Methods for Producing Carbohydrate Targeting Moiety, NAG-25, and Intermediates Thereof

Abstract

Provided herein are improved methods for preparing carbohydrate targeting moieties and intermediates thereof (I). Described herein are methods for preparing NAG-25 that arise from the recognition of the need for more efficient, streamlined, environmentally friendly, and / or economical methods for producing NAG-25, such as methods that result in improved purity and / or yields of NAG-25 and its intermediates. Such methods do not include chromatography steps, minimize or prevent the formation of impurities, reduce or remove reactive impurities, and / or improve the isolation point and physical properties of the intermediates. JPEG2025515631000161.jpg38169

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 337,316, filed May 2, 2022, U.S. Provisional Patent Application No. 63 / 373,823, filed August 29, 2022, and U.S. Provisional Patent Application No. 63 / 385,615, filed November 30, 2022, each of which is incorporated by reference in its entirety.

[0002] The present disclosure relates to improved carbohydrate targeting moieties and intermediates thereof, as well as methods for preparing any of the aforementioned compositions, which are useful for conjugation to therapeutic compounds to direct therapeutic targeting in vivo. [Background technology]

[0003] For a certain compound to have a therapeutic effect (e.g., in vivo) or be useful for diagnostic purposes, the compound must be delivered to a specific location, such as a target cell. Efficient delivery of a compound to a specific location or target can also reduce or eliminate unintended consequences (e.g., off-target effects) caused by administration of the compound. Targeting moieties (e.g., carbohydrate targeting moieties) can be linked to such compounds, such as therapeutic compounds (e.g., oligomeric compounds), to direct therapy to a target in vivo. An example of an oligomeric compound is small interfering RNA (siRNA), which can regulate the expression of a target nucleic acid, thereby altering the translation of the target nucleic acid into a protein. For example, siRNA can regulate the expression of a target gene to inhibit the translation or expression of a protein.

[0004] Targeting moieties containing N-acetylgalactosamine (GalNAc or NAG) (e.g., tri-GalNAc or triantennary-GalNAc) facilitate delivery of therapeutic compounds to hepatocytes via binding to the asialoglycoprotein receptor. An exemplary GalNAc-containing targeting moiety for delivery of oligomeric compounds to the liver (e.g., the compound designated NAG-25 (or NAG25)) is described in U.S. Pat. No. 10,246,709. U.S. Pat. No. 10,246,709 describes the following structure: [ka] One method for preparing NAG-25 is disclosed, which has the formula: 3 ) and i-Pr is isopropyl (or 1-methylethyl). Summary of the Invention [Means for solving the problem]

[0005] Described herein are methods for preparing NAG-25 that arose from the recognition of the need for more efficient, streamlined, environmentally friendly, and / or economical methods for producing NAG-25, such as methods that result in improved purity and / or yields of NAG-25 and its intermediates. Such methods do not include chromatography steps, minimize or prevent the formation of impurities, reduce or remove reactive impurities, and / or improve the isolation point and physical properties of the intermediates. Thus, described herein are methods by which NAG-25 is prepared without the use of column chromatography. Further described herein are methods by which NAG-25 is prepared using a crystalline form of the triacid intermediate. Further novel and improved methods are further described herein. As a result of the described process steps, there is improved isolation and yield of NAG-25 intermediates, reduced impurities, and reduced waste. Further described herein are compositions comprising NAG-25 or one of its intermediates (e.g., t-butyl core, triacid or salt thereof, alcohol-Z, NAG-Z, NAG-H or salt thereof, triol, unprotected triol, TGZ, TG amine or salt thereof, TG PEG, and / or a salt of any of the foregoing) having reduced levels of impurities, such as the impurities and percentages of impurities described herein.

[0006] Disclosed herein is a t-butyl core [ka] 1. A method for preparing GluZ, comprising: [ka] and GluOtBu or a salt thereof: [ka] and reacting the t-butyl core with GluOtBu or a salt thereof to form a t-butyl core. In some embodiments, the GluOtBu or a salt thereof is GluOtBu hydrochloride: [ka] In some embodiments, the reaction is carried out in the presence of a coupling reagent, a base, and a solvent. In some embodiments, the coupling reagent is EDC / Oxyma, TFFH, PyOxim, CDI, PivCl, T3P, or COMU. In some embodiments, the coupling reagent is T3P or PivCl. In some embodiments, the coupling reagent is PivCl. In some embodiments, the base is N-methylmorpholine (NMM). In some embodiments, the solvent is IPAc, MeTHF (also referred to as 2-MeTHF), MIBK, or MTBE. In some embodiments, the solvent is MTBE. In some embodiments, the antisolvent is heptane. In some embodiments, the solvent is MTBE and the antisolvent is heptane. In some embodiments, a first solution containing GluZ and NMM in a solvent is added to a second solution containing a solvent and PivCl. In some embodiments, PivCl is present in excess. In some embodiments, GluOtBu is present in excess, hi some embodiments, the conversion to the t-butyl core is greater than about 90% based on the amount of GluZ.

[0007] Disclosed herein is a triacid: [ka] The method of preparing the compound of formula (I) comprises reacting a t-butyl core with an acid. In some embodiments, the acid is phosphoric acid (H 3 PO 4 ), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid. In some embodiments, the acid is phosphoric acid (H 3 PO 4). In some embodiments, the reaction is carried out in a solvent selected from 2-MeTHF, acetonitrile, THF, DMA, sulfolane, and DME. In some embodiments, the solvent is 2-MeTHF, THF, or acetonitrile. In some embodiments, the solvent is 2-MeTHF or THF. In some embodiments, the solvent is 2-MeTHF. In some embodiments, the triacid is isolated as a crystalline solid. In some embodiments, the triacid is greater than or equal to 95% pure as determined by LC. In some embodiments, the solvent further comprises water. In some embodiments, the solvent is a mixture of 2-MeTHF and water. In some embodiments, about 3.0-4.0 volumes of 2-MeTHF and about 0.5-2 volumes of water are used. In some embodiments, the reaction is carried out at a temperature of about 40-60° C. In some embodiments, the reaction is carried out at a temperature of about 45-55° C. In some embodiments, the reaction is carried out at a temperature of about 50° C. In some embodiments, the method further comprises removing phosphoric acid with at least one organic / aqueous wash. In some embodiments, the organic / aqueous wash comprises isopropyl acetate (iPAC) as the organic layer and ammonium sulfate as the aqueous layer. In some embodiments, about 8-18 volumes of iPAC and about 3-7 volumes of 20 wt% ammonium sulfate are used. In some embodiments, about 10 volumes of iPAC and about 5 volumes of 20 wt% ammonium sulfate are used. In some embodiments, the method further comprises crystallizing the triacid using acetone / toluene, 2-MeTHF / IPAc, 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile. In some embodiments, the method further comprises crystallizing the triacid using acetone / toluene. In some embodiments, the acetone / toluene is in a ratio of 2:3, 1:1, 3:2, or 1:2. In some embodiments, the acetone / toluene is in a ratio of 2:3, 1:1, or 1:2.In some embodiments, the method produces a crystalline form of the triacid characterized by an X-ray powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2.

[0008] Disclosed herein is crystalline Form I of the triacid characterized by an X-ray powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2. In some embodiments, crystalline Form I of the triacid is characterized by an X-ray powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, and 21.0±0.2. In some embodiments, crystalline Form I of the triacid is characterized by an X-ray powder diffractogram substantially similar to that of FIG. 2. In some embodiments, crystalline Form II of the triacid is characterized by an X-ray powder diffractogram substantially similar to that of FIG. 3.

[0009] Disclosed herein is TGZ [ka] The method for preparing NAG-H is carried out by reacting a triacid with NAG-H in the presence of a coupling reagent and a base: [ka] or a salt thereof. In some embodiments, the NAG-H or salt thereof is a TFA salt of NAG-H: [ka] In some embodiments, the coupling reagent is TBTU, HATU, or TCFH. In some embodiments, the coupling reagent is TBTU. In some embodiments, the base is DIPEA, NMI, NMM, or TMP. In some embodiments, the base is NMI. In some embodiments, the method further comprises at least one buffer wash. In some embodiments, the buffer wash is a phosphate buffer. In some embodiments, the buffer wash is about pH 5-7. In some embodiments, the buffer wash is about pH 6. In some embodiments, the method is carried out in a solvent selected from DCM, DMF, MeCN, and DMAc, or a combination thereof. In some embodiments, the solvent is DMAc. In some embodiments, the solvent is DCM. In some embodiments, the method further comprises adding an anti-solvent to the solution comprising TGZ and the solvent. In some embodiments, the anti-solvent is an ether solvent. In some embodiments, the ether solvent is DME, 2-MeTHF, or MTBE. In some embodiments, the solvent is DCM and the anti-solvent is MTBE. In some embodiments, TGZ is prepared with low impurities. In some embodiments, the impurity by-product is des-acyl. In some embodiments, the method further comprises precipitating TGZ from the solvent / anti-solvent system. In some embodiments, the solvent / anti-solvent system is DCM / MTBE. In some embodiments, TGZ is obtained in at least 95% purity without the use of column chromatography. In some embodiments, NAG-H or a salt thereof is subsequently sent as a solution from a previous reaction to react with a triacid without isolating NAG-H or a salt thereof from the previous reaction. In some embodiments, NAG-H or a salt thereof is prepared in a previous reaction comprising hydrogenation of NAG-Z in the presence of a Pd / C catalyst and in DCM or dimethylacetamide (DMAc). In some embodiments, the DCM is from about 3 volumes to about 6 volumes. In some embodiments, the DMAc is from about 3 volumes to about 5 volumes.In some embodiments, the DMAc is about 4 volumes. In some embodiments, the NAG-H or salt thereof is NAG-H TFA. In some embodiments, the Pd / C catalyst is 5% Pd / C. In some embodiments, the NAG-H TFA is reacted with 1,2-dichlorophenyl ether according to the following scheme: [ka] Prepared in a previous reaction containing the reagents in

[0010] Disclosed herein is a methyl core: [ka] 1. A method for preparing zL-Glu-OMe: [ka] and L-glutamic acid dimethyl ester (di(OMe)Glu): [ka] to produce a methyl core.

[0011] In some embodiments, the reaction is carried out in the presence of a base and a coupling reagent. In some embodiments, the base is added to a solution of zL-GluOMe before the coupling reagent and di(OMe)Glu are added to the reaction. In some embodiments, the coupling reagent is added to a solution of zL-Glu-OMe and a base before di(OMe)Glu is added to the reaction. In some embodiments, the reaction further comprises a solvent. In some embodiments, the base is N-methylmorpholine (NMM) or diisopropylethylamine. In some embodiments, the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC. In some embodiments, the solvent is THF or MeTHF. In some embodiments, the reaction temperature before and during the reaction with the coupling reagent is about -20°C to about -10°C. In some embodiments, the reaction temperature is about -18°C to about -13°C. In some embodiments, the reaction temperature after addition of di(OMe)Glu is increased to about 5° C. to about 15° C. In some embodiments, the reaction temperature is increased to about 7° C. to about 12° C. In some embodiments, the reaction further comprises an additional charge of coupling reagent, base, and / or zL-Glu-OMe. In some embodiments, the reaction further comprises washing the reaction with aqueous acid followed by washing the reaction with aqueous base. In some embodiments, the reaction further comprises crystallization by addition of anti-solvent. In some embodiments, the method comprises the following scheme: [ka] The present invention includes reagents and conditions used in the present invention.

[0012] Disclosed herein is a triol: [ka] A method for preparing a compound according to the present invention comprising the steps of: [ka] to produce a triol. In some embodiments, the method is carried out in solvent-free 2-(2-aminoethoxy)ethanol. In some embodiments, the reaction temperature is about 25° C. to about 35° C. In some embodiments, the reaction temperature is about 30° C. In some embodiments, the reaction further comprises adding an anti-solvent to precipitate the triol. In some embodiments, the anti-solvent is ethyl acetate, methyl-tert-butyl ether, or iso-propyl acetate. In some embodiments, the method further comprises adding triol seed crystals. In some embodiments, the method comprises the following scheme: [ka] The present invention includes reagents and conditions used in the present invention.

[0013] Disclosed is an alternative method for preparing TGZ, comprising the steps of: [ka] and Beta-D-Galactosamine Pentaacetate: [ka] to produce TGZ.

[0014] In some embodiments, beta-D-galactosamine pentaacetate is first reacted with a silyl-triflate to produce an oxazoline solution. In some embodiments, the silyl-triflate is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or triisopropylsilyl trifluoromethanesulfonate (TIPSOTf). In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is dichloroethane (DCE) or dichloromethane (DCM). In some embodiments, the reaction temperature is about 35° C. to about 45° C. In some embodiments, the reaction temperature is about 40° C. In some embodiments, the reaction temperature is reduced to about 20° C. to about 28° C. In some embodiments, the reaction temperature is about 23° C. In some embodiments, the triol is reacted with sodium bicarbonate (NaHCO 3 ) and triol and NaHCO 3 After forming a slurry mixture of the above, beta-D-galactosamine pentaacetate or its oxazoline solution is added. In some embodiments, the triol and NaHCO 3 The slurry mixture of further comprises a solvent. In some embodiments, the solvent is dichloromethane (DCM), dichloroethane, or acetonitrile. In some embodiments, the oxazoline solution is dissolved in a triol and NaHCO 3 In some embodiments, the reaction temperature is about 20° C. to about 30° C. In some embodiments, the reaction temperature is about 25° C. In some embodiments, the TGZ is further precipitated from the solvent / anti-solvent system. In some embodiments, an anti-solvent is added to the solution containing TGZ and the solvent. In some embodiments, the anti-solvent is dimethoxyethane. In some embodiments, the method comprises the steps of: [ka] The present invention includes reagents and conditions used in the present invention.

[0015] In some embodiments, the reaction further comprises any one of the claims relating to step A-2 or A-1.

[0016] Disclosed herein are compounds useful for preparing the TGZ described herein. [ka] where R is H or a Cbz protecting group.

[0017] Disclosed herein are methods of preparing NAG-25, including any one of the embodiments for preparing a methyl core or triol using the alternative methods described. Disclosed herein are alternative methods of preparing NAG-25, including a triol intermediate. In some embodiments, the alternative method further includes any one of the embodiments for step 5a, step 5b, or step 6. In some embodiments, the alternative method includes reacting zL-Glu-OMe with L-glutamic acid dimethyl ester to form a methyl core, and converting the methyl core to NAG-25. In some embodiments, the alternative method includes reacting the methyl core with 2-(2-aminoethoxy)ethanol to form a triol, and converting the triol to NAG-25. In some embodiments, the alternative method includes reacting the triol with beta-D-galactosamine pentaacetate to form TGZ, and converting the TGZ to NAG-25. In some embodiments, the TGZ is further precipitated from a solvent / anti-solvent system as described herein.

[0018] Disclosed herein is a TG amine [ka] or a salt thereof, comprising the steps of: A method comprising forming a TG amine or a salt thereof by high pressure hydrogenolysis for the carboxybenzyl deprotection of TGZ. In some embodiments, the method includes a palladium source for hydrogenolysis. In some embodiments, the palladium source is a palladium on carbon (Pd / C) catalyst. In some embodiments, the palladium source is 5% Pd / C.4d. In some embodiments, the catalyst loading is less than about 10%, 9%, 8%, 7%, 6% Pd / C. In some embodiments, the catalyst used is 5% Pd / C. In some embodiments, the hydrogenolysis is carried out in the presence of an acid. In some embodiments, the acid is TFA, oxalic acid, HCl, AcOH, H 3 PO 4, citric acid. In some embodiments, the acid is TFA or oxalic acid. In some embodiments, the acid is TFA. In some embodiments, the hydrogenolysis is performed in a solvent, and the solvent is DCM, IPAc, or MeOH. In some embodiments, the solvent is DCM. In some embodiments, this method reduces the formation of deacylated impurities. In some embodiments, the TG amine or salt thereof is a TG amine acid salt. In some embodiments, the TG amine acid salt is a phosphate, a formate, an acetate, a trifluoroacetate, or an oxalate. In some embodiments, the TG amine acid salt is a trifluoroacetate or an oxalate. In some embodiments, the TG amine acid salt is a trifluoroacetate. In some embodiments, this method provides a high purity TG PEG product of step 5b, as well as a reduced amount of TG amine acetamide by-product. In some embodiments, the TG amine or salt thereof contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of acetamide by-product. In some embodiments, the TG amine or salt thereof produced from the improved step 5a process has a purity greater than or equal to about 97%, 98%, or 99% as measured by LC. In some embodiments, the TG amine or salt thereof is incorporated into step 5b without isolating the TG amine or salt thereof. In some embodiments, the method comprises the steps of: [ka] The present invention includes reagents in the above.

[0019] Disclosed herein is a TG PEG [ka] The method comprises treating a solution of TG amine and PEG acid with TBTU. In some embodiments, the solution further comprises a base. In some embodiments, the base is N,N-diisopropylethylamine (DIPEA). In some embodiments, the TBTU is added over a period of about 30 minutes to about 1.5 hours. In some embodiments, the TBTU is added over a period of about 1 to 1.5 hours. In some embodiments, the TG PEG is produced with a purity greater than or equal to about 90% as measured by LC. In some embodiments, at the end of the reaction, the TG PEG dimer impurity is present in an amount less than 10% as measured by LC. In some embodiments, the method comprises the following scheme: [ka] The present invention includes reagents in the above.

[0020] Disclosed herein is a method of preparing NAG-25, comprising treating TG PEG with an activating agent and a phosphitylating reagent. In some embodiments, the activating agent is tetrazole, 4,5-dicyanoimidazole (DCI), ETT, or benzothiotetrazole (BTT). In some embodiments, the activating agent is tetrazole, DCI, or ETT. In some embodiments, the activating agent is tetrazole. In some embodiments, the tetrazole is added at about 0.2eq to 1.2eq. In some embodiments, the activating agent is DCI. In some embodiments, the DCI is added at about 0.02eq to 1.0eq. In some embodiments, the method is carried out in the presence of a base. In some embodiments, the base is NMI. In some embodiments, the phosphitylation reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. In some embodiments, the method further comprises extraction, filtration, precipitation, and drying. In some embodiments, the precipitation step comprises precipitation of NAG-25 from DCM / heptane.

[0021] Disclosed herein is NAG-25: [ka] wherein the method comprises any one of the embodiments disclosed herein for steps 1, 2, 3, 4a, 4, 5, or 6. In some embodiments, the method comprises preparing a t-butyl core according to any one of the embodiments disclosed herein for preparing a t-butyl core, and converting the t-butyl core to NAG-25. In some embodiments, the method comprises preparing a triacid according to any one of the embodiments disclosed herein for preparing a triacid, and converting the triacid to NAG-25. In some embodiments, the method comprises preparing TGZ according to any one of the embodiments disclosed herein for preparing TGZ, and converting the TGZ to NAG-25. In some embodiments, the method comprises preparing a TG amine according to any one of the embodiments disclosed herein for preparing a TG amine, and converting the TG amine to NAG-25. In some embodiments, the method includes preparing TG PEG according to any one of the embodiments disclosed herein for TG PEG, and converting the TG PEG to NAG-25.

[0022] Disclosed herein is NAG-25: [ka] 2. A method for preparing TG PEG by treating a mixture of TG amine and PEG acid with TBTU. [ka] To obtain and converting TG PEG to NAG-25. Any of the above embodiments and further embodiments described herein relating to treating a mixture of TG amine and PEG acid with TBTU to obtain TG PEG may be useful for preparing NAG-25.

[0023] Disclosed herein is a method for preparing NAG-25 by high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to give TG amine: [ka] or forming a salt thereof; and converting TG amine or a salt thereof to NAG-25. Any of the above embodiments and further embodiments described herein relating to forming TG amine by high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ will be useful for preparing NAG-25.

[0024] Disclosed herein is a method for preparing NAG-25, comprising reacting a triacylglycerol (III) derivative with a triacylglycerol (III) derivative in the presence of a coupling reagent and a base. [ka] with NAG-H or a salt thereof to form TGZ, and converting TGZ to NAG-25. In some embodiments, TGZ is further precipitated from the solvent / anti-solvent system. Any of the above embodiments and further embodiments described herein relating to reacting a triacid with NAG-H or a salt thereof may be useful for preparing NAG-25.

[0025] Disclosed herein is a method of preparing NAG-25, comprising reacting a t-butyl core with an acid to form a triacid and converting the triacid to NAG-25. In some embodiments, the triacid is crystalline. In some embodiments, the triacid is in crystalline form I. Any of the foregoing embodiments and additional embodiments described herein relating to reacting a t-butyl core with an acid to form a triacid may be useful for preparing NAG-25.

[0026] Disclosed herein is a method of preparing NAG-25, comprising reacting GluZ with GluOtBu or a salt thereof to form a t-butyl core, and converting the t-butyl core to NAG-25. Any of the above embodiments and further embodiments described herein relating to a method comprising reacting GluZ with GluOtBu or a salt thereof to form a t-butyl core may be useful for preparing NAG-25.

[0027] Disclosed herein is a method for preparing NAG-25, the method comprising precipitation of TGZ from a solvent / anti-solvent system. Any of the above-mentioned embodiments and further embodiments described herein relating to precipitation of TGZ from a solvent / anti-solvent system may be useful for preparing NAG-25.

[0028] Disclosed herein are methods of preparing NAG-25, comprising a triacid intermediate that is crystalline. In some embodiments, the crystalline triacid is crystalline form I of the triacid characterized by an X-ray powder diffractogram having at least signals at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2. In some embodiments, the crystalline triacid is crystalline form I of the triacid characterized by an X-ray powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, and 21.0±0.2. In some embodiments, the crystalline triacid is crystalline form I of the triacid characterized by an X-ray powder diffractogram substantially similar to that of FIG. 2. In some embodiments, the crystalline triacid is crystalline Form II of the triacid, characterized by an X-ray powder diffractogram substantially similar to that of Figure 3. Any of the foregoing embodiments of the crystalline triacid and additional embodiments described herein may be useful in preparing NAG-25.

[0029] Disclosed herein is a method for preparing a t-butyl core, comprising reacting GluZ with GluOtBu or a salt thereof to produce a t-butyl core, the reacting being carried out in the presence of a coupling reagent, a base, and a solvent; optionally, a) the coupling reagent is EDC / Oxyma, TFFH, PyOxim, CDI, PivCl, T3P, or COMU; or b) the base is N-methylmorpholine (NMM); and / or c) the solvent is IPAc, MeTHF, MIBK, or MTBE.

[0030] Disclosed herein is a method for preparing a triacid, comprising reacting a t-butyl core with an acid, where the acid is phosphoric acid (HPO), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

[0031] Disclosed herein is a method for preparing NAG-Z, comprising reacting an acyl GalNAc with an alcohol-Z to produce NAG-Z, wherein the reaction is carried out in the presence of an acid.

[0032] Disclosed herein is a method for preparing NAG-H or a salt thereof, comprising hydrogenation of NAG-Z in the presence of a Pd / C catalyst and a solvent, optionally wherein the solvent is dimethylacetamide (DMAc) or DCM.

[0033] Disclosed herein is a method for preparing TGZ, comprising reacting a triacid with NAG-H or a salt thereof in the presence of a coupling reagent and a base, and precipitating TGZ from a solvent / anti-solvent system.

[0034] Disclosed herein is a process for preparing a TG amine or a salt thereof, comprising high pressure hydrogenolysis of TGZ to form a TG amine or a salt thereof, wherein the hydrogenolysis is carried out in the presence of an acid.

[0035] Disclosed herein is a method for preparing TG PEG, comprising reacting a solution of a TG amine or salt thereof, and a PEG acid with a coupling reagent, wherein the coupling reagent is added to the solution of the PEG acid and the TG amine or salt thereof over a period of about 30 minutes to about 1.5 hours.

[0036] Disclosed herein is a method for preparing NAG-25, comprising reacting TG PEG with an activating agent and a phosphitylation reagent, where the activating agent is tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), or benzothiotetrazole (BTT); and / or the phosphitylation reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, or 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.

[0037] Further aspects and advantages will be apparent to those of ordinary skill in the art upon review of the following detailed description in conjunction with the drawings, in which: The following description herein includes specific embodiments, with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein. [Brief description of the drawings]

[0038] [Figure 1] 1 shows crystals of the triacid Form I formed during acetone / toluene crystallization. [Diagram 2] 1 shows the XRPD pattern of the triacid crystalline form I after toluene / acetone crystallization. [Diagram 3] 1 shows the XRPD pattern of the triacid crystalline form II. [Figure 4] Figure 5 shows the results of step 5b TG PEG conversion between three solvents (DCM, DMF, MeCN) under various conditions. [Diagram 5]The following enzyme screening results are shown: (A) HPLC results from selected reactions in MeCN; (B) HPLC results from selected reactions in THF; (C) HPLC results from selected reactions under solvent-free conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] definition As used herein, the terms "including" and "comprising," as well as variations such as "include," "includes," "comprise," or "comprises," are used in an open-ended manner and, as such, should be interpreted to mean "including, but not limited to." These terms will also be understood to mean the inclusion of the specified integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps.

[0040] The use of the terms "a," "an," "the," and similar referents in the context of this disclosure (especially in the context of the claims) should be construed to encompass both the singular and the plural, unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. Any examples provided herein, or the use of exemplary language (e.g., "etc.") are intended to better illustrate the disclosure herein, and are not a limitation on the scope of the disclosure herein, unless otherwise indicated. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure herein.

[0041] As used herein, the terms "crystal form", "crystalline form" and "Form" refer synonymously to a crystalline structure (or polymorph) having a particular molecular packing arrangement within a crystal lattice. Crystal forms can be identified and distinguished from one another by one or more characterization techniques (e.g., X-ray powder diffraction (XRPD), single crystal X-ray diffraction, differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), and / or thermogravimetric analysis (TGA)). Thus, as used herein, the terms "Form I" and "Form II" refer to unique crystalline forms that can be identified and distinguished from one another by one or more characterization techniques (e.g., X-ray powder diffraction (XRPD), single crystal X-ray diffraction, differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), and / or thermogravimetric analysis (TGA)). In some embodiments, the novel crystalline forms are characterized by an X-ray powder diffractogram having one or more signals at one or more particular degree two theta values ​​(°2θ).

[0042] As used herein, the term "XRPD" refers to the analytical characterization method of X-ray powder diffraction. XRPD patterns may be recorded at ambient conditions using a diffractometer in transmission or reflection geometry.

[0043] As used herein, the terms "X-ray powder diffractogram", "X-ray powder diffraction pattern", "XRPD pattern" refer interchangeably to a pattern that plots signal position (on the abscissa) versus signal intensity (on the ordinate). In the case of an amorphous material, the X-ray powder diffractogram may include one or more broad signals. In the case of a crystalline material, the X-ray powder diffractogram may include one or more signals, each identified by an angle value as measured in degrees two-theta (°2θ), plotted on the abscissa of the X-ray powder diffractogram, and may be represented as "a signal at... degrees 2-theta", "a signal at a 2-theta value of...", and / or "at least a signal at a... 2-theta value selected from...". The term "X-ray powder diffractogram having a signal at... 2-theta value" as used herein refers to an XRPD pattern that includes a signal or peak at a specified position (°2θ).

[0044] A "signal" or "peak" as used herein refers to a point in an XRPD pattern where the intensity, as measured in counts, is a local maximum. Those skilled in the art will recognize that one or more signals (or peaks) in an XRPD pattern may overlap and may not be apparent, for example, to the naked eye. Indeed, those skilled in the art will recognize that several art-recognized methods, such as the Rietveld method, are capable of and suitable for determining whether a signal is present in a pattern.

[0045] The reproducibility of the angle values ​​is within ±0.2 degrees 2θ, i.e. the angle value can be the recited angle value +0.2 degrees 2 theta, angle value -0.2 degrees 2 theta, or any value between these two end points (angle value +0.2 degrees 2 theta and angle value -0.2 degrees 2 theta).

[0046] The terms "signal intensity" and "peak intensity" refer interchangeably to relative signal intensities within a given X-ray powder diffractogram. Factors that can affect relative signal intensity or peak intensity include sample thickness and preferred orientation (e.g., crystalline grains are not randomly distributed).

[0047] As used herein, an X-ray powder diffractogram is "substantially similar to that of a [particular] figure" if at least 90% (e.g., at least 95%, at least 98%, or at least 99%) of the signals in the two diffractograms appear at overlapping positions in degrees 2-theta. In determining "substantial similarity," variations in intensity and / or signal positions in XRPD diffractograms may occur even for identical crystalline forms. Thus, signal maxima in XRPD diffractograms (expressed herein in degrees 2-theta (°2θ)) generally refer to the reported value ±0.2 degrees 2θ of the reported value (a variation recognized in the art).

[0048] As used herein, a crystalline form of a compound is "substantially pure" if it comprises 90% or more by weight of the sum of all solid forms of the compound in a sample as determined by methods according to the art, such as quantitative XRPD. In some embodiments, a solid form is "substantially pure" if it comprises 95% or more by weight of the sum of all solid forms of the compound in a sample. In some embodiments, a solid form is "substantially pure" if it comprises 99% or more by weight of the sum of all solid forms of the compound in a sample.

[0049] As used herein, the term "solvent" refers to any liquid in which a compound is at least partially soluble (eg, solubility of product greater than 1 g / L).

[0050] As used herein, the term "anti-solvent" refers to any liquid in which a compound is insoluble or at most slightly soluble (e.g., solubility of product less than 1 mg / mL, less than 2 mg / mL, less than 3 mg / mL, or less than 0.01 mol / L).

[0051] As used herein, the term "overall yield" refers to the cumulative yield of a combination of steps. For example, the yields of each step are multiplied together to get the overall yield of steps 1, 2, and 3.

[0052] As used herein, the Environmental Impact Factor ("E-Factor") is a measure of green chemistry that evaluates the total waste for a product. A high E-Factor indicates more waste generated and a greater negative impact on the environment.

[0053] The terms "about" and "approximately," when used in connection with an amount or range, include the value of the amount or range and include an amount or range that would be recognized by one of ordinary skill in the art to have an effect equivalent to that obtained from the amount or range. In some embodiments, the term "about" modifies the specified number by +10% or -10%. In some embodiments, the term "about" modifies the specified number by +5% or -5%. In some embodiments, the term "about" modifies the specified number by +2% or -2%. In some embodiments, the term "about" modifies the specified number by +1% or -1%.

[0054] As used herein, when "about" is followed by a series of numbers, it is understood that "about" applies to all the numbers that follow. Similarly, when a unit (e.g., %, time, equivalents) is listed at the end of a series of numbers or a range of numbers, the unit applies to all the series of numbers or ranges of numbers that precede the unit. For example, "about 3, 4, or 5%" is the same as "about 3%, about 4%, or about 5%", "about 5 to 10 equivalents" is the same as about 5 equivalents to about 10 equivalents", "about 3 to 10 hours, 3 to 6 hours, 4 to 9 hours" is the same as "about 3 to 10 hours, about 3 to 6 hours, about 4 to 9 hours", or "about 50 to 70 minutes or 60 minutes" is the same as "about 50 to 70 minutes or about 60 minutes".

[0055] As used herein, an "oligomeric" compound refers to a compound that includes at least one oligonucleotide containing about 10-100 nucleotides. In some embodiments, the oligomeric compound has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell. In some embodiments, the oligomeric compound, when delivered to a cell expressing the gene, is capable of inhibiting expression of the endogenous gene, and is referred to herein as an "expression-inhibiting oligomeric compound." This gene expression may be inhibited in vitro or in vivo. "Oligomeric compounds" include, but are not limited to, oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates.

[0056] As used herein, the term "oligonucleotide" means a polymer of linked nucleosides, each of which can be independently modified or unmodified.

[0057] As used herein, the term "oligonucleotide" means a polymer of linked nucleosides, each of which can be independently modified or unmodified.

[0058] As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomeric compound having a sequence at least partially complementary to a target mRNA, capable of hybridizing to the target mRNA via hydrogen bonding or Watson-Crick base pairing under physiological conditions in a mammal (or equivalent conditions in vitro). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0059] As used herein, "RNAi construct" refers to an agent that comprises an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specifically degrading or inhibiting translation of a messenger RNA (mRNA) transcript of a target gene. As used herein, an RNAi construct may operate via an RNA interference mechanism (i.e., inducing RNA interference via interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism or pathway. RNAi constructs include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), and dicer substrates. RNAi constructs suitable for conjugation to the targeting ligands (e.g., NAG-25) described herein are composed of oligonucleotides having a strand that is at least partially complementary to the mRNA to be targeted. In some embodiments, the RNAi construct is double-stranded and is composed of an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi construct may be composed of modified nucleotides and / or one or more non-phosphodiester linkages. In some embodiments, the RNAi construct suitable for conjugation to the targeting ligand (e.g., NAG-25) described herein is single-stranded.

[0060] As used herein, "LCAP," which stands for "liquid chromatography area percentage," means the ratio of the peak area of ​​the compound of interest to the total area of ​​the peak.

[0061] As used herein, the "salt" of the compounds described herein can be prepared, for example, by reacting the free base form of the compound with a suitable organic or inorganic acid and isolating the salt thus formed, if necessary.Non-limiting examples of suitable salts of any one or more of the compounds described herein include: hydrobromide, hydrochloride, sulfate, bisulfate, sulfonate, camphorsulfonate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, amino acid salts, and the like.

[0062] The following description relates to various exemplary embodiments of the method of the present disclosure. However, the disclosed embodiments should not be interpreted or used as limiting the scope of the present disclosure, including the claims. In addition, those skilled in the art will understand that the following description has broad application, and the description of any embodiment is meant only as an example of that embodiment, and the scope of the present disclosure, including the claims, is not limited to that embodiment.

[0063] Methods for preparing NAG-25 and its intermediates Provided herein are methods for preparing carbohydrate targeting moieties, NAG-25, and the following intermediates of NAG-25: t-butyl core, triacid, alcohol-Z, NAG-Z, NAG-H, triol, methyl core, TGZ, TG amine, TG PEG, and salts of any of the foregoing. The methods described herein produce NAG-25 and the aforementioned intermediates with low levels of impurity by-products, a reduced number of unit operations, and / or streamlined work-up / isolation used in multi-step NAG-25 processes. Also provided herein are methods for isolating and / or crystallizing certain intermediates, such as the triacid.

[0064] The processes, intermediates, and products described herein have the following structure: [ka] The present invention is useful for preparing therapeutic compounds such as siRNA molecules conjugated to NAG-25, a carbohydrate targeting moiety having the formula:

[0065] As further described herein, NAG-25 can be prepared starting from any one of the steps described herein. As further described, the compound 1-tert-butyl N-carbobenzoxy-L-glutamate (GluZ) and L-glutamic acid di-tert-butyl ester (GluOtBu) or a salt thereof (e.g., GluOtBu hydrochloride) can be conjugated to obtain a t-butyl core (step 1), followed by tri-ester deprotection to generate a triacid (step 2). The triacid can then be conjugated with three units of NAG-H or a salt thereof (e.g., NAG-H TFA) to generate TGZ (step 4b). NAG-H TFA can be synthesized by reductive deprotection of the Cbz group from NAG-Z (step 4a). NAG-Z can be synthesized by direct glycosylation of alcohol-Z with acyl GalNAc (step 3b). Optionally, alcohol Z can be prepared from amino protection of an amino alcohol as described herein (step 3a). TG amine or its salt can be obtained by reductive deprotection of the Cbz group from TGZ (step 5a). TG amine or its salt can be conjugated with PEG acid to obtain TG PEG (step 5b). Finally, NAG-25 can be synthesized by phosphoramidite formation from TG PEG (step 6).

[0066] As further described herein, NAG-25 may be produced using an alternative method for producing TGZ. This alternative method utilizes a variety of protecting group strategies. This alternative method includes amide coupling (step A-1), aminolysis (step A-2), and glycosylation (step A-3). In this alternative method, TGZ is prepared using a triol intermediate as further described herein. After TGZ isolation using the alternative method as described herein, TGZ may be converted to NAG-25 using steps 4, 5, and / or 6 as described herein.

[0067] The present NAG-25 process includes one or more steps in which one or more impurities and / or by-products described herein are reduced, absent, or substantially absent.

[0068] Described herein are compositions comprising NAG-25 or one of its intermediates as further described herein, which have reduced levels of one or more impurities. The disclosed methods provide NAG-25 with high chemical purity. In various embodiments, the chemical purity of NAG-25 prepared according to the disclosed methods is 90% or greater as determined by liquid chromatography. For example, in various embodiments, the chemical purity of NAG-25 is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% as determined by liquid chromatography.

[0069] In contrast to the previous method described in U.S. Pat. No. 10,246,709, the method disclosed herein includes improvements at each step (e.g., the option to avoid column chromatography at the end of step 4b (where TGZ is produced), which can be expensive, wasteful and very time-consuming in large-scale synthesis) and a new crystalline form of the triacid isolated at the end of step 2, which helps to eliminate or remove impurities in a more controlled manner.

[0070] Method for preparing NAG-25 Disclosed herein is a method for preparing NAG-25, the method comprising the processes of steps 1, 2, and / or 4b. The combination of processes 1, 2, and 3 described herein improves the overall yield (e.g., from about 47% to about 67%) and / or reduces the environmental impact factor (E factor) (e.g., from about 1000 to about 540).

[0071] Disclosed herein is a method for preparing NAG-25, the method including a process in step 1 that achieves near complete reaction conversion, optionally without the need for additional amounts (or additional charges) of one or more reagents or starting materials (also known as a kicker charge).

[0072] Disclosed herein is a method for preparing NAG-25, comprising a process in step 2, which results in a novel crystalline triacid (eg, triacid crystalline Form I).

[0073] Disclosed herein is a method for preparing NAG-25, comprising a process step 4b in which TGZ is isolated and purified using precipitation from a solvent / anti-solvent system without the use of column chromatography.

[0074] Disclosed herein is a method for preparing NAG-25, comprising a process of step 3b, which results in NAG-Z, one of the starting materials for NAG-25. The process of step 3b may be carried out without isolation of the oxazoline intermediate, resulting in improved yields with reduced impurities. Further disclosed herein is a process of step 3a, which results in alcohol-Z, used as a starting material for NAG-Z, with a more streamlined process (fewer unit operations), improved yields, and / or reduced impurities.

[0075] Disclosed herein is a method for preparing NAG-25, comprising steps A-1, A-2, and / or A-3 for producing TGZ and its alternate intermediates (methyl core and triol) as further described herein. Also disclosed is a triol intermediate (benzyl((10S,15S)-1,22-dihydroxy-10-((2-(2-hydroxyethoxy)ethyl)carbamoyl)-7,12,16-trioxo-3,20-dioxa-6,11,17-triazadocosane-15-yl)carbamate) useful for preparing TGZ. Provided herein is a composition comprising the triol intermediate and an unprotected triol. Disclosed herein is a method for preparing NAG-25, comprising a triol intermediate.

[0076] Disclosed herein are methods for preparing NAG-25, comprising the processes of steps 5a, 5b, and / or 6. The combination of the processes of steps 4, 5, and 6 described herein streamlines unit operations, reduces solvent volume, and / or improves purity. The improvements described herein include improved isolation points, and improved physical properties of intermediates, and reduction or elimination of reaction impurities. The improved processes of steps 4-6 described herein reduce unit operations and solvent volume.

[0077] Disclosed herein is a method of preparing NAG-25, comprising a process step 5b in which a coupling reagent (e.g., TBTU) is added over a period of about 1 to 1.5 hours.

[0078] Disclosed herein is a method of preparing NAG-25, comprising step 1, step 2, step 4a, step 3b, step 3a, step 4b, step 5a, step 5b, step 6, and / or any one of these embodiments, as further described herein.

[0079] Disclosed herein is a method of preparing NAG-25, the method including step A-1, step A-2, step A-3, step 5a, step 5b, step 6, and / or any one of their embodiments, as further described herein.

[0080] Overall, the improved method of preparing NAG-25 disclosed herein includes improved steps 1-6, which improve the overall NAG-25 yield (e.g., an increase of about 6%) and reduce the E-factor (e.g., a reduction in the E-factor of about 24%). The process improvements described herein allow for the production of NAG-25 on a larger scale and more efficiently.

[0081] Step 1: t-Butyl core Disclosed herein is a method for preparing a t-butyl core (Step 1 process), which involves conjugating 1-tert-butyl N-carbobenzoxy-L-glutamate (GluZ) and L-glutamic acid di-tert-butyl ester (GluOtBu) to obtain a t-butyl core. [ka]

[0082] Challenges with the past Step 1 process included: reaction stalls that required additional reagent charges (kicker charges) to drive full conversion; and crystallization from hexane, which can be environmentally unfriendly. To address these challenges, experiments were conducted to explore: alternative coupling reagents that would achieve near full conversion without the use of the kicker charge used in the past process; modes of reagent addition to enhance conversion to the t-butyl core; and ways to streamline the Step 1 process and the crystallization of the t-butyl core (e.g., by eliminating the solvent exchange used in the past Step 1 process).

[0083] Disclosed herein is a step 1 process for producing t-butyl core that utilizes a single charge of reagents while providing higher reaction conversion and / or higher isolated purity. In this improved step 1 process, various coupling reagents, solvents, addition orders, and reagent amounts are used, resulting in improved reaction conversion to t-butyl core and improved purity of t-butyl core. Step 1 also includes reverse addition of reagents that is believed (without being bound by theory) to favor the formation of mixed anhydride intermediates over symmetric anhydride intermediates. Additionally, crystallization of the t-butyl core product has been made more streamlined by changing the reaction solvent (e.g., from THF to MTBE), eliminating a solvent exchange step prior to crystallization, and reducing the amount of solvent for crystallization.

[0084] In one embodiment of step 1, a t-butyl core: [ka] The method for preparing GluZ: [ka] And, GluOtBu: [ka] or a salt thereof to produce a t-butyl core. In some embodiments, GluOtBu or a salt thereof is used in excess. In some embodiments, GluOtBu or a salt thereof is a GluOtBu salt. In some embodiments, GluOtBu salt is a hydrochloride salt. In some embodiments of step 1, the process includes the use of a coupling reagent, a base, and a solvent.

[0085] In some embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / ethyl cyanohydroxyiminoacetate (Oxyma) (EDC / Oxyma), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), [ethylcyano(hydroxyimino)acetato-O 2 ]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), 1,1'-carbonyldiimidazole (CDI), trimethylacetyl chloride (PivCl), 1-propanephosphonic anhydride solution (T3P), or (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU). In some embodiments, the coupling reagent is T3P or PivCl. In some embodiments, the coupling reagent is PivCl. In some embodiments, PivCl is added in excess. In some embodiments, the base is N-methylmorpholine (NMM). In some embodiments, the coupling reagent is T3P or PivCl and the base is NMM. In some embodiments, the coupling reagent is PivCl and the base is NMM.

[0086] In some embodiments, the solvent is isopropyl acetate (IPAc), 2-methyltetrahydrofuran (MeTHF), methyl isobutyl ketone (MIBK), or tert-butyl methyl ether (also known as methyl tert-butyl ether or MTBE). In some embodiments, the solvent is MeTHF or MTBE. In some embodiments, the solvent is MTBE. In some embodiments, an anti-solvent is used to induce crystallization of the product (t-butyl core). In some embodiments, the anti-solvent is heptane. In some embodiments, the anti-solvent is a 1:2 ratio of IPA:water. In some embodiments, the solvent is MTBE and the anti-solvent is heptane.

[0087] The process of step 1 may use either normal addition or inverse addition of GluZ to the coupling reagent. In normal addition, a solution of the coupling reagent in a solvent is added to a solution of GluZ, base, and solvent. In some embodiments, the process of step 1 includes normal addition, where a solution of the coupling reagent in a solvent is added to a solution of GluZ and base in a solvent. Inverse addition, where a solution of GluZ, base, and solvent is added to a solution of the coupling reagent in a solvent. Without being bound by theory, it is believed that the inverse addition forms a mixed anhydride, which helps reduce the formation of GluZ symmetric anhydrides. In some embodiments, the process of step 1 includes inverse addition, where a solution of GluZ and base in a solvent is added to another solution of the coupling reagent in a solvent.

[0088] A solution of GluZ and base in a solvent is prepared and added to a solution of PivCl in the solvent. In some embodiments, the solvent is MTBE, and a solution of GluZ and NMM in MTBE is added to a solution of MTBE and PivCl. The addition of GluZ and NMM is carried out over a period of at least about 50 minutes, 50-70 minutes, or 60 minutes. After stirring for about an additional 20-40 minutes, GluOtBu is added in small portions about every 10-20 minutes, and the reaction is allowed to proceed at 0° C. for about 20-40 minutes (e.g., 30 minutes). The reaction is monitored (e.g., by LC) until adequate conversion is achieved.

[0089] In some embodiments, about 1 eq to about 1.5 eq of each of PivCl and GluOtBu or a salt thereof is used, independently. In some embodiments, about 1.1, 1.2, or 1.3 eq of PivCl is used, and about 1.2, 1.3, or 1.4 eq of GluOtBu or a salt thereof is used. In some embodiments, about 1.2 eq of PivCl is used, and about 1.3 eq of GluOtBu or a salt thereof is used. Equivalents of PivCl and GluOtBu or a salt thereof (herein, "eq" or "equiv" are relative to GluZ).

[0090] In some embodiments, the NMM used is about 3.0 to about 3.5 eq. In some embodiments, the NMM used is about 3.3 to about 3.7 eq. In some embodiments, the NMM used is at least about 3.0 eq. In some embodiments, the NMM used is at least about 3.5 eq. In some embodiments, the NMM used is about 3.5 eq.

[0091] In some embodiments, PivCl is about 1.1-1.4 eq, GluOtBu is about 1.1-1.5 eq, and NMM is about 3.3-3.7 eq. In some embodiments, PivCl is about 1.2 eq, GluOtBu is about 1.3 eq, and NMM is about 3.5 eq. These equivalents are relative to GluZ.

[0092] In some embodiments, the step 1 process for preparing the t-butyl core comprises reacting GluZ with GluOtBu or a salt thereof in the presence of PivCl, NMM, and MTBE to produce a t-butyl core. In some embodiments, the step 1 process for preparing the t-butyl core comprises reacting GluZ with GluOtBu or a salt thereof in the presence of PivCl (about 1.1-1.4 eq), NMM (at least 3.0 eq), and MTBE (about 8-12 volumes) to produce a t-butyl core.

[0093] In some embodiments, the process of step 1 comprises: a) combining a first solution of GluZ, a base, and a solvent with a second solution of a coupling reagent and a solvent to form a third solution of GluZ, a base, a solvent, and a coupling reagent; and b) adding GluOtBu or a salt thereof to the third solution of GluZ, base, solvent, and coupling reagent; Includes.

[0094] In some embodiments, the process of step 1 comprises: a) combining a first solution of GluZ, NMM, and MTBE with a second solution of PivCl and MTBE to form a third solution of GluZ, NMM, MTBE, PivCl; and b) adding GluOtBu or a salt thereof to the third solution of GluZ, NMM, MTBE, and PivCl; Includes.

[0095] In some embodiments, the GluOtBu or a salt thereof is GluOtBu hydrochloride.

[0096] The improved step 1 process described herein reduces impurities such as Piv-Glu-OH and the tripeptide impurity shown below. [ka]

[0097] In some embodiments, step 1 produces t-butyl core in greater than about 85% or about 90% yield. In some embodiments, step 1 produces t-butyl core in greater than about 91, 92, 93, 94, 95, 96, or 97% yield.

[0098] In some embodiments, step 1 produces a t-butyl core with a purity of greater than about 95% as measured by liquid chromatography (LC). In some embodiments, the purity of the t-butyl core is greater than about 96, 97, 98, or 99% as measured by LC.

[0099] In some embodiments, the step 1 process for preparing the t-butyl core can be represented by the following scheme: [ka] In this scheme, GluZ is reacted with GluOtBu HCl in the presence of PivCl, base, and MTBE. In some embodiments of the above scheme, PivCl is about 1.2 eq, NMM is about 3.5 eq, and MTBE is about 10 volumes. In some embodiments of the above scheme, step 1 is carried out at about 0° C. In some embodiments, the reaction involves the inverse addition of a solution of GluZ and NMM in MTBE to a solution of PivCl in MTBE.

[0100] In some embodiments, one, two, or three of the t-butyl groups of the t-butyl core, GluZ, and GluOtBu can be replaced with one, two, or three other types of alkyl groups (e.g., methyl, ethyl, propyl, and / or butyl).

[0101] In some embodiments of step 1, the process further comprises work-up (eg, extractive washes), distillation, crystallization, filtration, rinsing, and / or drying.

[0102] In some embodiments, the extractive work-up step includes adding a hydrochloric acid solution (e.g., about 0.3-0.7M or 0.5M HCl) followed by stirring followed by phase separation, adding a sodium carbonate solution (e.g., 0.3-0.7M or 0.5M sodium carbonate) followed by stirring followed by phase separation, and adding a brine solution (e.g., a 4-6 wt% or 5 wt% brine solution) followed by stirring followed by phase separation. The organic layer can be carried forward to crystallization or can be held at 20° C. overnight if desired.

[0103] Further provided herein is a method of crystallizing t-butyl cores, comprising adding an anti-solvent to a solution of t-butyl cores in a solvent. In some embodiments, t-butyl core seeds are also added (e.g., before, after, or together with the anti-solvent). In some embodiments, the anti-solvent is added over a period of at least about 2, 3, 4, or 5 hours, or over a period of about 2-5, 2-4, 3-4, or 2.5-3.5 hours, or over a period of about 3, 3.5, or 4 hours. In some embodiments, the anti-solvent is added until a final amount of about 30, 40, 50, 60, 70, 80% anti-solvent is reached. In some embodiments, the anti-solvent is added to a solution of t-butyl cores in a solvent that is held at about 30-40, 35-45, or 33-37°C. In some embodiments, after adding the anti-solvent, the solution temperature is lowered to about 15-25, 18-22, or 20° C. In some of the above embodiments, the solvent is MTBE and the anti-solvent is heptane.

[0104] Step 2: Triacid or its salt Disclosed herein is a method for preparing a triacid or a salt thereof (Process Step 2) that involves triester deprotection of the t-butyl core to generate the triacid or a salt thereof. [ka]

[0105] Previously, in step 2 of the process, formic acid was used to deprotect the three tert-butyl groups of the t-butyl core. After completion of this reaction, the formic acid was removed by a series of solvent exchange / distillation procedures and the triacid was isolated as an oil.

[0106] To improve upon the previous Step 2 process, experiments were conducted to investigate the following: isolation of solid triacid that allows for improved purity; more efficient work-up procedures (e.g., extractive washes); and development of reaction conditions that increase the purity of the triacid or its salt, such as the use of alternative acids in the reaction.

[0107] Disclosed herein is an improved step 2 process that provides high purity triacid or its salt. Examples of triacid salts include phosphate, sodium, and calcium salts. In one aspect of step 2, the method of preparing triacid or its salt includes reacting a t-butyl core with an acid as a deprotection reagent. The reaction can be carried out in the presence of a solvent or in the presence of a solvent mixed with water. When present with a solvent, water acts as a tert-butyl scavenger, efficiently producing the triacid from the tert-butyl core in high yield.

[0108] In some embodiments, the acid is phosphoric acid (H 3 PO 4 ), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid. In some embodiments, the acid is phosphoric acid (H 3 PO 4 ), TFA, or HCl. In some embodiments, the acid is phosphoric acid (H 3 PO 4 ). In some embodiments, the reaction of step 2 proceeds in a solvent selected from 2-MeTHF, acetonitrile, THF, DMA, sulfolane, and DME. In some embodiments, the solvent is 2-MeTHF, THF, or acetonitrile. In some embodiments, the solvent is 2-MeTHF or THF. In some embodiments, the solvent is 2-MeTHF. In some embodiments, the solvent is mixed with water. In some embodiments, the solvent is 2-MeTHF and mixed with water. In some embodiments, the solvent is about 3.0-4.0 volumes of 2-MeTHF and mixed with about 0.3-1, 0.4-0.6, or 0.5-2 volumes of water. In some embodiments, the solvent is about 3.0-4.0 volumes of 2-MeTHF and mixed with about 0.4-0.6 volumes of water. In some embodiments, the improved step 2 uses phosphoric acid (H) as the acid. 3 PO 4) and 2-MeTHF as the solvent that is mixed with water. In some embodiments, the improved process 2 uses phosphoric acid (H 3 PO 4 ) and 2-MeTHF as the solvent mixed with water, where phosphoric acid is present in about 3 volumes, 2-MeTHF is present in about 3.5 volumes, and water is about 0.5 volumes.

[0109] In some embodiments, the reaction is carried out at a temperature of about 40-60° C. In some embodiments, the temperature is about 45-55° C. In some embodiments, the temperature is about 50° C. In some embodiments, the reaction is allowed to proceed for about 3-10 hours, 3-6 hours, 4-9 hours, 4-7 hours, or 4-5 hours.

[0110] In some embodiments, the process for preparing the triacid or salt thereof in step 2 comprises: a) combining the t-butyl core in a solvent with an acid; b) adding water; and c) heating the reaction to a certain temperature In some embodiments, the solvent is 2-MeTHF and the acid is H 3 PO 4 and this temperature is about 50°C.

[0111] In some embodiments, the step 2 process for preparing the triacid or a salt thereof can be represented by the following scheme: [ka] As shown in.

[0112] In some embodiments, the step 2 process for preparing the triacid or a salt thereof can be represented by the following scheme: [ka] As shown in.

[0113] The reaction is complete when the amount of diacid is less than 3% as determined by LC. In some embodiments of step 2, after the reaction is terminated, the process further comprises work-up (e.g., extractive washes), distillation, crystallization, filtration, and / or drying.

[0114] In some embodiments, the acid (e.g., phosphoric acid) is efficiently removed by aqueous workup, thereby avoiding lengthy formic acid distillation sequences. In some embodiments, step 2 further comprises removing phosphoric acid with at least one organic / aqueous wash. In some embodiments of the aqueous workup, the reaction mixture is diluted with isopropyl acetate and washed with 20 wt% aqueous ammonium sulfate. In some embodiments of the aqueous workup step, the reaction mixture is diluted with 2-methyltetrahydrofuran and / or washed with 20 wt% aqueous ammonium sulfate. The organic layer is then washed with water.

[0115] In some embodiments, this organic / aqueous wash includes isopropyl acetate (iPAc) as the organic layer and ammonium sulfate as the aqueous layer. In some embodiments, about 8-18 volumes of iPAc and about 3-8 volumes of 20 wt% ammonium sulfate are used. In some embodiments, about 9-11 volumes of iPAc and about 4-6 volumes of 20 wt% ammonium sulfate are used. In some embodiments, about 10 volumes of iPAc and about 5 volumes of 20 wt% ammonium sulfate are used.

[0116] In some embodiments, this organic / aqueous wash includes 2-methyltetrahydrofuran (MetHF) as the organic layer and ammonium sulfate as the aqueous layer. In some embodiments, about 6-18 volumes of MeTHF and about 3-20 volumes of 20 wt% ammonium sulfate are used. In some embodiments, about 8-10 volumes of MeTHF and about 12-18 volumes of 20 wt% ammonium sulfate are used. In some embodiments, about 9 volumes of MeTHF and about 15 volumes of 20 wt% ammonium sulfate are used. In some embodiments, the volume amounts recited herein may be split into 2-4 (e.g., 3) separate smaller wash steps. After this wash, an optional water wash (e.g., 1, 2, or 3 volumes) may be used.

[0117] Additionally, in some embodiments, a solvent / anti-solvent crystallization system may be used to produce the final product as a high purity crystalline material. In some embodiments, the solvent / anti-solvent is acetone / toluene, 2-MeTHF / IPAc, 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile. In some embodiments, the solvent is a combination of one or more of any of the listed solvents. In some embodiments, the solvent is a combination of MeTHF and acetone. In some embodiments, the solvent is a combination of MeTHF and acetone, and the anti-solvent is toluene. In some embodiments, the solvent / anti-solvent is acetone / toluene. In some embodiments, the solvent is about 2-4 volumes of MeTHF and 3-5 volumes of acetone, and the toluene is about 13 volumes. In some embodiments, the solvent is about 3 volumes of MeTHF and 4 volumes of acetone, and the toluene is about 13 volumes. In some embodiments, the toluene is present in a ratio of 2:3, 1:1, 3:2, or 1:2. In some embodiments, acetone / toluene is in a ratio of 2:3, 1:1, or 1:2. In some embodiments, the solvent / anti-solvent is acetone / toluene. In some embodiments, acetone / toluene is in a ratio of 2:3, 1:1, 3:2, or 1:2. In some embodiments, acetone / toluene is in a ratio of 2:3, 1:1, or 1:2. In some embodiments, toluene is about 40, 50, 60, 70%, or about 40-70, 40-50, 45-55, 50-60, 55-65, or 60-70% of the total volume of the solvent / anti-solvent system, with the remainder of the volume percentage being one or more solvents. In some embodiments, the one or more solvents are acetone or MeTHF in combination with toluene.

[0118] In some embodiments, the triacid or a salt thereof is isolated as a crystalline solid. In some embodiments, the crystalline triacid is not a salt.

[0119] In some embodiments, the triacid or salt thereof has a purity greater than or equal to about 95%, 96%, 97%, 98%, or 99% as measured by LC. In some embodiments, the triacid or salt thereof has a purity greater than or equal to about 95% as measured by LC. In some embodiments, the method for preparing the triacid or salt thereof from the t-butyl core comprises crystallization of the triacid or salt thereof using a solvent / anti-solvent system that is acetone / toluene, MeTHF / IPAc, MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile. In some embodiments, the method for preparing the triacid or salt thereof from the t-butyl core comprises crystallization of the triacid or salt thereof from acetone / toluene. In some embodiments, the crystallization includes seeding with about 3:2, 1:1, or 5:6 acetone / toluene and further charging toluene until the final acetone / toluene ratio is about 2:3, 1:2, or 1:3. This seeding can be done with triacid seeds (e.g., 1 wt%). In some embodiments, the final volume percentage of toluene in the acetone / toluene mixture is about 40-70% toluene. In some embodiments, the final volume percentage of toluene in the acetone / toluene mixture is about 55-70% toluene. All of the above embodiments are applicable to triacids (i.e., the free triacid, not a salt).

[0120] In some embodiments, any one of the embodiments of step 2 described herein results in a crystalline triacid, characterized by an X-ray powder diffractogram as further described below.

[0121] Disclosed herein are crystalline forms of the triacid. In some embodiments, the process in step 2 produces the triacid crystalline form I. In some embodiments, the process in step 2 produces the triacid crystalline form II.

[0122] In some embodiments, the crystalline solid of the triacid is crystalline Form I. In some embodiments, the triacid crystalline Form I is in a substantially pure form. In some embodiments, the triacid crystalline Form I is characterized by an X-powder diffractogram generated by X-powder diffraction analysis with an incident beam of Cu Kα radiation.

[0123] In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 7.4±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 9.2±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 21.0±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 10.2±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 14.6±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 18.3±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form I is characterized by an X powder diffractogram with a signal at 19.3±0.2 degrees 2-theta. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram with a signal at 22.9±0.2 degrees 2-theta. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram with a signal at 11.0±0.2 degrees 2-theta. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram with a signal at 11.3±0.2 degrees 2-theta.

[0124] In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form I is optionally further characterized by an X powder diffractogram having additional signals at 2-theta values ​​of 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is optionally further characterized by an X powder diffractogram having additional signals at 2-theta values ​​of 11.0±0.2 or 11.3±0.2.

[0125] In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at seven 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at six 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at five 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at four 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at two 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least a signal at one 2-theta value selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.

[0126] In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at seven 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at six 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at five 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at four 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least signals at two 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least a signal at one 2-theta value selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.0±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.

[0127] In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at seven 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at six 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at five 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at four 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least signals at two 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least a signal at one 2-theta value selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 10.2±0.2, 11.3±0.2, 14.6±0.2, 18.3±0.2, 19.3±0.2, and 22.9±0.2.

[0128] In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least signals at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least signals at two 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form I is characterized by an X powder diffractogram having at least signals at two 2-theta values ​​of 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2.

[0129] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffractogram substantially similar to that of FIG.

[0130] In some embodiments, the crystalline solid of the triacid is crystalline Form II. In some embodiments, the triacid crystalline Form II is in a substantially pure form. In some embodiments, the triacid crystalline Form II is characterized by an X-ray powder diffractogram generated by X-ray powder diffraction analysis with an incident beam of Cu Kα radiation.

[0131] In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 7.8±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 9.7±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 15.7±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 13.3±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 17.1±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 18.9±0.2 degrees 2-theta. In some embodiments, the triacid crystalline form II is characterized by an X powder diffractogram with a signal at 20.2±0.2 degrees 2-theta. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having a signal at 21.0±0.2 degrees 2-theta.

[0132] In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.8±0.2, 9.7±0.2, and 15.7±0.2. In some embodiments, the triacid crystalline Form II is optionally further characterized by an X powder diffractogram having additional signals at 2-theta values ​​of 13.3±0.2, 17.1±0.2, and / or 18.9±0.2. In some embodiments, the triacid crystalline Form II is optionally further characterized by an X powder diffractogram having additional signals at 2-theta values ​​of 20.2±0.2, and / or 21.0±0.2.

[0133] In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at seven 2-theta values ​​selected from 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at six 2-theta values ​​selected from: 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at five 2-theta values ​​selected from: 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at four 2-theta values ​​selected from: 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at three 2-theta values ​​selected from: 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at two 2-theta values ​​selected from 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2.In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having at least a signal at one 2-theta value selected from 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2.

[0134] In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at three 2-theta values ​​selected from 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at least at two 2-theta values ​​selected from 7.8±0.2, 9.7±0.2, and 15.7±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram having signals at 2-theta values ​​of 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, and 18.9±0.2. In some embodiments, the triacid crystalline Form II is characterized by an X powder diffractogram with signals at 2-theta values ​​of 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, and 20.2±0.2.

[0135] In some embodiments, the triacid crystalline Form II is characterized by an X-ray powder diffractogram substantially similar to that of FIG.

[0136] Step 3a: Alcohol Z Disclosed herein is a method for preparing alcohol-Z (process step 3a), which comprises reacting benzyl chloroformate with an aminoalcohol to give alcohol-z: [ka] This includes forming

[0137] The reaction is carried out in the presence of a solvent and a base. Non-limiting solvents include polar aprotic solvents, aprotic solvents, and ether solvents. In some embodiments, the solvent is dichloromethane (DCM), dichloroethane, ethyl acetate, or methyl THF. In some embodiments, the solvent is a polar aprotic solvent. In some embodiments, the solvent is DCM or dichloroethane. In some embodiments, the solvent is DCM. In some embodiments, the solvent is ethyl acetate or methyl THF. Non-limiting bases include tertiary amine bases, such as triethylamine (TEA), DIPEA, N-methylmorpholine, 1-methylimidazole, and pyridine. In some embodiments, the base is a tertiary amine base. In some embodiments, the base is triethylamine (TEA), DIPEA, N-methylmorpholine, 1-methylimidazole, and pyridine. In some embodiments, the base is TEA. In some embodiments, the solvent is DCM and the base is TEA. In some embodiments, the amino alcohol is present at about 1.0-1.2 equivalents relative to the benzyl chloroformate. In some embodiments, the amino alcohol is present at about 1.1 equivalents relative to the benzyl chloroformate. After cooling the amino alcohol solution (and solvent and base) to about -5-5°C, CbzCl is added to the amino alcohol solution over a period of time. In some embodiments, CbzCl is added to the amino alcohol over a period of about 50-70 minutes or 60 minutes with the temperature maintained at about 0-12°C, 3-10°C, 8-11°C, 10°C, 5°C, or 0°C, as appropriate. After addition of CbzCl, the reaction temperature is adjusted to about 28-42°C. In some embodiments, the reaction temperature is adjusted to about 30-40°C, 33-37°C, or 35°C. In some embodiments, the reaction temperature is 35°C. In some embodiments, the amino alcohol is present in about 1.1 equivalents relative to the benzyl chloroformate and the reaction temperature is about 20-30°C, 30-40°C, 33-37°C, or 35°C.In some embodiments, the amino alcohol is present at about 1.1 equivalents relative to the benzyl chloroformate and the reaction temperature is about 34-36° C. In some embodiments, the amino alcohol is present at about 1.1 equivalents relative to the benzyl chloroformate and the reaction is maintained at about 35° C.

[0138] In some embodiments, the process of step 3a for preparing alcohol-Z comprises reacting benzyl chloroformate (CbzCl) with an amino alcohol in the presence of DCM as a solvent and TEA as a base. After addition of CbzCl over a period of about 1 hour, the reaction temperature is adjusted from about 0-5 or 3-12° C. to about 35° C. In some embodiments, DCM is present in about 3-5 volumes. In some embodiments, TEA is present in about 0.8-1.2 eq. The reaction is continued for at least 12 hours, for example, about 10-12 hours. In some embodiments, the process of step 3a for preparing alcohol-Z is depicted in the following scheme: [ka] In some embodiments, the temperature during addition of the reagents is about 5° C., and the reaction temperature is allowed to increase to about 35° C.

[0139] After the reaction, the alcohol-Z product can be isolated by cooling to about room temperature (e.g., 20-25° C.), quenching (e.g., with water), extraction with a solvent (e.g., DCM), optional washing (e.g., 10% NaCl), and then distillation. In some embodiments, the yield of alcohol-Z is greater than or equal to about 95, 96, 97, 98, or 99%.

[0140] The process of alcohol Z step 3a described also provides benzyl alcohol, dibenzyl carbonate, benzyl chloroformate, and / or doubly protected alcohol-Z as shown below: [ka] One or more impurities such as are reduced.

[0141] In some embodiments, step 3a produces alcohol-Z with a purity of greater than about 90 or 95% as measured by liquid chromatography (LC). In some embodiments, the purity is greater than about 96, 97, 98, or 99% as measured by LC. In some embodiments, impurities present at the end of the reaction are less than or equal to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% as measured by LC.

[0142] Process 3b:NAG-Z Disclosed herein is a method for preparing NAG-Z (process step 3b), which comprises the direct glycosylation of alcohol-Z with acyl GalNAc (also referred to herein as DGalNAc): [ka] Includes.

[0143] Without being bound by theory, the oxazoline intermediate is formed in situ, eliminating the need for a step of isolating the oxazoline intermediate, thus increasing the yield and reducing the number of unit operations.A method for preparing NAG-Z (chemical name (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate) includes reacting acyl GalNAc (chemical name: (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate) with alcohol-Z (chemical name: benzyl(2-(2-hydroxyethoxy)ethyl)carbamate) to produce NAG-Z.

[0144] Provided herein is a method for crystallizing NAG-Z, the method comprising a solvent and an anti-solvent. In some embodiments, the solvent is acetonitrile and the anti-solvent is water, methyl tert-butyl ether (MTBE), toluene, IPAc, or IPA. The described method also provides DGalOH and / or Ac-Z, as shown below: [ka] In some embodiments, the purity of NAG-Z is greater than about 96, 97, 98, or 99% as measured by LC. In some embodiments, the impurities present at the end of the reaction are less than or equal to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% as measured by LC.

[0145] In some embodiments, the NAG-Z reaction is carried out in the presence of an acid. In some embodiments, the acid is a Lewis acid. In some embodiments, the acid is TMSOTf. In some embodiments, the acid is not TMSOTf. TMSOTf is commonly used in glycosylation reactions, but it has been discovered that under certain conditions, TMSOTf can decompose to the NAG-Z product, thereby reducing the yield of NAG-Z. Non-limiting examples of acids include: trifluoromethanesulfonic acid (trifluoromethanesulfonate (CF 3 SO 3 ) or triflate (Tf), silyl triflates (e.g., TBSOTf or TIPSOTf), metal triflates (e.g., bismuth triflate, indium triflate, copper triflate, scandium triflate, neodymium triflate, dysprosium triflate, ytterbium triflate, samarium triflate, lanthanum triflate, potassium triflate, magnesium triflate, or aluminum triflate), metal chlorides (e.g., FeCl 3 , AlCl3 , InCl 3 , or BiCl 3 ), trifluoroacetic acid (TFA), and BF 3 OEt 2 In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ) 、 Boron trifluoride etherate (BF 3 OEt 2 ), tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), indium trifluoromethanesulfonate (In(OTf) 3 ), or copper trifluoromethanesulfonate (Cu(OTf) 2 In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ) 、 Indium trifluoromethanesulfonate (In(OTf) 3 ), copper trifluoromethanesulfonate (Cu(OTf) 2 ), or tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf). In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ) 、 Indium trifluoromethanesulfonate (In(OTf) 3 ), or copper trifluoromethanesulfonate (Cu(OTf) 2 In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ), or copper trifluoromethanesulfonate (Cu(OTf) 2 In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 In some embodiments, the acid is copper trifluoromethanesulfonate (Cu(OTf) 2 In some embodiments, the acid is indium trifluoromethanesulfonate (In(OTf) 3In some embodiments, the acid is indium trifluoromethanesulfonate (In(OTf) 3 ), or copper trifluoromethanesulfonate (Cu(OTf) 2 ). In some embodiments, the acid is tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf). In some embodiments, the acid is present in about 0.01-0.1, 0.01-0.04, 0.05-0.1, 0.1-0.6, 0.1-0.4, 0.3-0.6, or 0.5-0.7 equivalents relative to alcohol-Z. In some embodiments, the acid is present in about 0.1-0.2 or 0.13-0.17 equivalents relative to alcohol-Z. In some embodiments, the acid is present in about 0.15 equivalents relative to alcohol-Z. In some embodiments, the acid is present in about 0.01-0.04 or 0.02-0.03 equivalents relative to alcohol-Z. In some embodiments, the acid is present in about 0.025 equivalents (or 2.5 mol %) relative to alcohol-Z. In some embodiments, the acid is present at about 1-10, 1-4, or 2-3 mol % relative to alcohol-Z.

[0146] In some embodiments, the reaction is carried out in the presence of a solvent. Non-limiting examples of solvents that may be used include acetonitrile, dichloroethane, dichloromethane, dimethylformamide (DMF), and DMSO. In some embodiments, the solvent is acetonitrile. In some embodiments, the solvent is dichloroethane or dichloromethane. The solvent volumes listed herein are relative to the alcohol-Z. In some embodiments, the solvent is present in a total of about 6-10 or 8-12 volumes. In some embodiments, the solvent is present in a total of 8 volumes. In some embodiments, the solvent is present in a total of 10 volumes. In some embodiments, the acyl GalNAc is added to the reaction mixture as an acyl GalNAc solution that includes acyl GalNAc, acid, and solvent. In some embodiments, the reaction mixture already includes Alc-Z produced at the end of step 3a. In some embodiments, the solvent in the acyl GalNAc solution is about 2-4 or 4-6 volumes. In some embodiments, the solvent in the acylGalNAc solution is about 2-4 or 4-5 volumes, or about 2, 3, 4, or 5 volumes. In some embodiments, alcohol-Z is added to the reaction as an alcohol-Z solution that includes dried (e.g., azeotropic or desiccant-dried) alcohol-Z and a solvent. In some embodiments, the solvent in the alcohol-Z solution is about 5 volumes.

[0147] In some embodiments, the acyl GalNAc is present in about 1.0-2.0 equivalents, 1.3-1.7 equivalents, or 1.4-1.6 equivalents relative to alcohol-Z. In some embodiments, the acyl GalNAc is present in about 1.5 equivalents relative to alcohol-Z.

[0148] After combining the acyl GalNAc, alcohol-Z, acid, and solvent, the reaction temperature is heated to about 55-65, 65-75, or 70-90° C. In some embodiments, the reaction temperature is about 70-90° C. In some embodiments, the reaction temperature is about 75-85° C. In some embodiments, the reaction temperature is about 80° C. In some embodiments, the reaction temperature is about 58-62° C. In some embodiments, the reaction temperature is about 60° C. In some embodiments, the reaction time is about 10-20 hours. In some embodiments, the reaction time is about 12-18, 10-14, or 16-20 hours, or at least about 12, 14, 16, or 18 hours. The reaction is then cooled to about room temperature (e.g., about 20° C. or 15-25° C.), filtered (e.g., polish filtration), and treated with activated charcoal. In some embodiments, the amount of activated carbon added is about 3-7 weight (wt)%, 5-10 wt%, 8-15 wt%, 13-25 wt%, 20-35 wt%, 30-50 wt%, 40-60 wt%, or 50-70 wt%. In some embodiments, the amount of activated carbon added is about 3-7 weight (wt)%, 5-10 wt%, or 8-15 wt%. In some embodiments, the amount of activated carbon added is about 30-50 wt%, 40-60 wt%, or 50-70 wt%. In some embodiments, the amount of activated carbon added is about 40 wt%, 50 wt%, or 60 wt%.

[0149] After charcoal treatment of the NAG-Z solution, the charcoal is filtered and an anti-solvent is added to crystallize the NAG-Z. In some embodiments, the anti-solvent is MTBE, water, toluene, IPAc, or IPA. In some embodiments, the anti-solvent is MTBE, water, or toluene. In some embodiments, the anti-solvent is MTBE. In some embodiments, the anti-solvent is added over a period of about 20-40 minutes or 30 minutes. In some embodiments, the anti-solvent is added over a period of about 50-70 minutes or 60 minutes. Optionally, NAG-Z seeds are added to the solution and optionally aged overnight. In some embodiments, additional anti-solvent is added as needed and crystallization is allowed to proceed for an additional period of about 70-100 minutes or 90 minutes. The resulting wet cake is rinsed with additional solvent and anti-solvent.

[0150] In some embodiments, the process of step 3b for preparing NAG-Z can be represented as follows: [ka] As shown in.

[0151] In some embodiments, the method for preparing NAG-Z is shown in the following scheme: [ka] As shown in.

[0152] Step 4a: NAG-H or a salt thereof Disclosed herein is a method for producing NAG-H or a salt thereof (process step 4a), which comprises hydrogenating NAG-Z in the presence of a Pd / C catalyst and a solvent (e.g., dimethylacetamide) to prepare NAG-H or a salt thereof. [ka]

[0153] In some embodiments, NAG-H or its salt is optionally delivered as a solution to the amide coupling reaction of the process of step 4b described herein. The advantage of this improved process is the avoidance of isolation of NAG-H or its salt as a solid, which is hygroscopic and difficult to handle. In addition, the amount of solvent used in this improved process is reduced from previous processes.

[0154] In some embodiments, NAG-H or a salt thereof is NAG-H trifluoroacetate (TFA) salt, NAG-H oxalate, NAG-H tartrate, or NAG-H citrate. In some embodiments, NAG-H or a salt thereof is NAG-H TFA (also referred to herein as NAG-H TFA salt).

[0155] In some embodiments, the process of step 4a comprises combining NAG-Z with a Pd / C catalyst in a solvent, adding an acid, and pressurizing with hydrogen.

[0156] In some embodiments, the Pd / C catalyst is 10, 9, 8, 7, 6, 5, 4, or 3% palladium on carbon. In some embodiments, the Pd / C catalyst is 5% palladium on carbon.

[0157] In some embodiments, the solvent is IPA, EtOH, DMAc, or DCM. In some embodiments, the solvent is DMAc. In some embodiments, the solvent is DCM. In some embodiments, the solvent volume used is about 3 volumes to about 6 volumes. In some embodiments, the solvent volume used is about 3 volumes to about 5 volumes. In some embodiments, about 3 volumes to about 5 volumes of DMAc are used. In some embodiments, about 4 volumes of DMAc are used. In some embodiments, about 3 volumes to about 6 volumes of DCM are used. In some embodiments, about 3 to 5 or 4 to 6 volumes of DCM are used.

[0158] In some embodiments, the acid is TFA, acetic acid (AcOH), pivalic acid (PivOH). In some embodiments, the acid is TFA. In some embodiments, the acid is present at about 0.8-1.2 eq relative to NAG-Z. In some embodiments, TFA is present at about 0.8-1.2 eq or 1.0 eq relative to NAG-Z.

[0159] In some embodiments, the reaction is pressurized with hydrogen at about 30-60 psig or 40-50 psig. In some embodiments, the reaction is pressurized with hydrogen at about 35, 40, 45, 50, or 55 psig. In some embodiments, the reaction is pressurized with hydrogen at about 45 psig.

[0160] In some embodiments, step 4a is carried out at a temperature of about 20° C. to about 25° C. In some embodiments, the temperature is about 20° C. to about 24° C. In some embodiments, the temperature is about 20° C. to about 23° C. In some embodiments, the temperature is about 20° C. to about 22° C.

[0161] In some embodiments, the reaction is carried out for about 15-25 hours or 18-20 hours.

[0162] Process 4b: TGZ Described herein is a method for preparing TGZ (process step 4b). TGZ is synthesized by conjugating three units of NAG-H to a triacid via simultaneous amide coupling. [ka]

[0163] In past processes of step 4b, tri-GalNAc-Cbz (TGZ) is isolated and purified by a solvent-intensive and low-yielding chromatography step. Disclosed herein is an improved step 4b process that includes chromatography-free isolation and purification of the TGZ intermediate by precipitation from a solvent. Advantages of this improved step 4b process are increased TGZ yield (e.g., greater than about 85%) due to elimination of chromatography, as well as time and money savings, for example, by reducing solvent usage and / or avoiding specialized manufacturing equipment.

[0164] The improved TGZ purification process further provides more efficient amide coupling. The improved step 4b process includes NAG-H coupling to the triacid using a coupling reagent in a given solvent (e.g., TBTU in dimethylacetamide), which allowed for lowering the equivalents of NAG-H used in the reaction (e.g., from about 3.5 to about 3-3.3 or 3.2). The improved step 4b also includes a mild buffer wash that minimizes the formation of impurity by-products (e.g., deacylation by-products). The improved step 4b uses a mild buffer (e.g., sodium phosphate buffer or potassium phosphate buffer) instead of a strong acid / base aqueous wash. Finally, a single precipitation of TGZ from a solvent system (e.g., DCM / MTBE) has been performed, which allowed for more efficient and higher yield (e.g., greater than about 80-90%) isolation of TGZ as an amorphous solid with higher purity (e.g., greater than about 95% purity as measured by LC).

[0165] In some embodiments, the process of step 4b comprises combining the triacid and NAG-H or a salt thereof, optionally in a solvent, adding a base, and adding a coupling reagent. In some embodiments of step 4b, the process further comprises extraction / work-up, distillation, crystallization, precipitation, filtration, and / or drying. In some embodiments, the NAG-H or a salt thereof is NAG-H TFA.

[0166] In some embodiments, the coupling reagent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), chloro-N,N,N',N'-tetramethylformamidinium Hexafluorophosphate (TCFH), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), (3-dimethylamino-propyl)-ethyl-carbodiimide (EDC), ethyl cyano(hydroxyimino)acetate (Oxyma), 1-hydroxybenzotriazole (HOBt), [ethyl cyano(hydroxyimino)acetate-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), propylphosphonic anhydride (T3P), phosphoric acid Bis(2-oxooxazolidinium) chloride (BOPCl), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 7-azabenzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyAOP), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N'N'-tetramethyluronium tetrafluoroborate (TOTU), and N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU). In some embodiments, the coupling reagent is TBTU or HATU. In some embodiments, the coupling reagent is TCFH. In some embodiments, the coupling reagent is HATU.In some embodiments, the coupling reagent is TBTU.

[0167] In some embodiments, the base is triethylamine, diisopropylethylamine (DIPEA), N-methylimidazole (NMI), N-methylmorpholine (NMM), or 2,2,6,6-tetramethylpiperidine (TMP). In some embodiments, the base is DIPEA, NMI, NMM. In some embodiments, the base is NMI.

[0168] In some embodiments, the coupling reagent is TBTU and the base is NMI. In some embodiments, the coupling reagent is TCFH and the base is NMI.

[0169] In some embodiments, the solvent is dichloromethane (DCM), dimethylformamide (DMF), acetonitrile (MeCN), dimethylacetamide (DMAc), or a combination thereof. In some embodiments, the solvent is DMF or DMAc. In some embodiments, the solvent is DCM or DMAc. In some embodiments, the solvent is DCM. In some embodiments, the solvent is DMF. In some embodiments, the solvent is DMAc.

[0170] In some embodiments, the coupling reagent is TBTU and the solvent is DMF. In some embodiments, the coupling reagent is TBTU and the solvent is DMAc. In some embodiments, the coupling reagent is TBTU and the solvent is DCM.

[0171] In some embodiments, the NAG-H or salt thereof is about 3-4 equivalents. In some embodiments, the NAG-H or salt thereof is about 3-3.7 equivalents. In some embodiments, the NAG-H or salt thereof is about 3.1-3.3 equivalents. In some embodiments, the NAG-H or salt thereof is NAG-H TFA. The aforementioned equivalents are relative to the triacid.

[0172] In some embodiments, the base equivalents are about 6-15 eq, 8-12 eq, or 10-12 eq. In some embodiments, the base equivalents are about 10-12 eq. In some embodiments, the base equivalents are about 11.5-12.5 eq. In some embodiments, the base equivalents are about 11-12 eq. In some embodiments, the base equivalents are about 12 eq. In some embodiments of the above-mentioned embodiments of the base, the base is NMI. In some embodiments of the above-mentioned embodiments of the base, the base is 12 equivalents of NMI. The above-mentioned equivalents are relative to the triacid.

[0173] In some embodiments, the step 4b process for preparing TGZ comprises reacting the triacid with 3.2 equivalents of NAG-H or a salt thereof, 3.5 equivalents of TBTU, and 12.0 equivalents of NMI, the aforementioned equivalents being relative to the triacid.

[0174] In some embodiments, the process of step 4b for preparing TGZ can be represented by the following scheme: [ka] As shown in.

[0175] In some embodiments, the process of step 4b described herein produces high purity TGZ with a low percentage of deacylated impurities. In some embodiments, the TGZ has a purity of greater than about 93, 94, 95%. In some embodiments, the resulting TGZ contains less than about 1, 0.5, or 0.2% deacylated impurities. In some embodiments, the resulting TGZ contains more than about 1% deacylated impurities.

[0176] In some embodiments, the process of step 4b further comprises at least 1, 2, or 3 washes with an acid followed by at least 1, 2, or 3 washes with a base. In some embodiments, the acid is HCl and the base is NH 4 In some embodiments, when the coupling reagent is TCFH, the base is NMI, and the solvent is DCM, the HCl wash and the NH 4 Use OH wash.

[0177] In some embodiments, the process of step 4b further comprises at least one, two, or three washes with a buffer. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer has a pH of about 4-8. In some embodiments, the buffer has a pH of about 5-7. In some embodiments, the buffer has a pH of about 6. In some embodiments, at least two or three washes with a phosphate buffer at pH 6 are performed. In some embodiments, three washes with a phosphate buffer at pH 6 are performed. In some embodiments, the buffer is sodium phosphate or potassium phosphate. In some embodiments, the buffer is potassium phosphate. In some embodiments, the buffer is about 20-550 mM potassium phosphate. In some embodiments, the buffer is about 25-500 mM potassium phosphate. In some embodiments, the buffer is about 40-60 mM potassium phosphate. In some embodiments, the buffer is about 50 mM potassium phosphate. In some embodiments, the buffer is about 50 mM potassium phosphate at about pH 5-7. In some embodiments, the buffer is about 50 mM potassium phosphate at about pH 6.

[0178] In some embodiments, the process of step 4b comprises azeotropic distillation of the organic phase with a solvent, hi some embodiments, the solvent for distillation is DCM.

[0179] In some embodiments, the process of step 4b further comprises precipitating TGZ from the solvent using an anti-solvent. In some embodiments, the solvent is DCM and the anti-solvent is an ether solvent. In some embodiments, the ether solvent is dimethoxyethane (DME), 2-methyltetrahydrofuran (MeTHF or 2-MeTHF), or methyl tert-butyl ether (MTBE). In some embodiments, the solvent is DCM and the anti-solvent is MTBE. In some embodiments, the solvent is DCM and the anti-solvent is isopropyl acetate (IPAc) or ethyl acetate (EtOAc). In some embodiments, the solvent is DCM and the anti-solvent is DME. In some embodiments, the solvent is DMSO or dimethylacetamide (DMAc) and the anti-solvent is 2-MeTHF. In some embodiments, the solvent is DMF and the anti-solvent is isopropyl acetate (IPAc). In some embodiments, a final solvent ratio of about 1:1 ratio of solvent:anti-solvent is used to precipitate TGZ. In some embodiments, a final solvent ratio of about 1:1 of DCM:MTBE is used to precipitate TGZ, hi some embodiments, the addition of the anti-solvent (e.g., MTBE) was carried out over about 1.5-2.5, 2-3, 2.5-3.5, or 3-4 hours, or over about 2, 3, or 4 hours.

[0180] In some embodiments, TGZ is obtained in a yield of at least about 88%, 89%, 90%. In some embodiments, TGZ is obtained in a purity of at least about 95% as measured by LC (95 LCAP). In some embodiments, TGZ is obtained in a purity of at least about 96%, 97%, 98% as measured by LC.

[0181] In some embodiments, the process for preparing TGZ, step 4b, comprises triacid (1.0 eq), NAG-H TFA (3.5 eq), DMAc, NMI (12 eq), TBTU (4.0 eq). In some embodiments, the process for step 4b further comprises washing with 50 mM pH 6 phosphate buffer and washing with DCM. In some embodiments, step 4b further comprises precipitating TGZ by addition of MTBE to a solution comprising DCM.

[0182] The improved step 4b process reduces, minimizes or eliminates the amount of by-products of this process, including monoNAG, diNAG, diNAG-AZLACTONE, des-Acyl (also called des-acetate), diNAG-OH, diNAG-OAc, NAG-H guanidine, and NAG-H trifluoroacetamide, each of which is shown below. [ka]

[0183] In some embodiments, the process of step 4b results in TGZ having less than about 5%, 3%, 2%, 1%, or 0.5% monoNAG, diNAG, and / or diNAG-azlactone as measured by LC. In some embodiments, the process of step 4b results in TGZ having less than about 2% NAG-H guanidine, less than about 2% diNAG-OH, less than about 1% diNAG, less than about 1% deacylation, and less than about 1% diNAG-OAc. Disclosed are compositions comprising TGZ in which any of the described impurities are less than or equal to 5% (e.g., 5, 4, 3, 2, 1%) as measured by LC.

[0184] In some embodiments, the process results in a final TGZ yield of greater than about 85, 86, 87, 88, 89, or 90%. In some embodiments, the process results in a final TGZ yield of greater than about 90%.

[0185] Alternative methods to TGZ: Step A-1, Step A-2, Step A-3 Step A-1: ​​Step A-1 produces the methyl core (chemical name: dimethyl((S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanoyl)-L-glutamate) shown below, and Step A-1 is an amide coupling between ZL-GluOMe (chemical name: (S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanoic acid) and di(OMe)Glu (chemical name: dimethyl L-glutamic acid hydrochloride): [ka] Includes.

[0186] In some embodiments, the reaction is carried out in the presence of a base and a coupling reagent. In some embodiments, the base is added to a solution of zL-GluOMe before adding the coupling reagent and di(OMe)Glu to the reaction. In some embodiments, the base is N-methylmorpholine (NMM) or di-isopropylethylamine. In some embodiments, the base is NMM.

[0187] In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is an ethereal solvent. In some embodiments, the solvent is THF or MeTHF. In some embodiments, the solvent is THF. In some embodiments, the solvent is added to the reaction under a positive pressure of nitrogen.

[0188] In some embodiments, the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC. In some embodiments, the coupling reagent is IBCF.

[0189] In some embodiments, the reaction temperature prior to and during the reaction with the coupling reagent is about -20°C to about -10°C. In some embodiments, the reaction temperature is about -18°C to about -13°C. In some embodiments, the reaction temperature is about -15°C.

[0190] In some embodiments, after the reaction temperature is reached, a coupling reagent is added to the solution of zL-Glu-OMe and base, and then di(OMe)Glu is added to the reaction.

[0191] In some embodiments, the reaction time after di(OMe)Glu is about 50-70 minutes, in some embodiments, the reaction time is about 60 minutes.

[0192] In some embodiments, di(OMe)Glu is added to the reaction. In some embodiments, the amount of di(OMe)Glu is added in 4-6 equal portions. In some embodiments, the amount of di(OMe)Glu is added in 5 equal portions.

[0193] In some embodiments, the reaction temperature after addition of di(OMe)Glu is increased to about 5° C. to about 15° C. In some embodiments, the reaction temperature is increased to about 7° C. to about 12° C. In some embodiments, the reaction temperature is increased over a period of 2 hours.

[0194] In some embodiments, the reaction time is about 12 to 24 hours. In some embodiments, the reaction time is about 14 to 18 hours.

[0195] In some embodiments, the reaction further comprises an additional charge of coupling reagent, base, and / or zL-Glu-OMe. In some embodiments, the reaction further comprises an additional charge of IBCF, NMM, and ZL-Glu-OMe. In some embodiments, the additional charge is about 8-12 mol %. In some embodiments, the additional charge is about 10 mol %.

[0196] In some embodiments, the equivalents (eq.) of di(OMe)Glu relative to zL-GluOMe is about 0.8eq to about 1.3eq, the NMM is about 2.3eq to about 2.7eq, and the IBCF is about 0.8eq to about 1.3eq. In some embodiments, the equivalents of di(OMe)Glu relative to zL-GluOMe is about 1eq, the NMM is about 2.5eq, and the IBCF is about 1eq.

[0197] Upon completion, the reaction is diluted with an organic solvent and then washed successively with an aqueous acid and then an aqueous base. In some embodiments, the organic solvent is an acetate solvent. In some embodiments, the organic solvent is ethyl acetate (EtOAc). In some embodiments, the aqueous acid is HCl. In some embodiments, the aqueous base is NaOH. In some embodiments, the aqueous washes are performed about 2 to about 4 times. Residual water is then removed from the organic solution prior to crystallization.

[0198] Anti-solvent crystallization is performed by slow cooling gradient to room temperature. In some embodiments, the anti-solvent is heptane. In some embodiments, the solvent / anti-solvent is EtOAc / heptane. In some embodiments, the solvent / anti-solvent is 1:1, 1:2, or 1:3 EtOAc / heptane. In some embodiments, the solvent / anti-solvent is 1:1 EtOAc / heptane. The resulting slurry was filtered to isolate the methyl core solid. The solid was washed with an organic solvent and then dried under nitrogen.

[0199] In some embodiments, the process of step A-1 for preparing the methyl core can be represented by the following scheme: [ka] As shown in.

[0200] Step A-2: Disclosed herein is a triol compound (chemical name: benzyl ((10S,15S)-1,22-dihydroxy-10-((2-(2-hydroxyethoxy)ethyl)carbamoyl)-7,12,16-trioxo-3,20-dioxa-6,11,17-triazadocosane-15-yl)carbamate) and a method for preparing the triol. Also disclosed herein is a compound having the formula: -NH 2 Step A-2 is an unprotected triol that does not have a CBz protecting group on it. Step A-2 produces the triol shown below, which is an aminolysis between a methyl core (dimethyl((S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanoyl)-L-glutamate) and an amino alcohol (chemical name: 2-(2-aminoethoxy)ethanol or 2-(2-aminoethoxy)ethan-1-ol): [ka] Includes.

[0201] In some embodiments, the 2-(2-aminoethoxy)ethanol is solvent-free and the reaction does not use a solvent, hi some embodiments, a solvent additive is used.

[0202] In some embodiments, the amount of 2-(2-aminoethoxy)ethanol is about 25-30 equivalents relative to the methyl core.

[0203] In some embodiments, the reaction temperature is about 25° C. to about 35° C. In some embodiments, the reaction temperature is about 30° C. In some embodiments, the reaction time is about 22 to 26 hours. In some embodiments, the reaction time is about 24 hours.

[0204] In some embodiments, step A-2 is not mediated by an enzyme. In some embodiments, step A-2 is mediated by an enzyme. In some embodiments, the enzyme is: Novozym 5103 (liquid), Lipozyme CALB L (liquid), Resinase HT (liquid), Lipozyme TL (liquid), Novozym 435 (solid supp.), Novozym 40086 (solid supp.), Lipozyme TL (solid supp.), Papain (lyophilized), Trypsin (lyophilized), Amano PS (powder), Novozym 5103 (liquid), Lipozyme CALB L (liquid), Resinase HT (liquid), Aldolase (lyophilized), Papain (lyophilized), Trypsin (lyophilized), or Palatase (liquid). In some embodiments, the enzyme is Lipozyme CALB L (liquid), Lipozyme TL (liquid), Novozym 435 (solid supported), Lipozyme TL (solid supported). In some embodiments, the enzyme is Lipozyme CALB L (liquid). In some embodiments, the enzyme is Lipozyme TL (liquid). In some embodiments, the enzyme is Novozym 435 (solid supported). In some embodiments, the enzyme is Lipozyme TL (solid supported).

[0205] In some embodiments, an anti-solvent is added to precipitate the triol. In some embodiments, the anti-solvent is ethyl acetate, methyl-tert-butyl ether, or isopropyl acetate. In some embodiments, the anti-solvent is ethyl acetate.

[0206] In some embodiments, optionally, seed crystals of the triol are added to the reaction. In some embodiments, about 1-3 weight percent (wt%) seed crystals are added. In some embodiments, about 2 weight percent (wt%) seed crystals are added.

[0207] The reaction temperature is reduced to about room temperature. In some embodiments, the reaction temperature is reduced to about 20° C. to about 25° C. The triol slurry is aged for about 16-20 hours or about 18 hours.

[0208] The slurry is then filtered, the crude product solids are washed with an organic solvent, and then redissolved in an organic solvent and reslurried at temperature. The slurry is filtered, the product solids are washed with an organic solvent, and then dried under nitrogen. In some embodiments, the organic solvent is ethyl acetate.

[0209] In some embodiments, the process of step A-2 for preparing the triol can be represented by the following scheme: [ka] As shown in.

[0210] Step A-3: Step A-3 produces TGZ and involves glycosylation between a triol and BD-galactosamine pentaacetate. [ka]

[0211] BD-galactosamine pentaacetate is converted to the oxazoline solution shown below (i.e., the oxazoline derivative of BD-galactosamine pentaacetate). [ka]

[0212] In some embodiments, the oxazoline derivative of BD-galactosamine pentaacetate is prepared in the same reaction vessel as the triol, rather than being prepared separately.

[0213] In some embodiments, BD-galactosamine pentaacetate is first converted to an oxazoline solution (ie, the oxazoline derivative of BD-galactosamine pentaacetate) before being added to a solution of the triol.

[0214] An oxazoline solution is produced by reacting BD-galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf). In some embodiments, about 1 to about 1.5 eq of TMSOTf is used. In some embodiments, about 1.2 eq of TMSOTf is used. These equivalents are relative to the amount of BD-galactosamine pentaacetate. In some embodiments, the reaction temperature is about 35° C. to about 45° C. In some embodiments, the reaction temperature is about 40° C. In some embodiments, the reaction time is about 70 to about 110 minutes. In some embodiments, the reaction time is about 90 minutes.

[0215] In some embodiments, the reactants for preparing the oxazoline solution further comprise a solvent. In some embodiments, the solvent is dichloroethane (DCE) or dichloromethane (DCM). In some embodiments, the solvent is DCE.

[0216] In some embodiments, the reaction temperature of the oxazoline solution is reduced to about 20° C. to about 28° C. In some embodiments, such reaction temperature is about 23° C.

[0217] In some embodiments, the triol is prepared by dissolving in sodium bicarbonate (NaHCO 3 ) and triol and NaHCO 3 is then added to the beta-D-galactosamine pentaacetate or its oxazoline solution. In some embodiments, the triol and NaHCO 3The slurry mixture further comprises a solvent. In some embodiments, the solvent of the slurry mixture is dichloromethane (DCM), dichloroethane, or acetonitrile. In some embodiments, the solvent of the slurry mixture is dichloromethane (DCM).

[0218] In some embodiments, the oxazoline solution is added to a slurry mixture of triol and NaHCO. In some embodiments, the oxazoline solution is added to a mixture of triol and NaHCO at a rate of about 0.8 to about 1.2 equivalents of oxazoline solution / hour to about 4 to 5 equivalents / hour. In some embodiments, the oxazoline solution is added to a mixture of triol and NaHCO at a rate of about 1.0 equivalent of oxazoline solution / hour to about 4.5 equivalents / hour. 3 Add to the mixture.

[0219] The reaction is then allowed to proceed for about 18 to about 30 hours, or overnight. In some embodiments, the reaction is allowed to proceed for about 24 hours. In some embodiments, the reaction temperature is about 20° C. to about 30° C. In some embodiments, the reaction temperature is about 25° C.

[0220] An additional charge of remetered oxazoline solution may be added to drive reaction conversion. The reaction is quenched with an organic base followed by an aqueous base. In some embodiments, the organic base is Et 3 In some embodiments, the aqueous base is NaHCO 3 It is.

[0221] The organic phase is washed with a weak acid aqueous wash. In some embodiments, the acid is NH 4 Cl. The organic solution was dried to remove residual water.

[0222] The resulting TGZ product is precipitated from the solvent / anti-solvent system. In some embodiments, the solvent is DCM and the anti-solvent is dimethoxyethane (DME). The TGZ slurry is filtered and the product solid is washed with an organic solvent and dried under nitrogen. In some embodiments, the organic solvent is DCM / DME 1:1. To achieve higher purity, the solid can be reslurried with an organic solvent at temperature, filtered, washed with an organic solvent, and then dried under nitrogen.

[0223] In some embodiments, the process of step A-3 for preparing TGZ can be represented by the following scheme: [ka] As shown in.

[0224] Step 5a: TG amine or its salt Disclosed herein is a method for preparing TG amine or a salt thereof (process step 5a), which comprises high pressure hydrogenolysis of TGZ to produce TG amine or a salt thereof. [ka]

[0225] In past step 5a processes, the hydrogenolysis of TGZ to TG amine or its salts had several undesirable factors, such as high catalyst loading (e.g., 10%) of expensive Pd source for the catalyst, or MeOH, a flammable reaction solvent (when exposed to hydrogen).

[0226] Disclosed herein is an improved process involving high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to form TG amine or its salt. The improvements include the use of DCM as the reaction solvent to eliminate isolation, and reaction kinetic analysis to determine optimal catalyst loading and stirring speed. The TG amine or its salt can be incorporated (without isolation) into the next step 5b process described herein.

[0227] In some embodiments, the process of step 5a includes combining TGZ with a Pd / C catalyst and pressurizing with hydrogen. In some embodiments, the process further includes an acid. In some embodiments, the process includes combining TGZ with a solvent, adding a Pd / C catalyst, adding an acid, and pressurizing with hydrogen. In some embodiments, the process of step 5a includes combining TGZ with a Pd / C catalyst, adding an acid and a solvent, and pressurizing with hydrogen. After the reaction is complete, the catalyst may be removed by filtration. In some embodiments of step 5a, the process further includes filtration.

[0228] A palladium source is used for the hydrogenolysis. In some embodiments, the palladium source is a Pd / C catalyst. In some embodiments, the palladium on carbon (Pd / C) catalyst used is less than about 10%, 9%, 8%, 7%, 6% Pd / C. In some embodiments, the Pd / C catalyst is about 4-6% Pd / C. In some embodiments, the Pd / C catalyst is 5% Pd / C.

[0229] In some embodiments, the process includes the use of an acid. In some embodiments, the acid is TFA, oxalic acid, HCl, AcOH, H 3 PO 4 , citric acid. In some embodiments, the acid is TFA or oxalic acid. In some embodiments, the acid is TFA. In some embodiments, the acid is oxalic acid. In some embodiments, the acid is added in an amount of about 0.8-1.1 or 1.0 eq, or not more than about 1.0 or 1.1 eq. Addition of too much acid in the process in step 5a will generally result in higher levels of the TG amine-TFA acetamide impurity in the process in step 5b.

[0230] In some embodiments, the solvent is DCM, IPAc, or MeOH. In some embodiments, the solvent is DCM or IPAc. In some embodiments, the solvent is DCM. In some embodiments, the solvent is IPAc.

[0231] In some embodiments, the reaction is maintained at a temperature of about 20-25° C. In some embodiments, the reaction is stirred for at least about 15, 16, 17, or 18 hours.

[0232] In some embodiments, the reaction reduces the formation of one or more impurities, such as TG amine deAc, diNAG-OH TG amine, TG amin guanidine, TG amine Ac, TG acetamide, and / or NAG-H guanidine, each of which is shown below. [ka]

[0233] In some embodiments, the NAG-H guanidine impurity is present at less than or equal to 2 or 1% as measured by LC. In some embodiments, the TG amine diNAG-OH impurity is present at less than or equal to 2 or 1% as measured by LC. In some embodiments, the reaction reduces the formation of TG amine deacylation impurity. In some embodiments, the TG amine deacylation impurity is present at 5, 4, 3, 2, 1% or less as measured by LC.

[0234] In some embodiments, the TG amine or its salt is a TG amine salt. In some embodiments, the TG amine salt is a phosphate, a formate, an acetate, a trifluoroacetate, or an oxalate. In some embodiments, the TG amine salt is a trifluoroacetate or an oxalate. In some embodiments, the TG amine salt is a trifluoroacetate.

[0235] The improved step 5a process results in a high purity TG PEG product in step 5b, and also reduces the amount of TG acetamide by-product. In some embodiments, the TG amine or salt thereof contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of acetamide by-product. In some embodiments, the TG amine or salt thereof contains less than about 5% of acetamide by-product. In some embodiments, the TG amine or salt thereof contains less than about 2% of acetamide by-product.

[0236] In some embodiments, the TG amine or salt thereof produced from the improved step 5a process has a purity greater than or equal to about 97%, 98%, or 99% as measured by LC. In some embodiments, the TG amine or salt thereof produced from the improved step 5a process has a yield greater than or equal to about 97%, 98%, or 99%.

[0237] In some embodiments, the process of step 5a for preparing TG amine TFA salt can be represented by the following scheme: [ka] As shown in.

[0238] In some embodiments, the process of step 5a for preparing TG amine TFA salt can be represented by the following scheme: [ka] As shown in.

[0239] After reaction and filtration, the TG amine or its salt in the resulting solution can be directly incorporated into the process of step 5b described herein. Advantages include the elimination of unit operations involving distillation and precipitation. In some embodiments, the TG amine or its salt is incorporated into step 5b without isolating the TG amine or its salt. In some embodiments, the TG amine or its salt is a salt that is TG amine TFA.

[0240] Step 5b: TG PEG Disclosed herein is a method for preparing TG PEG (process step 5b). TG PEG is prepared by conjugating TG amine or its salt with PEG acid. [ka]

[0241] This improvement reduces impurities during the amide coupling process, eliminates column chromatography, and reduces the amount of solvent. This improved process includes controlled addition of a coupling reagent (e.g., TBTU). By controlling the rate of addition of the coupling reagent (e.g., TBTU) to the PEG acid, this improved process reduces the formation of a PEG dimer impurity, called TG PEG dimer. In addition, the improved step 5b includes extraction of the crude TG PEG product into an aqueous solution, followed by back-extraction to remove the orthogonal impurity.

[0242] The improved step 5b process comprises reacting a TG amine or a salt thereof with a PEG acid in the presence of a coupling reagent. In some embodiments, the process further comprises a base. In some embodiments, the process further comprises a solvent. In some embodiments, the coupling reagent is TBTU or HATU. In some embodiments, the coupling reagent is TBTU. In some embodiments, the solvent is DCM or DMF. In some embodiments, the solvent is DCM. In some embodiments, the coupling reagent is TBTU and the solvent is DCM. In some embodiments, the coupling reagent is HATU and the solvent is DMF.

[0243] In some embodiments, the process of step 5b comprises adding TBTU to a solution comprising TG amine or a salt thereof and PEG acid.

[0244] In some embodiments, step 5b comprises: a) combining a TG amine or a salt thereof with a PEG acid; b) adding a base; and c) TBTU is added over a period of time. In some embodiments, the TG amine or salt thereof is added directly from the end of the process in step 5a. In some embodiments in step 5b, the process further comprises extraction / work-up, distillation, precipitation, filtration, and / or drying.

[0245] TBTU may be added in small increments or may be added using a slurry transfer. In some embodiments, the addition of TBTU to the solution of PEG acid and TG amine or salt thereof is over a period of about 80-100 or 90 minutes. In some embodiments, the addition of TBTU to the solution of PEG acid and TG amine or salt thereof is over a period of about 50-70 or 60 minutes.

[0246] In some embodiments, the process of step 5b further comprises the use of a base. In some embodiments, the base is N,N-diisopropylethylamine (DIPEA), triethylamine, or N-methylmorpholine (NMM). In some embodiments, the base is N,N-diisopropylethylamine (DIPEA).

[0247] The improved method for preparing TG PEG reduces at least one impurity, such as TG PEG dimer impurity. During NAG-25 conjugation to oligonucleotide, this TG PEG dimer impurity may be reactive in the phosphitylation step and conjugate to the oligonucleotide. Examples of impurities are shown below. [ka]

[0248] In some embodiments, the process of step 5b produces TG PEG containing less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% TG PEG dimer. Disclosed are compositions that contain TG PEG with less than or equal to 10% (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) TG PEG dimer as measured by LC.

[0249] In some embodiments, the TG amine or salt thereof is a TG amine TFA salt. In some embodiments, the process of step 5b of preparing TG PEG comprises reacting TG amine TFA salt with PEG acid, TBTU, and DIPEA in a solvent. In some embodiments, the solvent is DCM, DMF, DMAc, and MeCN. In some embodiments, the solvent is DCM. In some embodiments, the solvent is DMF. In some embodiments, the process of step 5b begins at about -10°C to about 5°C and is allowed to warm up to about 17-27°C during the course of the reaction. In some embodiments, the process of step 5b begins at about -10°C to about 4°C and is allowed to warm up to about 17-27°C during the course of the reaction. In some embodiments, the process of step 5b begins at about -8°C and is allowed to warm up to about 17-27°C during the course of the reaction. In some embodiments, the process of step 5b begins at about -8°C and is allowed to warm up to room temperature during the course of the reaction.

[0250] In some embodiments, the process of step 5b for preparing TG PEG can be represented by the following scheme: [ka] where TG amine is the TG amine TFA salt.

[0251] In some embodiments, the process of step 5b for preparing TG PEG includes combining TG amine and PEG acid. In some embodiments, the process includes DCM as the solvent. In some embodiments, the temperature is maintained at about 20-25° C. In some embodiments, the temperature is cooled to −5-5° C., DIPEA is added, and then TBTU is added. In some embodiments, TBTU is added in small portions or as a slurry over a period of about 60-90 minutes. In some embodiments, the reaction temperature is then slowly increased to about 0-5° C. over a period of about 1 hour. In some embodiments, the reaction is further maintained at 0-5° C. for about 1-5 hours. In each of the foregoing embodiments, PEG acid is added at about 0.9-1.3 eq, TBTU is added at about 1-1.5 eq, and DIPEA is added at about 2.5-3.5 eq relative to the TG amine in solution (from step 5a). In the above embodiment, each of them is added with about 1.1 eq of PEG acid, about 1.3 eq of TBTU, and about 3.0 eq of DIPEA relative to the TG amine in solution (from step 5a). Additional amounts of PEG acid and TBTU can be added to increase the reaction conversion, if necessary.

[0252] After the reaction is complete, the crude TG PEG product is extracted into an aqueous solution and then back-extracted to remove one or more impurities. In some embodiments of step 5b, the process further comprises extraction / work-up, distillation, precipitation, filtration, and / or drying.

[0253] In some embodiments, the process of step 5b includes an aqueous extraction wash sequence to remove the TG PEG dimer impurity, which in some embodiments includes a water (e.g., DI water) extraction, ammonium sulfate salting out (e.g., 13 wt % ammonium sulfate), back extraction into a solvent (e.g., DCM back extraction), an acidic buffer wash (e.g., pH 4-6), and a basic buffer wash (e.g., pH 7.5-9).

[0254] In some embodiments, the acidic buffer wash comprises a mixture of 0.5 M sodium monophosphate pH 6.0 and saturated brine. 2 PO 4 The ratio of ethanol to brine is about 4.5-5:3-3.5 volumes, in some embodiments, the ratio is about 4.8:3.2 volumes.

[0255] In some embodiments, the basic buffer wash comprises a mixture of saturated sodium bicarbonate and saturated brine solution. 3 The ratio of ethanol to brine is about 3.5-4.5 vol:3.5-4.5 vol. In some embodiments, the ratio is 4.0 vol:4.0 vol.

[0256] After the aqueous extraction wash sequence, the TG PEG solution contains TG PEG that is at least 90% pure and has less than 1% each of one or more impurities (e.g., diNAG-OH TG PEG, TG PEG DesAc, TG PEG dimer, TG PEG Des2Ac, TG Amine Ac, TG acetamide, and / or TG Amine Guanidyl).

[0257] In some embodiments, the process of step 5b comprises azeotropic distillation with a solvent, hi some embodiments, the solvent for this distillation is DCM.

[0258] After washing and distillation, the process further comprises precipitation by adding an anti-solvent to the mixture. In some embodiments, the anti-solvent is MTBE or IPAc. In some embodiments, the anti-solvent is MTBE. In some embodiments, the precipitation is carried out at a temperature of about -5 to 5°C.

[0259] In some embodiments, the process of step 5b produces TG PEG with a purity greater than or equal to about 90% or 95% as measured by LC, hi some embodiments, the process of step 5b produces TG PEG with a yield greater than or equal to about 80, 81, 82, 83, 84, 85, or 90%.

[0260] Process 6:NAG-25 Disclosed herein is a method for preparing NAG-25 (process step 6). The method involves phosphorosamide formation from TG PEG to prepare NAG-25. [ka]

[0261] The improvements include changing the precipitation solvent, which significantly reduces the solvent volume from past processes (e.g., from 130-170 volumes to about 10-20, 20-30, 25-35, 30-50, or 40-60 volumes), improving the stability of NAG-25, and / or reducing impurities overall (e.g., from 90-93 or 92% purity to about 95-98 or 97% purity).

[0262] In some embodiments, the method of preparing NAG-25 includes combining TG PEG, an activating agent, and a P reagent. In some embodiments, the phosphitylation reagent ("P reagent") is 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphoroamidite. In some embodiments, the phosphitylation reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite.

[0263] In some embodiments, the activator is tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), or benzothiotetrazole (BTT). In some embodiments, the activator is tetrazole, DCI, or ETT. In some embodiments, the activator is tetrazole or DCI.

[0264] In some embodiments, the activator is tetrazole. In some embodiments, the tetrazole is added at about 0.2-1.2 eq. In some embodiments, the tetrazole is added at about 0.4-0.8 eq. In some embodiments, the tetrazole is added at about 0.6 eq. The equivalents of tetrazole in the foregoing embodiments are relative to TG PEG.

[0265] In some embodiments, the activator is DCI. In some embodiments, DCI is added at about 0.01-0.05 eq. In some embodiments, DCI is added at about 0.02-0.04 eq. In some embodiments, DCI is added at about 0.02-0.03 eq. The equivalents of DCI in the foregoing embodiments are relative to TG PEG.

[0266] In some embodiments, the reaction in step 6 further comprises an additive. In some embodiments, optionally, step 6 comprises adding an additive to the TG PEG. In some embodiments, the additive is N-methylimidazole (NMI). In some embodiments, the reaction in step 6 does not comprise an additive.

[0267] In some embodiments, the P reagent is added at about 0.75-2 eq. In some embodiments, the P reagent is added at about 1-1.5 eq. The equivalents in the foregoing embodiments are relative to the TG PEG.

[0268] Improvements to this method include the order of addition of the reagents. In some embodiments, a solution containing the P reagent and activator is added to a solution of TG PEG, optionally containing additives. The solution of P reagent and activator is added at about 0-5° C. After addition is complete, the reaction mixture is allowed to warm to room temperature (e.g., about 20-25° C.) and allowed to proceed for about 2-4 hours.

[0269] In some embodiments, a solution of TG PEG in a solvent (TG PEG solution) is added to a separate solution of the P reagent and activating agent in a solvent. The TG PEG solution may be added over a period of time. In some embodiments, the period of time is about 2-10, 3-7, 3-5, 4-5, 4-6, or 5-6 hours, or about 3, 4, 5, 6, or 7 hours. In some embodiments, the reaction is allowed to proceed at a temperature of about 35-45° C., or about 38, 39, 40, 41, 42, 43, 44, or 45° C. In some embodiments, the reaction is allowed to proceed for about 8-12, 9-11, or 10-14 hours, or at least 8, 9, 10, 11, 12, 13, or 14 hours. In some embodiments, the P reagent is 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite. In some embodiments, the activating agent is DCI. In some embodiments, the solvent is DCM. In some embodiments, the solvent is anhydrous DCM. The equivalents of P reagent, activator, are described in the previous embodiment. In some embodiments, a solution of TG PEG in DCM is added over a period of 4.5-5.5 hours (e.g., about 5 hours) to a separate solution of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite and DCI in DCM (e.g., anhydrous DCM), and then reacted at 35-45°C (e.g., about 40°C) for at least 10 hours. In some embodiments, the method includes 1-1.5 eq of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite, 0.02-0.03 eq of DCI (each relative to TG PEG).

[0270] The improved step 6 process further includes the ability to identify, control, and / or reduce reactive impurities compared to other NAG-25 processes. The improved step 6 process also includes removing impurities related to reactive phosphorus. Examples of impurities from step 6 include NAG-25 dimers: [ka] [ka] [ka] [ka] Examples include:

[0271] In some embodiments, the impurities are H-phos, oxidized NAG-25, NAG-25 dimer, and / or NAG-25 PEG dimer. In some embodiments, H-phos, oxidized NAG-25, NAG-25 dimer, and / or NAG-25 PEG dimer are present at the end of the reaction in an amount less than or equal to about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% as measured by LC. In some embodiments, H-phos, oxidized NAG-25, NAG-25 dimer, and / or NAG-25 PEG dimer are present in an amount less than or equal to about 8, 7, 6, 5, 4, 3, 2, or 1% at the end of the reaction as measured by LC. In some embodiments, NAG-25 dimer is present in an amount less than or equal to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% at the end of the reaction as measured by LC. In some embodiments, NAG-25 dimer is present in an amount less than or equal to about 5, 4, 3, 2, or 1% at the end of the reaction as measured by LC. In some embodiments, NAG-25 dimer is present at about 4 or 3%, or an amount equal to about 4 or 3%, at the end of the reaction, as measured by LC. In some embodiments, NAG-25 dimer is present at about 2 or 1%, or an amount equal to about 2 or 1%, at the end of the reaction, as measured by LC.

[0272] Disclosed are compositions comprising NAG-25 in which any of the described impurities are less than or equal to 10% (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) as measured by LC. Disclosed are compositions comprising NAG-25 in which NAG-25 dimer is less than or equal to 10% (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) as measured by LC. In some embodiments, the composition comprises NAG-25 in which NAG-25 dimer is less than or equal to 5, 4, 3, 2, or 1% as measured by LC.

[0273] In some embodiments, the process of step 6 further comprises an extraction (eg, water extraction), a distillation, a precipitation, a filtration step, and / or a drying step.

[0274] In some embodiments, the NAG-25 product solution is dried. In some embodiments, the drying step uses molecular sieves. In some embodiments, the drying step uses azeotropic distillation.

[0275] In some embodiments, the precipitation step uses normal precipitation, where an anti-solvent is added to a NAG-25 solution, hi some embodiments, the precipitation step uses reverse precipitation, where an NAG-25 solution is added to an anti-solvent.

[0276] In some embodiments, the anti-solvent used in the precipitation is MTBE or heptane, hi some embodiments, the anti-solvent used in the precipitation is heptane.

[0277] In some embodiments, NAG-25 is precipitated from DCM / heptane as a solvent / anti-solvent.

[0278] In some embodiments, the process in step 6 comprises reacting TG PEG with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the presence of tetrazole and NMI in dichloromethane.

[0279] In some embodiments, the process of step 6 can be performed according to the following scheme: [ka] In some embodiments of the above scheme, DCM is about 15V, NMI is about 0.2eq, tetrazole is about 0.6eq, and P reagent is about 1.25eq. In some embodiments, the TG PEG solution is added to the solution of P reagent and activator for a period of about 2-10 hours and at a temperature of about 35-45°C.

[0280] In some embodiments of step 6, the process further comprises extraction / work-up, crystallization, precipitation, filtration, and / or drying. In some embodiments of step 6, the NAG-25 extraction and water wash are optional.

[0281] In some embodiments, the process of step 6 includes a solution of TG PEG in a solvent that is added to a solution of the P reagent and activating agent in the solvent. In some embodiments, the P reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite. In some embodiments, the process does not include an additive. In some embodiments, the solvent is DCM. In some embodiments, the activating agent is DCI. The TG PEG solution is added to the solution of the P reagent and activating agent over a period of time. In some embodiments, the period of time is about 4-6 hours, 4.75-5.25 hours, or 4.5-5.5 hours. After the addition of the TG PEG solution, the two solutions are allowed to react for at least 8-12 hours. In some embodiments, the reaction time is about 9-11 hours. In some embodiments, the reaction is carried out at about 35-45°C. In some embodiments, the reaction is carried out at about 38-42°C. After the reaction is completed, an aqueous extraction with water may be performed if necessary, and the NAG-25 may be azeotropically dried (e.g., azeotropically dried at 25-35° C. under vacuum), followed by filtration. In some embodiments, the NAG-25 is precipitated without aqueous extraction. For precipitation, the NAG-25 solution may be added to anhydrous anti-solvent (e.g., heptane) over 90 minutes. The solid is filtered off and washed twice with anti-solvent:solvent (e.g., anhydrous heptane:DCM). In some embodiments, the anti-solvent:solvent ratio is about 4:1, 3:1, 2:1, or 1:1. In some embodiments, the heptane:DCM ratio is about 4:1, 3:1, 2:1, or 1:1. In some embodiments, the heptane:DCM ratio is about 4:1 or 3:1. In some embodiments, the volume percentage of solvent in the antisolvent:solvent solution is about 20-50, 20-30, 30-40, or 40-50%, with the remaining percentage being made up of the volume percentage of antisolvent, hi some embodiments, the proportion of DCM is about 20-50% or about 20, 30, 40, 50% of the volume of the antisolvent / solvent solution.

[0282] Linking to Oligomeric Compounds NAG-25 and NAG-25-containing compounds are useful as targeting ligands by linking them to therapeutic compounds such as oligomeric compounds (e.g., RNA). NAG-25 facilitates targeted delivery of NAG-25-conjugated therapeutic compounds to hepatocytes for endocytosis of the therapeutic compounds, and the therapeutic compounds can modulate expression of target nucleic acids, resulting in altered translation of the target nucleic acids. For example, the therapeutic compounds can modulate expression of target genes to inhibit protein translation or expression.

[0283] In some embodiments, the targeting ligand is linked to the therapeutic compound via an additional linker and / or a cleavable moiety, which is then linked to the therapeutic compound, hi some embodiments, the targeting ligand is linked to the therapeutic compound itself.

[0284] In some embodiments, the therapeutic compound is an expression-inhibiting oligomeric compound. In some embodiments, the expression-inhibiting oligomeric compound is an RNAi construct. In some embodiments, the expression-inhibiting oligomeric compound is a double-stranded RNAi construct. In some embodiments, the expression-inhibiting oligomeric compound is a single-stranded oligonucleotide. The expression-inhibiting oligomeric compound can be synthesized using methods commonly used in the art.

[0285] In some embodiments, the targeting ligand is directly or indirectly linked to the 5' end of the sense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly linked to the 3' end of the sense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly linked to the 5' end or the 3' end of the antisense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly linked to the 5' end or the 3' end of the single-stranded RNAi construct.

[0286] In some embodiments, the targeting ligand is linked to the double-stranded RNAi construct at the 5' end of the terminal nucleoside of the sense strand of the double-stranded RNAi construct via a phosphate group, a phosphonate group, a phosphorothioate group, or other internucleoside linkage group.

[0287] In some embodiments, the targeting ligands disclosed herein comprise a cleavable moiety. In some embodiments, the cleavable moiety comprises or consists of a phosphate group or other internucleoside linking group that can be cleaved. In some embodiments, the targeting moiety is linked to the therapeutic compound via the cleavable moiety.

[0288] In some embodiments, the targeting ligands disclosed herein are linked to an additional group that includes a cleavable moiety, hi some embodiments, the targeting ligand is linked to a cleavable moiety that is in turn linked to an expression-inhibiting oligomeric compound.

[0289] In some embodiments, the targeting ligand is a phosphoramidite compound (also referred to herein as a "phosphoramidite-containing compound"). The phosphoramidite compounds containing the targeting ligands described herein can be useful for easily attaching the targeting ligand to a therapeutic compound or other group using methods generally known in the art for phosphoramidite synthesis. In some embodiments, the phosphoramidite compounds containing the targeting ligand are linked to an expression-inhibiting oligomeric compound using methods generally known in the art. In some embodiments, the phosphoramidite containing the targeting ligand is linked to the 5' end of the sense strand of the double-stranded RNAi construct.

[0290] In some embodiments, the expression-inhibiting oligomeric compound linked to targeting ligand comprises a single-stranded oligonucleotide.In some embodiments, this single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.In some embodiments, this targeting ligand is directly linked to the single-stranded antisense oligonucleotide.In some embodiments, an additional group is inserted between the targeting ligand and the single-stranded oligonucleotide.

[0291] In some embodiments, the expression-inhibiting oligomeric compound linked to any of the targeting ligands disclosed herein includes an RNAi construct. In some embodiments, the targeting ligands disclosed herein are linked to the RNAi construct either directly or indirectly.

[0292] In some embodiments, the targeting ligand disclosed herein is directly linked to the RNAi construct.In some embodiments, the targeting ligand disclosed herein is indirectly linked to the RNAi construct, because an additional group is inserted between the RNAi construct and the linker of the targeting ligand.In some embodiments, another linker is included between the linker and the therapeutic compound (e.g., the RNAi construct).

[0293] Other embodiments A list of exemplary embodiments includes the following:

[0294] 1. t-Butyl core [ka] 1. A method for preparing GluZ, comprising: [ka] and GluOtBu or a salt thereof: [ka] to produce a t-butyl core.

[0295] 2. GluOtBu or a salt thereof is GluOtBu hydrochloride: [ka] 2. The method of embodiment 1, wherein

[0296] 3. The method of embodiment 1 or 2, wherein the reacting is carried out in the presence of a coupling reagent, a base, and a solvent.

[0297] 4. The method of any one of embodiments 1 to 3, wherein the coupling reagent is EDC / Oxyma, TFFH, PyOxim, CDI, PivCl, T3P, or COMU.

[0298] 5. The method of any one of embodiments 1 to 4, wherein the coupling reagent is T3P or PivCl.

[0299] 6. The method of any one of embodiments 1 to 5, wherein the coupling reagent is PivCl.

[0300] 7. The method of any one of the preceding embodiments, wherein the base is N-methylmorpholine (NMM).

[0301] 8. The method of any one of the preceding embodiments, wherein the solvent is IPAc, MeTHF, MIBK, or MTBE.

[0302] 9. The method of any one of the preceding embodiments, wherein the solvent is MTBE.

[0303] 10. The method of any one of the preceding embodiments, wherein the anti-solvent is heptane.

[0304] 11. The method of any one of the preceding embodiments, wherein the solvent is MTBE and the anti-solvent is heptane.

[0305] 12. A method according to any one of embodiments 1 to 11, wherein a first solution comprising GluZ and NMM in a solvent is added to a second solution comprising a solvent and PivCl.

[0306] 13. The method of any one of embodiments 1 to 12, wherein PivCl is present in excess.

[0307] 14. The method of any one of embodiments 1 to 13, wherein GluOtBu is present in excess.

[0308] 15. The method of any one of embodiments 1 to 14, wherein the conversion to t-butyl core is greater than about 90% based on the amount of GluZ.

[0309] 16. Triacids: [ka] 13. A method for preparing a tert-butyl core comprising reacting the tert-butyl core with an acid.

[0310] 17. The acid is phosphoric acid (H 3 PO 4 ), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

[0311] 18. The acid is phosphoric acid (H 3 PO 4 17. The method of embodiment 16, wherein

[0312] 19. The method of any one of embodiments 16-18, wherein the reaction is carried out in a solvent selected from 2-MeTHF, acetonitrile, THF, DMA, sulfone, and DME.

[0313] 20. The method of embodiment 19, wherein the solvent is 2-MeTHF, THF, or acetonitrile.

[0314] 21. The method of embodiment 19, wherein the solvent is 2-MeTHF or THF.

[0315] 22. The method of embodiment 19, wherein the solvent is 2-MeTHF.

[0316] 23. The method of any one of embodiments 16-22, wherein the triacid is isolated as a crystalline solid.

[0317] 24. The method of any one of embodiments 16 to 23, wherein the triacid has a purity greater than or equal to 95% as determined by LC.

[0318] 25. The method of any one of embodiments 16-24, wherein the solvent further comprises water.

[0319] 26. The method of any one of embodiments 16-25, wherein the solvent is a mixture of 2-MeTHF and water.

[0320] 27. The method of any one of embodiments 16-26, wherein about 3.0-4.0 volumes of 2-MeTHF and about 0.5-2 volumes of water are used.

[0321] 28. The method of any one of embodiments 16 to 27, wherein the reaction is carried out at a temperature of about 40 to 60° C.

[0322] 29. The method of any one of embodiments 16 to 28, wherein the reaction is carried out at a temperature of about 45 to 55° C.

[0323] 30. The method of any one of embodiments 16 to 29, wherein the reaction is carried out at a temperature of about 50° C.

[0324] 31. The method of any one of embodiments 16-30, further comprising removing the phosphoric acid with at least one organic / aqueous wash.

[0325] 32. The method of any one of embodiments 16-31, wherein the organic / aqueous wash comprises isopropyl acetate (iPAC) as the organic phase and ammonium sulfate as the aqueous phase.

[0326] 33. The method of any one of embodiments 16-32, using about 8-18 volumes of iPAC and about 3-7 volumes of 20 wt % ammonium sulfate.

[0327] 34. The method of any one of embodiments 16-33, wherein about 10 volumes of iPAC and about 5 volumes of 20 wt % ammonium sulfate are used.

[0328] 35. The method of any one of embodiments 16-34, further comprising crystallization of the triacid using acetone / toluene, 2-MeTHF / IPAc, 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile.

[0329] 36. The method of any one of embodiments 16-35, further comprising crystallizing the triacid using acetone / toluene.

[0330] 37. The method of any one of embodiments 16-36, wherein the acetone / toluene is in a ratio of 2:3, 1:1, 3:2, or 1:2.

[0331] 38. The method of any one of embodiments 16-37, wherein the acetone / toluene is in a ratio of 2:3, 1:1, or 1:2.

[0332] 39. The method of any one of embodiments 16 to 38, wherein the triacid is obtained in a crystalline form characterized by an X-ray powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2.

[0333] 40. Crystalline form I of the triacid, characterized by an X-ray powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2.

[0334] 41. Crystalline form I of the triacid characterized by an X-ray powder diffractogram with signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, and 21.0±0.2.

[0335] 42. Crystalline form I of the triacid, characterized by an X-ray powder diffractogram substantially similar to that of FIG.

[0336] 43. Crystalline form II of the triacid, characterized by an X-ray powder diffractogram substantially similar to that of FIG.

[0337] 44. TGZ [ka] The method for preparing NAG-H comprises reacting a triacid with NAG-H in the presence of a coupling reagent and a base: [ka] or a salt thereof.

[0338] 45. The method of embodiment 44, wherein the coupling reagent is TBTU, HATU, or TCFH.

[0339] 46. ​​The method of embodiment 45, wherein the coupling reagent is TBTU.

[0340] 47. The method of any one of embodiments 44 to 46, wherein the base is DIPEA, NMI, NMM, or TMP.

[0341] 48. The method of embodiment 47, wherein the base is NMI.

[0342] 49. The method of any one of embodiments 44 to 48, further comprising at least one buffer wash.

[0343] 50. The method of embodiment 49, wherein the buffer wash is a phosphate buffer.

[0344] 51. The method of embodiment 49 or 50, wherein the buffer wash is at a pH of about 5 to 7.

[0345] 52. The method of embodiment 51, wherein the buffer wash is at about pH 6.

[0346] 53. The method of any one of embodiments 44-52, wherein the reacting is carried out in a solvent selected from DCM, DMF, MeCN, and DMAc, or a combination thereof.

[0347] 54. The method of embodiment 53, wherein the solvent is DMAc.

[0348] 55. The method of embodiment 53 or 54, further comprising adding an anti-solvent to the solution comprising TGZ and the solvent.

[0349] 56. The method of embodiment 55, wherein the antisolvent is an ether solvent.

[0350] 57. The method of embodiment 56, wherein the ether solvent is DME, 2-MeTHF, or MTBE.

[0351] 58. The method of any one of embodiments 53-57, wherein the solvent is DCM and the anti-solvent is MTBE.

[0352] 59. The method of any one of embodiments 44-58, wherein TGZ is prepared with low impurities.

[0353] 60. The method of embodiment 59, wherein the impurity is deacylated.

[0354] 61. The method of any one of embodiments 44-60, further comprising precipitating TGZ from the solvent / anti-solvent system.

[0355] 62. The method of embodiment 61, wherein the solvent / anti-solvent system is DCM / MTBE.

[0356] 63. The method of any one of embodiments 44-62, wherein TGZ is obtained with a purity of at least 95% without the use of column chromatography.

[0357] 64. The method of any one of embodiments 44 to 63, wherein NAG-H or a salt thereof is subsequently sent as a solution from a previous reaction to react with the triacid without isolating NAG-H or a salt thereof from the previous reaction.

[0358] 65. The method according to embodiment 64, wherein NAG-H or a salt thereof is prepared in a previous reaction comprising hydrogenation of NAG-Z in the presence of a Pd / C catalyst and in dimethylacetamide (DMAc).

[0359] 66. The method of any one of embodiments 44-65, wherein the DMAc is from about 3 volumes to about 5 volumes.

[0360] 67. The method of any one of embodiments 44 to 65, wherein the DMAc is about 4 volumes.

[0361] 68. The method of any one of embodiments 44 to 67, wherein the NAG-H or a salt thereof is NAG-H TFA.

[0362] 69. The method of any one of embodiments 44 to 66, wherein the Pd / C catalyst is 5% Pd / C.

[0363] 70. NAG-H TFA was reacted with 1,2-dichlorophenyl ether according to the following scheme: [ka] 70. The method according to any one of embodiments 44 to 69, wherein the compound is prepared in a previous reaction comprising a reagent in

[0364] 71. NAG-Z: [ka] 1. A method for preparing an acylGalNAc comprising the steps of: [ka] and Alcohol-Z: [ka] to produce NAG-Z.

[0365] 72. The method of embodiment 71, wherein the reaction is carried out in the presence of an acid.

[0366] 73. The acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ) 、 Boron trifluoride etherate (BF 3 OEt 2 ), tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), indium triflate (In(OTf) 3 ), or copper triflate (Cu(OTf) 2 73. The method of embodiment 72, wherein

[0367] 74. The acid is bismuth trifluoromethanesulfonate (Bi(OTf) 3 ) 、 Boron trifluoride etherate (BF 3 OEt 2 ), indium triflate (In(OTf) 3 ), or copper triflate (Cu(OTf) 2 74. The method of embodiment 73, wherein

[0368] 75. The method of any one of embodiments 72-74, wherein the acid is present in an amount of 0.1 to 0.2 equivalents relative to alcohol-Z.

[0369] 76. The method of any one of embodiments 72-74, wherein the acid is present in an amount of 0.13 to 0.17 equivalents relative to alcohol-Z.

[0370] 77. The method of any one of embodiments 71-76, wherein the reaction is carried out in the presence of a solvent selected from acetonitrile, dichloromethane, and dichloroethane.

[0371] 78. The method of embodiment 77, wherein the solvent is present in a total of 8 to 12 volumes.

[0372] 79. The method of any one of embodiments 71 to 78, wherein the acylGalNAc is present in 1.0 to 2.0 equivalents relative to alcohol-Z.

[0373] 80. The method of embodiment 79, wherein the acylGalNAc is present in 1.3 to 1.7 equivalents relative to alcohol-Z.

[0374] 81. The method of any one of embodiments 71 to 80, wherein the reaction temperature is heated to about 55 to 65° C. or 58 to 62° C.

[0375] 82. The method comprises the following scheme: [ka] 82. The method according to any one of embodiments 71 to 81, comprising the reagents and conditions in

[0376] 83. The method of any one of embodiments 71-82, wherein alcohol-Z is prepared by reacting benzyl chloroformate with an amino alcohol in the presence of a solvent and a base.

[0377] 84. The method of embodiment 83, wherein the solvent is dichloromethane (DCM).

[0378] 85. The method of embodiment 83 or 84, wherein the base is triethylamine (TEA).

[0379] 86. The method of any one of embodiments 83-85, wherein the amino alcohol is present in 1.1 equivalents relative to the benzyl chloroformate.

[0380] 87. The method of any one of embodiments 73 to 86, wherein the reaction temperature is about 30 to 40° C.

[0381] 88. The method of embodiment 86, wherein the reaction temperature is about 33 to 37°C.

[0382] 89. The method of any one of embodiments 77-87, wherein the solvent is acetonitrile and additional water is added to crystallize NAG-Z.

[0383] 90. TG Amine [ka] or a salt thereof, comprising the steps of: A process comprising high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to form TG amine or a salt thereof.

[0384] 91. The method of embodiment 90, comprising a source of palladium for hydrogenolysis.

[0385] 92. The method of embodiment 91, wherein the palladium source is a palladium on carbon (Pd / C) catalyst.

[0386] 93. The method of embodiment 92, wherein the source of palladium is 5% Pd / C.4d. The method of any one of embodiments 4a-4c, wherein the catalyst loading is less than about 10%, 9%, 8%, 7%, 6% Pd / C. In some embodiments, the catalyst used is 5% Pd / C.

[0387] 94. The method of any one of embodiments 90-93, wherein the hydrogenolysis is carried out in the presence of an acid.

[0388] 95. Acids include TFA, oxalic acid, HCl, AcOH, H 3 PO 4 , citric acid.

[0389] 96. The method of embodiment 94, wherein the acid is TFA or oxalic acid.

[0390] 97. The method of embodiment 94, wherein the acid is TFA.

[0391] 98. The method of any one of embodiments 90-97, wherein the hydrogenolysis is carried out in a solvent, and the solvent is DCM, IPAc, or MeOH.

[0392] 99. The method of embodiment 98, wherein the solvent is DCM.

[0393] 100. The method of any one of embodiments 90-99, wherein the method reduces the formation of deacylated impurities.

[0394] 101. The method of any one of embodiments 90-100, wherein the TG amine or salt thereof is a TG amine acid salt.

[0395] 102. The method of any one of embodiments 90-101, wherein the TG amine salt is a phosphate, formate, acetate, trifluoroacetate, or oxalate.

[0396] 103. The method of any one of embodiments 90-102, wherein the TG amine salt is a trifluoroacetate or oxalate salt.

[0397] 104. The method of embodiment 103, wherein the TG amine salt is a trifluoroacetate salt.

[0398] 105. The method of any one of embodiments 90-104, wherein the method results in a highly pure TG PEG product of step 5b and a reduced amount of TG amine acetamide by-product.

[0399] 106. The method of any one of embodiments 90-105, wherein the TG amine or salt thereof contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% acetamide by-product.

[0400] 107. The method of any one of embodiments 90-106, wherein the TG amine or salt thereof produced from the improved step 5a process has a purity greater than or equal to about 97%, 98%, or 99% as measured by LC.

[0401] 108. The method of any one of embodiments 90-107, wherein the TG amine or salt thereof is incorporated into step 5b without isolating the TG amine or salt thereof.

[0402] 109. The method comprises the following scheme: [ka] The method according to any one of embodiments 90 to 108, comprising the reagent according to any one of embodiments 90 to 108.

[0403] 110. TG PEG [ka] 13. A method for preparing a compound comprising treating a solution of TG amine and PEG acid with TBTU.

[0404] 111. The method of embodiment 110, wherein the solution further comprises a base.

[0405] 112. The method of embodiment 111, wherein the base is N,N-diisopropylethylamine (DIPEA).

[0406] 113. The method of any one of embodiments 110-112, wherein the TBTU is added over a period of about 30 minutes to about 1.5 hours.

[0407] 114. The method of any one of embodiments 110-112, wherein the TBTU is added over a period of about 1 to 1.5 hours.

[0408] 115. The method of any one of embodiments 110-114, wherein the TG PEG is produced with a purity greater than or equal to about 90% as measured by LC.

[0409] 116. The method of any one of embodiments 110-115, wherein at the end of the reaction, the TG PEG dimer impurity is present in an amount of less than 10% as measured by LC.

[0410] 117. The method comprises the following scheme: [ka] 117. The method of any one of embodiments 110 to 116, comprising the reagent of

[0411] 118. A method for preparing NAG-25, comprising treating TG PEG with an activating agent and a phosphitylating reagent.

[0412] 119. The method of embodiment 118, wherein the activator is tetrazole, DCI, ETT, or benzothiotetrazole (BTT).

[0413] 120. The method of embodiment 118, wherein the activating agent is tetrazole, DCI, or ETT.

[0414] 121. The method of embodiment 118, wherein the activating agent is tetrazole.

[0415] 122. The method of embodiment 121, wherein the tetrazole is added in about 0.2 eq to 1.2 eq.

[0416] 123. The method of any one of embodiments 118 to 122, wherein the method is carried out in the presence of a base.

[0417] 124. The method of any one of embodiments 118 to 123, wherein the base is NMI.

[0418] 125. The method of any one of embodiments 118 to 124, wherein the phosphitylation reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.

[0419] 126. The method of any one of embodiments 118-125, further comprising extraction, filtration, precipitation, and drying.

[0420] 127. The method of embodiment 126, wherein the precipitation comprises precipitation of NAG-25 from DCM / heptane.

[0421] 128. NAG-25 [ka] The method of preparing the compound of formula (I) comprising any one of the embodiments relating to steps 1, 2, 3, 4a, 3b, 3a, 4, 5, or 6.

[0422] 129. The method of embodiment 128, wherein the method comprises preparing a t-butyl core by the process of step 1, and converting the t-butyl core to NAG-25.

[0423] 130. The method of embodiment 128, wherein the method comprises preparing a triacid by the process of step 2, and converting the triacid to NAG-25.

[0424] 131. The method of embodiment 128, wherein the method comprises preparing TGZ by the process of step 4b, and converting TGZ to NAG-25.

[0425] 132. The method of embodiment 128, wherein the method comprises preparing NAG-Z by the process of step 3b, and converting NAG-Z to NAG-25.

[0426] 133. The method of embodiment 128, wherein the method comprises preparing TG amine by the process of step 5a, and converting the TG amine to NAG-25.

[0427] 134. The method of embodiment 128, wherein the method comprises preparing TG PEG by the process of step 5b, and converting the TG PEG to NAG-25.

[0428] 135. NAG-25: [ka] 2. A method for preparing TG PEG by treating a mixture of TG amine and PEG acid with TBTU. [ka] To obtain A method comprising converting TG PEG to NAG-25.

[0429] 136. A method for preparing NAG-25, comprising the steps of: high pressure hydrogenolysis for benzyl deprotection of TGZ to give TG amine: [ka] or forming a salt thereof; and converting the TG amine or a salt thereof to NAG-25.

[0430] 137. A method for preparing NAG-25, comprising reacting a triacid with a tert-butyl ether in the presence of a coupling agent and a base. [ka] with NAG-H or a salt thereof to form TGZ, and converting TGZ to NAG-25.

[0431] 138. The method of embodiment 137, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0432] 139. A method for preparing NAG-25, comprising reacting a t-butyl core with an acid to form a triacid, and converting the triacid to NAG-25.

[0433] 140. The method of embodiment 139, wherein the triacid is crystalline.

[0434] 141. The method of embodiment 140, wherein the triacid is in crystalline form I.

[0435] 142. A method for preparing NAG-25, comprising reacting GluZ with GluOtBu, or a salt thereof, to form a t-butyl core, and converting the t-butyl core to NAG-25.

[0436] 143. A method for preparing NAG-25, comprising precipitation of TGZ from a solvent / anti-solvent system.

[0437] 144. A method for preparing NAG-25, comprising a triacid intermediate that is crystalline.

[0438] 145. The method of embodiment 144, wherein the crystalline triacid is crystalline form I of the triacid characterized by an X-ray powder diffractogram having signals at least at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2.

[0439] 146. The method of embodiment 144, wherein the crystalline triacid is crystalline form I of the triacid, characterized by an X-ray powder diffractogram having signals at 2-theta values ​​of 7.4±0.2, 9.2±0.2, and 21.0±0.2.

[0440] 147. The method of any one of embodiments 144-166, wherein the crystalline triacid is crystalline form I of the triacid, characterized by an X-ray powder diffractogram substantially similar to that of FIG. 2.

[0441] 148. The method of any one of embodiments 144-147, wherein the crystalline triacid is crystalline form II of the triacid, characterized by an X-ray powder diffractogram substantially similar to that of FIG. 3.

[0442] 149. Methyl core: [ka] 1. A method for preparing zL-Glu-OMe: [ka] and L-glutamic acid dimethyl ester (di(OMe)Glu): [ka] to produce a methyl core.

[0443] 150. The method of embodiment 149, wherein the reaction is carried out in the presence of a base and a coupling reagent.

[0444] 151. The method of embodiment 150, wherein a base is added to the zL-GluOMe solution, followed by addition of a coupling reagent and di(OMe)Glu to the reaction.

[0445] 152. The method of embodiment 151, wherein a coupling reagent is added to a solution of zL-Glu-OMe and base, followed by addition of di(OMe)Glu to the reaction.

[0446] 153. The method of any one of embodiments 149-152, further comprising a solvent.

[0447] 154. The method of any one of embodiments 150-153, wherein the base is N-methylmorpholine (NMM) or diisopropylethylamine.

[0448] 155. The method of any one of embodiments 150-154, wherein the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC.

[0449] 156. The method of any one of embodiments 153 to 155, wherein the solvent is THF or MeTHF.

[0450] 157. The method of any one of embodiments 149 to 156, wherein the reaction temperature before and during the reaction with the coupling reagent is from about -20°C to about -10°C.

[0451] 158. The method of embodiment 157, wherein the reaction temperature is from about -18°C to about -13°C.

[0452] 159. The method of embodiment 157 or 158, wherein the reaction temperature after addition of di(OMe)Glu is increased to about 5°C to about 15°C.

[0453] 160. The method of embodiment 159, wherein the reaction temperature is increased to about 7°C to about 12°C.

[0454] 161. The method of any one of embodiments 149-160, further comprising additional inputs of coupling reagent, base, and / or zL-Glu-OMe.

[0455] 162. The method of any one of embodiments 149-161, further comprising washing the reaction with an aqueous acid, followed by washing the reaction with an aqueous base.

[0456] 163. The method of any one of embodiments 149-162, further comprising crystallization by adding an antisolvent.

[0457] 164. The method comprises the following scheme: [ka] The method according to any one of embodiments 149 to 163, comprising the reagents and conditions according to any one of embodiments 149 to 163.

[0458] 165. Triol: [ka] A method for preparing a compound according to the present invention comprising the steps of: [ka] to produce a triol.

[0459] 166. The method of embodiment 165, wherein the method is carried out with neat 2-(2-aminoethoxy)ethanol.

[0460] 167. The method of embodiment 165 or 166, wherein the reaction temperature is about 25°C to about 35°C.

[0461] 168. The method of embodiment 167, wherein the reaction temperature is about 30°C.

[0462] 169. The method of any one of embodiments 165-168, further comprising adding an anti-solvent to precipitate the triol.

[0463] 170. The method of embodiment 169, wherein the anti-solvent is ethyl acetate, methyl tert-butyl ether, or iso-propyl acetate.

[0464] 171. The method of any one of embodiments 165-170, further comprising adding triol seed crystals.

[0465] 172. The method comprises the following scheme: [ka] The method according to any one of embodiments 165 to 171, comprising the reagents and conditions according to any one of embodiments 165 to 171.

[0466] 173. A method for preparing TGZ, comprising the steps of: [ka] and Beta-D-Galactosamine Pentaacetate: [ka] to produce TGZ.

[0467] 174. The method of embodiment 173, wherein beta-D-galactosamine pentaacetate is first reacted with a silyl-triflate to produce an oxazoline solution.

[0468] 175. The method of embodiment 174, wherein the silyl-triflate is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or triisopropylsilyl trifluoromethanesulfonate (TIPSOTf).

[0469] 176. The method of embodiment 175, further comprising a solvent.

[0470] 177. The method of embodiment 176, wherein the solvent is dichloroethane (DCE) or dichloromethane (DCM).

[0471] 178. The method of any one of embodiments 173 to 177, wherein the reaction temperature is from about 35°C to about 45°C.

[0472] 179. The method of embodiment 178, wherein the reaction temperature is about 40° C.

[0473] 180. The method of embodiment 178 or 179, wherein the reaction temperature is reduced to about 20°C to about 28°C.

[0474] 181. The method of embodiment 180, wherein the reaction temperature is about 23° C.

[0475] 182. Triol is dissolved in sodium bicarbonate (NaHCO 3 ) and triol and NaHCO 3 The method of embodiment 173, wherein after forming the slurry mixture of the above, beta-D-galactosamine pentaacetate or its oxazoline solution is added.

[0476] 183. Triol and NaHCO 3 183. The method of embodiment 182, wherein the slurry mixture of further comprises a solvent.

[0477] 184. The method of embodiment 183, wherein the solvent is dichloromethane (DCM), dichloroethane, or acetonitrile.

[0478] 185. Oxazoline solution is dissolved in triol and NaHCO 3 185. The method of any one of embodiments 173 to 184, wherein the slurry mixture is added to the

[0479] 186. The method of embodiment 173 or 185, wherein the reaction temperature is about 20°C to about 30°C.

[0480] 187. The method of embodiment 186, wherein the reaction temperature is about 25° C.

[0481] 188. The method of any one of embodiments 173 to 187, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0482] 189. The method of any one of embodiments 173-188, wherein an anti-solvent is added to the solution containing TGZ and the solvent.

[0483] 190. The method of embodiment 188 or 189, wherein the anti-solvent is dimethoxyethane.

[0484] 191. The method of any one of embodiments 173-190, further comprising any one of embodiments 129-144, or 145-152, or embodiments 149-164, or 165-172.

[0485] 192. The method comprises the following scheme: [ka] The method according to any one of embodiments 173 to 191, comprising the reagents and conditions according to any one of embodiments 173 to 191.

[0486] 193. A method for preparing NAG-25, comprising any one of embodiments 149-164 for step A-1, any one of embodiments 165-172 for step A-2, any one of embodiments 173-192 for step A-3, or any one of embodiments 71-89 for step 3b.

[0487] 194. The method of embodiment 193, further comprising any one of embodiments 90 to 109 relating to step 5a, embodiments 110 to 117 relating to step 5b, or embodiments 118 to 127 relating to step 6.

[0488] 195. A method for preparing NAG-25, comprising reacting zL-Glu-OMe with L-glutamic acid dimethyl ester to form a methyl core, and converting the methyl core to NAG-25.

[0489] 196. A method for preparing NAG-25, comprising reacting a methyl core with 2-(2-aminoethoxy)ethanol to form a triol, and converting the triol to NAG-25.

[0490] 197. A method for preparing NAG-25, comprising reacting a triol with beta-D-galactosamine pentaacetate to form TGZ, and converting TGZ to NAG-25.

[0491] 198. The method of embodiment 197, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0492] 199. A method for preparing NAG-25, comprising a triol intermediate.

[0493] 200. Compound [ka] wherein R is H or a Cbz protecting group.

[0494] This disclosure should be read in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. For example, as shown in the following examples, the examples are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. EXAMPLES

[0495] Example 1: Step 1 An example of step 1 is shown below. [ka]

[0496] The GluZ solution is prepared as follows: 1.0 eq GluZ, 3.5 eq NMM, and 5 volumes MTBE are charged to a clean inert reactor 1. In a separate clean inert reactor 2, 1.2 eq PivCl and 5 volumes MTBE are charged at 0° C. Using reverse addition, the GluZ solution in reactor 1 is added to reactor 2 over 1 hour. A white slurry is formed and stirred for at least 30 minutes. Then, 1.3 eq GluOtBu hydrochloride is charged in small portions every 15 minutes (exothermic). The slurry is then held at 0° C. for at least 30 minutes. When the conversion is below 97%, 0.5 eq NMM, 0.4 eq PivCl, and 0.45 eq GluOtBu HCl are charged. If necessary, the reaction mixture can be held at 0° C. for 24 hours. Slowly add 12.5 volumes of 0.5N HCl to the reaction mixture at 0° C. (exothermic) and stir for 30 minutes. Allow the phases to settle and then separate. Wash the organic layer with 10 volumes of 1N sodium carbonate. Allow the phases to settle and then separate. Wash the organic layer with water (10 volumes). Allow the phases to settle and then separate. If necessary, the organic layer can be kept overnight at 0° C. Concentrate the organic layer to 6 volumes at 35° C. and charge heptane (10 volumes) all at once at 35° C. Set the reactor jacket temperature (Tj) to 50° C. and stir until completely dissolved. Then set Tj to 40° C. and charge 0.5 wt % seeds. The t-butyl cores that can be used as seeds can be prepared as described in U.S. Pat. No. 10,246,709 or by methods known to those skilled in the art. The slurry is stirred for at least 30 minutes. Then 15 volumes of heptane are charged at 40° C. over 3 hours. Once heptane addition is complete, the Tj is slowly cooled to 20° C. and held overnight. The slurry is stirred overnight at 20° C. The slurry is filtered and the filter cake is washed twice with heptane (3 volumes). The t-butyl core solids are dried under nitrogen overnight at 20° C.

[0497] 1H NMR(500MHz,DMSO-d6)δ ppm 1.40(d,J=1.30Hz,27H)1.72-1.85(m,2H)1.89-2.02(m,2H)2.21-2.32(m,4H)4.14-4.19(m,1H)4.97-5.13(m ,2H)7.29-7.43(m,8H)7.48(d,J=7.27Hz,2H)7.53(t,J=7.60Hz,2H)7.59-7.68(m,2H)8.01(d,J=7.59Hz,2H)

[0498] Reaction conversion was also improved when combined with 2.4 eq of NMM instead of 3.5 eq.

[0499] Screening of PivCl and GluOtBu HCl [ka]

[0500] An equivalent screening with PivCl and GluOtBu HCl was performed to further increase the reaction conversion and yield. With higher equivalents of PivCl and GluOtBu HCl, the conversion increased; however, the level of impurities increased as well. The goal was to find an amount of PivCl that would result in high conversion to the t-butyl core without increasing the level of impurities produced. The highest conversion was achieved with 1.2 eq of PivCl and 1.3 eq of GluOtBu HCl.

[0501] The table below shows the LCAPs of the t-butyl core from the PivCl equivalent screen.

[0502] [Table 4]

[0503] Base Equivalence Screening [ka]

[0504] Equivalent amounts of NMM were explored using 1.2eq PivCl and 1.3eq GluOtBu HCl. Experiments showed that at the end of the reaction, unreacted mixed anhydride was still present. Determination of the amount of NMM allowed additional GluOtBu HCl to become the free base and allow the reaction to proceed. Excess NMM (3-3.5eq) did not leave any mixed anhydride. Higher equivalent amounts (4-5eq NMM) were explored, but the reaction did not improve further.

[0505] The table below shows the LCAP of the t-butyl core for NMM equivalent screening.

[0506] [Table 5]

[0507] Comparison of forward and reverse addition Another study was performed comparing forward and reverse addition, since it was hypothesized that forward addition may lead to symmetric anhydride formation. A comparison between forward and reverse addition was performed using GluOtBu (1.3eq), NMM (3.5eq), and PivCl (1.2eq). In the forward addition, a solution of PivCl in MTBE was added to a solution of GluZ, NMM, and MTBE, and the reaction was cooled to 0°C and held for 30 min. GluOtBu hydrochloride was then added in three portions every 15 min, and the reaction was then held for 30 min, with samples taken at various time points and measured by liquid chromatography (LC).

[0508] The table below shows the following conditions: [ka] 4 shows a comparison of reverse addition and forward addition for step 1 using

[0509] The table below shows that the forward addition converted the t-butyl core by about 88.7%, while the reverse addition showed about 97.5% or 96.1% conversion.

[0510] [Table 6]

[0511] Solvent / antisolvent screening for crystallization A high-throughput experimental (HTE) solubility screen was performed to determine the solvent / anti-solvent combinations for crystallization, matching the reaction solvent with the crystallization solvent. Solubility studies of the t-butyl core at 20 °C were performed for various percentages of heptane. For 2-MeTHF, MIBK, and IPAc, the t-butyl core was found to have a steep solubility cliff from 50 mg / mL to 10 mg / mL between 80-90% heptane, making it suboptimal for crystallization design. This solubility screen showed that MTBE / heptane is an example of a useful solvent mixture for crystallization due to its moderate solubility curve.

[0512] Example 2: Step 2 An example of step 2 is shown below. [ka]

[0513] 3 Volume of H 3 PO 4 is added to a mixture of t-butyl core (1 eq), 2-MeTHF (3.5 vol), and 0.5 vol water, and the mixture is heated to 50° C. for 4.5 h. Phosphoric acid is efficiently removed by aqueous extraction as follows: the reaction mixture is diluted with isopropyl acetate (iPAC) (10 vol) and 20 wt % ammonium sulfate ((NH 4 ) 2 SO 4) aqueous solution (5 vol). The organic layer is then washed with 1.5 vol water. After azeotropic distillation, acetone (4 vol) is charged and the mixture is polish filtered to remove residual salts. Acetone (2 vol) is rinsed through the filter into the reactor. The mixture is cooled to 45° C. and the reactor is charged with toluene (6 vol). 1 wt % triacid seeds are charged at 45° C. The slurry is stirred at 45° C. After holding for 6-18 hours, toluene (6 vol) is charged over 2.5 hours and the solution is stirred until the mother liquor concentration reaches less than 6 mg / ml. The reactor is cooled to 20° C. and stirred for 0.5 hours or more. The solid product is filtered and washed once with premixed 2:1 toluene:acetone (3 vol). The cake is vacuum dried under a nitrogen stream at 20° C. For the triacid prepared by a similar procedure as described above, the mass spectrum from UPLC-MS was m / z (ESI, positive ion): 411.27 (M+H) + The triacid isolated from this preparation is crystalline form I.

[0514] Instead of using IPAc, an aqueous extraction was performed with MeTHF by washing the crude reaction mixture with 20 wt% ammonium sulfate (5 vol) and water (2 vol). After adding MeTHF (6-10 vol), the mixture was distilled and the addition of MeTHF by distillation was repeated up to two more times. The next step was followed by acetone and polish filtration as described above.

[0515] While investigating the deprotection step, it was found that the addition of water as a tert-butyl acceptor improved the rate and conversion of the deprotection reaction (Table 1) compared to MeTHF as the sole solvent (Table 1; conditions = 2 volumes of H 3 PO 4 Additional experiments showed that 3 volumes of H at 55 or 50 °C 3 PO 4 and 0.5 volumes of water, with a purity of 94-95% by liquid chromatography (LC) and 2 volumes of H 3 PO 4 It was found that the triacid obtained had fewer impurities than the / 60°C system.

[0516] [Table 1]

[0517] Preparation and XRPD Characterization of Triacid Crystalline Form I During process development, high throughput screening led to the discovery of acetone / toluene as a crystallization system. In additional experiments, the triacid crystalline Form I was produced by seeding a 49:51 toluene:acetone mixture at 45° C. and allowing desaturation at 45° C. at which the crystalline triacid formed. Figure 1 is an example of the triacid crystalline Form I. The resulting solids are typically formed at greater than 99% purity by LC.

[0518] Crystal preparation: The triacid crystalline form I was formed by charging the distilled crude reaction stream (after extractive workup, distillation, and two additions-removals of acetone) with acetone (4.9 volumes). The mixture was heated to 48° C. and charged with 4.7 volumes of toluene. At 45° C., seeds were charged. Stirring was continued at 45° C. for about 20 hours, after which an additional 3.5 volumes of toluene were charged. The reactor was cooled to 20° C. over 1 hour, and the solids were filtered off, washed once with 3:2 toluene:acetone, and then dried under vacuum.

[0519] X-ray powder diffraction: X-ray powder diffraction data were obtained on a PANalytical X'Pert PRO X-ray diffraction system equipped with a RTMS detector. Samples were scanned in continuous mode from 5 to 45° (2θ) with a step size of 0.0334° at 45 kV and 40 mA using CuKα radiation (1.54 Å). The incident beam path included 0.02 Radsolar slits, a 15 mm mask, a 4° fixed anti-scatter slit, and a programmable divergence slit. The diffracted beam included 0.02 Radsolar slits, a programmable anti-scatter slit, and a 0.02 mm nickel filter. Samples were prepared on a low background sample holder and placed on a rotating stage with a rotation time of 2 seconds. The XRPD pattern of the toluene / acetone form of the triacid crystalline form I is shown in Figure 2 and the XRPD peaks are listed in Table 2 below.

[0520] [Table 2]

[0521] Preparation of triacid crystalline form II 2 g of the triacid was placed in a 40 mL vial containing a stir bar. 1V (2 mL) of 2-MeTHF and 1.5V (3 mL) of iPAc were added. The mixture was heated to 50° C. with stirring to dissolve the triacid. To this mixture was added 13.5V of iPAc (27 mL). Upon addition of the iPAc, the solution became cloudy and some oiliness was observed. The temperature was increased until an internal temperature of 50° C. was reached. Upon heating and stirring, the oil turned to a brittle solid. The slurry was held at 50° C. for 15 minutes. A sample of the mother liquor concentration was measured at 58 mg / ml triacid. The mixture was cooled to 45° C., resulting in a very viscous slurry, which was then cooled to room temperature. The mother liquor concentration was measured at 5 mg / ml triacid. At this point, the slurry was transferred to a filter. 10V iPAc (20 mL) was charged to loosen the remaining solids in the vial, and this mixture was also charged to the filter. Finally, the vial was rinsed with 2.5V iPAc (5 mL), and this mixture was added to the filter. The solids were dried under vacuum with a stream of nitrogen overnight. 1.55 g of solid was isolated. Quantitative NMR (using maleic acid internal standard, d6-DMSO solvent) showed the material to be 86 wt% triacid and 11 wt% iPAc. The XRPD pattern of triacid crystalline Form II is shown in FIG. 3. The resulting triacid crystalline Form II can be used as a triacid seed in a series of crystallizations using the example of Step 2 described above to produce triacid crystalline Form I.

[0522] Example 3: Step 3a Step 3a: An example of step 3a is shown below. [ka]

[0523] A solution of amino alcohol (1.1 eq), TEA (1.0 eq), and DCM (4 volumes) is cooled to 5° C. CbzCl (1.0 eq) is added over 1 h, and upon completion of the addition, the solution is heated to 35° C. for 12 h. The solution is cooled to 20° C. and quenched by the addition of water (2 volumes). The aqueous layer is collected and back-extracted with DCM (2 volumes). The combined organic layers are washed twice with 10% NaCl (2 volumes) and solvent exchanged into ACN (5 volumes). The Alcohol-Z (abbreviation: Alc-Z) product is then isolated as a clear oil or stored in ACN (5 volumes) for use in the next step. 1 H NMR (400 MHz, chloroform-d) δ 3.41 (q, J = 5.11 Hz, 2H) 3.53-3.60 (m, 4H) 3.69-3.76 (m, 2H), 5.11 (s, 2H), 5.35 (br s, 1H) 7.29-7.43 (m, 5H).

[0524] Example 4: Step 3b Step 3b: An example of step 3b is shown below. [ka]

[0525] In an inert flask, add acyl GalNAc (1.5 eq), Bi(OTf) 3(0.15 eq), and ACN (5 vol). A solution of Alcohol-Z (1.0 eq) in ACN (vol) is azeotropically dried and charged into the flask. The solution is heated to 60°C and stirred for 18 hours. After cooling to 20°C, activated carbon (50 wt%) is charged and stirred at room temperature for 12 hours. The activated carbon is then filtered off and the filtrate is charged with water (18.6 vol). The resulting solution is seeded with NAG-Z (0.5 wt%) and stirred for 30 minutes. If the seeds are not retained, the seeding process is repeated until they are retained. If the seeds are retained, water (38 vol) is added over 1 hour and the resulting slurry is stirred for 12 hours. The resulting product is filtered off, washed with two water washes (2 vol) and vacuum dried under a nitrogen stream. The NAG-Z product is then obtained as a white solid (71.27%, potency adjusted). 1 H NMR(600MHz,DMSO-d6)δ 7.78(d,J=9.20Hz,1H),7.35-7.37(m,2H),7.32-7.35(m,2H),7.30-7.32(m,1H),7.24(br t,J=5.60Hz,1H),5.21(d,J=3.40Hz,1H),5.01(s,2H),4.99(dd,J=3.40,11.20Hz,1H ),4.56(d,J=8.40Hz,1H),3.99-4.04(m,1H),3.98-4.07(m,3H),3.88(ddd,J=8.40,9. 20,11.10Hz,1H),3.74-3.81(m,1H),3.55-3.63(m,1H),3.45-3.55(m,2H),3.38-3.4 3(m,2H),3.14(q,J=5.70Hz,2H),2.10(s,3H),1.99(s,3H),1.89(s,3H),1.77(s,3H).

[0526] Lewis Acid Screening for Step 3b: Certain Lewis acids commonly used in glycosylation reactions gave low yields for the NAG-Z process. Conditions as described below: [ka] A Lewis acid screen was performed below. Lewis acids of moderate strength were found to be useful for NAG-Z formation. Some Lewis acids were weak and did not achieve complete conversion of alcohol-Z, while others were too strong and degraded the resulting NAG-Z into acylated and deacylated products. The results of two related experiments are summarized in the table below. Column A: NAG-Z LCAP shows the LCAP of NAG-Z obtained after 24 hours at the given reaction conditions, and Column B: NAG-Z Decomposition shows the decomposition seen after exposing NAG-Z to the corresponding Lewis acid for 24 hours.

[0527] [Table 7]

[0528] AcylGalNAc Equivalent Screening for Step 3b: Equivalents of acylGalNAc were tested based on the underlying hydrolysis mechanism shown below. [ka] Due to the hygroscopic nature of the bismuth catalyst, it was difficult to avoid hydrolysis during drying, so alcohol-Z was used as the limiting reagent to improve the conversion, and the hydrolysis impurity DGalOH could be removed during NAG-Z crystallization. The conditions used for the equivalent screening are shown below (DGalNAc is the same as acylGalNAc). [ka] The results of this equivalent screening and the main impurities found (acyl GalNAc, DGalOH, alcohol-Z) are summarized below.

[0529] [Table 8]

[0530] Solvent volumes for step 3b: Less solvent volume generally reduces volume (better from a green chemistry / sustainability perspective) and generally results in less product loss to the mother liquor. Therefore, several reaction volumes of acetonitrile were screened. The table below shows the results for Alcohol-Z, NAG-Z, and Ac-Z impurities: [ka] This section provides an overview of LCAP.

[0531] [Table 9]

[0532] Example 5: Step 4a Step 4a: An example of step 4a is shown below. [ka]

[0533] A 5 L pressure vessel was charged with NAG-Z (356.0 g; 97.5 wt%, 610.4 mmol), followed by Pd / C (18.5 g; 5 wt%), DMAc (1.4 L; 4 V), and finally TFA (47 mL; 610.4 mmol). The reactor was sealed and purged with nitrogen gas. The headspace was then purged with hydrogen gas and set to 46 psi (3.1 bar) hydrogen. The reaction was stirred under these conditions for 19 hours at 21.6° C. The catalyst was removed by filtration and washed with DMAc (350 ml; 1 V). The resulting clear solution was assayed and contained 256.9 g (96.7% yield) of NAG-H at 97.1 LCAP.

[0534] Example 6: Step 4b Step 4b: An example of step 4b is shown below. [ka] Note: All equivalents / volumes are relative to the triacid

[0535] A 5 L jacketed reactor equipped with a nitrogen inlet and temperature probe was charged with the triacid (55.0 g, 1.0 equiv) and NAG-H TFA solution (13.27 wt% in DMAc, 3.5 equiv). The mixture was stirred for 10 min to dissolve the triacid and then cooled to Tj=10° C. To the cooled solution was added N-methylimidazole (115 mL, 12.0 equiv) via addition funnel followed by solid TBTU (154 g, 4.0 equiv) in one portion. The jacket temperature was set to 20° C. and the reaction was allowed to warm for 1 h. The reaction was then quenched by slow addition of 50 mM potassium phosphate pH 6 buffer (1100 mL, 20 ml / g triacid). DCM (1100 mL, 20 mL / g) was added and the mixture was stirred for at least 10 min. The phases were separated and the aqueous phase was extracted with additional DCM (1100 mL, 20 mL / g). The combined organic layers were washed with 50 mM pH 6 potassium phosphate buffer (2×1100 mL). The DCM solution was dried to 20V by azeotropic distillation and then supplemented with fresh DCM (1100 mL, 20 mL / g). The reaction stream in 40 mL / g DCM was heated to Tj=30° C. and then MTBE (2200 mL, 40 mL / g) was charged over 3 h. The slurry was cooled to 25° C., aged for up to 4 h, and filtered. The cake was washed with 1:1 DCM:MTBE (2×1100 mL, 5.0 mL / g TGZ) and dried under vacuum to give TGZ as a white solid (88% yield). 1 H NMR (600MHz, DMSO-d 6)δ ppm 7.91(m,3H),7.80(d,J=9.2Hz,4H),7.36(m,5H),7.31(dt,J=8.4,2.7Hz,1H),5.22(d,J=3.4Hz,3H),5.02(s,2 H),4.99(m,3H),4.56(d,J=8.7Hz,2H),4.55(d,J=8.4Hz,1H),4.18(td,J=8.2,5.4Hz,1H),4.03(m,9H),3.94( td,J=8.3,5.7Hz,1H),3.88(m,3H),3.78(m,3H),3.58(m,3H),3.50(m,6H),3.39(m,6H),3.21(m,3H),3.17(m, 3H),2.18(m,2H),2.10(s,11H),1.99(m,9H),1.89(s,9H),1.83(m,2H),1.78(s,6H),1.77(s,3H),1.70(m,2H).

[0536] Screening experiments were performed to determine various embodiments of the reaction conditions described herein. The results of one example of screening are shown in the table below. TBTU and HATU gave high conversion to the TGZ product (LCAP >95).

[0537] [Table 10]

[0538] After two rounds of HTE screening and hit validation, TBTU in DMF led to complete conversion of the intermediate to TGZ with a well-defined reaction profile. While all bases included in this screen were effective, NMI led to the highest LCAP TGZ (96.36 LCAP) with 0.15 LCAP deacylation. These conditions were then validated on a gram scale with complete conversion in 1 h. The reaction was also demonstrated to work equally well using dimethylacetamide (DMAc) as the solvent instead of DMF.

[0539] Additional reagent equivalence screening experiments were also performed. The effect of reaction components on reaction rate is shown in the table below.

[0540] [Table 11]

[0541] Example 7: Step A-1 Step A-1: ​​Amide coupling to prepare the methyl core. An example of this step is shown below. [ka]

[0542] A 1 L reactor was charged with zL-Glu-OMe (50 g, 1.0 equiv.) via a powder addition funnel. THF (10 vol.) was charged under positive nitrogen pressure and completely dissolved in less than 3 min. The reaction was cooled to -15°C. While the reaction was cooling, N-methylmorpholine (NMM) (41.8 mL, 2.5 equiv.) was charged to the reaction. The addition port was rinsed with 0.5 vol. THF and once the reaction temperature reached -15°C, isobutyl chloroformate (IBCF) (22 mL, 1.1 equiv.) was charged and a white precipitate formed immediately. The internal temperature was maintained below -10°C during the addition. The reaction was aged at -15°C for 1 h. L-glutamic acid dimethyl ester (di(OMe)Glu) was charged in 5 equal portions (36.2 g). The reaction was allowed to warm to 10° C. over 2 hours where it was aged for 16 hours. Additional 10 mol % charges of IBCF, NMM, and ZL-Glu-OMe could be added if necessary. The reaction was allowed to continue for an additional 3 hours. Once the reaction reached full conversion, the reaction was allowed to continue until workup.

[0543] Charged 6 volumes of EtOAc, followed by 8 volumes of 0.5M aqueous HCl, separated the phases, and drained the aqueous phase. Charged 8 volumes of 0.5M aqueous NaOH, separated the phases, and drained the aqueous phase. Charged 8 volumes of saturated aqueous brine solution, separated the phases, and drained the aqueous phase. Drained the cloudy organic layer and polish filtered. Returned the organic stream to the clean 1 L reactor. Distilled the organic solution under vacuum (45° C., 170 mmHg) to 400 mL. Two successive azeotropic distillations with EtOAc were performed, and charged an additional 300 mL (total organic approx. 800 mL) and distilled to approx. 400 mL (45° C., 200 mmHG). At 40° C., 4 volumes of heptane were added dropwise. Charged 2.0 wt % methyl core seeds and held at temperature for 30 minutes, then cooled slowly to 23° C. An additional 6 volumes of heptane was added dropwise and the slurry was aged overnight.

[0544] The slurry was drained into a Schott bottle and filtered through a 800 mL medium porosity fritted funnel. The cake was washed with 2×5 volumes of EtOAc / heptane (1:1). The cake was dried under a nitrogen / vacuum stream for 4 hours.

[0545] Example 8: Step A-2 Step A-2: Aminolysis to Prepare Triol An example of this step is shown below. [ka]

[0546] A 100 mL reactor was charged with methyl core (97 wt%, 3.5 g, 1.0 equiv.). The flask was charged with 2-(2-aminoethoxy)ethanol (6 volumes, 21 mL) under positive nitrogen pressure. The temperature was set to 30° C. and aged at this temperature for 24 hours. The reactor flask was charged with 20 volumes of ethyl acetate (70 mL) at 30° C. 2 wt% triol seeds were charged, making sure the seeds were held at temperature. The reaction was cooled to 23° C. and the slurry was aged for 18 hours. The slurry was filtered through a 300 mL filter funnel keeping the cake under a steady nitrogen flow. The cake was washed with 10 volumes of ethyl acetate (EtOAc). The triol cake was collected and returned to a clean and dry 100 mL reactor for reslurry. Charged 15 volumes of EtOAc and warmed the slurry to 40° C.; held for 3 hours; cooled to 23° C. and aged an additional 3 hours. The slurry was filtered through a 500 mL filter funnel. The cake was washed with 10 volumes of EtOAc and dried under nitrogen / vacuum bag for 24 hours. The isolated triol cake was 2.75 g, 55% yield.

[0547] Step A-2: Initial hydrolase screening: A preliminary enzyme screen based on hydrolases was carried out using 10 different lipases, one peptide, one protease, and one aldolase. The reactions were set up at 50 mg scale with the methyl core as the limiting reagent. Enzyme loading was based on the physical state of the reagent: liquid-based enzymes were loaded at 5 μL, lyophilized enzymes were loaded at 5 mg, and solid-supported enzymes were loaded at 50 mg, regardless of unit number. The enzymes were screened against three different solvent systems: MeCN, and THF (20 V), and neat in amino alcohol (8 eq.). The enzyme screening reaction conditions are shown below. [ka]

[0548] The reactions were set up in 2 mL vials and agitated on a shaker plate at 45° C. for 48 h. The crude reaction mixtures were then analyzed by HPLC, and no subsequent workup or isolation was performed. Enzyme reactivity was significantly affected by the reaction medium, and in general, reaction outcomes followed the following trend: solvent-free conditions > THF > MeCN. For all reactions carried out under solvent-free conditions, the triol was the major crude reaction component, and the methyl core, as well as the intermediate mono- and di-addition products, were completely consumed, which may be an inherent result of the high reaction concentration and the higher equivalent loading of amino alcohol. Alternatively, most reactions yielded complex mixtures with unreacted starting material, identifying MeCN as a poor solvent for biocatalytic aminolysis. Reactions in THF showed productive conversion of starting materials and intermediates, although the extent of conversion varied with the enzyme ( FIG. 5 ). It should be noted that although some enzymes (especially liquid-based variants) performed better in THF, the solid-supported enzymes showed optimal results under solvent-free conditions (Table 3). The advantage of these immobilized enzymes is that an aqueous workup is no longer necessary. The triol product is water-soluble, and if aqueous washes are required to remove enzyme residues from the organic product, there is a risk that the desired triol will be lost in the same aqueous phase. Enzymes immobilized on solid supports allow this reagent to be filtered out of the organic reaction solvent and subsequently reused, minimizing enzyme degradation. In addition, once the enzyme reagent is removed from the reaction mixture, the triol may be isolated by precipitation from most organic solvents, including solvent-free reaction conditions. Of the most successful reactions between THF and solvent-free conditions, triols were obtained at the high end of the 50-65% LCAP range, with Novozym 51032, Lipozyme CALB L, Resinase HT, Novozym 435, and Lypozyme TL (both solid-supported and liquid-based) emerging as superior enzymes for the conversion (Table 3). These results indicate that lipases may be the most suitable subclass of hydrolases for the desired reaction.The diol impurity was generally formed at less than 10% liquid chromatography area percentage (LCAP) for all reactions, supporting our hypothesis that lowering the reaction temperature could prevent diol formation. The major impurity formed in all reactions (15-30% LCAP) was not isolated or fully identified, and LCMS analysis of the unknown impurity showed it had the same mass as the triol product and is hypothesized to be a stereoisomer of the triol.

[0549] [Table 3]

[0550] These experiments demonstrated the feasibility of biocatalytic aminolysis reactions to afford triols. Conditions were identified that allowed complete conversion of the starting materials and intermediate mono- and di-amides to afford triols as the major reaction products.

[0551] Example 9: Step A-3 Step A-3: Triple glycosylation to prepare TGZ. An example of this step is shown below. [ka]

[0552] A 100 mL reactor flask was charged with beta-D-galactosamine pentaacetate (10.1 g). The flask was charged with DCE (37 mL, 0.7 M) under positive nitrogen pressure. TMSOTf (6.0 mL) was charged slowly first due to some initial vapor evolution. The reaction was warmed to 40° C. over 1.5 hours and then cooled to 23° C. During this time, the reaction flask containing the triol was prepared. A 50 mL reactor was charged with triol (95 w%, 2.5 g, 1.0 equiv.), followed by solid NaHCO 3 (0.2 equiv.). The flask was sealed and the headspace was inerted with nitrogen flush.3 To the mixture was charged DCM (6 vol). The oxazoline solution was charged portionwise (1.0 eq oxazoline solution / hr, max 4.5 eq) to the triol / DCM slurry at 25° C. The reaction was aged overnight (24 hr) at 25° C. Et 3 The reaction was quenched by charging N (3.0 equiv.) and aging for 20 min.

[0553] saturated (sat.) aqueous (aq.) NaHCO 3 (10 vol); separate phases, drain DCM / DCE phase, remove aqueous phase and recharge DCM / DCE solution; rinse with 2 vol DCM. 4 Cl (10 vol) was charged; the phases were separated, the DCM / DCE layer was drained and the aqueous layer was removed. The organic stream was washed with MgSO 4 The precipitate was dried at 40° C. and filtered through a filter funnel; the dry solid was rinsed with 2 volumes of DCM. A clean 100 mL reactor was charged with the DCM / DCE organic solution (approximately 35 mL of organic solution, 14 volumes relative to starting material). DME (17 volumes, 42 mL) was added dropwise to the organic solution over 1 hour to give a 1:1.2 ratio of solvent:anti-solvent. The precipitate was aged overnight (20 hours).

[0554] The slurry was filtered through a medium porosity fritted funnel; 5 volumes of DCM / DME (1:1) were used to rinse the reactor and wash the cake. The cake was dried under nitrogen / vacuum flow for 2 hours. The solid was collected and placed in a 100 mL reactor. 20 volumes (relative to starting material) of DCM / DME solution (1:1) were charged. The slurry was warmed to 40° C.; held for 3 hours; then cooled to 25° C. and aged for an additional 2 hours. The slurry was filtered through a filter funnel; the cake was washed with 5 volumes (DCM / DME 1:1). The analysis in the filtrate mother liquor was 3.5 mg / mL for TGZ. The TGZ cake was 92.6 LCAP. The cake was dried under nitrogen / vacuum bag to give TGZ as a solid.

[0555] Example 10: Step 5a An example of step 5a is shown below. [ka]

[0556] Charge the high pressure reactor with TGZ (1.0 eq.) and add 7% Pd / C catalyst (5 wt% loading). Charge DCM solvent (10 volumes) followed by TFA (1.0 eq.). Seal the reactor and purge the reactor with nitrogen gas three times, start agitation during charging and stop agitation during venting. Purge the reactor with H2 gas three times at 50 or 60 psi without agitation. Set the reaction agitation speed to 300 rpm, jacket temperature to 23°C, and H2 pressure to 45 psi. Age the reaction overnight. Purge the headspace with nitrogen gas three times and then remove the lid. Filter the heterogeneous solution through a 0.45 micron PTE filter, rinse the reactor with 3 volumes of DCM, and add the rinses to the filter. Do not allow the Pd / C cake to dry out. Assay the solution for TG amine potency and store the TG amine-TFA solution in DCM below 10°C. TG amine solution purity = 97.2% Mass analysis: expected mass = 1525.64147, observed m / z: 1525.63930.

[0557] Example 11: Step 5b An example of step 5b is shown below. [ka]

[0558] The TG amine solution in DCM is adjusted to 12 volumes of DCM per LC assay determination of TG amine (1.0 eq.). The reactor headspace is purged with N2, the reactor is set under positive N2 pressure, the jacket temperature is set to -5°C, and stirring is started. PEG acid (1.1 eq.), measured by mass, is added, followed by DIPEA (3.0 eq.). TBTU (1.3 eq.) solid is added to the reactor in small portions over 1 h under a N2 sweep. Once the TBTU solid is completely dissolved (approximately 1 h), the jacket temperature is set to a ramp to 0°C over 30 min.

[0559] The reaction is charged with 12 volumes of DI water and the internal temperature is set to 10°C. The biphasic mixture is stirred for 30 minutes, then the layers are separated, the DCM waste is drained, and the reactor contents are warmed to 23°C. 13 wt% (NH4)2SO4 (relative to water charge) is added to the aqueous TG PEG solution, followed by 10 volumes of DCM. The layers are mixed for 30 minutes, then the layers are allowed to separate. The layers are separated and the organic layer is washed with a mixture of 0.5M sodium monophosphate pH 6.0 and saturated brine (4.8 vol:3.2 vol). The layers are mixed for 30 minutes, then the layers are separated. The organic layer is washed with a mixture of saturated sodium bicarbonate and saturated brine solution (4.0 vol:4.0 vol). The layers are mixed for 30 minutes, then the layers are separated.

[0560] Charge the reactor with the TG PEG / DCM solution and distill to 5 vol. Add another 10 vol. of fresh DCM and distill again to 5 vol.

[0561] The concentrated TG PEG / DCM solution is drained and the reactor is rinsed with 0.5 volumes of DCM. The reactor is charged with 20 volumes of MTBE and the internal temperature is set to 0° C. The TG PEG / DCM solution is added to the cold MTBE over 1 hour. Once the addition is complete, the mixture is filtered. The filter cake is washed with 3 volumes of MTBE (×2). The solids are then dried with a nitrogen sweep for at least 24 hours.

[0562] TG PEG isolated yield = 79.8% potency adjusted. HRMS: Exact mass = 1861.81989, Found m / z: 931.41 [M+2H]+

[0563] High-throughput experiments on reaction conditions were carried out using 96-well plates. Example conditions for screening were as follows: 1.0 equiv TG amine TFA salt, 1.1 equiv PEG acid, 3.0 equiv base, and 1.1 equiv coupling reagent. Each combination was evaluated in DCM, DMF, and MeCN (see results in Figure 4). The bases evaluated were DIPEA, NMI, NMM, and TMP. Results were evaluated based on the LCAP of TG PEG. From this screening, HATU and TBTU provided TG PEG with an LCAP of over 75 with complete conversion of starting materials and intermediates. Order of addition experiments were also performed, in which each of the components (DIPEA, PEG acid, TG amine TFA solution, TBTU) was added last, and the percentage of impurities (e.g., deacylation or TG PEG dimer) was measured by LC. The addition of TBTU resulted in the lowest amount of overall impurities (eg, reduction of TG PEG dimers) and the highest TG PEG yield.

[0564] Example 12: Step 6 An example of step 6 is shown below. [ka]

[0565] NAG-25 was prepared from TG PEG using a process description similar to the above scheme, which is further described in the steps below. HRMS: expected mass = 2078.95400, observed m / z: 2078.95428 [M+NH4]+

[0566] The reactor is charged with TG PEG (1.0 eq), DCM (20V), followed by NMI (0.2 eq) and distilled to less than 200 ppm water. The reaction volume is then reduced to 15V and then cooled to 0-5°C before adding tetrazole (0.6 eq) and P reagent 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite (1.25 eq). Upon completion of addition at 0-5°C, the reaction is then warmed to 20±5°C and aged for 2.5 hours before sampling for conversion of starting TG PEG. Upon completion, the reaction is cooled to 10°C and then washed twice with 10V DI water. The organic solution is dried over molecular sieves and filtered under nitrogen protection. The resulting filtrate is concentrated to 8V at 20°C and then precipitated into 30V heptane by reverse addition. The resulting solid is isolated by filtration, washed twice with 10 V of heptane, and then vacuum dried under nitrogen.

[0567] An alternative example for the preparation of NAG-25 from TG PEG is as follows: A solution of TG PEG (24.87 g, 13.36 mmol, 1 equiv.) was prepared in anhydrous DCM (186.5 mL, 0.07 M). A separate solution containing the P reagent 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite (5.57 g, 16.70 mmol, 1.25 equiv.) and 4,5-dicyanoimidazole (0.039 g, 0.33 mmol, 0.025 equiv.) was prepared in anhydrous DCM (186.5 mL, 0.07 M). The TG PEG solution was charged to the P reagent / DCI solution over 5 hours and allowed to react at 40° C. for at least 10 hours. After the reaction was complete, three aqueous extractions with DI water (3×248.7 mL) were performed. The NAG-25 solution was then azeotropically dried under vacuum at 30° C. After filtration of the solution, anhydrous heptane (745 mL) was added to the NAG-25 solution over 90 min. The solid was filtered off, washed twice with anhydrous 4:1 heptane:DCM (198.9 mL heptane / 49.7 mL DCM) and concentrated under vacuum / N 2 It was dried overnight under

[0568] Experiments were performed with respect to reaction conditions such as activator and amount, P reagent and amount, solvent volume, reaction time, addition order, and addition rate. At various time points, reaction samples were taken and run by liquid chromatography to determine LCAP for TG PEG, H-Phos, NAG-25 dimer, NAG-25, and other impurities. LC results showed that the addition of TG PEG to the P reagent / DCI over a period of 2.5-10 hours was useful in reducing NAG-25 dimer formation. In addition, 1.25 eq of P reagent increased NAG-25 conversion. Results also showed an overall reduction in H-PHOS after precipitation.

[0569] While examples of certain specific embodiments have been provided herein, it will be apparent to those skilled in the art that various changes and modifications may be made. Such modifications are also intended to fall within the scope of the appended claims.

[0570] The foregoing descriptions are set forth merely for clarity of understanding, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art.

[0571] Throughout this specification, when a composition is described as comprising ingredients or raw materials, it is contemplated that the composition may also consist essentially of or consist of any combination of the listed ingredients or raw materials, unless otherwise stated. Similarly, when a method is described as comprising specific steps, it is contemplated that the method may also consist essentially of or consist of any combination of the listed steps, unless otherwise stated. The invention illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0572] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0573] The implementation of the methods disclosed herein, and their individual steps, may be performed manually and / or with the aid of automation provided by electronic equipment. Although the methods are described with respect to specific embodiments, those skilled in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various steps may be altered without departing from the scope or spirit of the methods, unless otherwise indicated. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

Claims

1. NAG-25: 【Chemical 124】 1. A method for preparing a TG PEG: 【Chemistry 125】 with an activating agent and a phosphitylation reagent, wherein the activating agent is tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), or benzothiotetrazole (BTT); and / or the phosphitylation reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.

2. 2. The process of claim 1, wherein the process is carried out in the presence of a base, and optionally the base is N-methylimidazole (NMI).

3. a) the TG PEG is present in a solution, and an activating agent and a phosphitylating reagent are combined in a separate solution; b) adding said solution of TG PEG to said solution of activator and P reagent over a period of time, optionally said period of time being about 2.5-10 hours, or optionally reacting said solution of TG PEG with said solution of P reagent and activator at about 35-45° C.; The method according to claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein NAG-25 is precipitated into an antisolvent using reverse precipitation.

5. The method of any one of claims 1 to 4, wherein the precipitation comprises precipitation of NAG-25 from dichloromethane (DCM) / heptane as a solvent / anti-solvent system.

6. TG PEG: 【Chemistry 126】 1. A method for preparing a TG amine comprising the steps of: 【Chemistry 127】 or a salt thereof, and a PEG acid with a coupling reagent, wherein the coupling reagent is added to the solution of the PEG acid and the TG amine or a salt thereof over a period of about 30 minutes to about 1.5 hours.

7. The method of claim 6, wherein the solution further comprises a base, optionally wherein the base is N,N-diisopropylethylamine (DIPEA).

8. The method according to any one of claims 6 to 7, wherein the coupling reagent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU).

9. 9. The method of any one of claims 6 to 8, wherein the TG PEG is produced in a purity greater than or equal to about 90% as measured by LC, or wherein the TG PEG dimer impurity is present in an amount of less than 10% at the end of the reaction as measured by LC.

10. The method according to any one of claims 6 to 9, wherein the solution of TG amine or a salt thereof is passed on from a separation process without isolation of the TG amine or a salt thereof, and optionally the TG amine or a salt thereof is a TG amine trifluoroacetate (TFA) salt.

11. The method of any one of claims 6 to 10, further comprising converting the TG PEG to NAG-25.

12. TG Amine: 【Chemistry 128】 or a salt thereof, comprising the steps of: A process comprising high pressure hydrogenolysis of TGZ to form said TG amine or a salt thereof, said hydrogenolysis being carried out in the presence of an acid.

13. 13. The method of claim 12, comprising a source of palladium for hydrogenolysis, optionally the source of palladium is a palladium on carbon (Pd / C) catalyst, optionally the source of palladium is 5% Pd / C.

14. The acid is trifluoroacetic acid (TFA), oxalic acid, hydrochloric acid (HCl), acetic acid (AcOH), phosphoric acid (H ３ P.O. ４ ), or citric acid, optionally wherein the acid is about 1.1 eq or less.

15. 15. The method of any one of claims 12 to 14, wherein the hydrogenolysis is carried out in a solvent, optionally the solvent being DCM, isopropyl acetate (IPAc) or methanol (MeOH).

16. 16. The method of any one of claims 12 to 15, wherein the method reduces the formation of one or more impurities which are TG amine deacylation, TG amine diNAG-OH, TG amin guanidinyl, TG amine acyl, TG acetamide, and / or NAG-H guanidine.

17. The method according to any one of claims 12 to 16, wherein the TG amine or its salt is sent to another process for preparing TG PEG as a solution of the TG amine or its salt and without isolating the TG amine or its salt.

18. The method according to any one of claims 12 to 17, wherein the TG amine or a salt thereof is a TG amine acid salt, optionally a phosphate, formate, acetate, trifluoroacetate or oxalate.

19. The method of any one of claims 12 to 18, further comprising converting the TG amine or salt thereof to NAG-25.

20. T.G.Z.: 【Chemistry 129】 The method for preparing the compound of formula (I) comprises reacting a triacid with NAG-H in the presence of a coupling reagent and a base: 【Chemistry 130】 or a salt thereof; and precipitating TGZ from the solvent / anti-solvent system.

21. 21. The method of claim 20, wherein the coupling reagent is TBTU, HATU, or chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TCFH), optionally wherein the coupling reagent is TBTU.

22. 22. The method of claim 20 or 21, wherein the base is diisopropylethylamine (DIPEA), N-methylimidazole (NMI), N-methylmorpholine (NMM), or 2,2,6,6-tetramethylpiperidine (TMP), optionally wherein the base is NMI.

23. 23. The method of any one of claims 20-22, further comprising at least one buffer wash, optionally wherein the buffer wash is at about pH 5-7.

24. The method of any one of claims 20 to 23, wherein the reacting is carried out in a solvent selected from DCM, DMF, MeCN, and DMAc, or a combination thereof.

25. The method according to any one of claims 20 to 24, wherein an anti-solvent is added to the solution comprising TGZ and the solvent.

26. 26. The method of any one of claims 24 to 25, wherein the anti-solvent is an ethereal solvent, optionally the ethereal solvent is DME, 2-MeTHF, or MTBE.

27. 27. The method of any one of claims 20-26, wherein TGZ is prepared with reduced levels of one or more impurities, said impurities being mono-NAG, di-NAG, di-NAG-AZLACTONE, deacylated, di-NAG-OH, di-NAG-OAc, NAG-H guanidine, or NAG-H trifluoroacetamide.

28. The method according to any one of claims 20 to 27, wherein TGZ is obtained in a purity of at least 95% without the use of column chromatography.

29. The method according to any one of claims 20 to 28, wherein the NAG-H or a salt thereof is a solution transferred from another process without isolating the NAG-H or a salt thereof.

30. 30. The method of any one of claims 20 to 29, further comprising converting the TGZ to NAG-25.

31. A method for preparing NAG-H or a salt thereof, comprising reacting NAG-Z in the presence of a Pd / C catalyst and a solvent: 【Chemistry 131】 and optionally wherein the solvent is dimethylacetamide (DMAc) or DCM.

32. 32. The method of claim 31 , wherein the DMAc or DCM is from about 3 volumes to about 6 volumes.

33. 33. The method of any one of claims 31 to 32, further comprising an acid, optionally wherein the acid is trifluoroacetic acid (TFA), acetic acid (AcOH), or pivalic acid (PivOH), and optionally wherein the acid is present in an amount of 0.01 to 0.04 equivalents relative to alcohol-Z.

34. The NAG-H or a salt thereof is NAG-H TFA: 【Chemistry 132】 The method according to any one of claims 31 to 33, wherein

35. 35. The method of any one of claims 31 to 34, wherein the Pd / C catalyst is 5-10% Pd / C, optionally the Pd / C is 5% Pd / C.

36. The method of any one of claims 31 to 35, further comprising converting the NAG-H or a salt thereof to NAG-25.

37. NAG-Z: 【Chemistry 133】 A method for preparing an acylGalNAc: 【Chemistry 134】 and Alcohol-Z: 【Chemistry 135】 to produce NAG-Z, wherein the reaction is carried out in the presence of an acid.

38. The acid is bismuth trifluoromethanesulfonate (Bi(OTf) ３ ), boron trifluoride etherate (BF ３ OEt ２ ), tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), indium triflate (In(OTf) ３ ), or copper triflate (Cu(OTf) ２ 38. The method of claim 37, wherein

39. A process according to any one of claims 37 to 38, wherein the reaction is carried out in the presence of a solvent selected from acetonitrile, dichloromethane and dichloroethane.

40. 40. The method of any one of claims 37-39, wherein the acylGalNAc is present in about 1.0-2.0 equivalents relative to alcohol-Z; or the acid is present in about 0.01-0.1 equivalents relative to alcohol-Z.

41. 41. The process of any one of claims 37 to 40, wherein the reaction temperature is heated to about 55-65°C or 58-62°C.

42. The method according to any one of claims 37 to 41, wherein the solvent is acetonitrile and an anti-solvent is added to crystallize the NAG-Z, and optionally the anti-solvent is water or MTBE.

43. The method of any one of claims 37 to 42, further comprising converting the NAG-Z to NAG-25.

44. A process for preparing alcohol-Z, comprising reacting benzyl chloroformate with an amino alcohol in the presence of a solvent and a base.

45. 45. The method of claim 43 or 44, wherein the solvent is dichloromethane (DCM) and the base is triethylamine (TEA).

46. 46. ​​The method of any one of claims 43 to 45, wherein the amino alcohol is present in 1.1 equivalents relative to the benzyl chloroformate and / or the reaction temperature is about 30-40°C.

47. Crystalline Form I of the triacid, characterized by an X-ray powder diffractogram having at least signals at three 2-theta values ​​selected from: 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2, or characterized by an X-ray powder diffractogram substantially similar to that of FIG.

48. Crystalline Form II of the triacid, characterized by an X-ray powder diffractogram having at least signals at three 2-theta values ​​selected from: 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.2, or characterized by an X-ray powder diffractogram substantially similar to that of FIG.

49. Triacid: 【Chemistry 136】 The method of preparing the compound of formula (I) comprises reacting a t-butyl core with an acid, the acid being phosphoric acid (H ３ P.O. ４ ), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

50. 50. The method of claim 49, wherein the reaction is carried out in a solvent selected from 2-MeTHF, acetonitrile, THF, DMA, sulfolane, and DME.

51. A method according to any one of claims 49 to 50, wherein the solvent is mixed with water, and optionally the solvent is 2-MeTHF.

52. 52. The method of any one of claims 49-51, wherein the solvent is about 3.0-4.0 volumes of 2-MeTHF mixed with about 0.5-2 volumes of water.

53. 53. The process of any one of claims 49 to 52, wherein the reaction is carried out at a temperature of about 40 to 60°C.

54. 54. The method of any one of claims 49 to 53, wherein the triacid is isolated as a crystalline solid.

55. 55. The method of any one of claims 49 to 54, further comprising crystallization of the triacid using acetone / toluene, 2-MeTHF / IPAc, 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile.

56. 56. The method of any one of claims 49 to 55, wherein the triacid is obtained in a crystalline form characterized by an X-ray powder diffractogram having at least signals at three 2-theta values ​​selected from 7.4±0.2, 9.2±0.2, 21.0±0.2, 14.6±0.2, 18.3±0.2, and 19.3±0.2, or characterized by an X-ray powder diffractogram having at least signals at three 2-theta values ​​selected from 7.8±0.2, 9.7±0.2, 13.3±0.2, 15.7±0.2, 17.1±0.2, 18.9±0.2, 20.2±0.2, and 21.0±0.

2.

57. 57. The method of any one of claims 49 to 56, further comprising converting the triacid to NAG-25.

58. t-Butyl Core: 【Chemistry 137】 GluZ: 【Chemistry 138】 and GluOtBu or a salt thereof: 【Chemistry 139】 to produce a t-butyl core, said reacting being carried out in the presence of a coupling reagent, a base, and a solvent; optionally a) the coupling reagent is EDC / Oxyma, TFFH, PyOxim, CDI, PivCl, T3P, or COMU; or b) the base is N-methylmorpholine (NMM); and / or c) the solvent is IPAc, MeTHF, MIBK, or MTBE.

59. GluOtBu or a salt thereof is GluOtBu hydrochloride: 【Chemistry 140】 59. The method of claim 58, wherein:

60. 60. The method of claim 58 or 59.

61. 61. The method of any one of claims 58 to 60, wherein the coupling reagent is PivCl and the solvent is MTBE; optionally, the GluOtBu is about 1-1.5 eq relative to GluZ; or the PivCl is about 1-1.5 eq; or the NMM is about 3-4 eq; or the MTBE is about 8-12 volumes, respectively.

62. 62. The method of any one of claims 58 to 61, wherein a first solution comprising GluZ and NMM in a solvent is added to a second solution comprising said solvent and a coupling reagent; optionally, said coupling reagent is present in excess and / or GluOtBu is present in excess, and optionally, said coupling reagent is PivCl.

63. 63. The method of any one of claims 58 to 62, further comprising the addition of an anti-solvent, optionally over a period of about 2 to 5 hours.

64. 64. The method of any one of claims 58-63, further comprising converting the t-butyl core to NAG-25.

65. Methyl core: 【Chemistry 141】 The method for preparing z-L-Glu-OMe: 【Chemistry 142】 and L-glutamic acid dimethyl ester (di(OMe)Glu): 【Chemistry 143】 to produce a methyl core.

66. 66. The method of claim 65, wherein a) the base is added to a solution of z-L-GluOMe prior to adding the coupling reagent and di(OMe)Glu to the reaction, or b) the coupling reagent is added to the solution of z-L-Glu-OMe and base prior to adding di(OMe)Glu to the reaction.

67. 67. The method of any one of claims 65 to 66, further comprising a solvent, optionally wherein the solvent is THF or MeTHF.

68. 68. The method of any one of claims 65 to 67, wherein the base is N-methylmorpholine (NMM) or di-isopropylethylamine, and / or the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC.

69. 69. The method of any one of claims 65-68, further comprising converting the methyl core to NAG-25.

70. Compound: 【Chemistry 144】 wherein R is H (unprotected triol) or a Cbz protecting group (triol).

71. Triol: 【Chemistry 145】 A method for preparing a compound of formula (I) comprising reacting a methyl core with 2-(2-aminoethoxy)ethanol: 【Chemistry 146】 to produce a triol, and optionally converting the triol to NAG-25.

72. A method for preparing TGZ, comprising the steps of: 【Chemistry 147】 and beta-D-galactosamine pentaacetate: 【Chemistry 148】 to produce TGZ, and optionally converting said TGZ to NAG-25.

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