Process for preparing NAG-25 carbohydrate targeting moieties and intermediates thereof

Abstract

Provided herein are improved processes for the preparation of carbohydrate targeting moieties and intermediates (I) thereof. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 337,316 filed on May 02, 2022, U.S. Provisional Patent Application No. 63 / 373,823 filed on August 29, 2022, and U.S. Provisional Patent Application No. 63 / 385,615 filed on November 30, 2022, each of which is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to improved methods for preparing carbohydrate targeting moieties and intermediates thereof, and compositions of any of the foregoing.Carbohydrate targeting moieties can be used for conjugation with therapeutic compounds to direct therapy to a target in vivo. Background Art

[0004] In order to make certain compounds have therapeutic effects (for example, in vivo) or can be used for diagnostic purposes, it is necessary to deliver the compound to a specific location, such as a target cell. Effectively delivering the compound to a specific location or target can also reduce or eliminate the unexpected results (for example, off-target effects) caused by the administration of the compound. The targeting moiety (for example, a carbohydrate targeting moiety) can be connected to such a compound, such as a therapeutic compound (for example, an oligomeric compound), to guide therapy to a target in vivo. An example of an oligomeric compound is a small interfering RNA (siRNA), which can regulate the expression of a target nucleic acid, resulting in a change in the translation of the target nucleic acid to a protein. For example, siRNA can regulate the expression of a target gene to inhibit protein translation or expression.

[0005] Targeting moieties containing N-acetylgalactosamine (GalNAc or NAG) (e.g., triGalNAc or triantennary GalNAc) facilitate delivery of therapeutic compounds to hepatocytes via binding to asialoglycoprotein receptors. U.S. Pat. No. 10,246,709 describes exemplary GalNAc-containing targeting moieties for delivering oligomeric compounds to the liver, including a compound designated NAG-25 (or NAG25). U.S. Pat. No. 10,246,709 discloses a method for preparing NAG-25 having the following structure:

[0006]

[0007] wherein Ac is acetyl (or -(C=O)CH3), and i-Pr is isopropyl (or 1-methylethyl). Summary of the invention

[0008] Described herein are methods for preparing NAG-25 that arise from the recognition of a need for more efficient, simplified, environmentally friendly and / or economical methods for making NAG-25, including methods that produce NAG-25 and its intermediates with increased purity and / or yield. Such methods do not contain chromatography steps, minimize or prevent the formation of impurities, reduce or eliminate reactive impurities, and / or improve the separation points and physical properties of intermediates. Thus, described herein are methods for preparing NAG-25 thereby without the use of column chromatography. Further described herein are methods for preparing NAG-25 thereby using a crystalline form of the triacid intermediate. Further described herein are additional new and improved methods. As a result of the method steps, the separation and yield of NAG-25 intermediates are improved, impurities are reduced, and waste is reduced. Further described herein are compositions comprising NAG-25, or one of its intermediates (e.g., a tert-butyl core, a triacid or a salt thereof, an alcohol-Z, NAG-Z, NAG-H or a salt thereof, a triol, an unprotected triol, TGZ, a TG amine or a salt thereof, TG PEG, and / or a salt of any of the foregoing), having reduced levels of impurities, such as the impurities and impurity percentages described herein.

[0009] Disclosed herein is a method for preparing a tert-butyl core

[0010]

[0011] The method comprises causing GluZ to:

[0012]

[0013] With GluOtBu or its salts:

[0014]

[0015] Reaction to produce a tert-butyl core. In some embodiments, GluOtBu or a salt thereof is GluOtBu hydrochloride:

[0016] 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 anti-solvent is heptane. In some embodiments, the solvent is MTBE, and the anti-solvent is heptane. In some embodiments, a first solution of GluZ and NMM contained in a solvent is added to a second solution containing the solvent and PivCl. In some embodiments, PivCl is excessive. In some embodiments, GluOtBu is excessive. In some embodiments, based on the amount of GluZ, the conversion rate of the tert-butyl core is greater than about 90%.

[0017] Disclosed herein is a method for preparing triacids:

[0018]

[0019] The method includes reacting the tert-butyl core with an acid. In some embodiments, the acid is phosphoric acid (H3PO4), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid. In some embodiments, the acid is phosphoric acid (H3PO4). In some embodiments, the reaction is carried out in a solvent selected from the following: 2-MeTHF, acetonitrile, THF, DMA, cyclopentane, 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 separated into a crystalline solid. In some embodiments, the triacid has a purity greater than or equal to 95%, as measured 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°C-60°C. In some embodiments, the reaction is carried out at a temperature of about 45°C-55°C. In some embodiments, the reaction is carried out at a temperature of about 50°C. In some embodiments, the method further includes removing phosphoric acid with at least one organic / aqueous washing liquid. In some embodiments, the organic / aqueous washing liquid comprises isopropyl acetate (iPAC) as an organic layer and ammonium sulfate as an aqueous layer. In some embodiments, about 8-18 volumes of iPAC and about 3-7 volumes of 20wt% ammonium sulfate are used. In some embodiments, about 10 volumes of iPAC and about 5 volumes of 20wt% ammonium sulfate are used. In some embodiments, the method further includes crystallizing the triacid using acetone / toluene, 2-MeTHF / IPAc / , 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile. In some embodiments, the method further includes crystallizing the triacid using acetone / toluene. In some embodiments, the ratio of acetone / toluene is 2:3, 1:1, 3:2, or 1:2. In some embodiments, the ratio of acetone / toluene is 2:3, 1:1, or 1:2. In some embodiments, these methods produce the triacid in crystalline form characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ 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.

[0020] Disclosed herein is a crystalline Form I of a triacid characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ values ​​selected from the group consisting 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. In some embodiments, the crystalline Form I of the triacid is characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 7.4±0.2, 9.2±0.2, and 21.0±0.2. In some embodiments, the crystalline Form I of the triacid is characterized by an X-ray powder diffraction pattern substantially similar to Figure 2 In some embodiments, the crystalline Form II of the triacid is characterized by an X-ray powder diffraction pattern substantially similar to Figure 3 X-ray powder diffraction pattern of .

[0021] This article discloses a method for preparing TGZ.

[0022]

[0023] The method comprises reacting a triacid with NAG-H in the presence of a coupling reagent and a base:

[0024] In some embodiments, NAG-H or its salt is a TFA salt of NAG-H: 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 buffered wash solution. In some embodiments, the buffered wash solution is a phosphate buffer. In some embodiments, the buffered wash solution is about pH 5-7. In some embodiments, the buffered wash solution is about pH 6. In some embodiments, the method is carried out in a solvent selected from the following or a combination thereof: DCM, DMF, MeCN, and DMAc. 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 a solution comprising TGZ and a 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, the prepared TGZ impurities are reduced. In some embodiments, the impurity by-product is deacylating. In some embodiments, the method further comprises precipitating TGZ from a solvent / anti-solvent system. In some embodiments, the solvent / anti-solvent system is DCM / MTBE. In some embodiments, TGZ is obtained with a purity of at least 95% without the use of column chromatography. In some embodiments, NAG-H, or a salt thereof, is continued as a solution from a previous reaction for reaction with the triacid without isolating NAG-H, or a salt thereof, from a previous reaction. In some embodiments, NAG-H, or a salt thereof, is prepared in a previous reaction comprising hydrogenating NAG-Z in the presence of a Pd / C catalyst and DCM or dimethylacetamide (DMAc). In some embodiments, DCM is about 3 volumes to about 6 volumes. In some embodiments, DMAc is about 3 volumes to about 5 volumes. In some embodiments, DMAc is about 4 volumes. In some embodiments, NAG-H or a salt thereof is NAG-H TFA. In some embodiments, the Pd / C catalyst is 5% Pd / C. In some embodiments, NAG-H TFA is prepared in a previous reaction of reagents contained in the following schemes:

[0025]

[0026] Disclosed herein is a method for preparing a methyl core,

[0027] The method comprises making zL-Glu-OMe:

[0028] With L-glutamic acid dimethyl ester (di(OMe)Glu):

[0029] reacts to produce a methyl core.

[0030] In some embodiments, the reaction is carried out in the presence of a base and a coupling agent. In some embodiments, a base is added to a zL-GluOMe solution, and then a coupling agent and two (OMe) Glu are added to the reaction. In some embodiments, a coupling agent is added to a solution of zL-Glu-OMe and a base, and then two (OMe) Glu are 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 agent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC. In some embodiments, the solvent is THF or MeTHF. In some embodiments, before and during the reaction with the coupling agent, the reaction temperature 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, after adding two (OMe) Glu, the reaction temperature 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 additionally charging a coupling reagent, a base, and / or zL-Glu-OMe. In some embodiments, the reaction further comprises washing the reaction with an aqueous acid and then washing the reaction with an aqueous base. In some embodiments, the reaction further comprises crystallizing by adding an antisolvent. In some embodiments, the method comprises the reagents and conditions in the following scheme:

[0031]

[0032] Disclosed herein is a method for preparing triols,

[0033]

[0034] The method involves reacting a methyl core with 2-(2-aminoethoxy)ethanol:

[0035] Reaction to produce a triol. In some embodiments, the method is carried out with pure 2-(2-aminoethoxy)ethanol. In some embodiments, the reaction temperature is about 25°C to 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 isopropyl acetate. In some embodiments, the method further comprises adding a triol seed. In some embodiments, the method comprises the reagents and conditions in the following scheme:

[0036]

[0037] An alternative process for preparing TGZ is disclosed, the process comprising reacting a triol:

[0038]

[0039] With β-D-galactosamine pentaacetate:

[0040] reaction to produce TGZ.

[0041] In certain embodiments, first β-D-galactosamine pentaacetate is reacted with silyl triflate to produce oxazoline solution. In certain embodiments, silyl triflate is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or triisopropylsilyl trifluoromethanesulfonate (TIPSOTf). In certain embodiments, reaction further comprises solvent. In certain embodiments, solvent is dichloroethane (DCE) or dichloromethane (DCM). In certain embodiments, reaction temperature is about 35 DEG C to about 45 DEG C. In certain embodiments, reaction temperature is about 40 DEG C. In certain embodiments, reaction temperature is reduced to about 20 DEG C to about 28 DEG C. In certain embodiments, reaction temperature is about 23 DEG C. In certain embodiments, triol is mixed with sodium bicarbonate (NaHCO3) to produce triol and NaHCO3 slurry mixture, then β-D-galactosamine pentaacetate or its oxazoline solution is added. In certain embodiments, triol and NaHCO3 slurry mixture further comprises solvent. In certain embodiments, solvent is dichloromethane (DCM), dichloroethane or acetonitrile. In some embodiments, the oxazoline solution is added to a slurry mixture of triol and NaHCO3. 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, TGZ is further precipitated from a solvent / anti-solvent system. In some embodiments, an anti-solvent is added to a solution comprising TGZ and a solvent. In some embodiments, the anti-solvent is dimethoxyethane. In some embodiments, the method comprises the reagents and conditions in the following scheme:

[0042]

[0043] In some embodiments, the reaction further comprises any of the claims directed to step A-2 or A-1.

[0044] Disclosed herein are compounds Wherein R is H or a Cbz protecting group, the compound can be used to prepare TGZ as described herein.

[0045] Disclosed herein are methods for preparing NAG-25, comprising using the alternative methods to prepare any of the embodiments of the methyl core or triol. Disclosed herein are alternative methods for preparing NAG-25, comprising a triol intermediate. In some embodiments, the alternative method further comprises any of the embodiments for step 5a, step 5b, or step 6. In some embodiments, the alternative method comprises reacting zL-Glu-OMe with dimethyl L-glutamate to form a methyl core, and converting the methyl core to NAG-25. In some embodiments, the alternative method comprises 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 comprises reacting the triol with β-D-galactosamine pentaacetate to form TGZ, and converting TGZ to NAG-25. In some embodiments, TGZ is further precipitated from the solvent / antisolvent system as described herein.

[0046] This article discloses the preparation of TG amine

[0047] or a salt thereof,

[0048] The method includes high pressure hydrogenolysis for deprotecting TGZ carboxybenzyl to form the TG amine or its salt. In some embodiments, the method includes a palladium source for hydrogenolysis. In some embodiments, the palladium source is a palladium carbon (Pd / C) catalyst. In some embodiments, the palladium source is 5% Pd / C.4d. In some embodiments, the catalyst loaded is less than about 10%, 9%, 8%, 7%, 6% Pd / C. In some embodiments, the catalyst used is 5% Pd / C. In some embodiments, hydrogenolysis is carried out in the presence of an acid. In some embodiments, the acid is TFA, oxalic acid, HCl, AcOH, H3PO4, 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 carried out in a solvent, and the solvent is DCM, IPAc, or MeOH. In some embodiments, the solvent is DCM. In some embodiments, the method reduces the formation of deacyl impurities. In some embodiments, TG amine or its salt 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 trifluoroacetate. In some embodiments, the method makes the TG PEG product of step 5b have high purity while reducing the amount of TG amine acetamide by-product. In some embodiments, TG amine or its salt contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% acetamide by-product. In some embodiments, the TG amine or its salt produced from the improved step 5a method has a purity greater than or equal to about 97%, 98% or 99%, as measured by LC. In some embodiments, without separating TG amine or its salt, TG amine or its salt is scaled into (telescoped into) step 5b. In some embodiments, the method includes the reagents in the following scheme:

[0049]

[0050] This article discloses a method for preparing TG PEG.

[0051]

[0052] 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, TBTU is added within a time period of about 30 min to about 1.5 hours. In some embodiments, TBTU is added within a time period of about 1-1.5 hours. In some embodiments, TG PEG is produced with a purity greater than or equal to about 90%, as measured by LC. In some embodiments, TG PEG dimer impurities are present at the end of the reaction in an amount less than 10%, as measured by LC. In some embodiments, the method comprises the reagents in the following scheme:

[0053]

[0054] Disclosed herein is a method for preparing NAG-25, the method comprising treating TGPEG with an activator and a phosphitylation agent. In some embodiments, the activator is tetrazole, 4,5-dicyanoimidazole (DCI), ETT, or benzothiotetrazole (BTT). In some embodiments, the activator is tetrazole, DCI, or ETT. In some embodiments, the activator is tetrazole. In some embodiments, tetrazole is added in about 0.2 equivalents to 1.2 equivalents. In some embodiments, the activator is DCI. In some embodiments, DCI is added in about 0.02 equivalents to 1.0 equivalents. 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 agent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphorodiamidite. In some embodiments, the method further comprises extraction, filtration, precipitation, and drying. In some embodiments, the precipitation step comprises precipitating NAG-25 from DCM / heptane.

[0055] Disclosed herein is a method for preparing NAG-25,

[0056]

[0057] The method comprises any of the embodiments disclosed herein for steps 1, 2, 3, 4a, 4, 5, or 6. In some embodiments, the method comprises preparing a tert-butyl core by any of the embodiments disclosed herein for preparing a tert-butyl core and converting the tert-butyl core to NAG-25. In some embodiments, the method comprises preparing a triacid by any of the embodiments disclosed herein for preparing a triacid and converting the triacid to NAG-25. In some embodiments, the method comprises preparing TGZ by any of the embodiments disclosed herein for preparing TGZ and converting TGZ to NAG-25. In some embodiments, the method comprises preparing TG amine by any of the embodiments disclosed herein for preparing TG amine and converting TG amine to NAG-25. In some embodiments, the method comprises preparing TG PEG by any of the embodiments disclosed herein for TG PEG and converting TG PEG to NAG-25.

[0058] Disclosed herein is a method for preparing NAG-25,

[0059]

[0060] The method comprises treating a mixture of TG amine and PEG acid with TBTU to obtain TG PEG

[0061]

[0062] as well as

[0063] Conversion of TG PEG to NAG-25. Any of the foregoing examples involving treatment of a mixture of TG amine and PEG acid with TBTU to yield TG PEG, as well as the additional examples described herein, will be useful in preparing NAG-25.

[0064] Disclosed herein is a method for preparing NAG-25, comprising deprotecting TGZ from a carboxybenzyl group to form TG amine:

[0065] or high pressure hydrogenolysis of its salts,

[0066] and converting TG amine, or a salt thereof, to NAG-25. Any of the foregoing embodiments involving high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to form TG amine, as well as the additional embodiments described herein, can be used to prepare NAG-25.

[0067] The present invention discloses a method for preparing NAG-25, which comprises reacting a triacid in the presence of a coupling agent and a base.

[0068]

[0069] Reacting 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 / antisolvent system. Any of the foregoing embodiments involving reacting the triacid with NAG-H or a salt thereof, as well as the additional embodiments described herein, will be useful in preparing NAG-25.

[0070] Disclosed herein is a method of preparing NAG-25 comprising reacting a tert-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 crystalline Form I. Any of the foregoing embodiments involving reacting a tert-butyl core with an acid to form a triacid, as well as the additional embodiments described herein, will be useful in preparing NAG-25.

[0071] Disclosed herein is a method for preparing NAG-25, the method comprising reacting GluZ with GluOtBu or a salt thereof to form a tert-butyl core, and converting the tert-butyl core to NAG-25. Any of the foregoing embodiments involving a method comprising reacting GluZ with GluOtBu or a salt thereof to form a tert-butyl core, as well as the additional embodiments described herein, will be useful for preparing NAG-25.

[0072] Disclosed herein is a method of preparing NAG-25 comprising precipitating TGZ from a solvent / antisolvent system. Any of the foregoing embodiments involving precipitation of TGZ from a solvent / antisolvent system, as well as any of the additional embodiments described herein, will be useful in preparing NAG-25.

[0073] Disclosed herein are methods of preparing NAG-25, the methods comprising a crystalline triacid intermediate. In some embodiments, the crystalline triacid is crystalline Form I of the triacid, characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ values ​​selected from the group consisting 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. In some embodiments, the crystalline triacid is crystalline Form I of the triacid, characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 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 diffraction pattern substantially similar to Figure 2 In some embodiments, the crystalline triacid is crystalline Form II of the triacid, characterized in that the X-ray powder diffraction pattern is substantially similar to Figure 3 Any of the foregoing examples involving crystalline triacids, as well as the additional examples described herein, will be useful in preparing NAG-25.

[0074] Disclosed herein is a method for preparing a tert-butyl core, the method comprising reacting GluZ with GluOtBu or a salt thereof to produce a tert-butyl core, wherein the reaction is carried out in the presence of a coupling reagent, a base and a solvent; optionally wherein 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.

[0075] Disclosed herein is a method for preparing triacids comprising reacting a tert-butyl core with an acid, wherein the acid is phosphoric acid (H3PO4), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

[0076] Disclosed herein is a method for preparing NAG-Z, the method comprising reacting acylGalNAc with alcohol-Z to produce NAG-Z, wherein the reaction is carried out in the presence of an acid.

[0077] Disclosed herein is a method for preparing NAG-H or a salt thereof, the method comprising hydrogenating NAG-Z in the presence of a Pd / C catalyst and a solvent, optionally wherein the solvent is dimethylacetamide (DMAc) or DCM.

[0078] 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 / antisolvent system.

[0079] Disclosed herein is a method for preparing TG amine or a salt thereof, the method comprising subjecting TGZ to high pressure hydrogenolysis to form the TG amine or a salt thereof, wherein the hydrogenolysis is performed in the presence of an acid.

[0080] Disclosed herein is a method for preparing TG PEG, the method comprising reacting a solution of TG amine or a salt thereof, and PEG acid with a coupling reagent, wherein the coupling reagent is added to the solution of PEG acid and TG amine or a salt thereof over a period of about 30 min to about 1.5 hours.

[0081] Disclosed herein is a method for preparing NAG-25, the method comprising reacting TG PEG with an activator and a phosphitylation agent, wherein the activator is tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), or benzothiotetrazole (BTT); and / or wherein the phosphitylation agent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphorodiamidite.

[0082] Other aspects and advantages will be apparent to those skilled in the art from a review of the following detailed description in conjunction with the accompanying drawings.The following description includes specific embodiments, it being understood that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Shown are crystals of triacid crystalline Form I formed during crystallization in acetone / toluene.

[0084] Figure 2 Shown is the XRPD pattern of triacid crystalline Form I after crystallization in toluene / acetone.

[0085] Figure 3 The XRPD pattern of triacid crystalline Form II is shown.

[0086] Figure 4 The results of TG PEG conversion between three solvents (DCM, DMF, MeCN) under different conditions in step 5b are shown.

[0087] Figure 5 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 neat conditions.

[0088] definition

[0089] As used herein, the terms "including" and "comprising," and variations such as "include," "includes," "comprise," or "comprises" are used in an open-ended manner, and thus should be interpreted to mean "including, but not limited to... " These terms will also be understood to mean the inclusion of stated integers or steps, or groups of integers or steps, but not the exclusion of any other integers or steps, or groups of integers or steps.

[0090] Unless otherwise indicated, the use of the terms "a / an", "the", and similar references in the context of the disclosure herein (especially in the context of the claims) should be interpreted to cover both the singular and the plural. Unless otherwise indicated herein, the description of the range of values ​​herein is intended only to be used as a shorthand method of referring to each individual value within the range individually, and each individual value is incorporated into the specification as if the value were individually described herein. Unless otherwise indicated, the use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended to better illustrate the disclosure herein and is not a limitation on the scope of the disclosure herein. The language in the specification should not be interpreted as indicating that any unclaimed element is necessary to practice the disclosure herein.

[0091] As used herein, the terms "crystalline form", "crystalline form", and "form" interchangeably refer to a crystal structure (or polymorph) having a specific molecular packing arrangement in the crystal lattice. Crystalline forms can be identified and distinguished from each other by one or more characterization techniques (including, for example, X-ray powder diffraction (XRPD), single crystal X-ray diffraction, differential scanning calorimetry (DSC), dynamic vapor sorption (DVS) and / or thermogravimetric analysis (TGA)). Therefore, as used herein, the terms "crystalline form I" and "crystalline form II" refer to unique crystalline forms that can be identified and distinguished from each other by one or more characterization techniques (including, for example, 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 form is characterized by an X-ray powder diffraction pattern having one or more signals at one or more specific degrees of 2θ values ​​(°2θ).

[0092] As used herein, the term "XRPD" refers to the analytical characterization method of X-ray powder diffraction. XRPD patterns can be recorded using a diffractometer in transmission or reflection geometry under ambient conditions.

[0093] As used herein, the terms "X-ray powder diffraction pattern", "X-ray powder diffraction pattern", "XRPD pattern" interchangeably refer to a pattern that plots signal positions (on the abscissa) relative to signal intensities (on the ordinate). For amorphous materials, an X-ray powder diffraction pattern may include one or more broad signals. For crystalline materials, an X-ray powder diffraction pattern may include one or more signals depicted on the abscissa of the X-ray powder diffraction pattern, each signal being identified by its angular value, as measured in degrees 2θ (°2θ), which may be expressed as "a signal at ... degrees 2θ", "one or more signals at one or more [a] 2θ values ​​of ...", and / or "at least one signal at one or more ... 2θ values ​​selected from ...". As used herein, the term "X-ray powder diffraction pattern having signals at ... 2θ values" refers to an XRPD pattern containing one or more signals or one or more peaks at a specific position (°2θ).

[0094] As used herein, a "signal" or "peak" refers to a point in an XRPD spectrum where the intensity, measured in counts, is at a local maximum. One of ordinary skill in the art will recognize that one or more signals (or one or more peaks) in an XRPD spectrum may overlap and may not be apparent to the naked eye, for example. In fact, one of ordinary skill in the art will recognize that some art-recognized methods are capable of and are applicable to determining whether a signal is present in a spectrum, such as Rietveld refinement.

[0095] The repeatability of the angle value is within the range of ±0.2° 2θ, that is, the angle value can be at the listed angle value +0.2° 2θ, the angle value -0.2° 2θ, or any value between these two endpoints (angle value +0.2° 2θ and angle value -0.2° 2θ).

[0096] The terms "signal intensity" and "peak intensity" interchangeably refer to the relative signal intensity in a given X-ray powder diffraction pattern. Factors that can affect the relative signal or peak intensity include sample thickness and preferred orientation (e.g., crystal particles are not randomly distributed).

[0097] As used herein, an X-ray powder diffraction pattern is "substantially similar to the diffraction pattern in [a particular] pattern" when at least 90% (e.g., at least 95%, at least 98%, or at least 99%) of the signals in the two diffraction patterns appear at overlapping positions expressed in degrees 2θ. In determining "substantially similar," the intensities and / or signal positions in an XRPD diffraction pattern may differ even for the same crystalline form. Thus, a signal maximum in an XRPD diffraction pattern (expressed in degrees 2θ (°2θ) as referred to herein) is generally meant to be reported as a value ±0.2 degrees 2θ of the reported value, which is an art-recognized variance.

[0098] As used herein, a crystalline form of a compound is "substantially pure" when it is equal to or greater than 90% by weight of the sum of all one or more solid forms of the compound in a sample (as determined by methods according to the art, such as quantitative XRPD). In some embodiments, the solid form is "substantially pure" when it is equal to or greater than 95% by weight of the sum of all one or more solid forms of the compound in a sample. In some embodiments, the solid form is "substantially pure" when it is equal to or greater than 99% by weight of the sum of all one or more solid forms of the compound in a sample.

[0099] As used herein, the term "solvent" refers to any liquid in which a compound is at least partially soluble (eg, solubility of the product > 1 g / L).

[0100] As used herein, the term "antisolvent" refers to any liquid in which a compound is insoluble or minimally soluble (eg, solubility of the product <1 mg / mL, <2 mg / mL, <3 mg / mL, or <0.01 mol / L).

[0101] As used herein, the term "total yield" is the cumulative yield of the combination of steps. For example, to obtain the total yield of steps 1, 2 and 3, the yields of each step are multiplied.

[0102] As used herein, the environmental impact factor ("E-factor") is an indicator of green chemistry and measures total waste relative to product. A high E-factor indicates more waste generation and a negative impact on the environment.

[0103] The terms "about" and "approximately", when used with a quantity or range, include the value of the quantity or range, and include quantities or ranges recognized by those of ordinary skill in the art to provide an effect equivalent to that obtained from the quantity or range. In some embodiments, the term "about" modifies a particular number by + or -10%. In some embodiments, the term "about" modifies a particular number by + or -5%. In some embodiments, the term "about" modifies a particular number by + or -2%. In some embodiments, the term "approximately" modifies a particular number by + or -1%.

[0104] As used herein, when "about" is followed by a series of numbers, it should be understood that "about" applies to all the numbers that follow. Similarly, when a unit (e.g., %, hours, equivalents) appears at the end of a series or range of numbers, the unit applies to all series or numbers before the unit. For example, "about 3%, 4%, or 5%" is the same as "about 3%, about 4%, or about 5%"; "about 5-10 equivalents" is the same as "about 5 equivalents to about 10 equivalents"; "about 3-10 hours, 3-6 hours, 4-9 hours" is the same as "about 3-10 hours, about 3-6 hours, about 4-8 hours"; or "about 50-70 minutes or 60 minutes" is the same as "about 50-70 minutes or about 60 minutes".

[0105] As used herein, "oligomeric compound" is a compound comprising at least one oligonucleotide containing about 10-100 nucleotides. In some embodiments, the oligomeric compound has a core base sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in the cell. In some embodiments, the oligomeric compound can inhibit the expression of the potential gene when delivered to the cell expressing the gene, and is referred to herein as "oligomeric compound that inhibits expression". Gene expression can be inhibited in vitro or in vivo. "Oligomeric compound" includes but is not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates.

[0106] As used herein, the term "oligonucleotide" means a polymer of linked nucleosides, each of which may independently be modified or unmodified.

[0107] As used herein, the term "oligonucleotide" means a polymer of linked nucleosides, each of which may independently be modified or unmodified.

[0108] As used herein, the term "single-stranded oligonucleotide" means a single-stranded oligomeric compound having a sequence that is at least partially complementary to a target mRNA, which can hybridize to the target mRNA by hydrogen bonding or Watson-Crick base pairing under mammalian physiological conditions (or equivalent conditions in vitro). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0109] As used herein, "RNAi construct" refers to an agent comprising RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the translation of messenger RNA (mRNA) transcripts of target genes in a sequence-specific manner. As used herein, RNAi constructs can act by RNA interference mechanisms (i.e., by inducing RNA interference by interacting with RNA interference pathway mechanisms (RNA-induced silencing complexes or RISC) of mammalian cells) or by any alternative one or more mechanisms or pathways. RNAi constructs include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. RNAi constructs suitable for conjugation with targeting ligands described herein (e.g., NAG-25) are composed of oligonucleotides having a strand at least partially complementary to the targeted mRNA. In some embodiments, the RNAi construct is double-stranded and consists of an antisense strand and a sense strand at least partially complementary to the antisense strand. The RNAi construct may be composed of modified nucleotides and / or one or more non-phosphodiester bonds.In some embodiments, the RNAi construct suitable for conjugation to a targeting ligand described herein (eg, NAG-25) is single-stranded.

[0110] As used herein, "LCAP" (standing for "liquid chromatography area percentage") means the percentage of the peak area of ​​the target compound to the total peak area.

[0111] As used herein, salts 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 optionally isolating the salt so formed. Non-limiting examples of suitable salts of any one or more of the compounds described herein include hydrobromates, hydrochlorides, sulfates, bisulfates, sulfonates, camphorsulfonates, phosphates, nitrates, acetates, valerates, oleates, palmitates, stearates, laurates, benzoates, lactates, phosphates, toluenesulfonates, citrates, maleates, fumarates, succinates, tartrates, naphthoates, mesylates, glucoheptonates, lactobionates, laurylsulfonates, and amino acid salts, etc. DETAILED DESCRIPTION

[0112] The following discussion relates to various exemplary embodiments of the disclosed methods. However, the disclosed embodiments should not be interpreted or otherwise used to limit the scope of the present disclosure, including the claims. In addition, it will be understood by those skilled in the art that the following description has broad application, and the discussion of any embodiment is intended only to exemplarily discuss that embodiment, and the scope of the present disclosure, including the claims, is not limited to that embodiment.

[0113] Processes for NAG-25 and its intermediates

[0114] Provided herein are methods for preparing NAG-25, which is a carbohydrate targeting moiety, and intermediates of NAG-25 (tert-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 provide NAG-25 and the foregoing intermediates with reduced levels of impurity byproducts, reduced number of unit operations, and / or simplified handling / separation for use in multi-step NAG-25 processes. Further provided herein are methods for isolating and / or crystallizing certain intermediates (such as triacids).

[0115] The methods, intermediates, and products described herein can be used to prepare therapeutic compounds, such as siRNA molecules conjugated to NAG-25, a carbohydrate targeting moiety having the following structure:

[0116]

[0117] As further described herein, NAG-25 can be produced starting from any of the steps described herein. As will be further described, the compound 1-tert-butyl N-benzyloxycarbonyl-L-glutamate (GluZ) and di-tert-butyl L-glutamate (GluOtBu) or a salt thereof (e.g., GluOtBu hydrochloride) are coupled to obtain a tert-butyl core (step 1), which can then be subjected to triester deprotection to produce a triacid (step 2). The triacid can then be coupled with three units of NAG-H or a salt thereof (e.g., NAG-HTFA) to produce TGZ (step 4b). NAG-HTFA can be synthesized from NAG-Z by reductive deprotection of the Cbz group (step 4a). NAG-Z can be synthesized by direct glycosylation of alcohol-Z with acyl GalNAc (step 3b). Alcohol-Z can be optionally prepared by amino protection of an amino alcohol, as described herein (step 3a). TG amine or a salt thereof can be obtained from TGZ by reductive deprotection of the Cbz group (step 5a). TG amine or its salt can be coupled with PEG acid to produce TG PEG (step 5b). Finally, NAG-25 can be synthesized by forming phosphoramidite from TG PEG (step 6).

[0118] As further described herein, an alternative method can be used to produce TGZ to produce NAG-25. The alternative method utilizes different protecting group strategies. The 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 isolating TGZ using the alternative method described herein, TGZ can be converted to NAG-25 using steps 4, 5, and / or 6 as described herein.

[0119] The NAG-25 process comprises one or more steps as described herein that result in the reduction of, or the absence or substantial absence of, one or more impurities and / or by-products.

[0120] Described herein are compositions comprising NAG-25, or one of its intermediates, having reduced levels of one or more impurities as further described herein. The disclosed methods provide NAG-25 of 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.

[0121] Compared to the previous method described in U.S. Pat. No. 10,246,709, the method disclosed herein includes improvements at each step, such as the option of avoiding column chromatography at the end of step 4b (which produces TGZ) which can be expensive, wasteful and time intensive for large-scale synthesis, and a new crystalline form of the triacid isolated at the end of step 2, which facilitates the exclusion or removal of impurities in a more controlled manner.

[0122] Method for preparing NAG-25

[0123] Disclosed herein is a method for preparing NAG-25, comprising step 1, step 2, and / or step 4b methods. The combination of the steps 1, 2, and 3 methods described herein results in an improved overall yield (e.g., from about 47% to about 67%) and / or a reduction in the environmental impact factor (E-factor) (e.g., from about 1000 to about 540).

[0124] Disclosed herein are methods of preparing NAG-25 comprising a step 1 process to achieve near complete reaction conversion, optionally in the absence of additional amounts (or charges) of one or more reagents or starting materials (also referred to as sparging).

[0125] Disclosed herein are methods for preparing NAG-25, which methods include a step 2 process that produces a new crystalline triacid (eg, triacid crystalline Form I).

[0126] Disclosed herein is a method for preparing NAG-25 comprising the step 4b method of isolating and purifying TGZ using precipitation from a solvent / antisolvent system without the use of column chromatography.

[0127] Disclosed herein is a method for preparing NAG-25, comprising a step 3b method for producing NAG-Z, which is one of the starting materials for NAG-25. The step 3b method can be performed without isolating the oxazoline intermediate and has reduced impurities and improved yields. Further disclosed herein is a step 3a method for producing alcohol-Z with a more simplified process (fewer unit operations), improved yields and / or reduced impurities, which is used as a starting material for NAG-Z.

[0128] Disclosed herein is a method for preparing NAG-25, the method comprising step A-1, step A-2, and / or step A-3 to produce TGZ and its alternative intermediates (methyl core and triol) further described herein. Further 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-triazadocosapentazone-15-yl)carbamate) that can be used to prepare TGZ. Provided herein is a composition comprising the triol intermediate and an unprotected triol. Disclosed herein is a method for preparing NAG-25, wherein the method comprises a triol intermediate.

[0129] Disclosed herein is a method for preparing NAG-25 comprising step 5a, step 5b, and / or step 6 methods. The combination of step 4, 5, and 6 methods described herein results in simplified unit operations, reduced solvent volume, and / or improved purity. The improvements described herein include improved separation points and physical properties of intermediates and reduction or elimination of reactive impurities. The improved step 4-6 methods described herein result in reduced unit operations and solvent volume.

[0130] Disclosed herein is a method for preparing NAG-25 comprising a step 5b process wherein a coupling reagent (eg, TBTU) is added over a period of about 1-1.5 hr.

[0131] Disclosed herein is a method for preparing NAG-25, comprising any of step 1, step 2, step 4a, step 3b, step 3a, step 4b, step 5a, step 5b, step 6, and / or embodiments thereof, further described herein.

[0132] Disclosed herein is a method for preparing NAG-25, comprising any of step A-1, step A-2, step A-3, step 5a, step 5b, step 6, and / or embodiments thereof, further described herein.

[0133] In general, the improved method for preparing NAG-25 disclosed herein includes improved steps 1-6 that improve the overall yield of NAG-25 (e.g., by about 6%) and reduce the E-factor (e.g., the E-factor is reduced by about 24%). The process improvements described herein allow NAG-25 to be manufactured more efficiently on a larger scale.

[0134] Step 1: tert-Butyl Core

[0135] Disclosed herein are methods for preparing tert-butyl cores (Step 1 methods). These methods include coupling 1-tert-butyl N-benzyloxycarbonyl-L-glutamate (GluZ) with di-tert-butyl L-glutamate (GluOtBu) to obtain the tert-butyl core:

[0136]

[0137] Previous Step 1 methods have challenges, including: reaction quenching, which requires additional reagent charging (sparge) to assist in complete conversion; and crystallization from hexane, which can be environmentally unfriendly. To address these challenges, experiments were conducted to investigate alternative coupling reagents that achieve closer to complete conversion without the need for sparge, which was used in previous methods; reagent addition patterns that increase conversion to the tert-butyl core; and methods to simplify the Step 1 method and crystallization of the tert-butyl core (e.g., by eliminating the solvent exchange used in previous Step 1 methods).

[0138] Disclosed herein is a step 1 method for producing a tert-butyl core, which provides a higher reaction conversion and / or a higher separation purity while using a single charge of reagents. The improved step 1 method uses different coupling reagents, solvents, addition orders and the amount of reagents, resulting in improved reaction conversion to the tert-butyl core, and improved purity of the tert-butyl core. Step 1 also includes the reverse addition of reagents, which (not subject to theoretical constraints) is believed to promote the formation of mixed anhydride intermediates exceeding symmetrical anhydride intermediates. In addition, by switching the reaction solvent (from THF to, for example, MTBE), removing the solvent exchange step in the crystallization and reducing the solvent volume of the crystallization, the crystallization of the tert-butyl core product is simplified.

[0139] In one aspect of step 1, for preparing the tert-butyl core: The method includes making GluZ:

[0140]

[0141] With GluOtBu:

[0142] or a salt thereof to produce a tert-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, the GluOtBu salt is a hydrochloride. In some embodiments of step 1, the method comprises using a coupling reagent, a base, and a solvent.

[0143] In some embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / ethyl cyanohydroxyiminoacetate (Oxyma) (EDC / Oxyma), tetramethylfluoroformamidine hexafluorophosphate (TFFH), [ethylcyano(hydroxyimino)acetate-O 2 ] tris-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-carbon 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.

[0144] 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 (tert-butyl core). In some embodiments, the anti-solvent is heptane. In some embodiments, the anti-solvent is IPA: water in a 1:2 ratio. In some embodiments, the solvent is MTBE, and the anti-solvent is heptane.

[0145] The step 1 method can use normal addition or reverse addition of GluZ to the coupling reagent. Under normal addition, a solution of the coupling reagent in a solvent is added to a solution of GluZ, a base, and a solvent. In some embodiments, the step 1 method includes normal addition, wherein a solution of the coupling reagent in a solvent is added to a solution of GluZ and a base in a solvent. Under reverse addition, a solution of GluZ, a base, and a solvent is added to a solution of the coupling reagent in a solvent. Without being bound by theory, reverse addition forms a mixed anhydride, which helps to reduce the formation of symmetrical anhydrides of GluZ. In some embodiments, the step 1 method includes reverse addition, wherein a solution of GluZ and a base in a solvent is added to another solution of the coupling reagent in a solvent.

[0146] Prepare a solution of GluZ and a base in a solvent and add it to a solution of PivCl in a solvent. In certain embodiments, the solvent is MTBE, and a solution of GluZ and NMM in MTBE is added to a solution of MTBE and PivCl. This addition of Gluz and NMM occurs over a period of about at least 50 minutes, 50-70 minutes, or 60 minutes. After stirring for about 20-40 minutes, GluOtBu is added in small amounts about every 10-20 minutes, and the reaction is carried out at 0°C for about 20-40 minutes (e.g., 30 minutes). Monitor the reaction (e.g., with LC) until a suitable conversion is achieved.

[0147] In certain embodiments, each of PivCl and GluOtBu or its salt is used independently with about 1 equivalent to about 1.5 equivalents. In certain embodiments, the PivCl used is about 1.1, 1.2 or 1.3 equivalents, and the GluOtBu or its salt used is about 1.2, 1.3 or 1.4 equivalents. In certain embodiments, the PivCl used is about 1.2 equivalents, and the GluOtBu or its salt used is about 1.3 equivalents. The equivalents of PivCl and GluOtBu or its salt (" equivalent (eq) " or " equivalent (equiv) " herein) are relative to GluZ.

[0148] In some embodiments, the NMM used is about 3.0 to about 3.5 equivalents. In some embodiments, the NMM used is about 3.3 to about 3.7 equivalents. In some embodiments, the NMM used is at least about 3.0 equivalents. In some embodiments, the NMM used is at least about 3.5 equivalents. In some embodiments, the NMM used is about 3.5 equivalents.

[0149] In some embodiments, PivCl is about 1.1-1.4 equivalents, GluOtBu is about 1.1-1.5 equivalents, and NMM is about 3.3-3.7 equivalents. In some embodiments, PivCl is about 1.2 equivalents, GluOtBu is about 1.3 equivalents, and NMM is about 3.5 equivalents. Equivalents are relative to GluZ.

[0150] In some embodiments, the step 1 method for preparing the tert-butyl core comprises reacting GluZ with GluOtBu or a salt thereof in the presence of PivCl, NMM, and MTBE to produce the tert-butyl core. In some embodiments, the step 1 method for preparing the tert-butyl core comprises reacting GluZ with GluOtBu or a salt thereof in the presence of PivCl (about 1.1-1.4 equivalents), NMM (at least 3.0 equivalents), and MTBE (about 8-12 volumes) to produce the tert-butyl core.

[0151] In some embodiments, step 1 of the method comprises:

[0152] 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

[0153] b) Adding GluOtBu or a salt thereof to the third solution of GluZ, base, solvent, and coupling reagent.

[0154] In some embodiments, step 1 of the method comprises:

[0155] 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

[0156] b) Adding GluOtBu or its salt to the third solution of GluZ, NMM, MTBE, PivCl.

[0157] In some embodiments, GluOtBu or a salt thereof is GluOtBu hydrochloride.

[0158] The improved Step 1 process described herein produces fewer impurities, such as Piv-Glu-OH and tripeptide impurities, as shown below.

[0159]

[0160] In some embodiments, the yield of the tert-butyl core produced in step 1 is greater than about 85% or about 90%. In some embodiments, the yield of the tert-butyl core produced in step 1 is greater than about 91%, 92%, 93%, 94%, 95%, 96%, or 97%.

[0161] In some embodiments, the purity of the tert-butyl core produced in step 1 is greater than about 95%, as measured by liquid chromatography (LC). In some embodiments, the purity of the tert-butyl core is greater than about 96%, 97%, 98%, or 99%, as measured by LC.

[0162] In some embodiments, the Step 1 method for preparing the tert-butyl core is shown in the following scheme:

[0163] Wherein GluZ is reacted with GluOtBu HCl in the presence of PivCl, base, MTBE. In some embodiments of the above schemes, PivCl is about 1.2 equivalents, NMM is about 3.5 equivalents, and MTBE is about 10 volumes. In some embodiments of the above schemes, step 1 is performed at about 0°C. In some embodiments, the reaction comprises adding a solution of GluZ and NMM in MTBE in reverse to a solution of PivCl in MTBE.

[0164] In some embodiments, one, two, or three other types of alkyl groups (eg, methyl, ethyl, propyl, and / or butyl) can replace one, two, or three of the tert-butyl groups in the tert-butyl core, GluZ, and GluOtBu.

[0165] In some embodiments of step 1, the method further comprises processing (eg, extractive washing), distillation, crystallization, filtration, washing, and / or drying.

[0166] In some embodiments, the extraction treatment step comprises: adding hydrochloric acid solution (e.g., about 0.3-0.7M or 0.5M HCl), stirring and then phase separation, adding sodium carbonate solution (e.g., about 0.3-0.7M or 0.5M sodium carbonate), stirring and then phase separation, adding brine solution (e.g., 4-6wt% or 5wt% brine solution), stirring and then layer separation. If desired, the organic layer can be used for crystallization or kept at 20°C overnight.

[0167] Further provided herein is a crystallization method for a tert-butyl core, the method comprising adding an antisolvent to a solution of the tert-butyl core in a solvent. In some embodiments, a seed of the tert-butyl core is also added (e.g., before, after, or with the antisolvent). In some embodiments, the antisolvent is added within a time period of at least about 2, 3, 4, or 5 hours; or within a time period of about 2-5, 2-4, 3-5, 3-4, or 2.5-3.5 hours; or within a time period of about 3, 3.5, or 4 hours. In some embodiments, the antisolvent is added until the final amount is about 30%, 40%, 50%, 60%, 70%, 80% of the antisolvent. In some embodiments, the antisolvent is added to a solution of the tert-butyl core in a solvent, the solution being maintained at about 30°C-40°C, 35°C-45°C, or 33°C-37°C. In some embodiments, after the antisolvent is added, the solution temperature is reduced to about 15°C-25°C, 18°C-22°C, or 20°C. In some of the above embodiments, the solvent is MTBE and the anti-solvent is heptane.

[0168] Step 2: Triacid or its salt

[0169] Disclosed herein are methods for preparing triacids or their salts (Step 2 methods). These methods include trying deprotection of a tert-butyl core to produce a triacid or its salt:

[0170]

[0171] In the previous step 2 method, formic acid was used to deprotect the three tert-butyl groups of the tert-butyl core. After the reaction was complete, the formic acid was removed after a series of solvent exchange / distillation operations and the triacid was isolated as an oil.

[0172] To improve the previous Step 2 process, experiments were conducted to investigate: the isolation of solid triacids that could be upgraded in purity; more efficient processing procedures (e.g., extractive washing); and the development of reaction conditions that would yield higher purity triacids or their salts (including the use of alternative acids in the reaction).

[0173] This article discloses an improved step 2 method leading to high purity triacids or their salts. Examples of triacids include phosphates, sodium salts, and calcium salts. In one aspect of step 2, the method for preparing triacids, or their salts, includes reacting the tert-butyl core with an acid as a deprotecting agent. The reaction can be carried out in the presence of a solvent or a solvent mixed with water. When present with a solvent, water acts as a tert-butyl scavenger and effectively produces triacids from the tert-butyl core in high yield.

[0174] In some embodiments, the acid is phosphoric acid (H3PO4), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid. In some embodiments, the acid is phosphoric acid (H3PO4), TFA, or HCl. In some embodiments, the acid is phosphoric acid (H3PO4). In some embodiments, step 2 is reacted in a solvent selected from the following: 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 (H3PO4) as an acid and 2-MeTHF as a solvent mixed with water. In some embodiments, the improved step 2 uses phosphoric acid (H3PO4) as an acid and 2-MeTHF as a solvent mixed with water, wherein phosphoric acid is present in about 3 volumes, 2-MeTHF is present in about 3.5 volumes, and water is about 0.5 volumes.

[0175] In some embodiments, the reaction is carried out at a temperature of about 40° C.-60° C. In some embodiments, the temperature is about 45° C.-55° C. In some embodiments, the temperature is about 50° C. In some embodiments, the reaction is carried out for about 3-10 hours, 3-6 hours, 4-9 hours, 4-7 hours, or 4-5 hours.

[0176] In some embodiments, a method for preparing a triacid, or a salt thereof, in step 2, the method comprising:

[0177] a) combining a tert-butyl core with an acid in a solvent;

[0178] b) adding water; and

[0179] c) heating the reaction to a certain temperature. In some embodiments, the solvent is 2-MeTHF, the acid is H3PO4, and the temperature is about 50°C.

[0180] In some embodiments, the step 2 method for preparing the triacid, or a salt thereof, is shown in the following scheme:

[0181]

[0182] In some embodiments, the step 2 method for preparing the triacid, or a salt thereof, is shown in the following scheme:

[0183]

[0184] The reaction is complete when the amount of diacid is less than 3%, as measured by LC. In some embodiments of step 2, after the reaction is complete, the method further comprises processing (eg, extractive washing), distillation, crystallization, filtration, and / or drying.

[0185] In some embodiments, the acid (e.g., phosphoric acid) is effectively removed by aqueous treatment, thereby avoiding a lengthy formic acid distillation process. In some embodiments, step 2 further comprises removing phosphoric acid with at least one organic / aqueous washing liquid. In some embodiments of the aqueous treatment step, the reaction mixture is diluted with isopropyl acetate and washed with a 20 wt % aqueous solution of ammonium sulfate. In some embodiments of the aqueous treatment step, the reaction mixture is diluted with 2-methyltetrahydrofuran and / or washed with a 20 wt % aqueous solution of ammonium sulfate. Then, the organic layer is washed with water.

[0186] 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 3-8 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.

[0187] In some embodiments, the organic / aqueous washing liquid comprises 2-methyltetrahydrofuran (MeTHF) as an organic layer and ammonium sulfate as an aqueous layer. In some embodiments, about 6-18 volumes of MeTHF and about 3-20 volumes of 20wt% ammonium sulfate are used. In some embodiments, about 8-10 volumes of MeTHF and about 12-18 volumes of 20wt% ammonium sulfate are used. In some embodiments, about 9 volumes of MeTHF and about 15 volumes of 20wt% ammonium sulfate are used. In some embodiments, the washing volumes listed herein can be divided into 2-4 (e.g., 3) separate smaller washing steps. After washing, an optional water washing liquid (e.g., 1, 2, or 3 volumes) can be used.

[0188] In addition, in some embodiments, a solvent / anti-solvent crystallization system can be used to provide a 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 toluene is about 13 volumes. In some embodiments, the solvent is about 3 volumes of MeTHF and 4 volumes of acetone, and toluene is about 13 volumes. In some embodiments, the ratio of toluene is 2:3, 1:1, 3:2 or 1:2. In some embodiments, the ratio of acetone / toluene is 2:3, 1:1 or 1:2. In some embodiments, the solvent / anti-solvent is acetone / toluene. In some embodiments, the ratio of acetone / toluene is 2:3, 1:1, 3:2, or 1:2. In some embodiments, the ratio of acetone / toluene is 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, wherein the remaining volume percentages are one or more solvents. In some embodiments, the one or more solvents are acetone or MeTHF combined with toluene.

[0189] 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.

[0190] In some embodiments, the purity of the triacid, or its salt, is greater than or equal to about 95%, 96%, 97%, 98%, or 99%, as measured by LC. In some embodiments, the purity of the triacid, or its salt, is greater than or equal to about 95%, as measured by LC. In some embodiments, the method for preparing the triacid, or its salt from the tert-butyl core includes crystallizing the triacid, or its salt, using a solvent / antisolvent system of acetone / toluene, MeTHF / IPAc, MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile. In some embodiments, the method for preparing the triacid, or its salt from the tert-butyl core includes crystallizing the triacid, or its salt, with acetone / toluene. In some embodiments, crystallization includes seeding acetone / toluene with a ratio of about 3:2, 1:1, or 5:6, and further filling toluene until the final ratio of acetone / toluene is about 2:3, 1:2, or 1:3. Inoculation can be completed with triacid seeds (e.g., 1wt%). 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 percent of toluene in the acetone / toluene mixture is about 55%-70% toluene.Any of the foregoing embodiments apply to the triacid (ie, the free triacid, not a salt).

[0191] In some embodiments, any of the Step 2 embodiments described herein produces a crystalline triacid characterized by an X-ray powder diffraction pattern as further described in the Examples below.

[0192] Crystal forms of triacids are disclosed herein. In some embodiments, the Step 2 method produces triacid crystalline Form I. In some embodiments, the Step 2 method produces triacid crystalline Form II.

[0193] In some embodiments, the triacid crystalline solid is crystalline Form I. In some embodiments, the triacid crystalline Form I is in substantially pure form. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern generated by X-ray powder diffraction analysis, wherein the incident beam is Cu Ka radiation.

[0194] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 7.4 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 9.2 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 21.0 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 10.2 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 14.6 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 18.3 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern with a signal at 19.3 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having a signal at 22.9 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having a signal at 11.0 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having a signal at 11.3 ± 0.2 degrees 2θ.

[0195] In some embodiments, the triacid crystalline form I is characterized by an X-ray powder diffraction pattern with signals at the following 2θ values: 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-ray powder diffraction pattern with additional signals at the following 2θ values: 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-ray powder diffraction pattern with additional signals at the following 2θ values: 11.0 ± 0.2 or 11.3 ± 0.2.

[0196] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 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-ray powder diffraction pattern having at least one signal at seven 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at six 2θ 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-ray powder diffraction pattern having at least one signal at five 2θ 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-ray powder diffraction pattern having at least one signal at four 2θ 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-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ 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-ray powder diffraction pattern having at least one signal at one 2θ 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.

[0197] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 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-ray powder diffraction pattern having at least one signal at seven 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at six 2θ 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-ray powder diffraction pattern having at least one signal at five 2θ 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-ray powder diffraction pattern having at least one signal at four 2θ 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-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at a 2θ value selected from the group consisting 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.

[0198] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 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-ray powder diffraction pattern having at least one signal at seven 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at six 2θ 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-ray powder diffraction pattern having at least one signal at five 2θ 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-ray powder diffraction pattern having at least one signal at four 2θ 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-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at a 2θ value selected from the group consisting 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.

[0199] In some embodiments, the triacid crystalline Form I is characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ 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-ray powder diffraction pattern having signals at the following 2θ values: 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.

[0200] In some embodiments, triacid crystalline Form I is characterized by an X-ray powder diffraction pattern substantially similar to Figure 2 X-ray powder diffraction pattern of .

[0201] In some embodiments, the triacid crystalline solid is crystalline Form II. In some embodiments, the triacid crystalline Form II is in substantially pure form. In some embodiments, the triacid crystalline Form II is characterized by an X-ray powder diffraction pattern generated by X-ray powder diffraction analysis, wherein the incident beam is Cu Ka radiation.

[0202] In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 7.8 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 9.7 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 15.7 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 13.3 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 17.1 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 18.9 ± 0.2 degrees 2θ. In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with a signal at 20.2 ± 0.2 degrees 2θ. In some embodiments, triacid crystalline Form II is characterized by an X-ray powder diffraction pattern having a signal at 21.0 ± 0.2 degrees 2Θ.

[0203] In some embodiments, the triacid crystalline form II is characterized by an X-ray powder diffraction pattern with signals at the following 2θ values: 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-ray powder diffraction pattern with additional signals at the following 2θ values: 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-ray powder diffraction pattern with additional signals at the following 2θ values: 20.2 ± 0.2 and / or 21.0 ± 0.2.

[0204] In some embodiments, the triacid crystalline Form II is characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 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-ray powder diffraction pattern having at least one signal at seven 2θ values ​​selected from the group consisting 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-ray powder diffraction pattern having at least one signal at six 2θ 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-ray powder diffraction pattern having at least one signal at five 2θ 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-ray powder diffraction pattern having at least one signal at four 2θ 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-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ 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-ray powder diffraction pattern having at least one signal at one 2θ 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.

[0205] In some embodiments, the triacid crystalline Form II is characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ 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-ray powder diffraction pattern having at least one signal at two 2θ 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-ray powder diffraction pattern having signals at the following 2θ values: 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-ray powder diffraction pattern having signals at the following 2θ values: 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.

[0206] In some embodiments, triacid crystalline Form II is characterized by an X-ray powder diffraction pattern substantially similar to Figure 3 X-ray powder diffraction pattern of .

[0207] Step 3a: Alcohol-Z

[0208] Disclosed herein are methods for preparing alcohol-Z (Step 3a method). These methods comprise reacting benzyl chloroformate (CbzCl) with an amino alcohol to form alcohol-z:

[0209]

[0210] The reaction occurs in the presence of a solvent and a base. Non-limiting solvents include polar aprotic solvents, aprotic solvents, and ether solvents. In certain embodiments, the solvent is dichloromethane (DCM), dichloroethane, ethyl acetate, or methyl THF. In certain embodiments, the solvent is a polar aprotic solvent. In certain embodiments, the solvent is DCM or dichloroethane. In certain embodiments, the solvent is DCM. In certain 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 certain embodiments, the base is a tertiary amine base. In certain embodiments, the base is triethylamine (TEA), DIPEA, N-methylmorpholine, 1-methylimidazole, and pyridine. In certain embodiments, the base is TEA. In certain embodiments, the solvent is DCM, and the base is TEA. In certain embodiments, relative to benzyl chloroformate, amino alcohols are present in about 1.0-1.2 equivalents. In certain embodiments, relative to benzyl chloroformate, amino alcohols are present in about 1.1 equivalents. After the amino alcohol solution (with solvent and base) is cooled to about -5°C to 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, optionally at a temperature that is maintained at about 0°C-12°C, 3°C-10°C, 8°C-11°C, 10°C, 5°C or 0°C. After adding CbzCl, the reaction temperature is adjusted to about 28°C-42°C. In some embodiments, the reaction temperature is adjusted to about 30°C-40°C, 33°C-37°C, or 35°C. In some embodiments, the reaction temperature is about 35°C. In some embodiments, the amino alcohol is present in about 1.1 equivalents relative to benzyl chloroformate, and the reaction temperature is about 20°C-30°C, 30°C-40°C, 33°C-37°C, or 35°C. In some embodiments, the amino alcohol is present at about 1.1 equivalents relative to 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 benzyl chloroformate, and the reaction is maintained at about 35° C.

[0211] In some embodiments, the step 3a method for preparing alcohol-Z includes reacting benzyl chloroformate (CbzCl) with an amino alcohol in the presence of DCM as a solvent and TEA as a base. After adding CbzCl over a period of about 1 hour, the reaction temperature is adjusted from about 0°C-5°C or 3°C-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 equivalents. The reaction lasts at least 12 hours, for example, about 10-12 hours. In some embodiments, the step 3a method for preparing alcohol-Z is shown in the following scheme:

[0212] In some embodiments, during the addition of reagents, the temperature is about 5°C, and the reaction temperature increases to about 35°C.

[0213] After the reaction, the alcohol-Z product can be isolated by cooling to about room temperature (e.g., 20° C.-25° C.), quenching (e.g., with water) and extracting with a solvent (e.g., DCM), and optionally washing (e.g., 10% NaCl), and then distilling. In some embodiments, the yield of alcohol-Z is greater than or equal to about 95%, 96%, 97%, 98% or 99% yield.

[0214] The alcohol-Z step 3a process also reduces one or more impurities including benzyl alcohol, dibenzyl carbonate, benzyl chloroformate, and / or doubly protected alcohol-Z, as shown below.

[0215]

[0216] In some embodiments, the purity of alcohol-Z produced in step 3a is 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, the impurity 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.

[0217] Step 3b: NAG-Z

[0218] Methods for preparing NAG-Z are disclosed herein (Step 3b Method). These methods include direct glycosylation of alcohol-Z with acylGalNAc (also referred to herein as DGalNAc):

[0219]

[0220] Without being bound by theory, the oxazoline intermediate is formed in situ and no step of isolating the oxazoline intermediate is required, resulting in increased yield and reduced 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.

[0221] 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 method also reduces one or more impurities, including DGalOH and / or Ac-Z, as shown below:

[0222] 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 impurity 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.

[0223] 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. Although TMSOTf is commonly used in glycosylation reactions, it has been found that TMSOTf can degrade the NAG-Z product under certain conditions, resulting in a reduced yield of NAG-Z. Non-limiting examples of acids include trifluoromethanesulfonic acid (also known as trifluoromethanesulfonate (CF3SO3) or triflate (Tf)), silyl triflate (e.g., TBSOTf, or TIPSOTf), metal triflate (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 chloride (e.g., FeCl3, AlCl3, InCl3, or BiCl3), trifluoroacetic acid (TFA), and BF3OEt2. In some embodiments, the acid is bismuth trifluoromethanesulfonate (Bi(OTf)3), trifluoroboron etherate (BF3OEt2), 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)3). In 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 in about 1-10 mol%, 1-4 mol%, or 2-3 mol% relative to alcohol-Z.

[0224] In some embodiments, the reaction is carried out in the presence of a solvent. Non-limiting examples of usable solvents 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 volume listed herein is relative to 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, acyl GalNAc is added to the reaction mixture as an acyl GalNAc solution comprising acyl GalNAc, acid and solvent. In some embodiments, the reaction mixture already contains the Alc-Z produced when step 3a ends. 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 acyl GalNAc 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 comprising dried (eg, azeotropically dried or dried with a drying agent) alcohol-Z and a solvent. In some embodiments, the solvent in the alcohol-Z solution is about 5 volumes.

[0225] In some embodiments, 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, acyl GalNAc is present in about 1.5 equivalents relative to alcohol-Z.

[0226] After combining the acyl GalNAc, alcohol-Z, acid, and solvent, the reaction temperature is heated to about 55°C-65°C, 65°C-75°C, or 70°C-90°C. In some embodiments, the reaction temperature is about 70°C-90°C. In some embodiments, the reaction temperature is about 75°C-85°C. In some embodiments, the reaction temperature is about 80°C. In some embodiments, the reaction temperature is about 58°C-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. Subsequently, the reaction is cooled to about room temperature (e.g., about 20°C or 15°C-25°C) for filtration (e.g., fine filtration) and charcoal treatment. In some embodiments, the added charcoal is about 3-7 weight (wt)%, 5-10wt%, 8-15wt%, 13-25wt%, 20-35wt%, 30-50wt%, 40-60wt%, or 50-70wt%. In some embodiments, the added charcoal is about 3-7 weight (wt)%, 5-10wt%, or 8-15wt%. In some embodiments, the added charcoal is about 30-50wt%, 40-60wt%, or 50-70wt%. In some embodiments, the added charcoal is about 40wt%, 50wt%, or 60wt%.

[0227] After charcoal treatment of the NAG-Z solution, the charcoal is filtered and the NAG-Z is crystallized by adding an anti-solvent. 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. Seeds of NAG-Z are optionally added to the solution and optionally aged overnight. In some embodiments, additional anti-solvents are optionally added 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.

[0228] In some embodiments, the step 3b method for preparing NAG-Z is shown in the following scheme:

[0229]

[0230] In some embodiments, methods of preparing NAG-Z are shown in the following scheme:

[0231]

[0232] Step 4a: NAG-H or its salt

[0233] Disclosed herein are methods for making NAG-H or its salts (Step 4a methods). These methods include hydrogenating NAG-Z in the presence of a Pd / C catalyst and a solvent (e.g., dimethylacetamide) to prepare NAG-H or its salts:

[0234]

[0235] In some embodiments, NAG-H or its salt is optionally used as a solution in the amide coupling reaction of step 4b described herein. The advantage of this improved method is that it avoids separating NAG-H or its salt into a hygroscopic and difficult to handle solid. In addition, the volume of solvent used in this improved method is reduced compared to the previous method.

[0236] In some embodiments, NAG-H or a salt thereof is NAG-H trifluoroacetic acid (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-HTFA salt).

[0237] In some embodiments, the step 4a method comprises combining NAG-Z with a Pd / C catalyst in a solvent, adding an acid, and pressurizing with hydrogen.

[0238] 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.

[0239] 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, DMAc is used with about 3 volumes to about 5 volumes. In some embodiments, DMAc is used with about 4 volumes. In some embodiments, DCM is used with about 3 volumes to about 6 volumes. In some embodiments, DCM is used with about 3-5 or 4-6 volumes.

[0240] 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 in about 0.8-1.2 equivalents relative to NAG-Z. In some embodiments, TFA is present in about 0.8-1.2 equivalents or 1.0 equivalents relative to NAG-Z.

[0241] 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.

[0242] In some embodiments, step 4a is performed 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.

[0243] In some embodiments, the reaction occurs over about 15-25 hours or 18-20 hours.

[0244] Step 4b: TGZ

[0245] A method for preparing TGZ is described herein (Step 4b Method). TGZ is synthesized by coupling three NAG-H units to a triacid via simultaneous amide coupling:

[0246]

[0247] In previous methods of step 4b, tri-GalNAc-Cbz (TGZ) has been separated and purified via solvent-intensive and low-yield chromatography steps. Improved step 4b methods are disclosed herein, which include chromatography-free separation and purification of the TGZ intermediate by precipitation from a solvent. The advantage of the improved step 4b method is the elimination of chromatography, thereby increasing the TGZ yield, e.g., to greater than about 85%, and saving time and money, e.g., by reducing solvent use and / or avoiding specialized manufacturing equipment.

[0248] The improved TGZ purification method additionally provides a more effective amide coupling. The improved step 4b method includes using a coupling reagent and coupling NAG-H to a triacid in a given solvent (e.g., TBTU in dimethylacetamide), which reduces the equivalent 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 buffered washing solution that minimizes the formation of impurity by-products (e.g., deacylating by-products). The improved step 4b uses a mild buffer (e.g., sodium phosphate or potassium phosphate buffer) instead of a stimulating acidic / alkaline aqueous washing solution. Finally, a single precipitation of TGZ from a solvent system (e.g., DCM / MTBE) provides a more effective and higher yield (e.g., greater than about 80%-90%) separation of TGZ, making TGZ an amorphous solid with a higher purity (e.g., greater than about 95% purity, as measured by LC).

[0249] In some embodiments, the step 4b method comprises: combining the triacid with NAG-H or a salt thereof, which may optionally be in a solvent, adding a base, and adding a coupling reagent. In some embodiments of step 4b, the method further comprises extraction / processing, distillation, crystallization, precipitation, filtration, and / or drying. In some embodiments, NAG-H or a salt thereof is NAG-H TFA.

[0250] In some embodiments, the coupling reagent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylammonium tetrafluoroborate (TBTU), Urea tetrafluoroborate (TATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), chloro-N,N,N',N'-tetramethylformamidine hexafluorophosphate (TCFH), (1-cyano-2-ethoxy-2-oxoethylenaminooxy)dimethylamino-morpholino-carbon hexafluorophosphate (COMU), (3-dimethylamino-propyl)-ethyl-carbodiimide (EDC), ethyl cyano(hydroxyimino)acetate (Oxyma), 1-hydroxybenzotriazole (HOBt), [ethyl cyano(hydroxyimino)acetoxy-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), propylphosphonic anhydride (T3P), bis(2-oxooxazolidine)phosphinoyl chloride (BOPCl), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] In some embodiments, NAG-H or a salt thereof is about 3-4 equivalents. In some embodiments, NAG-H or a salt thereof is about 3-3.7 equivalents. In some embodiments, NAG-H or a salt thereof is about 3.1-3.3 equivalents. In some embodiments, NAG-H or a salt thereof is NAG-H TFA. The foregoing equivalents are relative to the triacid.

[0256] In some embodiments, the base equivalent is about 6-15 equivalents, 8-12 equivalents, or 10-12 equivalents. In some embodiments, the base equivalent is about 10-12 equivalents. In some embodiments, the base equivalent is about 11.5-12.5 equivalents. In some embodiments, the base equivalent is about 11-12 equivalents. In some embodiments of the foregoing embodiments of the base, the base is NMI. In some embodiments of the foregoing embodiments of the base, the base is 12 equivalents of NMI. The foregoing equivalents are relative to the triacid.

[0257] In some embodiments, the step 4b method 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 foregoing equivalents are relative to the triacid.

[0258] In some embodiments, the step 4b method for preparing TGZ is shown in the following scheme:

[0259]

[0260] In some embodiments, the step 4b method described herein produces a high purity TGZ with a low percentage of deacyl impurities. In some embodiments, the purity of the TGZ is greater than about 93%, 94%, 95%. In some embodiments, the resulting TGZ contains less than about 1%, 0.5%, or 0.2% deacyl impurities. In some embodiments, the resulting TGZ contains less than about 1% deacyl impurities.

[0261] In some embodiments, the step 4b method further comprises washing with an acid at least once, twice, or three times, and then washing with a base at least once, twice, or three times. In some embodiments, the acid is HCl and the base is NH4OH. In some embodiments, when the coupling agent is TCFH, the base is NMI, and the solvent is DCM, HCl washing liquid and NH4OH washing liquid are used.

[0262] In some embodiments, the step 4b method further comprises washing with a buffer at least once, twice, or three times. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the pH of the buffer is about 4 to 8. In some embodiments, the pH of the buffer is about 5-7. In some embodiments, the pH of the buffer is about 6. In some embodiments, at least 2 or 3 pH 6 phosphate buffer washes are performed. In some embodiments, 3 pH 6 phosphate buffer washes 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 to 550mM potassium phosphate. In some embodiments, the buffer is about 25 to 500mM potassium phosphate. In some embodiments, the buffer is about 40 to 60mM potassium phosphate. In some embodiments, the buffer is about 50mM potassium phosphate. In some embodiments, the buffer is about 50mM potassium phosphate at about pH 5-7. In some embodiments, the buffer is about 50mM potassium phosphate at about pH 6.

[0263] In some embodiments, the step 4b method comprises azeotropically distilling the organic phase with a solvent. In some embodiments, the solvent used for distillation is DCM.

[0264] In some embodiments, the step 4b method further includes precipitating TGZ from a 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 solvent with a final solvent ratio of about 1:1 ratio is used: anti-solvent is used to precipitate TGZ. In some embodiments, a DCM:MTBE with a final solvent ratio of about 1:1 ratio is used to precipitate TGZ. In some embodiments, antisolvent (eg, MTBE) addition occurs within about 1.5-2.5, 2-3, 2.5-3.5, or 3-4 hours or within about 2, 3, or 4 hours.

[0265] In some embodiments, a yield of at least about 88%, 89%, 90% TGZ is obtained. In some embodiments, a purity of at least about 95% TGZ is obtained, as measured by LC (95LCAP). In some embodiments, a purity of at least about 96%, 97%, 98% TGZ is obtained, as measured by LC.

[0266] In some embodiments, the step 4b method for preparing TGZ comprises triacid (1.0 equivalent), NAG-HTFA (3.5 equivalent), DMAc, NMI (12 equivalent), TBTU (4.0 equivalent). In some embodiments, the step 4b method further comprises a 50 mM pH 6 phosphate buffer wash and a DCM wash. In some embodiments, step 4b further comprises precipitating TGZ after adding MTBE to a solution comprising DCM.

[0267] The improved step 4b process results in reduced, minimal or undetectable amounts of byproducts of the process. Byproducts of the process include mono-NAG, di-NAG, di-NAG-azlactone, deacylation (also known as des-acetate), di-NAG-OH, di-NAG-OAc, NAG-H guanidine, and NAG-H trifluoroacetamide, each of which is described below.

[0268]

[0269] In some embodiments, the step 4b method results in a TGZ having less than about 5%, 3%, 2%, 1%, or 0.5% mono-NAG, di-NAG, and / or di-NAG-azlactone as measured by LC. In some embodiments, the step 4b method results in a TGZ having less than about 2% NAG-H guanidine, less than about 2% di-NAG-OH, less than about 1% di-NAG, about less than 1% deacylation, and less than about 1% di-NAG-OAc. Disclosed are compositions comprising TGZ wherein any of the impurities is less than or equal to 5% (e.g., 5%, 4%, 3%, 2%, 1%) as measured by LC.

[0270] In some embodiments, the method results in a final TGZ yield greater than about 85%, 86%, 87%, 88%, 89%, or 90% yield. In some embodiments, the method results in a final TGZ yield greater than about 90% yield.

[0271] Alternative method for TGZ: Step A-1, Step A-2, Step A-3

[0272] 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 comprises 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-glutamate hydrochloride):

[0273]

[0274] In some embodiments, the reaction is carried out in the presence of a base and a coupling reagent. In some embodiments, a base is added to the zL-GluOMe solution, and then the coupling reagent and di(OMe)Glu are added to the reaction. In some embodiments, the base is N-methylmorpholine (NMM) or diisopropylethylamine. In some embodiments, the base is NMM.

[0275] In some embodiments, the reaction further comprises a solvent. In some embodiments, the solvent is an ether 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.

[0276] In some embodiments, the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC. In some embodiments, the coupling reagent is IBCF.

[0277] In some embodiments, the reaction temperature is about -20°C to about -10°C before and during the reaction with the coupling reagent. In some embodiments, the reaction temperature is about -18°C to about -13°C. In some embodiments, the reaction temperature is about -15°C.

[0278] In some embodiments, after the reaction temperature is reached, the coupling reagent is added to the solution of zL-Glu-OMe and base, and then di(OMe)Glu is added to the reaction.

[0279] 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.

[0280] 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.

[0281] In some embodiments, after adding the bis(OMe)Glu, the reaction temperature 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.

[0282] In some embodiments, the reaction time is about 12-24 hours. In some embodiments, the reaction time is about 14-18 hours.

[0283] In some embodiments, the reaction further comprises additionally charging a coupling reagent, a base, and / or zL-Glu-OMe. In some embodiments, the reaction further comprises additionally charging IBCF, NMM, and zL-Glu-OMe. In some embodiments, the additional charging is about 8-12 mol%. In some embodiments, the additional charging is about 10 mol%.

[0284] In some embodiments, the equivalent of di(OMe)Glu is about 0.8 equivalents to about 1.3 equivalents, the equivalent of NMM is about 2.3 equivalents to about 2.7 equivalents, and the equivalent of IBCF is about 0.8 equivalents to about 1.3 equivalents relative to zL-GluOMe. In some embodiments, the equivalent of di(OMe)Glu is about 1 equivalent, the equivalent of NMM is about 2.5 equivalents, and the equivalent of IBCF is about 1 equivalent relative to zL-GluOMe.

[0285] After completion, the reaction is diluted with an organic solvent, then washed with an aqueous acid in sequence, and then washed with an aqueous base. In certain embodiments, the organic solvent is an acetate solvent. In certain embodiments, the organic solvent is ethyl acetate (EtOAc). In certain embodiments, the aqueous acid is HCl. In certain embodiments, the aqueous base is NaOH. In certain embodiments, the aqueous washing is carried out for about 2 to about 4 times. Then, residual water is removed from the organic solution before crystallization.

[0286] Anti-solvent addition crystallization is carried out by slow cooling ramp down 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 is filtered to separate the methyl core solid. The solid is washed with an organic solvent and then dried under nitrogen.

[0287] In some embodiments, the Step A-1 method for preparing the methyl core is shown in the following scheme:

[0288]

[0289] Step A-2: Disclosed herein are triol compounds (chemical name: benzyl ((10S, 15S) -1,22-dihydroxy-10- ((2-(2-hydroxyethoxy) ethyl) carbamoyl) -7,12,16-trioxo-3,20-dioxa-6,11,17-triazacosane-15-yl) carbamate) and methods for preparing triols. Also disclosed herein are unprotected triols that do not have a CBz protecting group on -NH2. Step A-2 produces the triol shown below and includes 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) ethane-1-ol):

[0290]

[0291] In some embodiments, 2-(2-aminoethoxy)ethanol is pure and the reaction is performed without solvent. In some embodiments, a solvent additive is used.

[0292] In some embodiments, the 2-(2-aminoethoxy)ethanol is about 25-30 equivalents relative to the methyl core.

[0293] 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 22-26 hours. In some embodiments, the reaction time is about 24 hours.

[0294] In some embodiments, step A-2 is not enzyme-mediated. In some embodiments, step A-2 is enzyme-mediated. In some embodiments, the enzyme is Novozym 5103 (liquid), Lipozyme CALB L (liquid), Resinase HT (liquid), Lipozyme TL (liquid), Novozym 435 (solid support), Novozym 40086 (solid support), Lipozyme TL (solid support), papain (lyophilized), trypsin (lyophilized), Amano PS (powder), Novozym 5103 (liquid), Lipozyme CALB L (liquid), Reinase 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 support), Lipozyme TL (solid support). 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 support). In some embodiments, the enzyme is Lipozyme TL (solid support).

[0295] 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.

[0296] In some embodiments, triol seeds are optionally added to the reaction. In some embodiments, about 1-3 weight percent (wt%) of seeds are added. In some embodiments, about 2 weight percent (wt%) of seeds are added.

[0297] 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 slurry of triol is aged for about 16-20 hours or about 18 hours.

[0298] The slurry is filtered, and the crude product solid is then washed with an organic solvent and then redissolved in an organic solvent for reslurry at a certain temperature. The slurry is filtered, and the product solid is washed with an organic solvent and then dried under nitrogen. In some embodiments, the organic solvent is ethyl acetate.

[0299] In some embodiments, the Step A-2 method for preparing the triol is shown in the following scheme:

[0300]

[0301] Step A-3: Step A-3 produces TGZ and comprises glycosylation between triol and BD-galactosamine pentaacetate:

[0302]

[0303] BD-galactosamine pentaacetate was converted to an oxazoline solution (ie, an oxazoline derivative of BD-galactosamine pentaacetate) as shown below.

[0304]

[0305] 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.

[0306] In some embodiments, BD-galactosamine pentaacetate is first converted to an oxazoline solution (ie, an oxazoline derivative of BD-galactosamine pentaacetate) and then added to the solution of the triol.

[0307] The oxazoline solution is produced by reacting BD-galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf). In some embodiments, TMSOTf is about 1 to about 1.5 equivalents. In some embodiments, TMSOTf is used in about 1.2 equivalents. The equivalents are relative to 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.

[0308] In certain embodiments, the reaction of preparing oxazoline solution further comprises solvent. In certain embodiments, solvent is dichloroethane (DCE) or dichloromethane (DCM). In certain embodiments, solvent is DCE.

[0309] 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.

[0310] In certain embodiments, triol is mixed with sodium bicarbonate (NaHCO3) to produce triol and NaHCO3 slurry mixture, then add β-D-galactosamine pentaacetate or its oxazoline solution.In certain embodiments, triol and NaHCO3 slurry mixture further comprises solvent.In certain embodiments, the solvent of slurry mixture is dichloromethane (DCM), ethylene dichloride or acetonitrile.In certain embodiments, the solvent of slurry mixture is dichloromethane (DCM).

[0311] In certain embodiments, oxazoline solution is added to triol and NaHCO slurry mixture in. In certain embodiments, oxazoline solution is added to triol and NaHCO with about 0.8 to about 1.2 equivalents / hour of oxazoline solution in a ratio up to about 4-5 equivalents / hour in mixture. In certain embodiments, oxazoline solution is added to triol and NaHCO with about 1.0 equivalents / hour of oxazoline solution in a ratio up to about 4.5 equivalents / hour in mixture.

[0312] Subsequently, the reaction is carried out for about 18 to about 30 hours or overnight. In some embodiments, the reaction is carried out 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.

[0313] The quantitative oxazoline solution charged in addition can be added to help the reaction conversion. The reaction is quenched with an organic base, and then quenched with an aqueous base. In certain embodiments, the organic base is Et3N. In certain embodiments, the aqueous base is NaHCO3.

[0314] The organic phase is washed with a mild acidic aqueous wash. In some embodiments, the acid is NH4Cl. The organic solution is dried to remove residual water.

[0315] The obtained TGZ product is precipitated from a solvent / anti-solvent system. In some embodiments, the solvent is DCM and the anti-solvent is dimethoxyethane (DME). The TGZ slurry is filtered, the product solid is washed with an organic solvent, and dried under nitrogen. In some embodiments, the organic solvent is DCM / DME 1:1. In order to achieve higher purity, the solid can be re-slurried in an organic solvent at a certain temperature, filtered, washed with an organic solvent, and then dried under nitrogen.

[0316] In some embodiments, the step A-3 method for preparing TGZ is shown in the following scheme:

[0317]

[0318] Step 5a: TG amine or its salt

[0319] Disclosed herein are methods for preparing TG amine or its salt (Step 5a method). These methods include subjecting TGZ to high pressure hydrogenolysis to produce TG amine or its salt:

[0320]

[0321] In the previous step 5a method, the hydrogenolysis of TGZ to TG amine, or its salt, has some undesirable factors, such as expensive Pd source catalyst at high catalyst loading (eg, 10%) or flammable reaction solvent MeOH (when exposed to hydrogen).

[0322] Improved methods are disclosed herein, including high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to form TG amine or its salt. The improvement includes using DCM as the reaction solvent to eliminate separation and performing reaction kinetic analysis to determine the optimal catalyst loading and stirring speed. TG amine or its salt can be scaled (without separation) into the next step 5b method described herein.

[0323] In some embodiments, the step 5a method comprises: merging TGZ with a Pd / C catalyst and pressurizing with hydrogen. In some embodiments, the method further comprises an acid. In some embodiments, the method comprises the following steps: merging TGZ with a solvent, adding a Pd / C catalyst, adding an acid, and pressurizing with hydrogen. In some embodiments, the step 5a method comprises: merging TGZ with a Pd / C catalyst, adding an acid and a solvent, and pressurizing with hydrogen. After completing the reaction, the catalyst can be removed by filtering. In some embodiments of step 5a, the method further comprises filtering.

[0324] A palladium source is used for hydrogenolysis. In some embodiments, the palladium source is a Pd / C catalyst. In some embodiments, the palladium 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.

[0325] In some embodiments, the method includes using an acid. In some embodiments, the acid is TFA, oxalic acid, HCl, AcOH, H3PO4, 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 equivalents or in an amount not greater than about 1.0 or 1.1 equivalents. Excessive addition of acid in the step 5a method generally results in higher levels of TG amine-TFA acetamide impurities in the step 5b method.

[0326] 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.

[0327] In some embodiments, the reaction is maintained at a temperature of about 20° C.-25° C. In some embodiments, the reaction is stirred for at least about 15, 16, 17, or 18 hours.

[0328] In some embodiments, the reaction reduces the formation of one or more impurities (e.g., TG AmineDesAc, di-NAG-OH TG amine, TG amine guanidyl, TG amine acyl (TG Amine Ac), TG acetamide, and / or NAG-H guanidine, each of which is described below).

[0329]

[0330] In some embodiments, the NAG-H guanidine impurity is present at a minimum of less than or equal to 2% or 1%, as measured by LC. In some embodiments, the TG amine di-NAG-OH impurity is present at a minimum of less than or equal to 2% or 1%, as measured by LC. In some embodiments, the reaction reduces the formation of the TG amine deacyl impurity. In some embodiments, the TG amine deacyl impurity is present at a minimum of less than or equal to 5%, 4%, 3%, 2%, 1%, as measured by LC.

[0331] In some embodiments, the TG amine or its salt 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.

[0332] The improved step 5a method allows the TG PEG product of step 5b to have high purity while reducing the amount of TG acetamide byproduct. In some embodiments, the TG amine or its salt contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% acetamide byproduct. In some embodiments, the TG amine or its salt contains less than about 5% acetamide byproduct. In some embodiments, the TG amine or its salt contains less than about 2% acetamide byproduct.

[0333] In some embodiments, the TG amine or its salt produced from the improved step 5a method has a purity greater than or equal to about 97%, 98%, or 99%, as measured by LC. In some embodiments, the TG amine or its salt produced from the improved step 5a method has a yield greater than or equal to about 97%, 98%, or 99%.

[0334] In some embodiments, the step 5a method for preparing the TG amine TFA salt is shown in the following scheme:

[0335]

[0336] In some embodiments, the step 5a method for preparing the TG amine TFA salt is shown in the following scheme:

[0337]

[0338] After reaction and filtration, the resulting TG amine or its salt in solvent can be scaled directly into the step 5b method described herein. Advantages include elimination of unit operations involving distillation and precipitation. In some embodiments, the TG amine or its salt is scaled into step 5b without isolating the TG amine or its salt. In some embodiments, the TG amine or its salt is a salt of TG amine TFA.

[0339] Step 5b: TG PEG

[0340] A method for preparing TG PEG is disclosed herein (Step 5b method). TG amine or its salt is coupled with PEG acid to prepare TG PEG:

[0341]

[0342] Improvement reduces impurities during the amide coupling process, eliminates column chromatography, and reduces solvent volume. The improved method includes controlled addition of coupling reagents (e.g., TBTU). By controlling the ratio of coupling reagents (e.g., TBTU) added to the PEG acid, the improved method reduces the formation of PEG-dimer impurities called TG PEG dimers. In addition, the improved step 5b includes extraction of the TG PEG crude product into an aqueous solution, followed by stripping to achieve the exclusion of orthogonal impurities.

[0343] The improved step 5b method comprises reacting TG amine or its salt with PEG acid in the presence of a coupling reagent. In some embodiments, the method further comprises a base. In some embodiments, the method 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.

[0344] In some embodiments, step 5b method comprises adding TBTU to a solution comprising TG amine or a salt thereof and PEG acid.

[0345] In some embodiments, step 5b includes

[0346] a) combining TG amine or its salt with PEG acid;

[0347] b) adding a base; and

[0348] c) adding TBTU over a period of time. In some embodiments, TG amine or its salt is added directly from the end of the process of step 5a. In some embodiments of step 5b, the process further comprises extraction / treatment, distillation, precipitation, filtration, and / or drying.

[0349] TBTU can be added in batches or using slurry transfer. In some embodiments, TBTU is added to the solution of PEG acid and TG amine or its salt over a period of about 80-100 or 90 minutes. In some embodiments, TBTU is added to the solution of PEG acid and TG amine or its salt over a period of about 50-70 or 60 minutes.

[0350] In some embodiments, the step 5b method further comprises using 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).

[0351] The improved method of preparing TG PEG reduces at least one impurity, such as a TG PEG dimer impurity. During the conjugation of NAG-25 to an oligonucleotide, the TG PEG dimer impurity is reactive during the phosphitylation step and can conjugate to the oligonucleotide. Examples of impurities are shown below:

[0352]

[0353] In some embodiments, the step 5b method produces a TG PEG comprising less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% TG PEG dimer. A composition comprising TG PEG is disclosed, wherein the TG PEG dimer is less than or equal to 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%), as measured by LC.

[0354] In some embodiments, TG amine or its salt is TG amine TFA salt. In some embodiments, the step 5b method of preparing TG PEG includes 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 step 5b method starts at about -10°C to about 5°C and increases to about 17°C-27°C during the reaction. In some embodiments, the step 5b method starts at about -10°C to about -4°C and increases to about 17°C-27°C during the reaction. In some embodiments, the step 5b method starts at about -8°C and increases to about 17°C-27°C during the reaction. In some embodiments, the step 5b method starts at about -8°C and increases to room temperature during the reaction.

[0355] In some embodiments, the step 5b method for preparing TG PEG is shown in the following scheme:

[0356]

[0357] wherein TG amine is TG amine TFA salt.

[0358] In some embodiments, the step 5b method for preparing TG PEG includes combining TG amine with PEG acid. In some embodiments, the method includes DCM as a solvent. In some embodiments, the temperature is maintained at about 20°C-25°C. In some embodiments, the temperature is cooled to -5°C to 5°C, DIPEA is added, and then TBTU is added. In some embodiments, TBTU is added in batches 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°C-5°C over a period of about 1 hour. In some embodiments, the reaction is further maintained at 0°C-5°C for about 1-5 hours. In the foregoing embodiments, PEG acid is added in about 0.9-1.3 equivalents, TBTU is added in about 1-1.5 equivalents, and DIPEA is added in about 2.5-3.5 equivalents, each of which is relative to the TG amine in the solution (from step 5a). In the foregoing example, PEG acid was added in about 1.1 equivalents, TBTU was added in about 1.3 equivalents, and DIPEA was added in about 3.0 equivalents, each relative to the TG amine in solution (from step 5a). Additional amounts of PEG acid and TBTU may optionally be added to increase the reaction conversion.

[0359] After the reaction is completed, the crude TG PEG is extracted into an aqueous solution and then stripped to remove one or more impurities. In some embodiments of step 5b, the method further comprises extraction / treatment, distillation, precipitation, filtration, and / or drying.

[0360] In some embodiments, the step 5b method includes an aqueous extraction washing process for removing TG PEG dimer impurities. In some embodiments, the aqueous extraction washing process includes 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), acidic buffer washing solution (e.g., pH 4-6), and alkaline buffer washing solution (e.g., pH 7.5-9).

[0361] In some embodiments, the acidic buffered wash comprises a mixture of 0.5 M sodium monophosphate (pH 6.0) and saturated saline. In some embodiments, the ratio of NaH2PO4: saline is about 4.5-5 volumes: 3-3.5 volumes. In some embodiments, the ratio is about 4.8 volumes: 3.2 volumes.

[0362] In some embodiments, the alkaline buffered wash solution comprises a mixture of saturated sodium bicarbonate and saturated saline solution. In some embodiments, the ratio of NaHCO3: saline is about 3.5-4.5 volumes: 3.5-4.5 volumes. In some embodiments, the ratio is 4.0 volumes: 4.0 volumes.

[0363] After the aqueous extraction washing step, the TG PEG solution contains TG PEG with a purity of at least 90% and less than 1% of each of one or more impurities (e.g., di-NAG-OH TG PEG, TG PEG deacylation, TG PEG dimer, TG PEG des2Ac, TG amine acylation, TG acetamide, and / or TG amine guanidine).

[0364] In some embodiments, the step 5b method comprises azeotropic distillation with a solvent. In some embodiments, the solvent used for distillation is DCM.

[0365] After washing and distillation, the method further comprises precipitating 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, precipitation occurs at a temperature of about -5°C to 5°C.

[0366] In some embodiments, the step 5b method produces a TG PEG having a purity greater than or equal to about 90% or 95%, as measured by LC. In some embodiments, the step 5b method produces a TG PEG having a yield greater than or equal to about 80%, 81%, 82%, 83%, 84%, 85%, or 90%.

[0367] Step 6: NAG-25

[0368] Methods for preparing NAG-25 are disclosed herein (Step 6 Methods). These methods include the phosphoramide formation of NAG-25 from TG PEG:

[0369]

[0370] Improvements include changing the precipitation solvent, which results in a significant reduction in solvent volume compared to previous methods (e.g., from 130-170 volumes to about 10-20, 20-30, 25-35, 30-50, or 40-60 volumes), increased stability of NAG-25, and / or reduced total impurities (e.g., from 90%-93% or 92% purity to about 95%-98% or 97% purity).

[0371] In some embodiments, the method of preparing NAG-25 comprises combining TG PEG, an activator, and a P-agent. In some embodiments, the phosphitylation agent ("P-agent") is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphorodiamidite. In some embodiments, the phosphitylation agent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

[0372] 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.

[0373] In some embodiments, the activator is tetrazole. In some embodiments, the tetrazole is added in about 0.2-1.2 equivalents. In some embodiments, the tetrazole is added in about 0.4-0.8 equivalents. In some embodiments, the tetrazole is added in about 0.6 equivalents. The equivalents of tetrazole in the foregoing embodiments are relative to TG PEG.

[0374] In some embodiments, the activator is DCI. In some embodiments, DCI is added in an amount of about 0.01-0.05 equivalents. In some embodiments, DCI is added in an amount of about 0.02-0.04 equivalents. In some embodiments, DCI is added in an amount of about 0.02-0.03 equivalents. The equivalents of DCI in the foregoing embodiments are relative to TG PEG.

[0375] In some embodiments, the step 6 reaction further comprises an additive. In some embodiments, step 6 optionally comprises adding an additive to the TG PEG. In some embodiments, the additive is N-methylimidazole (NMI). In some embodiments, the step 6 reaction does not comprise an additive.

[0376] In some embodiments, the P-agent is added in about 0.75-2 equivalents. In some embodiments, the P-agent is added in about 1-1.5 equivalents. The equivalents in the foregoing embodiments are relative to TG PEG.

[0377] Improvements to the method include the order of addition of reagents. In some embodiments, a solution comprising a P-reagent and an activator is added to a solution of TG PEG optionally comprising an additive. The P-reagent and activator solution are added at about 0°C-5°C. After the addition is complete, the reaction mixture is warmed to room temperature (e.g., 20°C-25°C) and allowed to proceed for about 2-4 hours.

[0378] In some embodiments, a solution of TG PEG in a solvent (TG PEG solution) is added to another solution of P-reagent and activator in a solvent. The TG PEG solution can 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, 7 hours. In some embodiments, the reaction is carried out at a temperature of about 35°C-45°C or about 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. In some embodiments, the reaction lasts 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 activator is DCI. In some embodiments, the solvent is DCM. In some embodiments, the solvent is anhydrous DCM. The amount of equivalent P-agent, activator has been described in previous embodiments. In some embodiments, a solution of TG PEG in DCM is added to another solution of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite and DCI in DCM (e.g., anhydrous DCM) over a period of 4.5-5.5 hours (e.g., about 5 hours), and then reacted at 35°C-45°C (e.g., about 40°C) for at least 10 hours. In some embodiments, the method includes 1-1.5 equivalents of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, 0.02-0.03 equivalents of DCI, each equivalent being relative to TG PEG.

[0379] 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 the exclusion of reactive phosphorus-related impurities. Examples of impurities from Step 6 include NAG-25 dimers:

[0380]

[0381] NAG-25PEG dimer:

[0382]

[0383] Oxidized NAG-25:

[0384]

[0385] H-phos:

[0386]

[0387] In some embodiments, the impurity is 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 in an amount of less than or equal to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% at the end of the reaction, 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 of 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 of 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 of 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 in an amount of less than or equal to about 4% or 3% at the end of the reaction as measured by LC. In some embodiments, NAG-25 dimer is present in an amount of less than or equal to about 2% or 1% at the end of the reaction as measured by LC.

[0388] Disclosed are compositions comprising NAG-25, wherein any of the 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, wherein NAG-25 dimer is less than or equal to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as measured by LC. In some embodiments, the composition comprises NAG-25, wherein NAG-25 dimer is less than or equal to 5%, 4%, 3%, 2%, or 1% as measured by LC.

[0389] In some embodiments, the step 6 method further comprises extraction (eg, water extraction), distillation, precipitation, filtration steps, and / or drying steps.

[0390] 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.

[0391] In some embodiments, the precipitating step uses forward precipitation, where an antisolvent is added to the NAG-25 solution. In some embodiments, the precipitating step uses reverse precipitation, where the NAG-25 solution is added to the antisolvent.

[0392] In some embodiments, the anti-solvent used for precipitation is MTBE or heptane. In some embodiments, the anti-solvent used for precipitation is heptane.

[0393] In some embodiments, NAG-25 was precipitated from DCM / heptane as solvent / anti-solvent.

[0394] In some embodiments, the step 6 method comprises reacting TG PEG with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the presence of tetrazole and NMI in dichloromethane.

[0395] In some embodiments, the step 6 method is shown in the following scheme:

[0396]

[0397] In some embodiments of the above schemes, DCM is about 15V, NMI is about 0.2 equivalents, tetrazole is about 0.6 equivalents, and P-reagent is about 1.25 equivalents. In some embodiments, the TG PEG solution is added to the P-reagent and activator solution over a period of about 2-10 hours and a temperature of about 35°C-45°C.

[0398] In some embodiments of step 6, the method further comprises extracting / treating, crystallizing, precipitating, filtering, and / or drying. In some embodiments of step 6, extracting NAG-25 with a water wash is optional.

[0399] In some embodiments, the step 6 method comprises adding a solution of TG PEG in a solvent to a solution of P-reagent and activator in a solvent. In some embodiments, the P-reagent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite. In some embodiments, the method does not include an additive. In some embodiments, the solvent is DCM. In some embodiments, the activator is DCI. The TG PEG solution is added to the P-reagent and activator solution 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 adding 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°C-45°C. In some embodiments, the reaction is carried out at about 38°C-42°C. After the reaction is complete, aqueous extraction can be optionally performed with water, and NAG-25 is azeotropically dried (e.g., under vacuum at 25°C-35°C) and then filtered. In some embodiments, NAG-25 is precipitated without aqueous extraction. To precipitate, the NAG-25 solution can be added to an anhydrous antisolvent (e.g., heptane) over 90 minutes. The solid is filtered and washed twice with an antisolvent: solvent (e.g., anhydrous heptane: DCM). In some embodiments, the antisolvent: 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%, wherein the remaining percentage is composed of the antisolvent volume percentage. In some embodiments, the percentage of DCM is about 20%-50% or about 20%, 30%, 40%, 50% of the volume of the antisolvent / solvent solution.

[0400] Linking to oligomeric compounds

[0401] NAG-25 and compounds containing NAG-25 can be used as targeting ligands by linking to therapeutic compounds, such as oligomeric compounds (e.g., RNA). NAG-25 facilitates targeted delivery of therapeutic compounds to hepatocytes for endocytosis of NAG-25-conjugated therapeutic compounds, where the therapeutic compound can modulate the expression of a target nucleic acid, resulting in a translational change in the target nucleic acid. For example, the therapeutic compound can modulate the expression of a target gene to inhibit protein translation or expression.

[0402] In some embodiments, the targeting ligand is linked to the therapeutic compound via an additional linker and / or cleavable moiety, which is then linked to the therapeutic compound. In some embodiments, the targeting ligand is linked to the therapeutic compound itself.

[0403] In some embodiments, the therapeutic compound is an oligomeric compound that inhibits expression. In some embodiments, the oligomeric compound that inhibits expression is an RNAi construct. In some embodiments, the oligomeric compound that inhibits expression is a double-stranded RNAi construct. In some embodiments, the oligomeric compound that inhibits expression is a single-stranded oligonucleotide. The oligomeric compound that inhibits expression can be synthesized using methods commonly used in the art.

[0404] In some embodiments, the targeting ligand is directly or indirectly connected to the 5' end of the sense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly connected to the 3' end of the sense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly connected to the 5' end or 3' end of the antisense strand of the double-stranded RNAi construct. In some embodiments, the targeting ligand is directly or indirectly connected to the 5' end or 3' end of the single-stranded RNAi construct.

[0405] In some embodiments, the targeting ligand is linked to the double-stranded RNAi construct via a phosphate, phosphonate, phosphorothioate, or other internucleoside linking group at the 5' end of the terminal nucleoside of the sense strand of the double-stranded RNAi construct.

[0406] In some embodiments, the targeting ligands disclosed herein include a cleavable portion. In some embodiments, the cleavable portion includes or consists of a phosphate or other internucleoside linking group that can be cleaved. In some embodiments, the targeting ligand is connected to the therapeutic compound via a cleavable portion.

[0407] In some embodiments, the targeting ligands disclosed herein are linked to additional one or more groups including a cleavable moiety. In some embodiments, the targeting ligand is linked to a cleavable moiety, which is then linked to an oligomeric compound that inhibits expression.

[0408] In some embodiments, the targeting ligand is a phosphoramidite compound (also referred to herein as a "phosphoramidite-containing compound"). The phosphoramidite compounds comprising the targeting ligand described herein can be used to easily attach the targeting ligand to a therapeutic compound or other group using phosphoramidite synthesis methods generally known in the art. In some embodiments, the phosphoramidite compound comprising the targeting ligand is connected to an oligomeric compound that inhibits expression using methods generally known in the art. In some embodiments, the phosphoramidite containing the targeting ligand is connected to the 5' end of the sense strand of the double-stranded RNAi construct.

[0409] In certain embodiments, the oligomeric compound inhibiting expression connected to the targeting ligand includes a single-stranded oligonucleotide. In certain embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. In certain embodiments, the targeting ligand is directly connected to the single-stranded antisense oligonucleotide. In certain embodiments, another group is inserted between the targeting ligand and the single-stranded oligonucleotide.

[0410] In some embodiments, the expression-inhibiting oligomeric compound linked to any of the targeting ligands disclosed herein comprises an RNAi construct. In some embodiments, the targeting ligand disclosed herein is directly or indirectly linked to an RNAi construct.

[0411] In some embodiments, the targeting ligand disclosed herein is directly connected to the RNAi construct. In some embodiments, the targeting ligand disclosed herein is indirectly connected to the RNAi construct because an additional one or more groups are inserted between the RNAi construct and the linker of the targeting ligand. In some embodiments, a second linker is included between the linker and the therapeutic compound (e.g., RNAi construct).

[0412] Other embodiments

[0413] A list of exemplary embodiments includes:

[0414] 1. A method for preparing a tert-butyl core,

[0415]

[0416] The method comprises causing GluZ to:

[0417]

[0418] With GluOtBu or its salts:

[0419]

[0420] reaction to produce a tert-butyl core.

[0421] 2. The method of embodiment 1, wherein GluOtBu or its salt is GluOtBu hydrochloride:

[0422]

[0423] 3. The method of embodiment 1 or 2, wherein the reaction is carried out in the presence of a coupling reagent, a base and a solvent.

[0424] 4. The method of any one of embodiments 1-3, wherein the coupling reagent is EDC / Oxyma, TFFH, PyOxim, CDI, PivCl, T3P, or COMU.

[0425] 5. The method of any one of embodiments 1-4, wherein the coupling reagent is T3P or PivCl.

[0426] 6. The method of any one of embodiments 1-5, wherein the coupling reagent is PivCl.

[0427] 7. The method of any one of embodiments 1-6, wherein the base is N-methylmorpholine (NMM).

[0428] 8. The method of any one of embodiments 1-7, wherein the solvent is IPAc, MeTHF, MIBK, or MTBE.

[0429] 9. The method of any one of embodiments 1-8, wherein the solvent is MTBE.

[0430] 10. The method of any one of embodiments 1-9, wherein the anti-solvent is heptane.

[0431] 11. The method of any one of embodiments 1-10, wherein the solvent is MTBE and the anti-solvent is heptane.

[0432] 12. The method of any one of embodiments 1-11, wherein a first solution comprising GluZ and NMM in a solvent is added to a second solution comprising the solvent and PivCl.

[0433] 13. The method of any one of embodiments 1-12, wherein PivCl is in excess.

[0434] 14. The method of any one of embodiments 1-13, wherein GluOtBu is in excess.

[0435] 15. The method of any one of embodiments 1-14, wherein the conversion of the tert-butyl core is greater than about 90% based on the amount of GluZ.

[0436] 16. A method for preparing triacids:

[0437]

[0438] The method comprises reacting a tert-butyl core with an acid.

[0439] 17. The method of embodiment 16, wherein the acid is phosphoric acid (H3PO4), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

[0440] 18. The method of embodiment 16, wherein the acid is phosphoric acid (H3PO4).

[0441] 19. The method of any one of embodiments 16-18, wherein the reaction is carried out in a solvent selected from the group consisting of 2-MeTHF, acetonitrile, THF, DMA, sulfolane, and DME.

[0442] 20. The method of embodiment 19, wherein the solvent is 2-MeTHF, THF, or acetonitrile.

[0443] 21. The method of embodiment 19, wherein the solvent is 2-MeTHF or THF.

[0444] 22. The method of embodiment 19, wherein the solvent is 2-MeTHF.

[0445] 23. The method of any one of embodiments 16-22, wherein the triacid is isolated as a crystalline solid.

[0446] 24. The method of any one of embodiments 16-23, wherein the triacid has a purity greater than or equal to 95% as measured by LC.

[0447] 25. The method of any one of embodiments 16-24, wherein the solvent further comprises water.

[0448] 26. The method of any one of embodiments 16-25, wherein the solvent is a mixture of 2-MeTHF and water.

[0449] 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.

[0450] 28. The method of any one of embodiments 16-27, wherein the reaction is carried out at a temperature of about 40°C-60°C.

[0451] 29. The method of any one of embodiments 16-28, wherein the reaction is carried out at a temperature of about 45°C-55°C.

[0452] 30. The method of any one of embodiments 16-29, wherein the reaction is carried out at a temperature of about 50°C.

[0453] 31. The method of any of embodiments 16-30, further comprising removing phosphoric acid with at least one organic / aqueous wash solution.

[0454] 32. The method of any one of embodiments 16-31, wherein the organic / aqueous wash comprises isopropyl acetate (iPAC) as the organic layer and ammonium sulfate as the aqueous layer.

[0455] 33. The method of any one of embodiments 16-32, wherein about 8-18 volumes of iPAC and about 3-7 volumes of 20 wt % ammonium sulfate are used.

[0456] 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.

[0457] 35. The method of any one of embodiments 16-34, further comprising crystallizing the triacid using acetone / toluene, 2-MeTHF / IPAc, 2-MeTHF / CPME, acetone / heptane, or MeTHF / acetonitrile.

[0458] 36. The method of any one of embodiments 16-35, further comprising crystallizing the triacid using acetone / toluene.

[0459] 37. The method of any one of embodiments 16-36, wherein the acetone / toluene ratio is 2:3, 1:1, 3:2, or 1:2.

[0460] 38. The method of any one of embodiments 16-37, wherein the acetone / toluene ratio is 2:3, 1:1, or 1:2.

[0461] 39. The method of any one of embodiments 16-38, wherein the triacid is obtained in a crystalline form characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ values ​​selected from the group consisting 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.

[0462] 40. A crystalline Form I of a triacid characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ values ​​selected from the group consisting 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.

[0463] 41. A crystalline Form I of a triacid characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 7.4±0.2, 9.2±0.2, and 21.0±0.2.

[0464] 42. A crystalline form I of a triacid characterized by an X-ray powder diffraction pattern substantially similar to Figure 2X-ray powder diffraction pattern of .

[0465] 43. A crystalline form II of a triacid characterized by an X-ray powder diffraction pattern substantially similar to Figure 3 X-ray powder diffraction pattern of .

[0466] 44. A method for preparing TGZ,

[0467]

[0468] The method comprises reacting a triacid with NAG-H in the presence of a coupling reagent and a base:

[0469] or its salts

[0470] reaction.

[0471] 45. The method of embodiment 44, wherein the coupling reagent is TBTU, HATU or TCFH.

[0472] 46. ​​The method of embodiment 45, wherein the coupling reagent is TBTU.

[0473] 47. The method of any one of embodiments 44-46, wherein the base is DIPEA, NMI, NMM, or TMP.

[0474] 48. The method of embodiment 47, wherein the base is NMI.

[0475] 49. The method of any one of embodiments 44-48, further comprising at least one buffered wash solution.

[0476] 50. The method of embodiment 49, wherein the buffered wash solution is phosphate buffered saline.

[0477] 51. The method of embodiment 49 or 50, wherein the buffered wash solution is about pH 5-7.

[0478] 52. The method of embodiment 51, wherein the buffered wash solution is about pH 6.

[0479] 53. The method of any one of embodiments 44-52, wherein the reaction is carried out in a solvent selected from the group consisting of DCM, DMF, MeCN, and DMAc, or a combination thereof.

[0480] 54. The method of embodiment 53, wherein the solvent is DMAc.

[0481] 55. The method of embodiment 53 or 54, further comprising adding an antisolvent to the solution comprising TGZ and the solvent.

[0482] 56. The method of embodiment 55, wherein the anti-solvent is an ether solvent.

[0483] 57. The method of embodiment 56, wherein the ether solvent is DME, 2-MeTHF, or MTBE.

[0484] 58. The method of any one of embodiments 53-57, wherein the solvent is DCM and the anti-solvent is MTBE.

[0485] 59. The method of any one of embodiments 44-58, wherein the TGZ produced has reduced impurities.

[0486] 60. The method of embodiment 59, wherein the impurity is deacylated.

[0487] 61. The method of any one of embodiments 44-60, further comprising precipitating TGZ from the solvent / antisolvent system.

[0488] 62. The method of embodiment 61, wherein the solvent / anti-solvent system is DCM / MTBE.

[0489] 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.

[0490] 64. The method of any one of embodiments 44-63, wherein the NAG-H or its salt is continued to be used for the reaction with the triacid as a solution of the previous reaction without isolating the NAG-H or its salt from the previous reaction.

[0491] 65. The method of embodiment 64, wherein NAG-H, or a salt thereof, is prepared in a previous reaction comprising hydrogenating NAG-Z in the presence of a Pd / C catalyst and dimethylacetamide (DMAc).

[0492] 66. The method of any one of embodiments 44-65, wherein the DMAc is about 3 volumes to about 5 volumes.

[0493] 67. The method of any one of embodiments 44-65, wherein the DMAc is about 4 volumes.

[0494] 68. The method of any one of embodiments 44-67, wherein the NAG-H or a salt thereof is NAG-H TFA.

[0495] 69. The method of any one of embodiments 44-66, wherein the Pd / C catalyst is 5% Pd / C.

[0496] 70. The method of any one of embodiments 44-69, wherein NAG-HTFA is prepared in a reaction comprising the reagents in the following scheme:

[0497]

[0498] 71. A method for preparing NAG-Z,

[0499]

[0500] The method comprises making acylGalNAc:

[0501]

[0502] With alcohol-Z:

[0503]

[0504] reaction to produce NAG-Z.

[0505] 72. The method of embodiment 71, wherein the reaction is carried out in the presence of an acid.

[0506] 73. A method as described in Example 72, wherein the acid is bismuth trifluoromethanesulfonate (Bi(OTf)3), boron trifluoride etherate (BF3OEt2), tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), indium trifluoromethanesulfonate (In(OTf)3), or copper trifluoromethanesulfonate (Cu(OTf)2).

[0507] 74. The method of embodiment 73, wherein the acid is bismuth trifluoromethanesulfonate (Bi(OTf)3), boron trifluoride etherate (BF3OEt2), indium trifluoromethanesulfonate (In(OTf)3), or copper trifluoromethanesulfonate (Cu(OTf)2).

[0508] 75. The method of any one of embodiments 72-74, wherein the acid is present in 0.1-0.2 equivalents relative to alcohol-Z.

[0509] 76. The method of any one of embodiments 72-74, wherein the acid is present in 0.13-0.17 equivalents relative to alcohol-Z.

[0510] 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.

[0511] 78. The method of embodiment 77, wherein the solvent is present in a total of 8-12 volumes.

[0512] 79. The method of any one of embodiments 71-78, wherein the acylGalNAc is present in 1.0-2.0 equivalents relative to alcohol-Z.

[0513] 80. The method of embodiment 79, wherein the acylGalNAc is present in 1.3-1.7 equivalents relative to alcohol-Z.

[0514] 81. The method of any one of embodiments 71-80, wherein the reaction temperature is heated to about 55-65°C or 58-62°C.

[0515] 82. The method of any one of embodiments 71-81, wherein the method comprises the reagents and conditions of the following scheme:

[0516]

[0517] 83. The method of any one of embodiments 71-82, wherein the alcohol-Z is prepared by reacting benzyl chloroformate with an amino alcohol in the presence of a solvent and a base.

[0518] 84. The method of embodiment 83, wherein the solvent is dichloromethane (DCM).

[0519] 85. The method of embodiment 83 or 84, wherein the base is triethylamine (TEA).

[0520] 86. The method of any one of embodiments 83-85, wherein the amino alcohol is present in 1.1 equivalents relative to benzyl chloroformate.

[0521] 87. The method of any one of embodiments 73-86, wherein the reaction temperature is about 30°C-40°C.

[0522] 88. The method of embodiment 86, wherein the reaction temperature is about 33°C-37°C.

[0523] 89. The method of any one of embodiments 77-87, wherein the solvent is acetonitrile and additional water is added to crystallize the NAG-Z.

[0524] 90. A method for preparing TG amine

[0525] or a salt thereof,

[0526] The method involves high pressure hydrogenolysis for carboxybenzyl deprotection of TGZ to form the TG amine or a salt thereof.

[0527] 91. The method of embodiment 90, comprising a palladium source for hydrogenolysis.

[0528] 92. The method of embodiment 91, wherein the palladium source is a palladium-carbon (Pd / C) catalyst.

[0529] 93. The method of embodiment 92, wherein the palladium source is 5% Pd / C. 4d. The method of any one of embodiments 4a-4c, wherein the supported catalyst is less than about 10%, 9%, 8%, 7%, 6% Pd / C. In some embodiments, the catalyst used is 5% Pd / C.

[0530] 94. The method of any one of embodiments 90-93, wherein the hydrogenolysis is performed in the presence of an acid.

[0531] 95. The method of embodiment 94, wherein the acid is TFA, oxalic acid, HCl, AcOH, H3PO4, citric acid.

[0532] 96. The method of embodiment 94, wherein the acid is TFA or oxalic acid.

[0533] 97. The method of embodiment 94, wherein the acid is TFA.

[0534] 98. The method of any one of embodiments 90-97, wherein the hydrogenolysis is performed in a solvent, and the solvent is DCM, IPAc, or MeOH.

[0535] 99. The method of embodiment 98, wherein the solvent is DCM.

[0536] 100. The method of any one of embodiments 90-99, wherein the method reduces the formation of deacyl impurities.

[0537] 101. The method of any one of embodiments 90-100, wherein the TG amine or salt thereof is a TG amine acid salt.

[0538] 102. The method of any one of embodiments 90-101, wherein the TG amine acid salt is phosphate, formate, acetate, trifluoroacetate, or oxalate.

[0539] 103. The method of any one of embodiments 90-102, wherein the TG amine acid salt is trifluoroacetate or oxalate.

[0540] 104. The method of embodiment 103, wherein the TG amine acid salt is trifluoroacetate.

[0541] 105. The method of any one of embodiments 90-104, wherein the method results in a TG PEG product of step 5b of high purity while reducing the amount of TG amine acetamide byproduct.

[0542] 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 byproduct.

[0543] 107. The method of any one of embodiments 90-106, wherein the TG amine or salt thereof produced from the improved step 5a method has a purity greater than or equal to about 97%, 98%, or 99% as measured by LC.

[0544] 108. The method of any one of embodiments 90-107, wherein the TG amine or its salt is scaled into step 5b without isolating the TG amine or its salt.

[0545] 109. The method of any one of embodiments 90-108, wherein the method comprises the reagents of the following scheme:

[0546]

[0547] 110. A method for preparing TG PEG,

[0548]

[0549] The method involves treating a solution of TG amine and PEG acid with TBTU.

[0550] 111. The method of embodiment 110, wherein the solution further comprises a base.

[0551] 112. The method of embodiment 111, wherein the base is N,N-diisopropylethylamine (DIPEA).

[0552] 113. The method of any one of embodiments 110-112, wherein the TBTU is added over a period of about 30 min to about 1.5 hours.

[0553] 114. The method of any one of embodiments 110-112, wherein the TBTU is added over a period of about 1-1.5 hours.

[0554] 115. The method of any one of embodiments 110-114, wherein the TG PEG is produced at a purity greater than or equal to about 90% as measured by LC.

[0555] 116. The method of any one of embodiments 110-115, wherein the TG PEG dimer impurity is present in an amount less than 10% at the end of the reaction as measured by LC.

[0556] 117. The method of any one of embodiments 110-116, wherein the method comprises the reagents of the following scheme:

[0557]

[0558] 118. A method for preparing NAG-25, the method comprising treating TG PEG with an activating agent and a phosphitylation agent.

[0559] 119. The method of embodiment 118, wherein the activator is tetrazole, DCI, ETT, or benzothiotetrazolium (BTT).

[0560] 120. The method of embodiment 118, wherein the activator is tetrazole, DCI, or ETT.

[0561] 121. The method of embodiment 118, wherein the activator is tetrazole.

[0562] 122. The method of embodiment 121, wherein the tetrazole is added in about 0.2 to 1.2 equivalents.

[0563] 123. The method of any one of embodiments 118-122, wherein the method is performed in the presence of a base.

[0564] 124. The method of any one of embodiments 118-123, wherein the base is NMI.

[0565] 125. The method of any one of embodiments 118-124, wherein the phosphitylation agent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphorodiamidite.

[0566] 126. The method of any one of embodiments 118-125, further comprising extracting, filtering, precipitating, and drying.

[0567] 127. The method of embodiment 126, wherein precipitating comprises precipitating NAG-25 from DCM / heptane.

[0568] 128. A method for preparing NAG-25,

[0569]

[0570] The method includes any of the embodiments for steps 1, 2, 3, 4a, 3b, 3a, 4, 5, or 6.

[0571] 129. The method of embodiment 128, wherein the method comprises preparing a tert-butyl core by the method of step 1 and converting the tert-butyl core into NAG-25.

[0572] 130. The method of embodiment 128, wherein the method comprises preparing the triacid by the method of step 2 and converting the triacid to NAG-25.

[0573] 131. The method of embodiment 128, wherein the method comprises preparing TGZ by the method of step 4b and converting the TGZ into NAG-25.

[0574] 132. The method of embodiment 128, wherein the method comprises preparing NAG-Z by the method of step 3b and converting the NAG-Z into NAG-25.

[0575] 133. The method of embodiment 128, wherein the method comprises preparing TG amine by the method of step 5a and converting the TG amine to NAG-25.

[0576] 134. The method of embodiment 128, wherein the method comprises preparing TG PEG by the method of step 5b and converting the TG PEG into NAG-25.

[0577] 135. A method for preparing NAG-25:

[0578] method,

[0579] The method comprises treating a mixture of TG amine and PEG acid with TBTU to obtain TG PEG

[0580]

[0581] as well as

[0582] Conversion of TG PEG to NAG-25.

[0583] 136. A method for preparing NAG-25, the method comprising benzyl deprotecting TGZ to form the TG amine:

[0584] or high pressure hydrogenolysis of its salts,

[0585] and converting TG amine, or a salt thereof, into NAG-25.

[0586] 137. A method for preparing NAG-25, comprising reacting a triacid in the presence of a coupling agent and a base

[0587]

[0588] Reacting with NAG-H or a salt thereof to form TGZ, and converting TGZ to NAG-25.

[0589] 138. The method of embodiment 137, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0590] 139. A process for preparing NAG-25, the process comprising reacting a tert-butyl core with an acid to form the triacid, and converting the triacid to NAG-25.

[0591] 140. The method of embodiment 139, wherein the triacid is crystalline.

[0592] 141. The method of embodiment 140, wherein the triacid is in crystalline Form I.

[0593] 142. A method for preparing NAG-25, the method comprising reacting GluZ with GluOtBu or a salt thereof to form the tert-butyl core, and converting the tert-butyl core to NAG-25.

[0594] 143. A method for preparing NAG-25, the method comprising precipitating TGZ from a solvent / antisolvent system.

[0595] 144. A process for preparing NAG-25 comprising a triacid intermediate that is crystalline.

[0596] 145. The method of embodiment 144, wherein the crystalline triacid is crystalline Form I of the triacid, characterized by an X-ray powder diffraction pattern having at least one signal at three 2θ values ​​selected from the group consisting 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.

[0597] 146. The method of embodiment 144, wherein the crystalline triacid is crystalline Form I of the triacid, characterized by an X-ray powder diffraction pattern having signals at the following 2θ values: 7.4±0.2, 9.2±0.2, and 21.0±0.2.

[0598] 147. The method of any one of embodiments 144-166, wherein the crystalline triacid is crystalline Form I of the triacid, characterized in that the X-ray powder diffraction pattern is substantially similar to Figure 2 X-ray powder diffraction pattern of .

[0599] 148. The method of any one of embodiments 144-147, wherein the crystalline triacid is crystalline Form II of the triacid, characterized in that the X-ray powder diffraction pattern is substantially similar to Figure 3 X-ray powder diffraction pattern of .

[0600] 149. A method for preparing a methyl core,

[0601]

[0602] The method comprises making zL-Glu-OMe:

[0603]

[0604] With L-glutamic acid dimethyl ester (di(OMe)Glu):

[0605] reacts to produce a methyl core.

[0606] 150. The method of embodiment 149, wherein the reaction is carried out in the presence of a base and a coupling reagent.

[0607] 151. The method of embodiment 150, wherein the base is added to the zL-GluOMe solution and then the coupling reagent and bis(OMe)Glu are added to the reaction.

[0608] 152. The method of embodiment 151, wherein the coupling reagent is added to a solution of zL-Glu-OMe and base, and then di(OMe)Glu is added to the reaction.

[0609] 153. The method of any one of embodiments 149-152, further comprising a solvent.

[0610] 154. The method of any one of embodiments 150-153, wherein the base is N-methylmorpholine (NMM) or diisopropylethylamine.

[0611] 155. The method of any one of embodiments 150-154, wherein the coupling reagent is isobutyl chloroformate (IBCF), TBTU, HATU, EDC, or DCC.

[0612] 156. The method of any one of embodiments 153-155, wherein the solvent is THF or MeTHF.

[0613] 157. The method of any one of embodiments 149-156, wherein the reaction temperature is about -20°C to about -10°C before and during the reaction with the coupling reagent.

[0614] 158. The method of embodiment 157, wherein the reaction temperature is about -18°C to about -13°C.

[0615] 159. The method of embodiment 157 or 158, wherein after adding the bis(OMe)Glu, the reaction temperature is increased to about 5°C to about 15°C.

[0616] 160. The method of embodiment 159, wherein the reaction temperature is increased to about 7°C to about 12°C.

[0617] 161. The method of any one of embodiments 149-160, further comprising additional charging of a coupling reagent, a base, and / or zL-Glu-OMe.

[0618] 162. The method of any one of embodiments 149-161, further comprising washing the reaction with an aqueous acid and then washing the reaction with an aqueous base.

[0619] 163. The method of any one of embodiments 149-162, further comprising crystallizing by adding an anti-solvent.

[0620] 164. The method of any one of embodiments 149-163, wherein the method comprises the reagents and conditions of the following scheme:

[0621]

[0622] 165. A method for preparing triols,

[0623]

[0624] The method involves reacting a methyl core with 2-(2-aminoethoxy)ethanol:

[0625] Reaction to produce a triol.

[0626] 166. The method of embodiment 165, wherein the method is performed using pure 2-(2-aminoethoxy)ethanol.

[0627] 167. The method of embodiment 165 or 166, wherein the reaction temperature is about 25°C to 35°C.

[0628] 168. The method of embodiment 167, wherein the reaction temperature is about 30°C.

[0629] 169. The method of any one of embodiments 165-168, further comprising adding an anti-solvent to precipitate the triol.

[0630] 170. The method of embodiment 169, wherein the anti-solvent is ethyl acetate, methyl tert-butyl ether, or isopropyl acetate.

[0631] 171. The method of any one of embodiments 165-170, further comprising adding triol seeds.

[0632] 172. The method of any one of embodiments 165-171, wherein the method comprises the reagents and conditions of the following scheme:

[0633]

[0634] 173. A method for preparing TGZ, the method comprising:

[0635]

[0636] With β-D-galactosamine pentaacetate:

[0637] reaction to produce TGZ.

[0638] 174. The method of embodiment 173, wherein β-D-galactosamine pentaacetate is first reacted with silyl triflate to produce an oxazoline solution.

[0639] 175. The method of embodiment 174, wherein the silyl triflate is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or triisopropylsilyl trifluoromethanesulfonate (TIPSOTf).

[0640] 176. The method of embodiment 175, further comprising a solvent.

[0641] 177. The method of embodiment 176, wherein the solvent is dichloroethane (DCE) or dichloromethane (DCM).

[0642] 178. The method of any one of embodiments 173-177, wherein the reaction temperature is about 35°C to about 45°C.

[0643] 179. The method of embodiment 178, wherein the reaction temperature is about 40°C.

[0644] 180. The method of embodiment 178 or 179, wherein the reaction temperature is reduced to about 20°C to about 28°C.

[0645] 181. The method of embodiment 180, wherein the reaction temperature is about 23 °C.

[0646] 182. The method of embodiment 173, wherein the triol is mixed with sodium bicarbonate (NaHCO 3 ) to produce a slurry mixture of the triol and NaHCO 3 , and then β-D-galactosamine pentaacetate or an oxazoline solution thereof is added.

[0647] 183. The method of embodiment 182, wherein the slurry mixture of triol and NaHCO3 further comprises a solvent.

[0648] 184. The method of embodiment 183, wherein the solvent is dichloromethane (DCM), dichloroethane, or acetonitrile.

[0649] 185. The method of any one of embodiments 173-184, wherein the oxazoline solution is added to the slurry mixture of triol and NaHCO3.

[0650] 186. The method of embodiment 173 or 185, wherein the reaction temperature is about 20°C to about 30°C.

[0651] 187. The method of embodiment 186, wherein the reaction temperature is about 25°C.

[0652] 188. The method of any one of embodiments 173-187, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0653] 189. The method of any one of embodiments 173-188, wherein an antisolvent is added to the solution comprising TGZ and the solvent.

[0654] 190. The method of any one of embodiments 188 or 189, wherein the anti-solvent is dimethoxyethane.

[0655] 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.

[0656] 192. The method of any one of embodiments 173-191, wherein the method comprises the reagents and conditions of the following scheme:

[0657]

[0658] 193. A method for preparing NAG-25 comprising any of Examples 149-164 for step A-1, any of Examples 165-172 for step A-2, any of Examples 173-192 for step A-3, or any of Examples 71-89 for step 3b.

[0659] 194. The method of embodiment 193, further comprising any one of embodiments 90-109 for step 5a, embodiments 110-117 for step 5b, or embodiments 118-127 for step 6.

[0660] 195. A method for preparing NAG-25, the method comprising reacting zL-Glu-OMe with dimethyl L-glutamate to form the methyl core, and converting the methyl core to NAG-25.

[0661] 196. A method for preparing NAG-25, the method comprising reacting a methyl core with 2-(2-aminoethoxy)ethanol to form a triol, and converting the triol to NAG-25.

[0662] 197. A method for preparing NAG-25, the method comprising reacting a triol with β-D-galactosamine pentaacetate to form TGZ, and converting the TGZ to NAG-25.

[0663] 198. The method of embodiment 197, wherein TGZ is further precipitated from the solvent / anti-solvent system.

[0664] 199. A process for preparing NAG-25 comprising a triol intermediate.

[0665] 200. A compound Wherein R is H or a Cbz protecting group.

[0666] It should be understood that although the present disclosure is read in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. For example, as shown in the following examples, these examples are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0667] Examples

[0668] Example 1: Step 1

[0669] An example of step 1 is shown below.

[0670]

[0671] Prepare GluZ solution as follows: 1.0 equivalent of GluZ, 3.5 equivalents of NMM, and 5 volumes of MTBE are charged into a clean inert reactor 1. 1.2 equivalents of PivCl and 5 volumes of MTBE are charged into a separate clean inert reactor 2 at 0°C. Using reverse addition, the GluZ solution in reactor 1 is added to reactor 2 within 1 hour. A white slurry is formed and stirred for at least 30min. Then 1.3 equivalents of GluOtBu hydrochloride (exothermic) are charged in batches every 15min. Subsequently, the slurry is kept at 0°C for at least 30min. If the conversion is ≤97%, 0.5 equivalents of NMM, 0.4 equivalents of PivCl, and 0.45 equivalents of GluOtBu HCl are charged. If necessary, the reaction mixture can be kept at 0°C for 24h. 12.5 volumes of 0.5N HCl are slowly added to the reaction mixture at 0°C (exothermic) and stirred for 30min. These phases are allowed to stand and then separated. The organic layer is washed with 10 volumes of 1N sodium carbonate. These phases are allowed to stand and then separated. The organic layer is washed with water (10 volumes). These phases are allowed to stand and then separated. If necessary, the organic layer can be kept overnight at 0°C. The organic layer is concentrated to 6 volumes at 35°C and heptane (10 volumes) is charged at 35°C at one time. The jacket temperature (Tj) of the reactor is set to 50°C and stirred until completely dissolved. Then Tj is set to 40°C and 0.5wt% of seeds are charged. The tert-butyl core that can be used as a seed can be prepared as described in U.S. Patent No. 10,246,709 or by methods known to those of ordinary skill in the art. The slurry is stirred for at least 30min. Then, 15 volumes of heptane are charged within 3 hours at 40°C. Once the heptane addition is complete, Tj is slowly cooled to 20°C and kept 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 solid of the tert-butyl core was dried under nitrogen at 20 °C overnight.

[0672] 1 H NMR(500MHz,DMSO-d6)δppm 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)

[0673] Improved reaction conversions were also produced when paired with 2.4 equivalents of NMM instead of 3.5 equivalents of NMM.

[0674] PivCl and GluOtBu HCl screening

[0675]

[0676] PivCl and GluOtBu HCl were screened for equivalents to further improve the reaction conversion and yield. Conversion increased with higher equivalents of PivCl and GluOtBu HCl. However, the level of impurities also increased. The goal was to find the amount of PivCl that increased the conversion of the tert-butyl core without producing higher levels of impurities. The highest conversion was achieved with 1.2 equivalents of PivCl and 1.3 equivalents of GluOtBu HCl.

[0677] The following table provides the LCAP of the tert-butyl core from the PivCl equivalent screen:

[0678]

[0679]

[0680] Base equivalent screening

[0681]

[0682] The equivalents of NMM were checked using 1.2 equivalents of PivCl and 1.3 equivalents of GluOtBu HCl. The result was that there was still unreacted mixed anhydride at the end of the reaction. The determination of the amount of NMM will make the additional GluOtBu HCl a free base and allow the reaction to proceed. Having an excess of NMM (3-3.5 equivalents) resulted in no residual mixed anhydride. Higher equivalents (4-5 equivalents of NMM) were studied, but did not result in further reaction improvement.

[0683] The table below shows the LCAP of the tert-butyl core used for NMM equivalent screening

[0684]

[0685] Comparison of normal addition versus reverse addition

[0686] Since normal addition was assumed to have the potential for symmetrical anhydride formation, another study comparing normal addition versus reverse addition was performed. Both normal and reverse additions were compared using GluOtBu (1.3 eq.), NMM (3.5 eq.), PivCl (1.2 eq.). Under normal addition, a solution of PivCl in MTBE was added to a solution of GluZ, NMM, MTBE, and the reaction was cooled to 0°C and held for 30 minutes. Subsequently, GluOtBu hydrochloride was added in three portions every 15 minutes, and the reaction was then held for 30 minutes, with samples taken at different time points for liquid chromatography (LC) measurements.

[0687] The following table shows the comparison of reverse addition and normal addition of step 1 using the conditions shown below:

[0688]

[0689] The table below shows that normal addition resulted in about 88.7% conversion of the tert-butyl core, while reverse addition demonstrated about 97.5% or 96.1% conversion.

[0690] Added method GluZZ's LCAP LCAP with tert-butyl core Normal Add 11.3 88.7％ Add in reverse 2.5 97.5 Add in reverse 4.0 96.1

[0691] Screening of solvents / antisolvents for crystallization

[0692] A high throughput experiment (HTE) solubility screen was performed to determine the solvent / antisolvent combination for crystallization and to compare the reaction solvent to the crystallization solvent. A solubility study of the tert-butyl core at 20°C was performed for different percentages of heptane. For 2-MeTHF, MIBK, and IPAc, the tert-butyl core was found to have a steep solubility curve (cliff) from 50 mg / mL to 10 mg / mL between 80% and 90% heptane, which is not optimal when designing crystallization. The solubility screen showed that MTBE / heptane is an example of a useful solvent mixture for crystallization because its solubility curve is flat.

[0693] Example 2: Step 2

[0694] An example of step 2 is shown below.

[0695]

[0696] 3 volumes of H3PO4 were added to a mixture of tert-butyl core (1 equivalent), 2-MeTHF (3.5 volumes) and 0.5 volumes of water, and the mixture was heated to 50°C for 4.5 hours. Phosphoric acid was effectively removed by aqueous extraction: the reaction mixture was diluted with isopropyl acetate (iPAC) (10 volumes) and washed with 20 wt% ammonium sulfate ((NH4)2SO4) aqueous solution (5 volumes). Then, the organic layer was washed with 1.5 volumes of water. After azeotropic distillation, acetone (4 volumes) was charged and the mixture was finely filtered to remove residual salts. Acetone (2 volumes) was rinsed into the reactor through the filter. The mixture was heated to 45°C, and toluene (6 volumes) was charged into the reactor. 1 wt% triacid seeds were charged at 45°C. The slurry was stirred at 45°C. After keeping for 6–18h, toluene (6 volumes) was charged within 2.5 hours, and the solution was stirred until a mother liquor concentration of <6 mg / ml was reached. The reactor was cooled to 20°C and stirred for no less than 0.5 hr. The solid product was filtered and washed once with premixed 2:1 toluene:acetone (3 volumes). The filter cake was dried under vacuum at 20°C under a stream of nitrogen. For the triacid prepared in a similar manner as described above, the mass spectrum of UPLC-MS was m / z (ESI, positive ion): 411.27 (M+H) + The triacid isolated from this preparation is crystalline Form I.

[0697] As an alternative to using IPAc, the crude reaction mixture was washed with 20 wt% ammonium sulfate (5 volumes) and water (2 volumes) and aqueous extraction was performed with MeTHF. After adding MeTHF (6-10 volumes), the mixture was distilled and the MeTHF addition and distillation were repeated up to two more times. The next step continued with acetone and fine filtration as previously described.

[0698] When studying the deprotection step, it was found that the addition of water as the tert-butyl acceptor increased the rate and conversion of the deprotection reaction compared to MeTHF as the sole solvent (Table 1) (Table 1; conditions = 2 volumes H3PO4, 60°C). Additional experiments found that 3 volumes H3PO4 with 0.5 volumes water at 55°C or 50°C provided triacids with a purity of 94%-95% by liquid chromatography (LC) and with fewer impurities than the 2 volumes H3PO4 / 60°C system.

[0699] Table 1 Effect of water on deprotection characteristics.

[0700]

[0701] Preparation and XRPD Characterization of Triacid Crystalline Form I

[0702] During method development, acetone / toluene was discovered as a crystallization system via high throughput screening. In additional experiments, triacid crystalline Form I was generated by seeding a 49:51 toluene:acetone mixture at 45°C and allowing it to desaturate at 45°C, resulting in the formation of crystalline triacid. Figure 1 is an example of crystalline triacid Form I. The resulting solid is typically formed in >99% purity by LC.

[0703] Crystal preparation: Triacid crystalline form I is formed by charging acetone (4.9 volumes) into the distilled crude reaction stream (after extractive workup, distillation and two acetone additions and withdrawals). The mixture is heated to 48°C and charged with 4.7 volumes of toluene. At 45°C, seed is charged. Stirring is continued at 45°C for about 20 hours before charging with 3.5 volumes of additional toluene. The reactor is cooled to 20°C within 1 hour, the solid is filtered and washed once with 3:2 toluene:acetone, and then dried under vacuum.

[0704] X-ray powder diffraction: X-ray powder diffraction data were obtained on a PANalytical X'Pert PRO X-ray diffraction system with an RTMS detector. CuKα radiation was used in continuous mode from 5 to 45° (2θ) at 45 kV and 40 mA with a step size of 0.0334°. The sample was scanned. The incident beam path was equipped with a 0.02 radian Soller slit, a 15 mm mask, a 4° fixed anti-scatter slit, and a programmable divergence slit. The diffracted beam was equipped with a 0.02 radian Soller slit, a programmable anti-scatter slit, and a 0.02 mm nickel filter. The sample was prepared on a low background sample holder and placed on a rotating stage for 2 s. The XRPD pattern of the triacid crystalline form I in toluene / acetone form is shown in Figure 2 The XRPD peaks are shown in Table 2 below.

[0705] Table 2: XRPD signals of triacid crystalline form I

[0706]

[0707] Preparation of Triacid Crystalline Form II

[0708] 2g of triacid is charged into a 40mL vial with a stirring bar. 1V (2mL) of 2-MeTHF and 1.5V (3mL) of iPAc are added. The mixture is heated to 50°C with stirring to dissolve the triacid. 13.5V of iPAc (27mL) is added to the mixture. When iPAc is added, the solution is turbid and some oil is observed. The temperature is increased until an internal temperature of 50°C is reached. When heated and stirred, the oil becomes a loose solid. The slurry is kept at 50°C for 15min. The mother liquor sample concentration is measured to be 58mg / ml triacid. The mixture is cooled to 45°C to produce a very thick slurry, which is then cooled to room temperature. The concentration of the mother liquor is measured to be 5mg / mL triacid. At this point, the slurry is transferred to a filter. 10V of iPAc (20mL) is charged to loosen the residual solids in the vial, and the mixture is also charged into the filter. Finally, the vial was rinsed with 2.5V iPAc (5mL) and the mixture was added to the filter. The solid was dried overnight with a stream of nitrogen under vacuum. 1.55g of solid was isolated. Quantitative NMR (using maleic acid internal standard, d6-DMSO solvent) indicated that the material was 86wt% triacid and 11wt% iPAc. The XRPD pattern of triacid crystalline form II is Figure 3 The resulting triacid crystalline Form II can be used as a triacid seed in a series of crystallizations to produce triacid crystalline Form I using the Step 2 example described above.

[0709] Example 3: Step 3a

[0710] Step 3a: An example of step 3a is shown below.

[0711]

[0712] A solution of amino alcohol (1.1 equivalents), TEA (1.0 equivalents) and DCM (4 volumes) was cooled to 5°C. CbzCl (1.0 equivalents) was added within one hour, and once the addition was complete, the solution was heated to 35°C for 12 hours. The solution was cooled to 20°C and quenched by adding water (2 volumes). The collected aqueous layer was stripped with DCM (2 volumes). The combined organic layer was washed twice with 10% NaCl (2 volumes), and the solvent was exchanged into ACN (5 volumes). The alcohol-Z (abbreviation: Alc-Z) product was then separated into 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).

[0713] Example 4: Step 3b

[0714] Step 3b: An example of step 3b is shown below.

[0715]

[0716] AcylGalNAc (1.5 eq.), Bi(OTf)3 (0.15 eq.), and ACN (5 vol.) were added to an inert flask. A solution of alcohol-Z (1.0 eq.) was azeotropically dried and added to a flask in ACN (5 vol.). The solution was heated to 60°C and stirred for 18 hours. After cooling to 20°C, charcoal (50 wt. %) was added and stirred at room temperature for 12 hours. The charcoal was then filtered out and water (18.6 vol.) was charged to the filtrate. The resulting solution was inoculated with NAG-Z (0.5 wt. %) and stirred for 30 minutes. If the seed did not remain, the inoculation method was repeated until it remained. If the seed did remain, water (38 vol.) was added within one hour and the resulting slurry was stirred for 12 hours. The resulting product was filtered, washed twice with water wash (2 vol.), and vacuum dried under a stream of nitrogen. The NAG-Z product (71.27%, adjusted potency) was then obtained as a white solid. 1 HNMR(600MHz,DMSO-d6)δ7.78(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).

[0717] Lewis acid screening for step 3b: For the NAG-Z method, the yields of certain Lewis acids commonly used in glycosylation reactions are very low. Lewis acid screening was performed under the conditions described below:

[0718] Lewis acids of moderate strength were found to be useful for the formation of NAG-Z. Some Lewis acids were too weak to achieve complete conversion of alcohol-Z, while others were too strong and decomposed 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 under the given reaction conditions, and Column B: NAG-Z degradation shows the degradation observed after subjecting NAG-Z to the corresponding Lewis acid for 24 hours.

[0719] Lewis acid condition A:NAG-Z LCAP B: NAG-Z degradation <![CDATA[BiCl3]]> 0.15 equivalent, 60℃ 11.37 - <![CDATA[Fe(OTf)2]]> 0.15 equivalent, 60℃ 17.54 - <![CDATA[In(OTf)3]]> 0.15 equivalent, 60℃ 66.66 - <![CDATA[Cu(OTf)2]]> 0.15 equivalent, 60℃ 74.91 - <![CDATA[Bi(OTf)3]]> 0.15 equivalent, 60℃ 78.85 1.79％ <![CDATA[BF3OEt2]]> 1 equivalent, room temperature 46.57 5.43％ TBSOT 1 equivalent, room temperature 66.98 8.75％ TIPSOT 1 equivalent, room temperature 62.08 25.83％ TMSOT 1.1 equivalent, room temperature 55.02 64.03％

[0720] AcylGalNAc equivalent screening for step 3b: Due to the potential hydrolysis mechanism shown below, the equivalents of acylGalNAc were tested.

[0721]

[0722] Due to the hygroscopicity of the bismuth catalyst, hydrolysis is difficult to avoid by drying, so alcohol-Z is used as a limiting reagent to increase the conversion rate, and the hydrolysis impurity DGalOH can be purged during the crystallization of NAG-Z. The conditions used for equivalent screening (DGalNAc is the same as acyl GalNAc) are shown below.

[0723]

[0724] The results of the equivalent screening are summarized below, along with the major impurities found (acylGalNAc, DGalOH, alcohol-Z).

[0725]

[0726]

[0727] Solvent volume for step 3b: Lower volumes of solvent generally result in reduced volume (better from a green chemistry / sustainability perspective) and generally result in less product loss to the mother liquor. Therefore, several reaction volumes of acetonitrile were screened. The following table summarizes the LCAPs for the alcohol-Z, NAG-Z, and Ac-Z impurities:

[0728]

[0729] ACN Volume Alcohol-Z LCAP Ac-Z LCAP NAG-Z LCAP 3.0 3.13 3.04 73.22 6.0 3.10 1.10 69.95 7.5 2.50 2.56 61.72 8.0 2.51 1.17 60.88 8.5 2.92 2.31 60.64 9.0 2.60 0.38 63.29 10.0 2.32 0.50 67.87 20.0 43.89 0 42.33

[0730] Example 5: Step 4a

[0731] Step 4a: An example of step 4a is shown below.

[0732]

[0733] 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. The headspace was then purged with hydrogen and set to 46 psi (3.1 bar) of hydrogen. Under these conditions, the reaction was stirred at 21.6 C for 19 h. The catalyst was removed via filtration and washed with DMAc (350 ml; 1 V). The resulting clear solution was determined to contain 256.9 g NAG-H at 97.1 LCAP (96.7% yield).

[0734] Example 6: Step 4b

[0735] Step 4b: An example of step 4b is shown below.

[0736]

[0737] Note: All equivalents / volumes are relative to the triacid

[0738] A 5L jacketed reactor equipped with a nitrogen inlet and a temperature probe was charged with triacid (55.0 g, 1.0 eq.) and NAG-H TFA solution (13.27 wt %, 3.5 eq. in DMAc). The mixture was stirred for 10 minutes to dissolve the triacid and then cooled to Tj = 10 °C. N-methylimidazole (115 mL, 12.0 eq.) was added to the cooled solution via an addition funnel, followed by a one-time addition of solid TBTU (154 g, 4.0 eq.). The jacket temperature was set to 20 °C and the reaction was heated within 1 hour. The reaction was then quenched by slowly adding 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 minutes. The phases were separated and the aqueous phase was extracted with additional DCM (1100 mL, 20 mL / g). The organic layer merged is washed with 50mM pH 6 potassium phosphate buffer (2x1100mL).DCM solution is dried to 20V by azeotropic distillation, then fresh DCM (1100mL, 20mL / g) is supplemented.The reaction stream in 40mL / g DCM is heated to Tj=30°C, then MTBE (2200mL, 40mL / g) is charged in 3 hours.Slurries are cooled to 25°C, aged no more than 4 hours, and filtered.Filter cake is washed with 1:1DCM:MTBE (2x1100mL, 5.0mL / g TGZ), and dried under vacuum to provide TGZ (88% yield) as a white solid. 1H NMR(600MHz,DMSO-d6)δppm 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).

[0739] Screening experiments were performed to determine various embodiments of the reaction conditions described herein. The results of one example of the screening are shown in the table below. TBTU and HATU provided high conversion of TGZ product (>95 LCAP).

[0740]

[0741] After two rounds of HTE screening and hit validation, TBTU in DMF provided a complete conversion of the intermediate to TGZ with a clear reaction profile. While all bases included in the screen were effective, NMI provided the highest LCAP TGZ (96.36 LCAP) and 0.15 LCAP for deacylation. These conditions were subsequently validated on a gram scale, providing complete conversion within 1 hour. It was also demonstrated that the reaction was equally good using dimethylacetamide (DMAc) as a solvent instead of DMF.

[0742] Additional reagent equivalence screening experiments were also performed. The effects of reaction components on reaction ratios are shown in the table below.

[0743]

[0744] Example 7: Step A-1

[0745] Step A-1: ​​Amide coupling to prepare the methyl core. An example of this step is shown below.

[0746]

[0747] zL-Glu-OMe (50 g, 1.0 eq) was charged to a 1 L reactor via a powder addition funnel. THF (10 vol) was charged under positive nitrogen pressure and dissolution was complete in <3 min. The reaction was cooled to -15°C. While the reaction was cooling, N-methylmorpholine (NMM) (41.8 mL, 2.5 eq) was charged to the reaction. The addition port was flushed with 0.5 vol of THF and once the reaction temperature reached -15°C, isobutyl chloroformate (IBCF) (22 mL, 1.1 eq) was charged and a white precipitate formed immediately. The internal temperature was maintained < -10°C during the addition. The reaction was aged at -15°C for 1 h. Dimethyl L-glutamate (di(OMe)Glu) was charged in 5 equal portions (36.2 g). The reaction was warmed to 10°C over 2 h and aged there for 16 h. If necessary, an additional 10 mol% charge of IBCF, NMM and ZL-Glu-OMe can be added. The reaction is allowed to react for an additional 3 h. Once the reaction reaches full conversion, the reaction is worked up.

[0748] Charge 6 volumes of EtOAc, then charge 8 volumes of 0.5M HCl aqueous solution, divide the phases, and drain the water. Charge 8 volumes of 0.5M NaOH aqueous solution, divide the phases, and drain the water. Charge 8 volumes of saturated aqueous saline solution, divide the phases, and drain the water. Drain the turbid organic layer and perform fine filtration. Add the organic stream back into a clean 1L reactor. Distill the organic solution to 400mL under vacuum (45°C, 170mmHg). Perform two consecutive azeotropic distillations with EtOAc, charge an additional 300mL (total organic volume of about 800mL) and distill to about 400mL (45°C, 200mmHg). Charge 4 volumes of heptane dropwise at 40°C. Charge 2.0wt% methyl core seeds and keep the temperature for 30min before slowly cooling to 23°C. Charge another 6 volumes of heptane dropwise and age the slurry overnight.

[0749] The slurry was drained into an injection bottle and filtered through an 800 mL medium porosity sintered funnel. The filter cake was washed with 2x5 volumes of EtOAc / heptane (1:1). The filter cake was dried under nitrogen / vacuum flow for 4 hrs.

[0750] Example 8: Step A-2

[0751] Step A-2: Aminolysis to prepare triol An example of this step is shown below.

[0752]

[0753] The methyl core (97w%, 3.5g, 1.0 equivalent) was charged into a 100mL reactor. 2-(2-aminoethoxy)ethanol (6 volumes, 21mL) was charged into the flask under a positive pressure of nitrogen. The temperature was set to 30°C and aged at this temperature for 24h. 20 volumes of ethyl acetate (70mL) were charged into the reactor flask at 30°C. 2wt% triol seeds were charged and the seeds were checked to see if they were maintained at this temperature. The reaction was cooled to 23°C and the slurry was aged for 18h. The slurry was filtered in a 300mL filter funnel and the filter cake was kept under a steady stream of nitrogen. The filter cake was washed with 10 volumes of ethyl acetate (EtOAc). The triol filter cake was collected and added back into a clean and dry 100mL reactor for re-slurrying. 15 volumes of EtOAc were charged and the slurry was heated to 40°C; maintained for 3h; cooled back to 23°C and aged for another 3h. The slurry was filtered through a 500mL filter funnel. The filter cake was washed with 10 volumes of EtOAc and dried under nitrogen / vacuum bag for 24 h. The isolated triol filter cake was 2.75 g, 55% yield.

[0754] Step A-2: Initial hydrolase screening: Preliminary enzyme screening based on hydrolases was performed using 10 different lipases, a peptidase, a protease, and an aldolase. Reactions were set up on a 50 mg scale with a methyl core as a limiting reagent. Enzyme loading was based on the physical state of the reagent: regardless of the unit count, liquid-based enzymes were charged with 5 μL, freeze-dried enzymes were charged with 5 mg, and solid-supported enzymes were charged with 50 mg. These enzymes were screened for three different solvent systems: MeCN and THF (at 20 V) and pure (8 equivalents) in amino alcohols. Enzyme screening reaction conditions are shown below.

[0755]

[0756] Reactions were set up in 2 mL vials and stirred on an oscillator plate at 45 °C for 48 hr. The crude reaction mixture was then analyzed by HPLC without subsequent processing or separation. Enzyme reactivity was significantly affected by the reaction medium, and in general, the reaction results showed the following trend: pure conditions > THF > MeCN. For all reactions performed under pure conditions, the triol was the major crude reaction component, and the methyl core and intermediate mono- and di-addition products were completely consumed, although this may be an inherent result of the high reaction concentrations and higher amino alcohol equivalent loadings. Alternatively, MeCN was identified as a poor solvent for biocatalytic aminolysis as most reactions resulted in complex mixtures as well as unreacted starting materials. Reactions in THF showed efficient conversion of starting materials and intermediates, but the extent of conversion varied between enzymes ( Figure 5). It should be noted that some enzymes, especially liquid-based variants, perform better in THF, while solid-supported enzymes provide the best results under pure conditions (Table 3). The advantage of these immobilized enzymes is that they do not require aqueous treatment. The triol product is water-soluble, and if an aqueous wash is required to remove enzyme residues in the organic product, the desired triol is lost to the same aqueous phase. Enzymes immobilized on solid supports will allow the reagents to be filtered from the organic reaction solvent and then recycled, thereby providing minimal enzyme degradation. In addition, once the enzyme reagents are removed from the reaction mixture, the triols can be separated from most organic solvents (including pure reaction conditions) by precipitation. In the most successful reactions between THF and pure conditions, triols were obtained at the higher 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) becoming proficient enzymes for conversion (Table 3). These results indicate that lipases may be the subclass of hydrolases best suited for the desired reactions. For all reactions, the diol impurity was generally formed at <10% liquid chromatography area percentage (LCAP), supporting our hypothesis that lower reaction temperatures could prevent diol formation. The major impurity formed in all reactions (15%–30% LCAP) was not isolated or fully identified, and the unknown impurity had the same mass as the triol product by LCMS analysis and was assumed to be a stereoisomer of the triol.

[0757] Table 3: General results from enzyme screening in THF conditions and neat conditions:

[0758]

[0759]

[0760] These experiments demonstrate the feasibility of biocatalytic aminolysis to triols. Conditions were determined that resulted in complete conversion of the starting materials and intermediate mono- and diamides to give triols as the major reaction products.

[0761] Example 9: Step A-3

[0762] Step A-3: Triple glycosylation to prepare TGZ An example of this step is shown below.

[0763] β-D-galactosamine pentaacetate (10.1 g) was charged into a 100 mL reactor flask. DCE (37 mL, 0.7 M) was charged into the flask under a positive nitrogen pressure. TMSOTf (6.0 mL) was first slowly charged to prevent any initial vapor release. The reaction was heated to 40 ° C for 1.5 h; then cooled back to 23 ° C. At the same time, a reaction flask with triol was set up. Triol (95w%, 2.5 g, 1.0 eq) was charged into a 50 mL reactor, followed by solid NaHCO (0.2 eq). The sealed flask and inert headspace were flushed with nitrogen. DCM (6 volumes) was charged into the triol and NaHCO mixture. The oxazoline solution was charged into the triol / DCM slurry (1.0 eq of oxazoline solution / hr up to 4.5 eq) in batches at 25 ° C. The reaction was aged overnight (24 h) at 25 ° C. With high stirring, Et3N (3.0 equiv.) was charged and the quenched reaction was aged for 20 min.

[0764] Charge saturated (saturated / sat.) aqueous (aqueous / aq.) NaHCO3 (10 vol); separate the phases, drain the DCM / DCE layer, remove the aqueous, recharge the DCM / DCE solution; rinse with 2 vols of DCM. Charge saturated aqueous NH4Cl (10 vol); separate the phases, drain the DCM / DCE layer, and remove the aqueous. Dry the organic stream over MgSO4, filter through a filter funnel; rinse the dried solid with 2 vols of DCM. Charge the DCM / DCE organic solution to a clean 100 mL reactor (approximately 35 mL of organic solution, 14 vols wrt starting material). Charge DME (17 vols, 42 mL) dropwise to the organic solution over 1 hr to give a 1:1.2 ratio of solvent:antisolvent. Age the precipitate overnight (20 h).

[0765] The slurry was filtered through a medium porosity sintered funnel; the reactor was rinsed with 5 volumes of DCM / DME (1: 1) and the filter cake was washed. The filter cake was dried under a nitrogen / vacuum flow for 2 hours. The solid was collected and added to a 100 mL reactor. 20 volumes (relative to the starting material) of DCM / DME solution (1: 1) were charged. The slurry was heated to 40 ° C; maintained for 3 hr; then cooled to 25 ° C and aged for another 2 hr. The slurry was filtered through a filter funnel; the filter cake was washed with 5 volumes (DCM / DME 1: 1). The analyzed liquid in the filtered mother liquor was 3.5 mg / mL of TGZ. The TGZ filter cake was 92.6 LCAP. The filter cake was dried under a nitrogen / vacuum bag to give TGZ as a solid.

[0766] Example 10: Step 5a

[0767] An example of step 5a is shown below.

[0768]

[0769] TGZ (1.0 equivalent) was charged into a high pressure reaction vessel and 7% Pd / C catalyst (5wt% loading) was added. DCM solvent (10 volumes) was charged and then TFA (1.0 equivalent) was charged. The reactor was sealed and 3x reactor vessel purged with nitrogen, stirring was started during filling and stirring was stopped during discharge. 3x reactor vessel purged with H2 gas at 50 or 60psi without stirring. The reaction stirring rate was set to 300rpm, the jacket temperature was set to 23°C, and the H2 pressure was set to 45psi. The reaction was aged overnight. The headspace was purged 3x with nitrogen before the lid was removed. The heterogeneous solution was filtered through a 0.45 micron PTE filter, the reactor was rinsed with 3 volumes of DCM, and the rinse was added to the filter. Ensure that the Pd / C filter cake does not dry out. The effectiveness of the TG amine of the solution was determined and the TG amine-TFA solution in DCM was stored at <10°C. TG amine solution purity = 97.2% Mass spectrum: expected mass = 1525.64147, observed m / z: 1525.63930.

[0770] Example 11: Step 5b

[0771] An example of step 5b is shown below.

[0772]

[0773] The TG amine solution in DCM is adjusted to 12 volumes of DCM, quantitative (1.0 equivalent) relative to the LC determination of TG amine. The reactor headspace is purged with N2, and the reactor is arranged under N2 positive pressure, the jacket temperature is set to -5 ℃ and stirring is started. PEG acid (1.1 equivalents) (measured by mass) is added, followed by DIPEA (3.0 equivalents). TBTU (1.3 equivalents) solid is added to the reactor in batches under N2 purge within 1 hour. Once the TBTU solid is completely dissolved (about 1hr), the jacket temperature is set to ramp up to 0 ℃ in 30 minutes.

[0774] 12 volumes of DI water were charged into the reaction, and the internal temperature was set to 10°C. The biphasic mixture was stirred for 30min, and then the layers were separated, and the DCM waste was discharged. The reactor contents were heated to 23°C. 13wt% (NH4)2SO4 (charged with respect to water) was added to the aqueous TG PEG solution, and then 10 volumes of DCM were added. The layers were mixed in 30min, and then the layers were separated. The layers were separated, and the organic layer was washed with a mixture of 0.5M sodium monophosphate (pH 6.0) and saturated brine (4.8 volumes: 3.2 volumes). The layers were mixed for 30min, and then the layers were separated. The organic layer was washed with a mixture of saturated sodium bicarbonate and saturated brine solution (4.0 volumes: 4.0 volumes). The layers were mixed for 30min, and then the layers were separated.

[0775] The TG PEG / DCM solution was charged to the reactor and distilled to 5 volumes. An additional 10 volumes of fresh DCM were added and distilled a second time to 5 volumes.

[0776] Discharge concentrated TG PEG / DCM solution and rinse the reactor with 0.5 volume DCM. 20 volumes of MTBE are added to the reactor, and the internal temperature is set to 0 ° C. TG PEG / DCM solution is added to cold MTBE within 1 hour. Once the addition is complete, the mixture is filtered. The filter cake is washed with 3 volumes of MTBE (x2). The solid is then dried for at least 24 hours through a nitrogen purge.

[0777] TG PEG isolated yield = 79.8% adjusted potency. HRMS: exact mass = 1861.81989, observed m / z: 931.41 [M+2H]+

[0778] High throughput experiments were performed on the reaction conditions using 96-well plates. Examples of screening conditions are as follows: 1.0 equivalent of TG amine TFA salt, 1.1 equivalent of PEG acid, 3.0 equivalent of base, and 1.1 equivalent of coupling reagent. Each combination was evaluated in DCM, DMF, and MeCN (see Figure 4 The results in ( ). The bases evaluated were DIPEA, NMI, NMM, and TMP. The results were evaluated based on the LCAP of TG PEG. From this screening, HATU and TBTU provided TG PEG with> 75 LCAP, as well as fully converted starting materials and intermediates. An addition sequence experiment was also performed, in which each of these 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. Adding TBTU provided the least total impurity amount (e.g., TG PEG dimer reduction) and the highest TG PEG yield.

[0779] Example 12: Step 6

[0780] An example of step 6 is shown below.

[0781]

[0782] NAG-25 was prepared from TG PEG using a method similar to the above protocol, which is further described in the following steps. HRMS: expected mass = 2078.95400, observed m / z: 2078.95428 [M+NH4]+

[0783] TG PEG (1.0 equivalent), DCM (20V) were added to the reactor, followed by NMI (0.2 equivalent), and distilled to achieve ≤200ppm water. The reaction volume was then reduced to 15V, then cooled to 0°C-5°C, followed by tetrazole (0.6 equivalent) and P-reagent 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.25 equivalent). After the addition was completed at 0°C-5°C, the reaction was then warmed to 20°C±5°C and aged for 2.5h before sampling to convert the starting TG PEG. After completion, the reaction was cooled to 10°C and then washed twice with 10V DI water. The organic solution was dried over molecular sieves and filtered under nitrogen protection. The resulting filtrate was concentrated to 8V at 20°C and then precipitated into 30V of heptane via reverse addition. The obtained solid was separated by filtration and washed twice with 10V heptane, then dried under vacuum under nitrogen.

[0784] An alternative example of preparing NAG-25 from TG PEG is as follows. A solution of TG PEG (24.87 g, 13.36 mmol, 1 eq) was prepared in anhydrous DCM (186.5 mL, 0.07 M). Another solution containing the P-reagent 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (5.57 g, 16.70 mmol, 1.25 eq) and 4,5-dicyanoimidazole (0.039 g, 0.33 mmol, 0.025 eq) was prepared in anhydrous DCM (186.5 mL, 0.07 M). The TG PEG solution was charged into 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 were performed with DI water (3x248.7 mL). The NAG-25 solution was then azeotropically dried at 30°C under vacuum. After the solution was filtered, the NAG-25 solution was added to anhydrous heptane (745 mL) over 90 minutes. The solid was filtered, washed twice with anhydrous 4:1 heptane:DCM (198.9 mL heptane / 49.7 mL DCM), and dried overnight under vacuum / N2.

[0785] Experiments were conducted for reaction conditions including activator and amount thereof, P-reagent and amount thereof, solvent volume, reaction time, order of addition, and rate of addition. Reaction samples were taken at different time points for liquid chromatography measurements, and the LCAP of TG PEG, H-Phos, NAG-25 dimer, NAG-25, and other impurities were determined. LC results indicated that the rate of addition of TG PEG to P-reagent / DCI over a period of 2.5-10 hours can be used to reduce the formation of NAG-25 dimer. For addition mode experiments, results showed that the addition of TG PEG to P-reagent generally reduced NAG-25 dimer impurities. In addition, 1.25 equivalents of P-reagent increased NAG-25 conversion. Results also showed an overall reduction in H-PHOS after precipitation.

[0786] Although examples of certain specific embodiments are 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.

[0787] The foregoing description has been given for clearness of understanding only, and no unnecessary limitations should be understood as modifications within the scope of the invention will be apparent to one skilled in the art.

[0788] Throughout this specification, when compositions are described as comprising components or materials, unless otherwise described, it is contemplated that these compositions may also consist essentially of or consist of any combination of the recited components or materials. Similarly, when methods are described as comprising specific steps, unless otherwise described, it is contemplated that these methods may also consist essentially of or consist of any combination of the recited steps. The invention illustratively disclosed herein may be suitably practiced in the absence of any element or step not expressly disclosed herein.

[0789] As will be clear to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has different components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of events recited or in any other order that is logically possible.

[0790] The practice of the methods disclosed herein and individual steps thereof may be performed manually and / or with the assistance of electronic devices or automation provided therefrom. Although many methods have been described with reference to specific embodiments, it will be readily apparent to one of ordinary skill in the art that other ways of performing actions associated with these methods may be used. For example, unless otherwise described, the order of the steps may be changed without departing from the scope or spirit of the method. In addition, some individual steps may be combined, omitted, or further subdivided into additional steps.

Claims

1. A method for preparing NAG-25, The method comprises making TG PEG: Reacting with an activator and a phosphitylation agent, wherein the activator is tetrazole, 4,5-dicyanoimidazole (DCI), 5-ethylthio-1H-tetrazole (ETT), or benzothiotetrazole (BTT); and / or wherein the phosphitylation agent is 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite or 2-cyanoethyl N,N-diisopropylchlorophosphorodiamidite.

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

3. The method according to claim 1 or 2, wherein: a) the TG PEG is in solution, and wherein an activator and a phosphitylation agent are combined in another solution; and b) adding the solution of TG PEG to the solution of activator and P-agent over a period of time, optionally wherein the period of time is about 2.5-10 hours or optionally wherein the solution of TG PEG is reacted with the solution of P-agent and activator at about 35°C-45°C.

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

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

6. A method for preparing TG PEG, The method comprises making TG amine: or a salt thereof and PEG acid is reacted with a coupling reagent, wherein the coupling reagent is added to the solution of PEG acid and TG amine or a salt thereof over a period of about 30 min 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 of any one of claims 6-7, wherein the coupling reagent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU) or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethylammonium hexafluorophosphate N-oxide (HATU).

9. The method of any one of claims 6-8, wherein the TG PEG is produced at greater than or equal to about 90% purity, 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 of any one of claims 6-9, wherein the solution of TG amine or its salt comes from a different process and the TG amine or its salt is not isolated, optionally wherein the TG amine or its salt is TG amine trifluoroacetic acid (TFA) salt.

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

12. A method for preparing TG amine or its salt, The method comprises subjecting TGZ to high pressure hydrogenolysis to form the TG amine or a salt thereof, wherein the hydrogenolysis is performed in the presence of an acid.

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

14. The method of any one of claims 12-13, wherein the acid is trifluoroacetic acid (TFA), oxalic acid, hydrochloric acid (HCl), acetic acid (AcOH), phosphoric acid (H3PO4), or citric acid; optionally wherein the acid does not exceed about 1.1 equivalents.

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

16. The method of any one of claims 12-15, wherein the method reduces the formation of one or more impurities, the one or more impurities being TG amine deacylation, TG amine di-NAG-OH, TG amine guanidyl, TG amine acyl, TG acetamide, and / or NAG-H guanidine.

17. The method of any one of claims 12 to 16, wherein the TG amine or its salt is continued to be used in another method for preparing TG PEG as a solution of TG amine or its salt without isolating the TG amine or its salt.

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

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

20. A method for preparing TGZ, The method comprises reacting a triacid with NAG-H in the presence of a coupling reagent and a base: or its salt; precipitating TGZ from the solvent / antisolvent system.

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

22. The method of any one of claims 20-21, 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. The method of any one of claims 20-22, further comprising at least one buffered wash solution, optionally wherein the buffered wash solution is about pH-5-7.

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

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

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

27. The process of any one of claims 20-26, wherein TGZ is prepared with reduced levels of one or more impurities, wherein the impurities are mono-NAG, di-NAG, di-NAG-azlactone, deacylated, di-NAG-OH, di-NAG-OAc, NAG-H guanidine, or NAG-H trifluoroacetamide.

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

29. The process of any one of claims 20-28, wherein the NAG-H or a salt thereof is a solution from another process that is continued to be used without isolating the NAG-H, or a salt thereof.

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

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

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

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

34. The method of any one of claims 31-33, wherein the NAG-H or a salt thereof is NAG-HTFA:

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

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

37. A method for preparing NAG-Z, The method comprises making acylGalNAc: With alcohol-Z: Reaction to produce NAG-Z, wherein the reaction is carried out in the presence of an acid.

38. The method of claim 37, wherein the acid is bismuth trifluoromethanesulfonate (Bi(OTf)3), boron trifluoride etherate (BF3OEt2), tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), triisopropylsilyl trifluoromethanesulfonate (TIPSOTf), indium trifluoromethanesulfonate (In(OTf)3), or copper trifluoromethanesulfonate (Cu(OTf)2).

39. The method of any one of claims 37-38, wherein the reaction is carried out in the presence of a solvent selected from acetonitrile, dichloromethane, and dichloroethane.

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

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

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

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

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

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

47. A crystalline form I of a triacid, characterized in that An X-ray powder diffraction pattern having signals at at least three 2θ values ​​selected from the group consisting 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, or characterized by an X-ray powder diffraction pattern substantially similar to the X-ray powder diffraction pattern in FIG.

2.

48. A crystalline form II of a triacid, characterized in that An X-ray powder diffraction pattern having signals at at least three 2θ values ​​selected from the group consisting 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, or characterized by an X-ray powder diffraction pattern substantially similar to the X-ray powder diffraction pattern in Figure 3.

49. A method for preparing triacids, The method comprises reacting a tert-butyl core with an acid, wherein the acid is phosphoric acid (H3PO4), TFA, HCl, benzenesulfonic acid, or p-toluenesulfonic acid.

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

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

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

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

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

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

56. The process of any one of claims 49-55, wherein the triacid is obtained in a crystalline form characterized by an X-ray powder diffraction pattern having signals at at least three 2θ values ​​selected from the group consisting 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, or characterized by an X-ray powder diffraction pattern having signals at at least three 2θ values ​​selected from the group consisting 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.

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

58. A method for preparing a tert-butyl core, The method comprises causing GluZ to: With GluOtBu or its salts: Reaction to produce a tert-butyl core, wherein the reaction is carried out in the presence of a coupling reagent, a base and a solvent; optionally wherein 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. The method of claim 58, wherein GluOtBu or a salt thereof is GluOtBu hydrochloride:

60. The method of claim 58 or 59.

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

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

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

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

65. A method for preparing a methyl core, The method comprises making zL-Glu-OMe: With L-glutamic acid dimethyl ester (di(OMe)Glu): reacting to produce a methyl core, optionally in the presence of a base and a coupling reagent.

66. The method of claim 65, wherein: a) the base is added to a solution of zL-GluOMe and then the coupling reagent and di(OMe)Glu are added to the reaction; or b) wherein the coupling reagent is added to a solution of zL-Glu-OMe and base and then di(OMe)Glu is added to the reaction.

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

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

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

70. A compound: Where R is H (unprotected triol) or a Cbz protecting group (triol).

71. A method for preparing triols, The method involves reacting a methyl core with 2-(2-aminoethoxy)ethanol: reacting to produce a triol, optionally wherein the triol is converted to NAG-25.

72. A method for preparing TGZ, the method comprising subjecting a protected triol to: With β-D-galactosamine pentaacetate: reacting to produce TGZ, optionally wherein the TGZ is converted to NAG-25.

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