethylamino)ethyl)thio)acetyl)azandiyl)bis(ethane-2,1-diyl)ditetradecanoate in 93% yield.

Claims

1. Equation I 【Chemistry 1】 A method for synthesizing a compound (wherein n is an integer between 8 and 16), a) Equation II 【Chemistry 2】 Compounds of formula III (wherein R is a protecting group) and formula III 【Transformation 3】 The compounds (wherein n is an integer from 8 to 16 and X is a halogen) are reacted, Next, it is treated with methanesulfonic acid, and formula IV 【Chemistry 4】 Forms compounds of; b) Compounds of formula IV and formula V 【Transformation 5】 By reacting compounds of the form (wherein each Y is independently a halogen), we get formula VI 【Transformation 6】 Forms compounds of; c) The compound of formula VI is reacted with 2-(dimethylamino)ethanethiol·HCl under coupling conditions, followed by treatment with oxalic acid to obtain formula VII 【Transformation 7】 Forms compounds of; d) A method comprising the step of reacting a compound of formula VII with a base to produce a compound of formula I.

2. The method of claim 1, wherein the coupling condition in step b) includes the reaction of a compound of formula IV with a base.

3. The method of claim 2, wherein the base is triethylamine.

4. The method of claim 1, wherein the coupling condition in step c) includes the reaction of a compound of formula VI with a base.

5. The method of claim 4, wherein the base is triethylamine.

6. The method of claim 1, wherein n is 12.

7. The method of claim 1, wherein R is selected from the group consisting of carboxybenzyl, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, tosyl, trichloroethyl chloroformate, (4-nitrophenyl)sulfonyl, methyl, ethyl, propyl, n-butyl, t-butyl, succinimide, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol, trimethylsilyl, allyl, 1,1-dimethylallyl, 2,2,2-trifluoroethyl, phenyl, and 4-methoxybenzyl.

8. The method of claim 7, wherein R is t-butyloxycarbonyl.

9. The method of claim 1, wherein each Y is independently selected from Cl, Br, and I.

10. The method of claim 9, wherein each Y is Cl.

11. The method of claim 1, wherein X is selected from Cl, Br, and I.

12. The method of claim 11, wherein X is Cl.

13. The compound of formula VI is a compound 【Transformation 8】 The method according to claim 1.

14. The compound of formula I is a compound 【Chemistry 9】 The method according to claim 1.

15. The method of claim 1, wherein the yield of step a) is at least 80%.

16. The method according to claim 1, wherein the compound of formula I is produced from the compound of formula II in at least 60% yield.

17. Compound VIII 【Chemistry 10】 The method of claim 1, wherein the product of step d) is present at a concentration of less than 100 ppm.

18. The method according to claim 1, wherein the compound of formula VII is isolated as a crystalline solid.

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