Method for synthesizing lipids

Abstract

The present application provides methods for synthesizing lipids of Formula I that can be used to synthesize liposoluble compounds for targeting and enhancing the activity of therapeutic molecules, including siRNA.

Description

Technical Field

[0001] This application provides a method for synthesizing lipids, which can be used to synthesize lipid-soluble compounds for targeting and enhancing the activity of therapeutic molecules, including siRNA. Background Technology

[0002] Many techniques can be used to deliver therapeutic agents such as siRNA into cells, including the use of viral and non-viral transfection systems. Non-viral transfection systems can include, for example, polymers, lipids, liposomes, micelles, dendritic polymers, and nanomaterials. Examples of polymers previously studied for 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 produce high transfection efficiency but may be less safe than some non-viral systems. Additionally, the preparation of viral systems can be complex and / or expensive. Non-viral transfection systems, such as cationic polymers and / or lipids, have been reported to transfer plasmid DNA into cells. Cationic lipids offer many advantages.

[0003] One such example of a cationic lipid is ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanidinediyl)bis(ethane-2,1-diyl)bistetradecanoate (S104), as disclosed in U.S. Patent No. 8,308,267. Previous synthetic strategies for producing S104 and similar compounds have been plagued by low yields, inconvenient post-processing procedures, and a large number of byproducts.

[0004] There is still a need for synthetic methods for producing these cationic lipids that promote increased product yields, low byproduct yields, and use more readily available synthetic techniques. Summary of the Invention

[0005] In one respect, this application provides a method for producing S104.

[0006] In one embodiment, this application provides a method for synthesizing compounds of formula I.

[0007]

[0008] Where n is an integer between 8 and 16.

[0009] The method includes

[0010] a) Make the compound of formula II

[0011]

[0012] Where R is a protecting group;

[0013] Compounds of Formula III

[0014]

[0015] Where X is a halogen;

[0016] The reaction is followed by treatment with methanesulfonic acid to form a compound of formula IV;

[0017]

[0018] b) Under coupling conditions, the compound of formula IV is combined with the compound of formula V.

[0019]

[0020] Each Y is independently a halogen;

[0021] The reaction produces a compound of formula VI;

[0022]

[0023] c) React the compound of formula VI with 2-(dimethylamino)ethanethiol HCl under coupling conditions, and then treat with oxalic acid to form the compound of formula VII.

[0024]

[0025] as well as

[0026] d) React the compound of formula VII with a base to produce the compound of formula I.

[0027] In one embodiment, step b) coupling conditions include reacting the compound of formula IV with a base. In one embodiment, the base is trimethylamine.

[0028] In one embodiment, step c) coupling conditions include reacting the compound of formula VI with a base. In one embodiment, the base is trimethylamine.

[0029] In yet another implementation, n is 12.

[0030] In another embodiment, R is independently selected from carboxybenzyl, p-methoxybenzylcarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, toluenesulfonyl, trichloroethyl chloroformate, (4-nitrophenyl)sulfonyl, methyl, ethyl, propyl, n-butyl, tert-butyl, succinimide, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-ditert-butylphenol, trimethylsilyl, allyl, 1,1-dimethylallyl, 2,2,2-trifluoroethyl, phenyl, and 4-methoxybenzyl.

[0031] In yet another embodiment, R is a tert-butoxycarbonyl group.

[0032] In another embodiment, each Y is independently selected from Cl, Br, and I. In yet another embodiment, each Y is the same. In still another embodiment, each Y is Cl.

[0033] In another embodiment, X is independently selected from Cl, Br, and I. In yet another embodiment, X is Cl.

[0034] In yet another embodiment, the compound of formula VI is

[0035]

[0036] In yet another implementation, the yield of step a) is at least about 75%.

[0037] In yet another embodiment, the compound of formula I is

[0038]

[0039] In yet another embodiment, the compound of formula I is produced by the compound of formula II in a yield of at least about 70%.

[0040] In yet another implementation, compound VIII

[0041]

[0042] It exists in the product from step d) at a concentration of less than 500 ppm.

[0043] In yet another embodiment, compound VIII is present in the product from step d) at a concentration of less than 100 ppm.

[0044] In yet another embodiment, the compound of formula VII is isolated in solid form. In yet another embodiment, the compound of formula VII is isolated in crystalline solid form.

[0045] In one embodiment, without chromatographic purification, the purity of the resulting compound of formula VII, measured by liquid chromatography area percentage (LCAP), is between about 95% and 99.9%.

[0046] In yet another embodiment, the compound is not separable VI.

[0047] In another embodiment, the compound of formula I is prepared by the method of steps a)-d). Detailed Implementation

[0048] Throughout the specification and appended claims, the given chemical formula or name shall include all its stereo and optical isomers and racemates, where such isomers are present. Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of this invention. Many geometric isomers of C=C double bonds, C=N double bonds, cyclic systems, etc., may also be present in the compounds, and all such stable isomers are contemplated in this invention. Cis- and trans- (or E- and Z-) geometric isomers of the compounds of this invention are described and can be separated into mixtures of isomers or isolated isomeric forms. The compounds of this invention can be separated in optically active or racemic forms. Optically active forms can be prepared by resolving racemic forms or by synthesis from optically active starting materials. All methods used to prepare the compounds of this invention and the intermediates prepared therein are considered part of this invention. When preparing enantiomers or diastereomers, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization).

[0049] Depending on the method conditions, the end products of this invention can be obtained in free (neutral) or salt form. Both the free form and the salt of these end products are within the scope of this invention. If desired, one form of the compound can be converted to another. A free base or acid can be converted to a salt; a salt can be converted to a free compound or another salt; a mixture of isomers of the compounds of this invention can be separated into individual isomers. The compounds of this invention, their free forms, and salts can exist in a variety of tautomeric forms, wherein hydrogen atoms are transposed to other parts of the molecule, and thus the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all tautomeric forms, wherever they may exist, are included within the scope of this invention.

[0050] The term "stereoisomer" refers to isomers that have the same composition but differ in the spatial arrangement of their atoms. Enantiomers and diastereomers are examples of stereoisomers. The term "enantiomer" refers to one of a pair of molecular species that are mirror images of each other and cannot be superimposed. The term "diastereomer" refers to a stereoisomer that is not a mirror image. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomer species in equimolar amounts, wherein the composition is not optically active. It is well understood in the art that the stereochemistry of the product can be controlled by selecting the stereochemistry of the starting material, and that the stereochemistry of the product can be altered by changing the stereochemistry of the starting material. It is also well understood in the art how to separate racemic mixtures such that the stereochemical purity of the product is >99%.

[0051] The symbols “R” and “S” represent the configuration of the substituents surrounding one or more chiral carbon atoms. The isomer descriptors “R” and “S” are used as described herein to indicate one or more atomic configurations relative 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)).

[0052] The term "chirality" refers to a structural feature of a molecule that prevents it from superimposing itself on its mirror image. The term "isochirality" refers to the state of enantiomer purity. The term "optical activity" refers to the degree to which isochiral molecules or non-racemic mixtures of chiral molecules rotate the plane of polarized light.

[0053] The term "LCAP" refers to the percentage of liquid chromatographic area collected as in Waters Acquity HPLC.

[0054] The abbreviations used in this article are defined as follows: "°C" represents degrees Celsius, "eq" represents equivalent, "g" represents gram, "mg" represents milligram, "L" represents liter, "mL" represents milliliter, "μL" represents microliter, "N" represents equivalent concentration, "M" represents molar concentration, "mmol" represents millimole, "min" represents minute, "h" represents hour, "rt" represents room temperature, "RT" represents retention time, "conc." represents concentrate, "sat" or "saturated" represents saturation, "MW" represents molecular weight, "ee" represents enantiomer excess, "MS" or "Mass Spec" represents mass spectrometry, "ESI" represents electrospray ionization mass spectrometry, "HR" represents high resolution, "HRMS" represents high resolution mass spectrometry, "LCMS" represents liquid chromatography-mass spectrometry, "HPLC" represents high performance liquid chromatography, and "NMR" represents nuclear magnetic resonance spectroscopy. 1"H" represents the proton, and "D", "L", "α", "β", "R", "S", "E" and "Z" are stereochemical names familiar to those skilled in the art.

[0055] Step a)

[0056] In one implementation, step a) includes

[0057] Compounds of Formula II

[0058]

[0059] Where R is a protecting group;

[0060] Compounds of Formula III

[0061]

[0062] Where n is an integer between 8 and 16; and

[0063] X is a halogen;

[0064] The reaction is followed by treatment with methanesulfonic acid to form a compound of formula IV.

[0065]

[0066] In one embodiment, R is an amine protecting group. In another embodiment, R is selected from carboxybenzyl, p-methoxybenzylcarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, toluenesulfonyl, trichloroethyl chloroformate, and (4-nitrophenyl)sulfonyl. In one embodiment, R is tert-butoxycarbonyl.

[0067] In one embodiment, n is an integer from 8 to 16. In another embodiment, n is an integer from 8 to 9, 8 to 10, 8 to 11, 8 to 12, or 8 to 13. In one embodiment, n is an integer from 10 to 14. In another embodiment, n is an integer from 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, or 9 to 10. In another embodiment, n is an integer from 9 to 13 or 10 to 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.

[0068] In one embodiment, X is a halogen. In another embodiment, X is selected from fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At). In yet another embodiment, X is F. In yet another embodiment, X is Cl. In yet another embodiment, X is Br. In yet another embodiment, X is I.

[0069] In one embodiment, step a) is carried out in a solvent. In one embodiment, step a) is carried out in a solvent mixture. In one embodiment, at least one solvent is nonpolar. In one embodiment, the one or more solvents are 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.

[0070] In one embodiment, the compound of formula IV is separated in crystalline solid form. In one embodiment, step a) does not require chromatography for separation or purification.

[0071] In one embodiment, the compound of formula IV is separated in crystalline solid form. In one embodiment, step a) does not require chromatography for separation or purification. In one embodiment, without chromatographic purification, the purity of the resulting compound of formula IV, based on liquid chromatographic area percentage (LCAP), is between about 95% and 99.9%. In one embodiment, without chromatographic purification, the purity of the resulting compound of formula IV, based on LCAP, is at least about 95%.

[0072] In one embodiment, the crystalline compound of formula IV is produced from step a) in a yield between about 70% and 95%. In another embodiment, the compound of formula IV is produced in a yield between about 70% and 85%, or between about 70% and 75%. In yet another embodiment, the compound of formula IV is produced in a yield between about 80% and about 95%, or between about 90% and 95%. In yet another embodiment, the compound of formula IV is produced in a yield of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In yet another embodiment, the compound of formula IV is produced in a yield of about 80%. In one embodiment, the compound of formula IV is produced 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 produced from step a) in a yield of at least about 80%.

[0073] In one embodiment, step a) is a two-step process. The first reaction is the bismyristylation of the compound of formula I catalyzed by N-methylmorpholine. After the reaction is complete, the mixture is washed with an aqueous solution of 1N acetic acid to remove unreacted starting material and salt, and then subjected to dry distillation. The second reaction is the N-Boc-deprotection reaction followed by salt formation with methanesulfonic acid (MSA).

[0074] Step b

[0075] In one embodiment, step b includes reacting the compound of formula IV with the compound of formula V.

[0076]

[0077] Each Y is independently a halogen;

[0078] The reaction forms a compound of formula VI.

[0079]

[0080] In one implementation, n is as described with respect to step a). In one implementation, n is 12.

[0081] In one embodiment, step b) is carried out in a solvent. In one embodiment, at least one solvent is polar aprotic. In one embodiment, the one or more solvents are selected from dichloromethane (DCM), ethyl acetate (EtOAc), tetrahydrofuran (THF), acetone, N,N-dimethylformamide (DMF), acetonitrile, and dimethyl sulfoxide (DMSO). In one embodiment, step b) is carried out in EtOAc.

[0082] In one embodiment, step c) comprises reacting the 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.

[0083] In one embodiment, step b) is performed between about 0°C and about 30°C. In one embodiment, the process is performed between about 10°C and about 30°C or between about 20°C and about 30°C. In one embodiment, step b) is performed between about 0°C and about 25°C, about 0°C and about 15°C, or about 0°C and about 5°C. In one embodiment, step b) is performed between about 15°C and about 20°C. In one embodiment, step b) is performed at about 30°C, about 25°C, about 20°C, about 15°C, about 10°C, about 5°C, or about 0°C. In one embodiment, step b) is performed below about 30°C. In one embodiment, step b) is performed below about 25°C. In one embodiment, step b) is performed at about 20°C. In one embodiment, step b) is performed at about room temperature.

[0084] In one embodiment, step b) further comprises two aqueous washes (one with aqueous acetic acid and the other with KHCO3 / K2CO3 / salt water) to remove unreacted reagents and organic salt byproducts.

[0085] In one embodiment, the compound of formula VI is not separated. In one embodiment, step b) is the chloroacetylation of the compound of formula IV.

[0086] Step c

[0087] In one embodiment, step c) comprises reacting a compound of formula VI with 2-(dimethylamino)ethanethiol HCl under coupling conditions, followed by treatment with oxalic acid to form a compound of formula VII.

[0088]

[0089] In one implementation, n is the same as described in step a). In another implementation, n is 12.

[0090] In one embodiment, step c) is carried out in a solvent. In one embodiment, the solvent is a combination of solvents. In another embodiment, at least one solvent is polar proton. In another embodiment, at least one solvent is polar aproton. In another embodiment, at least one solvent is polar proton. In another embodiment, the one or more solvents are selected from 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 combination of acetonitrile and water.

[0091] 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.

[0092] In one embodiment, step c) is performed between about 0°C and about 30°C. In one embodiment, the process is performed between about 10°C and about 30°C, or between about 20°C and about 30°C. In one embodiment, step c) is performed between about 0°C and about 25°C, between about 0°C and about 15°C, or between about 0°C and about 5°C. In one embodiment, step c) is performed between about 15°C and about 25°C. In one embodiment, step c) is performed at about 30°C, about 25°C, about 20°C, about 15°C, about 10°C, about 5°C, or about 0°C. In one embodiment, step c) is performed below about 30°C. In one embodiment, step c) is performed at about 25°C.

[0093] In one embodiment, step c) further comprises two aqueous washes (acetic acid / salt water and KHCO3 / K2CO3 / salt water, respectively) followed by treatment with oxalic acid to remove unreacted reagents and organic salt byproducts.

[0094] In one embodiment, the compound of formula VII is separated in crystalline solid form. In one embodiment, step c) does not require chromatography for separation or purification. In one embodiment, without chromatographic purification, the purity of the resulting compound of formula VII, based on LCAP, is between about 95% and 99.9%. In one embodiment, without chromatographic purification, the purity of the resulting compound of formula VII, based on LCAP, is about 99%. In one embodiment, without chromatographic purification, the purity of the resulting compound of formula VII, based on LCAP, is at least about 95%.

[0095] In one embodiment, the crystalline compound of formula VII is produced from step c) in a yield between about 70% and 95%. In another embodiment, the compound of formula VII is produced in a yield between about 70% and 85%, or between about 70% and 75%. In yet another embodiment, the compound of formula VII is produced in a yield between about 80% and about 95%, or between about 90% and 95%. In yet another embodiment, the compound of formula VII is produced in a yield of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In yet another embodiment, the compound of formula VII is produced in a yield of about 80%. In one embodiment, the compound of formula VII is produced 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 another embodiment, formula VII is produced from step c) in a yield of at least about 80%.

[0096] In one embodiment, step c) is a coupling reaction. In one embodiment, step c) involves coupling 2-(dimethylamino)ethanethiol HCl with a compound of formula VI via CS bond formation, thereby forming an oxalate.

[0097] Step d

[0098] In one embodiment, step d) includes reacting the compound of formula VII with a base to form the compound of formula I.

[0099] In one embodiment, the crystalline compound of Formula I is produced from step d) in a yield between about 70% and 95%. In one embodiment, the compound of Formula VII is produced in a yield between about 70% and 85% or between about 70% and 75%. In another embodiment, the compound of Formula VII is produced in a yield between about 80% and about 95% or between about 90% and 95%. In another embodiment, the compound of Formula VII is produced in a yield of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In yet another embodiment, the compound of Formula VII is produced in a yield of about 80%. In one embodiment, the compound of Formula VII is produced 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 produced from step d) in a yield of at least about 80%.

[0100] In yet another implementation, compound VIII

[0101]

[0102] It is present in the product from step d) at a concentration of less than about 500 ppm. This byproduct is a contributing factor to the level of genotoxic impurities (GTI) in the previous synthetic route.

[0103] In another embodiment, compound VIII is present in the product from step d) at a concentration of less than about 300 ppm. In another embodiment, compound VIII is present in the product from step d) at a concentration of less than about 200 ppm. In another embodiment, compound VIII is present in the product from step d) at a concentration of less than about 100 ppm. In another embodiment, compound VIII is present in the product from step d) at a concentration of less than about 50 ppm. In yet another embodiment, compound VIII is not generated during the process.

[0104] Overall process

[0105] In one embodiment, the compound of Formula I is produced from the compound of Formula II in a total yield of about 40% to about 80%. In one embodiment, the compound of Formula I is produced from the compound of Formula III in a yield of about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, or about 40% to about 45%. In one embodiment, the compound of Formula I is produced from the compound of Formula II in a yield of about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 75% to about 80%, about 80% to about 80%, or about 85% to about 80%. In one embodiment, the compound of Formula I is produced from the compound of Formula II in a yield of about 55%. In one embodiment, the compound of Formula I is produced from the compound of Formula II in a yield of about 60%.

[0106] In one embodiment, the compound of formula I is generated 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 generated from the compound of formula II in a yield of at least about 50%. In one embodiment, the compound of formula I is generated from the compound of formula II in a yield of at least about 60%. In one embodiment, the compound of formula I is generated from the compound of formula II in a yield of at least about 65%.

[0107] Example

[0108] Example 1. Synthesis of aziridinedimethylbis(ethane-2,1-diyl)bistetradecanoate methanesulfonate methanesulfonate solvate

[0109]

[0110] Toluene (26.1 kg, 30.0 L, 30 L / kg) was charged into reactor 1, followed by N-Boc-diethanolamine (1.00 kg, 0.920 L). Observation: The presence of water may cause the myristylation reaction to stop and result in higher levels of impurities (myristic acid). The reactor should be thoroughly rinsed and dried before use. The jacket temperature was set to 15°C–25°C (target 20°C). N-methylmorpholine (1.68 kg, 1.82 L, 3.4 equivalents) was charged. Myristoyl chloride (2.65 kg, 2.94 L, 2.2 equivalents) was charged into the reactor after at least 30 minutes, maintaining the batch temperature <30°C (target 20°C). The reaction was aged at 15°C–25°C (target 20°C) for at least 5 hours.

[0111] Add 1N acetic acid (11.0 kg, 10 L, 10 L / kg) in a semi-saline solution to the reactor. Heat the reactants to 25°C–35°C (target 30°C) with stirring and age for 30 minutes. Stop stirring and allow the reaction to allow the phases to settle for at least 30 minutes. Separate the phases and send the bottom aqueous phase to the waste. Repeat this washing process.

[0112] The organic layer was distilled under vacuum (P < 100 mbar) to a final volume of 14 L / kg, maintaining the jacket setpoint temperature at 45°C. Observation: Distillation is typically performed using a put-and-take method; it is recommended to add a minimum volume of 20 L / kg of toluene to dry the stream. In subsequent steps, the presence of water may cause product decomposition. These impurities significantly increase product solubility, which in turn significantly reduces the yield. Furthermore, changes in solubility affect the seed point in step 28.

[0113] The reaction mixture was transferred to reactor 2 via a fine filter to remove precipitated NaCl. Toluene was loaded into reactor 1 using a spray nozzle as the vessel rinse solution and transferred to reactor 2 via a fine filter line (3.48 kg, 4.0 L, 4.0 L / kg). The reaction was heated to 30°C–40°C (target 35°C) under stirring and a nitrogen atmosphere. Methanesulfonic acid (0.936 kg, 0.693 L, 2.0 equivalent) was added after at least 30 minutes, maintaining the batch temperature at 30°C–40°C (target 35°C). Note: Anhydrous methanesulfonic acid (KF < 0.75 wt%) was used. Excess water may cause product decomposition. 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°C–40°C (target 35°C) for at least 1 hour, then cooled to 27°C–31°C (target 29°C).

[0114] 0.050 kg (5 wt%) of aziridine dibis(ethane-2,1-diyl)bistetradecanoic acid methanesulfonate methanesulfonate solvation seed was added to the reaction vessel. The reaction was then cooled to 10°C-20°C (target 15°C) for at least five hours.

[0115] Achieve the following cooling ramp:

[0116] i. After 30% of the total cooling time, from 29°C to 27.25°C.

[0117] ii. After 30% of the total cooling time, from 27.25°C to 24.5°C.

[0118] iii. After 40% of the total cooling time, from 24.5°C to 15°C.

[0119] Observation: Due to the effect of temperature on the solubility of the product in toluene / ethyl acetate, nonlinear cooling is implemented to help with filtration rate and impurity removal.

[0120] The reaction batch was then cooled at 10°C–20°C (target 15°C) for 1 hour and filtered. Tert-butyl methyl ether (MTBE, 2.22 kg, 3.0 L, 3.0 L / kg) was added to reactor 1 as crystallizer wash and cooled to 10°C–20°C (target 15°C). The product filter cake was then washed with MTBE wash from reactor 1, and the wash was filtered. The filter cake was dried under vacuum and nitrogen purging at a jacket setpoint of 20°C–30°C (target 25°C) to produce crystalline alkyldimethylbis(ethane-2,1-diyl)bistetradecanoate methanesulfonate methanesulfonic acid solvate in 90% yield. Observation: Increased temperature may cause the wet filter cake to melt / dissolve and lead to significant product degradation.

[0121] Example 2: Synthesis of ((2-chloroacetyl)azanidinediyl)bis(ethane-2,1-diyl)bistetradecanoate

[0122]

[0123] Add 1 kg (1 equivalent) of aziridine di(ethane-2,1-diyl)bistetradecanoate methanesulfonate methanesulfonic acid solvate to reactor 3, followed by EtOAc (10 L, 10.0 L / kg). Add triethylamine (0.457 kg, 3.25 equivalents). Then add chloroacetyl chloride (0.213 kg, 1.36 equivalents) to reactor 3. Note: The loading is exothermic. Load slowly while maintaining the batch temperature below 25°C. Age the reaction at 20°C for at least 1 hour.

[0124] After the reaction was complete, water (9.68 L, 9.68 L / kg) was added to reactor 3, followed by glacial acetic acid (0.29 L, 0.29 L / kg), and the mixture was allowed to stand at 25°C without stirring for at least 0.5 h. Observation: The acidic aqueous buffer solution will remove unreacted reagents and reaction byproducts. Aging was carried out at 25°C for at least 0.5 h. The phases were then separated, and the lower aqueous layer was sent to the waste.

[0125] Stir the organic layer and add a pH 9.5 carbonate buffer solution (10 L, 10 L / kg). Aged the reaction at 25°C for at least 0.5 h. Stop stirring and allow the reaction to stand at 25°C for at least 0.5 h. Then separate the phases, sending the lower aqueous layer from reactor 3 to the waste.

[0126] Example 3: Synthesis of ((2-((2-(dimethylamino)ethyl)thio)acetyl)azinediyl)bis(ethane-2,1-diyl)bistetradecanoate oxalate

[0127]

[0128] Add 0.375 kg of 2-(dimethylamino)ethanethiol HCl (1.90 equivalents) to reactor 4, followed by 10 L of acetonitrile (10.0 L / kg). Add water (0.16 L / kg) and age the reaction at 25°C for at least 0.5 h. Note: Visual confirmation of complete dissolution is required. If not, impurities will form at levels higher than typical for unrefined reactors.

[0129] Triethylamine (0.538 kg, 3.82 equivalents) was charged into reactor 3. The reaction mixture from reactor 3 was transferred to reactor 4 after at least 2 hours and aged for at least another 3 hours. Note: The ((2-chloroacetyl)azanidinediyl)bis(ethane-2,1-diyl)bistetradecanoate stream was charged slowly to maintain a high concentration of 2-(dimethylamino)ethanethiol relative to ((2-chloroacetyl)azanidinediyl)bis(ethane-2,1-diyl)bistetradecanoate in the solution to minimize impurity formation.

[0130] 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 brine (1.68 L, 1.68 L / kg) was added, and the reaction was aged at 25°C for at least 0.5 h. Stirring was stopped, and the reaction was allowed to stand at 25°C for at least 0.5 h. The phases were separated, and the lower aqueous layer from reactor 4 was sent to the waste.

[0131] Stir the remaining organic layer and add a pH 9.5 carbonate buffer solution (10 L, 10 L / kg). Aged the reaction at 25°C for at least 0.5 h. Stop stirring and allow the reaction to stand at 25°C for at least 0.5 h. Separate the phases, sending the lower aqueous layer from reactor 4 to the waste.

[0132] The organic layer is distilled at 150 mbar or lower, with a maximum batch temperature of 30°C, to a batch size of 5 L (target 5 L / kg). Toluene is added (10 L, 10.0 L / kg), and the reaction is concentrated to 5 L by distillation at 75 mbar or lower, with a maximum batch temperature of 30°C. Toluene is then added (2.5 L, 2.5 L / kg).

[0133] EtOAc (12.5 L, 12.5 L / kg) was added to reactor 4, and the reaction was heated to 38°C. Then, oxalic acid solution (5 L, 5.0 L / kg, 1.14 equivalents) was added over at least 2 hours. Note: The oxalic acid solution was added at a roughly constant rate over the addition time. If the addition rate changes drastically (i.e., from slow to rapid addition), the batch may become supersaturated, and there is a risk of secondary nucleation. The reaction was then cooled to 20°C and aged for 1 hour after 2 hours. The resulting slurry was filtered. Reactor 4 was then rinsed with EtOAc (5 L, 5.0 L / kg). The product filter cake was washed with the rinsing solution. The product filter cake was dried under vacuum at a jacket setpoint temperature ≤40°C to produce ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanidinediyl)bis(ethane-2,1-diyl)bistetradecanoate oxalate, with a yield of 83% from the azanidinediylbis(ethane-2,1-diyl)bistetradecanoate methanesulfonate methanesulfonic acid solvate.

[0134] Example 4: Synthesis of ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bistetradecanoate

[0135]

[0136] Add 20 L of EtOAc to reactor 1, followed by 1 kg of ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanyl)bis(ethane-2,1-diyl)bistetradecanoate oxalate. Then add 15 L of pH 9.5 carbonate buffer solution to reactor 1. Heat the solution to 25 °C and age for at least 0.5 h. Stop stirring and allow to stand at 20 °C for at least 0.5 h. Separate the phases and send the lower aqueous layer from reactor 1 to waste. Then add 8.32 L of water to reactor 1, followed by 1.75 L of 25% sodium chloride brine, and age the solution at 25 °C for at least 0.5 h. Stop stirring and allow to stand at 25 °C for at least 0.5 h. Separate the phases and send the lower aqueous layer to waste.

[0137] Concentrate the product phase to 10 L (target: 10 L / kg). Distill at 150 mbar, with a maximum feed temperature <30°C. The product may degrade at high temperatures in the presence of water. Charge EtOAc (6 L) into reactor 1 and concentrate to 10 L (target: 10 L / kg). Distill at 150 mbar, with a maximum feed temperature <30°C. The product may degrade at high temperatures in the presence of water. Charge EtOAc (6 L) into reactor 1 and concentrate to 5 L (target: 5 L / kg). Distill at 150 mbar, with a maximum feed temperature <30°C. The product may degrade at high temperatures in the presence of water.

[0138] The product is then filtered from reactor 1 to reactor 2. The solvent is exchanged for MeCN by distillation. Distillation should be carried out at a pressure of 150 mbar, with a maximum feed temperature <30°C. MeCN (13 L / kg) is charged into reactor 2 at 25°C. Reactor 2 is cooled to 23°C ± 0.5°C. The product (0.005 kg) is charged into reactor 2 and aged at 23°C for at least 1 hour. The following cooling gradient is applied:

[0139] The reactor temperature was brought up to 20°C over 2 hours.

[0140] The reactor temperature was adjusted to 2 to 10°C over 2.5 hours.

[0141] The reactor was brought to 2 to 0°C over 1.5 hours.

[0142] The reactor temperature was adjusted to -10°C over 1 hour.

[0143] It is crucial to strictly control the temperature ramp to prevent uncontrolled nucleation on the reactor walls, which leads to product loss. The product is aged at -10°C for at least 1 hour. The slurry is then transferred and filtered. The filter cake wash is cooled to -10°C in reactor 2 with stirring, and then transferred to a filter to wash the filter cake. The filter cake is dried under vacuum at ≤25°C to produce ((2-((2-(dimethylamino)ethyl)thio)acetyl)azanyl)bis(ethane-2,1-diyl)bistetradecanoate, in 93% yield.

Claims

1. A method for synthesizing compounds of formula I. Where n is an integer between 8 and 16. The method includes a) Make the compound of formula II Where R is a protecting group; Compounds of Formula III Where n is an integer between 8 and 16. And X is a halogen; The reaction is followed by treatment with methanesulfonic acid to form a compound of formula IV. b) Mixing the compound of formula IV with the compound of formula V Each Y is independently a halogen; The reaction forms a compound of formula VI. c) Reacting the compound of formula VI with 2-(dimethylamino)ethanethiol HCl under coupling conditions, followed by treatment with oxalic acid, to form Compounds of Formula VII, and d) React the compound of formula VII with a base to synthesize the compound of formula I.

2. The method according to claim 1, wherein step b) coupling conditions include reacting the compound of formula IV with a base.

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

4. The method according to claim 1, wherein step c) coupling conditions include reacting the compound of formula VI with a base.

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

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

7. The method according to claim 1, wherein R is selected from carboxybenzyl, p-methoxybenzylcarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, trifluoroacetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, toluenesulfonyl, trichloroethyl chloroformate, (4-nitrophenyl)sulfonyl, methyl, ethyl, propyl, n-butyl, tert-butyl, succinimide, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-ditert-butylphenol, trimethylsilyl, allyl, 1,1-dimethylallyl, 2,2,2-trifluoroethyl, phenyl, and 4-methoxybenzyl.

8. The method according to claim 7, wherein R is tert-butoxycarbonyl.

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 according to claim 1, wherein X is selected from Cl, Br and I.

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

13. The method according to claim 1, wherein the compound of formula VI is a compound 。 14. The method according to claim 1, wherein the compound of formula I is a compound 。 15. The method of claim 1, wherein the yield of a) is at least 80%.

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

17. The method of claim 1, wherein compound VIII It exists in the product from step d) at a concentration of less than 100 ppm.

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

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