Synthesis of ethyl-2-((((2-(2-amino-6-methoxy-9h-purin-9-YL)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate

Abstract

This disclosure provides a process for preparing a specific acyclic nucleotide phosponamidate and pharmaceutically acceptable salts thereof. This compound is highly active for the treatment of HPV infection and diseases associated with HPV infection, including intraepithelial neoplasia. Useful chemical intermediates are also provided.

Description

SYNTHESIS OF ETHYL-2-((((2-(2-AMINO-6-METHOXY-9H-PURIN-9-YL)-ETHOXY)- METHYL)-(BENZYLOXY)-PHOSPHORYL)-AMINO)-PROPIONATECROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 624,709, filed January 24, 2024, the entire contents of which are incorporated by reference herein.FIELD OF THE DISCLOSURE

[0002] This disclosure provides a process for preparing a specific acyclic nucleotide phosphonamidate and pharmaceutically acceptable salts thereof. This compound is highly active for the treatment of HPV infection and diseases associated with HPV infection, including intraepithelial neoplasia. Useful chemical intermediates are also provided.BACKGROUND

[0003] According to the U.S. Center for Disease Control, there is no direct cure for human papilloma virus. HPV is so common that most sexually active people have been infected at some point in their lives.

[0004] Papillomaviruses are a group of non-enveloped DNA viruses, which in humans infect keratinocytes of skin and mucous membranes including in the cervical area. HPV infections can cause cellular transformations in the human patient that have not yet progressed to cancer but have reached the stage of neoplasia. Forms of HPV-induced neoplasia include cervical intraepithelial neoplasia (“CIN”), anal intraepithelial neoplasia (“AIN”), perianal intraepithelial neoplasia (“PAIN”), vulvar intraepithelial neoplasia (“VIN”), penile intraepithelial neoplasia (“PIN”), and vaginal intraepithelial neoplasia (“VAIN”). Cancers caused by HPV include cervical, anal, perianal, penile, vaginal, vulvar, and oropharyngeal cancer.

[0005] Thus, HPV can cause viral infection, neoplasia and cancer. Most of the cancercausing HPV types are from the alpha-7 and alpha-9 species and include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The most common cancer-causing HPV types are 16 and 18. The causal role of HPV in cancer of the cervix has been firmly established biologicallyand epidemiologically. Persistent infection with high-risk HPV is necessary to promote progression of pre-malignant stages to invasive cancer. Oncogenic HPV types are detected in almost all cervical cancer specimens, with types 16 and 18 accounting for about 70% of cervical cancers and about 50% of high-grade lesions. The majority of venereal warts are caused by the low-risk HPV types 6 and 11.

[0006] The current therapeutic options for HPV and cervical intraepithelial neoplasia are adjunctive only. Commonly used drug therapies include trichloroacetic acid, 5-fluorouracil, imiquimod and podofilox. Imiquimod (Aldara™, Zyclara™) stimulates the immune system to clear the infection through toll-like receptor signaling and causes redness and swelling.Podofilox (Condylox®) destroys tissues by destabilizing microtubules which prevents host cell replication.

[0007] The cervical epithelium is composed of several layers of tissue and is referred to as stratified squamous epithelium. The layers are the superficial cell layer, the intermediate cell layer, the parabasal cell layer and the basal cell layer. It is essential that a topical drug for the treatment of cervical intraepithelial neoplasia is able to penetrate these multiple layers of tissue to adequately reach and treat the transformed cells. This is a formidable task because the cells are tightly bound and without blood vessels.

[0008] Cervical intraepithelial neoplasia is most often treated by observation (the wait and see approach) or by excision or ablation of the cervical transformation zone. Techniques include cryotherapy, laser therapy, loop electrosurgical procedure (LEEP) and cone biopsy. All of these surgical procedures damage the affected areas and can lead to scarring. The most common intervention, LEEP, is effective in 60-90% of cases, however, it can be a painful procedure and may be associated with a significantly increased risk of miscarriage, ectopic pregnancies, and negative psychological outcomes. Despite extensive research, no drug has been approved to replace or to be combined with these surgical methods.

[0009] Cervical high-grade squamous intraepithelial lesions (cHSIL), sometimes referred to as CIN2 and CIN3, is a disease caused by the abnormal hyperproliferation (dysplasia) of squamous cells in the cervical epithelium (Waxman, A. G., et al., 2012 “Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix” Obstet. Gynecol. 120, 1465-71). Hyperproliferation usually occurs where the simple columnar, endometrial-type epithelium of the endocervix transitions tothe stratified squamous epithelium of the ectocervix; this region is referred to as the “transformation zone” (Sellers, J. W. & Sankaranarayanan, R. 2003. An introduction to the anatomy of the uterine cervix. Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginners' Manual). Cervical HSIL is classified as a pre-cancerous condition because apoptosis is impaired in these hyperproliferating cells, which can lead to the accumulation of genetic alterations that transform the cells into cancer. The probability that the patient’s immune system will be sufficiently activated to clear lesions diminishes over time and is contingent upon a variety of factors including the patient’s immunocompetency, lifestyle (e.g., smoking), and nutritional status (Schiffman, M., et al., 2011 “Human papillomavirus testing in the prevention of cervical cancer”. J. Natl. Cancer Inst., 103, 368-83).

[0010] For women already infected with HPV, there is no approved drug therapy to treat cHSIL and thereby, to prevent the progression of HSIL to cervical cancer.

[0011] The Regents of the University of California, with Karl Hostetler, et. al, as named inventors, has filed a series of patents on various acyclic nucleotide derivatives including (i) U.S. Patent Nos. 9,156,867; 9,387,217; 9,629,860; 9,775,852; 10,076,532; 10,076,533; 10,195,222; and 10,449,207 with a priority date of March 15, 2013; (ii) U.S. Patent Nos. 9,493,493;9,801,884; 10,213,430; 10,702,532; 11,344,555, and PCT WO 2016 / 044281 with a priority date of September 15, 2014; and (iii) U.S. Patent No. 10,377,782 and 11,014,950 with a priority date of September 15, 2015.

[0012] Antiva Biosciences filed PCT / US2023 / 28218 on July 20, 2023, with a priority claim of July 21, 2022. The Regents patent filings in (ii) above, and the Antiva Biosciences PCT application describe processes for the preparation of Compound I (ethyl-2-((((2-(2-amino-6- methoxy-9h-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate) to treat papilloma infections.

[0013] Compound I has two chiral centers, one at the phosphorus atom and one in the amino acid moiety, either of which can be in the R or S stereoconfiguration. Therefore, Compound I exists as four stereoisomers, or two diastereomeric pairs: (Rp, Sc) / (Sp, Sc) and (Rp, Rc) / (Sp, RC . It has been discovered by Antiva (see PCT / US2023 / 28218) that the stereoisomer of Compound I with R- stereochemistry at the phosphorus and -stereochemistry at the amino acid carbon has advantageous properties over the other three stereoisomers (sometimes referred to herein as “(R,S)-Compound I”).

[0014] Given the importance of Compound I for the treatment of a range of medical conditions, it is a goal of this disclosure to provide new processes for its manufacture. In particular, there remains a need for scalable processes that have a good yield for the large-scale synthesis of Compound I, including (R,S)-Compound I. Thus, it is an object of the present disclosure to provide useful intermediates and improved processes for the synthesis of Compound I, including (R,S)-Compound I.SUMMARY

[0015] It has been discovered that ethyl-2-((((2-(2-amino-6-methoxy-9h-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Compound I) can be synthesized though an advantageous synthetic route which uses a new intermediate (2-3) which can be formed in a highly regioselective fashion. This advantageous cost-effective synthesis allows forhigher overall yield without column chromatography, reduced waste, increased throughput, and / or higher levels of purity than prior synthetic routes, facilitating production of Compound I on a large scale.

[0016] Previously described synthetic routes (see PCT / US2023 / 28218, which is incorporated herein by reference in its entirety) to prepare Compound I (see Scheme 1 below) start with the alkylation of 6-chloro-9H-purin-2-amine, followed by the subsequent steps.Scheme 1. Prior Synthesis of Compound I

[0017] In contrast, intermediate 2-3 can be prepared by selective nucleophilic substitution of a symmetric di chloropyrimidine 2-1 followed by cyclization with trimethyl orthoformate (also known as trimethoxymethane) and displacement of chloro group with methoxy group to provide the N9-functionalized purine 2-3 as the sole isomer (Scheme 2). Intermediate 2-3 allows for the selective preparation of intermediate 2-5’ (diethyl ((2-(2-amino- 6-methoxy-9H-purin-9-yl)ethoxy)methyl)phosphonate) without significant levels of undesired N7-functionalized regioisomers and without column chromatography. The intermediate 2-5’ is further converted to the desired (R, 5)-Compound I as shown in Scheme 2.Scheme 2. Synthetic Route to Prepare (R,S)-Compound I

[0018] Several improved methods for chiral separation have also been discovered to prepare (R,S)-Compound I. In certain aspects, an enzymatic hydrolysis is used to convert the (S,S) isomer of Compound I to (R,S)-Compound I, as shown in Scheme 3 below. In this chiral separation, an enzymatic hydrolysis is used to hydrolyze (S,S)-Compound I to the (R)- Phosphonic Acid. The (R)-Phosphonic Acid can then be recycled by using a Mitsunobu reaction, which occurs with inversion of stereochemistry to produce the desired (R,S)-Compound I.Scheme 3. Stereoselective Enzymatic Hydrolysis of Phosphonamidate Compound Iazodicarboxylate, PhsP

[0019] In certain embodiments, the (R)-Phosphonic Acid is separated from (R,S)-Compound I and then reacted with (S)-ethyl alaninate or a salt thereof. For example, the mixture resulting from stereoselective enzymatic hydrolysis may be extracted with an aqueous solution, for example an alkaline solution. In certain embodiments, (R,S)-Compound I is separated from the (R)-Phosphonic Acid extraction wherein the (R,S)-Compound I is in the organic phase and the (R)-Phosphonic Acid is in the aqueous phase. In other embodiments, the (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof without a separation step. In certain embodiments, the (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof in a Mitsunobu reaction to afford the (R, S)-Compound I by inversion of configuration.

[0020] (R,S)-Compound I can also be prepared selectively from a phosphonodiamidate. For example, in certain aspects, a phosphonodiamidate is prepared according to Scheme 4 and then selectively converted to (R,S)-Compound I according to Scheme 5.Scheme 4. Preparation of PhosphonodiamidateScheme 5. Stereoselective Enzymatic Hydrolysis of the Phosphonodiamidatestereoselective enzymatic hydrolysis

[0021] In certain embodiments, the enzymatic hydrolysis is used to stereoselectively hydrolyze one amino acid group of a phosphonodiamidate (Scheme 5). The resulting (R,S)- Phosphonamic Acid can then be reacted with a benzyl halide such as benzyl bromide to produce (R,S)-Compound I with the retention of configuration.

[0022] In certain embodiments, the (R,S)-Phosphonamic Acid is separated from phosphonodiamidate and then reacted with benzyl bromide to afford (R,S)-Compound I. For example, the mixture resulting from stereoselective enzymatic hydrolysis may be extracted with an aqueous solution, for example an alkaline solution. In certain embodiments, (R,S)-Compound I is extracted in the organic phase and the (R,S)-Phosphonamic Acid is extracted in the aqueous phase. In other embodiments, the (R,S)-Phosphonamic Acid is reacted with benzyl bromide without a separation step. In some embodiments, when the (R,S)-Phosphonamic Acid is reactedwith benzyl bromide without being separated from the phosphonodiamidite, the resultant (R,S)- Compound I is purified by chromatography.

[0023] (R,S)-Compound I can also be prepared selectively from a dibenzyl phosphonate ester. For example, in certain aspects a dibenzyl phosphonate ester is prepared according to Scheme 6 and then selectively converted to (R,S)-Compound I according to Scheme 7.Scheme 6. Preparation of a Dibenzyl Phosphonate EsterScheme 7. Stereoselective Enzymatic Hydrolysis of the Dibenzyl Phosphonate Estermonofumarate

[0024] In certain embodiments, the enzymatic hydrolysis is used to stereoselectively hydrolyze one ester group from a phosphonate ester (Scheme 7). The resulting (R)-Phosphonic Acid can be reacted with (S)-ethyl alaninate or a salt thereof in a Mitsunobu reaction to invert the chiral center and provide (R,S)-Compound I. The resulting (R,S)-Compound I is optionally reacted with fumaric acid to provide the monofumarate salt of (R,S)-Compound I.

[0025] The processes described in Schemes 2-7 above can be used to produce (R,S)- Compound I in a cost-effective manner with improved yield, increased throughput, a reduced number of chromatographic steps, and / or less waste.

[0026] The present disclosure thus includes at least the following embodiments:1. A process to prepare (R,S)-Compound I or a salt thereof comprising:(R, S)-Compound I a) reacting a mixture of (R,S) and (S,S)-Compound I with an enzyme, wherein nonlimiting illustrative examples of suitable enzymes include a lipase (for example a lipase from Candida antarcticans), an esterase (including but not limited to pig liver esterase) phosphatase, and phosphotriesterase, which affords a mixture of an (R)-Phosphonic Acid of the structure shown below and (R,S)-Compound I;(R)-Phosphonic Acid b) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I.2. The process of embodiment 1, wherein the (R)-Phosphonic Acid is separated from the mixture before the reaction with (S)-ethyl alaninate or a salt thereof.The process of embodiment 2, wherein an extraction is used for the separation and the (R)-Phosphonic Acid is in the aqueous phase. The process of embodiment 1, wherein the (R)-Phosphonic Acid is reacted with (S)- ethyl alaninate or a salt thereof without prior separation. A process to prepare (R,S)-Compound I or a salt thereof comprising:a) reacting a Phosphonodiamidate of the structure shown below with an enzyme to afford a mixture of an (R,S)-Phosphonamic Acid of the structure shown below and the Phosphonodiamidate;(R,S)-Phosphonamic Acid reacting the (R,S)-Phosphonamic Acid with a benzyl halide to afford (R,S)- Compound I; and b) optionally reacting the (R, S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound T.The process of embodiment 5, wherein the (R,S)-Phosphonamic Acid is separated from the Phosphonodiamidate before the reaction with benzyl halide. The process of embodiment 6, wherein an extraction is used for the separation and the (R,S)-Phosphonamic Acid is in the aqueous phase. The process of embodiment 5, wherein the (R,S)-Phosphonamic Acid is reacted with the benzyl halide without being separated from the Phosphonodiamidate. The process of any one of embodiments 5-8, wherein the benzyl halide is benzyl bromide. A process to prepare (R,S)-Compound I or a salt thereof comprising:( / ?, SJ-Compound I a) reacting a Dibenzyl Phosphonate Ester of the structure shown below with an enzyme to afford an (R)-Phosphonic Acid of the structure shown below;Dibenzyl Phosphonate Esterb) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I.11. A process to prepare a mixture of (R,S)-Compound I and (S,S)-Compound I comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated; c) reacting compound 2-3 with a compound of Formula 2-4 in the presence of a suitable base to afford a compound of Formula 2-5, wherein:0 the compound of Formula 2-4 is of structure.LG is a Leaving Group which is displaced by the primary hydroxy of compound 2-3; and the compound of Formula 2-5 is of structured) hydrolyzing the compound of Formula 2-5 in the presence of a suitable base to afforde) reacting compound 2-6 with (S)-ethyl alaninate or a salt thereof and benzyl alcohol to afford a mixture of (R,S)-Compound I and (S,S)-Compound I.12. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanolb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated; c) reacting compound 2-3 with a compound of Formula 2-4 in the presence of a suitable base to afford a compound of Formula 2-5, wherein:0 the compound of Formula 2-4 is of structureLG is a Leaving Group which is displaced by the primary hydroxy of compound 2-3; and the compound of Formulad) hydrolyzing the compound of Formula 2-5 to afforde) reacting compound 2-6 with (S)-ethyl alaninate or a salt thereof and benzyl alcohol to afford a mixture of (R,S)-Compound I and (S,S)-Compound I; f) performing a chiral separation to obtain (R, S)-Compound I from the mixture of (R,S)-Compound I and (S,S)-Compound I, optionally wherein the chiral separation is performed using chiral chromatography; and g) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I. A compound of structuresalt thereof. A process to prepare a compound of structure:or a salt thereof, comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated.DETAILED DESCRIPTIONTerminology

[0027] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0028] The terms “a” and “an” do not denote a limitation of quantity but rather denote the presence of at least one of the referenced item. The term “or” means “and / or”. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of examples, or exemplary language (e g., “such as”), is intended merely to better illustrate the embodiments and does not pose a limitation on the scope of the disclosure unless otherwise claimed. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0029] “Alkyl” is a branched or straight chain saturated hydrocarbon group. The term “alkyl” also encompasses cycloalkyl or carbocyclic groups. In one non-limiting embodiment, the alkyl group contains from 1 to about 6 carbon atoms, more generally from 1 to about 4 carbon atoms or from 1 to about 3 carbon atoms. In certain embodiments, the alkyl is C1-C2, C1-C3, Ci- C4, C1-C5, or Ci-Ce. The specified ranges as used herein indicate an alkyl group having each member of the range described as an independent species. For example, the term Ci-Ce alkyl asused herein indicates a straight or branched alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species. For example, the term C1-C4 alkyl as used herein indicates a straight or branched alkyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, / -butyl, n-pentyl, isopentyl, / ert-pentyl, neopentyl, n- hexyl, 2-methylpentane, 3 -methylpentane, 2,2-dimethylbutane, and 2,3 -dimethylbutane. For example, when a term is used that includes “alk” then “cycloalkyl” or “carbocyclic” can be considered part of the definition, unless unambiguously excluded by the context.AbbreviationsI. Synthesis of (R,S)-Compound I

[0030] It has been discovered that ethyl(((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy) methyl)(benzyloxy)-phosphoryl)-L-alaninate (Compound I) can be synthesized though an advantageous synthetic route. This advantageous cost-effective synthesis allows for higher overall yield, reduced waste, increased throughput, and / or higher levels of purity than prior synthetic routes, facilitating production of Compound I on a large scale. In some embodiments, (R,S)-Compound I and its monofumarate salt can be prepared as shown in Scheme 8.Scheme 8. Synthetic Route to Prepare (R,S)-Compound IMixture of (R, S) and (S, SJ-Compound IMonofumarate of (R,S)-Compound I

[0031] In Step 1, a symmetric di chloropyrimidine (2-1) is reacted with amino ethanol to prepare compound 2-2. In typical embodiments, this reaction is conducted in a solvent, for example a protic solvent such as ethanol, and conducted at a raised temperature, for example a temperature from about 80 °C to about 85 °C. In certain embodiments, this reaction is conducted for from about 7 to about 8 hours. In other embodiments, this reaction is conducted in a polar aprotic solvent, for example DMF (N,N-dimethylformamide), DMAc (N,N-dimethylacetamide), DMSO (dimethylsulfoxide), NMP (N-methyl-2-pyrrolidone), or mixtures thereof.

[0032] In certain embodiments, the reaction is conducted with an added base, such as inorganic bases or an organic base. Illustrative non-limiting inorganic bases include cesium carbonate, potassium carbonate, and sodium carbonate. Illustrative non-limiting organic bases include trialkylamine bases (such as tri ethylamine, diisopropylethylamine (Hunig’s base and the like), and guanidine bases (such as DBU (l,8-Diazabicyclo[5.4.0]undec-7-ene), 1, 1,3,3- tetram ethylguanidine, 2-tert-Butyl- 1,1, 3, 3 -tetramethylguanidine (Barton’s base) and the like).

[0033] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in Step 1 include ethanol, ACN (Acetonitrile), acetic acid, acetone, dioxane, DMAc, DCM (Dichloromethane), THF (Tetrahydrofuran), DMF, benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO, ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, n-propanol, isopropanol, MTBE (Methyl Tertiary Butyl Ether), NMP, pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent.

[0034] In certain embodiments, the reaction is conducted at a temperature above room temperature, for example, a temperature from about 30 °C to about 100 °C, from about 40 °C to about 100 °C, from about 50 °C to about 100 °C, from about 60 °C to about 100 °C, from about 70 °C to about 100 °C, from about 80 °C to about 100 °C, from about 50 °C to about 90 °C, from about 50 °C to about 80 °C, from about 50 °C to about 70 °C, or from about 60 °C to about 90 °C.

[0035] In alternative embodiments, the reaction is conducted at room temperature.Step 2

[0036] In Step 2, compound 2-2 is cyclized with tri methoxym ethane, optionally with added methanol, to provide the N9-functionalized purine 2-3 as the sole isomer. In typical embodiments, this reaction is conducted in the presence of an acid catalyst, for example a Bronsted acid such as methanesulfonic acid (MsOH). In certain embodiments, the reaction is conducted in the presence of a Lewis acid catalyst, for example a zinc salt such as zinc chloride or zinc triflate.

[0037] In certain embodiments, the reaction is conducted in neat trimethoxymethane. In certain embodiments, the reaction is carried out with a ratio of about 50:1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 4: 1, 3: 1, 2: 1, or 1 : 1 trimethoxymethane:methanol or a ratio of trimethoxymethane:methanolfrom about 1 : 1 to about 50: 1, from about 5: 1 to about 50: 1, from about 10: 1 to about 50: 1, from about 1 : 1 to about 25 : 1 , or from about 1 : 1 to about 15: 1.

[0038] In certain embodiments, the reaction is conducted at a temperature above room temperature (i.e., above about 25°C). In certain embodiments, the reaction is conducted at about 50 °C. In certain embodiments, the reaction is conducted at about 60 °C. In certain embodiments, the reaction is conducted at about 70 °C. In certain embodiments, the reaction is conducted at about 80 °C. In certain embodiments, the reaction is conducted at about 90 °C. In certain embodiments, the reaction is conducted at about 100 °C. In certain embodiments, the reaction is conducted at reflux. In certain embodiments, the reaction is conducted at the boiling point of the solvent or solvent mixture, for example at about 65 °C for solvent mixtures comprising methanol, or at about 100 °C for neat trimethoxy methane. In certain embodiments, the reaction is conducted in a sealed vessel at a temperature greater than the boiling point of the solvent or solvent mixture, for example from about 65 °C to about 150 °C, from about 65 °C to about 140 °C, from about 65 °C to about 130 °C, or from about 65 °C to about 120 °C.

[0039] Non-limiting examples of acids for use in Step 2 include TFA, acetic acid, aminomethanesulfonic acid, ascorbic acid, benzene sulfonic acid, benzoic acid, formic acid, lactic acid, malic acid, malonic acid, methanesulfonic acid, oxalic acid, phthalic acid, salicyclic acid, succinic acid, sulfamic acid, p-toluenesulfonic acid, trichloroacetic acid, trifluoromethanesulfonic acid, boric acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, iodic acid, nitric acid, perchloric acid, periodic acid, phosphinic acid, and phosphoric acid.

[0040] Non-limiting examples of Lewis acid catalysts for use in Step 2 include salts of zinc, aluminum, tin, titanium, zirconium, strontium, lanthanum, or copper, wherein the salt counterion is selected from chloride, bromide, triflate, tetrafluoroborate, and hexafluorophosphate. Additional non-limiting examples of Lewis acid catalysts include boron trifluoride or boron trifluoride-diethyl etherate.

[0041] In certain aspects, a compound is provided of structure:Step 3

[0042] In Step 3, compound 2-3 reacts with compound 2-4 in the presence of a suitable base to displace the Leaving Group of 2-4 and afford compound 2-5. In typical embodiments, this reaction is conducted in a solvent, for example an aprotic solvent such as NMP, and facilitated by a base, for example Mg(OtBu)2. In certain embodiments, this reaction is conducted in two solvents, such as NMP and toluene.

[0043] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in Step 3 include NMP, ACN (Acetonitrile), acetic acid, acetone, dioxane, DMAc (N,N-dimethylacetamide), DCM (Dichloromethane), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, ethanol, MTBE (Methyl Tertiary Butyl Ether), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent. In certain embodiments, the solvent is aprotic.

[0044] In alternative embodiments, the solvent is a protic solvent. Non-limiting examples of protic solvents for use in Step 3 include water, methanol, ethanol, propanol, and isopropanol.

[0045] Non-limiting examples of bases include Mg(OtBu) , DMAP, TEA, pyridine, ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, trimethylamine, tripropylamine, triisopropylamine, aniline, methylaniline, dimethylaniline, pyridine, azajulolidine, benzylamine, methylbenzylamine, dimethylbenzylamine, DABCO, l,5-diazabicyclo[4.3.0]non-5-ene, 2,6- lutidine, morpholine, piperidine, piperazine, Proton-sponge, l,5,7-Triazabicyclo[4.4.0]dec-5-ene, tripelennamine, ammonium hydroxide, triethanolamine, ethanolamine, NaHSCh, lutidine, NaOH, tBuOK, NaH, KH, and Trizma. A “suitable base” is a base that allows the reaction to occur. Insome embodiments, suitable bases include NaH and tBuOK. In some embodiments, suitable bases do not include CS2CO3, DBU, DIPEA, and LiHMDS.

[0046] In certain embodiments, the reaction mixture containing compound 2-5 is quenched with an aqueous solution. In some embodiments, the aqueous solution is basified. In some embodiments, the basified aqueous solution is extracted with an organic solvent.

[0047] In certain embodiments, the Leaving Group (LG) is -O-Ts. In certain embodiments, the Leaving Group (LG) is -O-Ns. Additional examples of Leaving Groups include Br, Cl, and -O-Ms.

[0048] Non-limiting examples of Ci-Ce alkyl include: methyl, ethyl, propyl, butyl, pentyl, and hexyl. Additional non-limiting examples of Ci-Ce alkyl include: isopropyl, isobutyl, isopentyl, isohexyl, sec-butyl, sec-pentyl, sec-hexyl, / e / 7-butyl, Ze / 7-pentyl, / e / 7-hexyl, neopentyl, 3-pentyl, and active pentyl. Additional non-limiting examples of Ci-Ce alkyl include: isopropyl, isobutyl, isopentyl, isohexyl, sec-butyl, sec-pentyl, sec-hexyl, terT-butyl, / e / 7-pentyl, / e / 7-hexyl, neopentyl, and 3-pentyl.

[0049] In certain embodiments, Ci-Ce alkyl is CH3. In certain embodiments, Ci-Ce alkyl is CH2CH3. In other aspects, the Ci-Ce alkyl group is a C3-C6 cycloalkyl. Non-limiting examples of C3-C6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0050] In Step 4, the compound of Formula 2-5 is hydrolyzed to afford compound 2-6. In certain embodiments, the reaction is conducted in a suitable base. In certain embodiments, the suitable base is tributylamine. In certain embodiments, the suitable base is not 2,6-lutidine.

[0051] In typical embodiments, this reaction is conducted in a solvent or mixture of solvents, for example acetonitrile and / or water, a reagent and or base may also be used to facilitate the hydrolysis for example TMSBr and / or NaHSCU

[0052] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in Step 4 include ethanol, ACN (Acetonitrile), acetic acid, acetone, dioxane, DMAc (N,N-dimethylacetamide), DCM (Dichloromethane), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, MTBE (Methyl Tertiary Butyl Ether), NMP (N-Methyl-2-Pyrrolidone), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent.

[0053] In certain embodiments, the solvent is protic. Non-limiting examples of protic solvents for use in Step 4 include water, methanol, ethanol, propanol, and isopropanol.

[0054] In alternative embodiments, the solvent is an aprotic solvent.

[0055] In certain aspects, the reaction is conducted in an organic solvent and then water is added.

[0056] Non-limiting examples of bases for use in Step 4 include NaHSC , lutidine, DIPEA, DMAP, DBU, TEA, pyridine, ammonia, methylamine, ethylamine, propylamine, isopropyl amine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, trimethylamine, tripropylamine, triisopropylamine, aniline, methylaniline, dimethylaniline, pyridine, azajulolidine, benzylamine, methylbenzylamine, dimethylbenzylamine, Mg(OtBu)2, DABCO, l,5-diazabicyclo[4.3.0]non-5-ene, 2,6-lutidine, morpholine, piperidine, piperazine, Proton-sponge, l,5,7-Triazabicyclo[4.4.0]dec-5-ene, tripelennamine, ammonium hydroxide, triethanolamine, ethanolamine, NaOH, tBuOK, NaH, KH, and Trizma.Step 5S)

[0057] In Step 5, compound 2-6 is reacted with (S)-ethyl alaninate or a salt thereof and benzyl alcohol (BnOH) to afford a mixture of (R,S) and (S,S)-Compound I. In typical embodiments, this reaction is conducted in a solvent, for example DCM, and one or more activating reagents and / or bases may also be used to facilitate the reaction, for example aldrithiol-2, triphenyl phosphine (PtoP), and triethylamine (EtsN).

[0058] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in Step 5 include DCM (Dichloromethane), ACN (Acetonitrile), acetic acid, acetone, dioxane, DMAc (N,N-dimethylacetamide), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, ethanol, MTBE (Methyl Tertiary Butyl Ether), NMP (N-Methyl-2 -Pyrrolidone), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent.

[0059] In certain embodiments, the solvent is protic. Non-limiting examples of protic solvents for use in Step 5 include water, methanol, ethanol, propanol, and isopropanol.

[0060] In alternative embodiments, the solvent is an aprotic solvent.

[0061] Non-limiting examples of bases for use in Step 5 include tri ethylamine, NaHSCN, lutidine, DIPEA, DMAP, DBU, pyridine, ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, trimethylamine, tripropylamine, triisopropylamine, aniline, methylaniline, dimethylaniline, pyridine, azajulolidine, benzylamine, methylbenzylamine, dimethylbenzylamine, Mg(OtBu)2, DABCO, l,5-diazabicyclo[4.3.0]non-5-ene, 2,6-lutidine, morpholine, piperidine, piperazine, Proton-sponge, l,5,7-Triazabicyclo[4.4.0]dec-5-ene, tripelennamine, ammonium hydroxide, triethanolamine, ethanolamine, NaOH, tBuOK, NaH, KH, and Trizma.Step 6 - Chiral Separations

[0062] Several improved methods for chiral separation have also been discovered to prepare (R,S)-Compound I. In certain aspects, Prep-LC (preparative liquid chromatography) is used to separate (R,S)-Compound I from (S,S)-Compound I. In certain aspects, an enzymatic hydrolysis is used to recycle the hydrolyzed (S,S)-Compound I, as shown in Scheme 9 below. In other aspects, an enzymatic hydrolysis is used to selectively prepare (R,S)-Compound I from aphosphonodiamidate, as shown in Scheme 10 below. Alternatively, an enzymatic hydrolysis is used to selectively prepare (R,S)-Compound I from an achiral dibenzyl phosphonate ester, as shown in Scheme 11 below.

[0063] Non-limiting illustrative examples of suitable enzymes that may be used in the chiral separation steps include a lipase (for example a lipase from Candida antarcticans), an esterase (including but not limited to pig liver esterase) phosphatase, and phosphotriesterase. Lipases from Candida antarctica are commercially available in a variety of different forms and preparations, including resin-immobilized, lyophilized, and suspended preparations. For example, suitable lipases from Candida antarctica are sold under the tradename Chirazyme by Roche Diagnostics Corp. The use of immobilized enzymes provides several advantages, including, but not limited to, improved enzyme stability, convenience of handling, and ease with which the enzyme can be recycled. In some embodiments, lipases from Candida antarctica type B are used. In some embodiments, lipases from Candida antarctica type A are used. In some embodiments, lipases from Candida antarctica type B are carrier-fixed. In some embodiments, lipases from Candida antarctica type A are carrier-fixed.Recycling of (S,S)-Compound I Stereoisomer

[0064] In this chiral separation, an enzymatic hydrolysis is used to hydrolyze (S,S)- Compound I to the (R)-Phosphonic Acid. (R)-Phosphonic Acid can then be recycled by Mitsunobu reaction which occurs with inversion of stereochemistry to produce the desired (R,S)- Compound I.Scheme 9. Stereoselective Enzymatic Hydrolysis of Phosphonamidate Compound I

[0065] In the first step of Scheme 9, a mixture of (R,S) and (S,S)-Compound I is selectively hydrolyzed with an enzyme to afford (R)-Phosphonic Acid. In typical embodiments, this reaction is conducted in a solvent with or without a catalytic amount of buffer.

[0066] In the second step of the reaction, the resulting (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof in a Mitsunobu reaction to invert the chiral center and afford (R,S)-Compound I. In typical embodiments, this reaction is conducted in a solvent which may be the same or different from the first step solvent and one or more activating reagents may also be used to facilitate the reaction, for example azodicarboxylate and triphenyl phosphine.

[0067] In certain embodiments, the (R)-Phosphonic Acid is separated from the reaction mixture by aqueous extraction before the Mitsunobu reaction, for example, the (R)-Phosphonic Acid may be extracted and isolated with an alkaline aqueous solution. In certain embodiments, (R,S)-Compound I is extracted in the organic phase and the (R)-Phosphonic Acid is extracted in the aqueous phase. In other embodiments, the (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof without a separation step.

[0068] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in the selective phosphonamidate cleavage orMitsunobu reaction include ACN (Acetonitrile), acetic acid, acetone, dioxane, DCM (Di chloromethane), DMAc (N,N-dimethylacetamide), THF (Tetrahydrofuran), DMF (N,N- dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, ethanol, MTBE (Methyl Tertiary Butyl Ether), NMP (N-Methyl-2-Pyrrolidone), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent. In certain embodiments, the solvent is protic. Non-limiting examples of protic solvents for use in the selective phosphonamidate cleavage or Mitsunobu reaction include water, methanol, ethanol, propanol, and isopropanol. In alternative embodiments, the solvent is an aprotic solvent.

[0069] In alternative aspects, an enzyme with the opposite selectivity may be used to hydrolyze the (R,S)-stereoisomer of Compound I. After performing the Mitsunobu reaction on the hydrolysis product, the (S,S)-Compound I would be formed.Phosphonodiamidate Route

[0070] The (R,S)-stereoisomer of Compound I can also be prepared selectively from a phosphonodiamidate. For example, in certain aspects a phosphonodiamidate is selectively converted to (R,S)-Compound I according to Scheme 10.Scheme 10. Stereoselective Enzymatic Hydrolysis of PhosphonodiamidateStereoselectiveEnzymatic Hydrolysis

[0071] In Scheme 10, in the first step of the reaction, the phosphonodiamidate is selectively hydrolyzed with an enzyme to afford (R,S)-Phosphonamic Acid. In typical embodiments, this reaction is conducted in a solvent.

[0072] In the second step of the reaction, the (R,S)-Phosphonamic Acid is reacted with a benzyl halide, for example benzyl bromide, to afford (R,S)-Compound I. In typical embodiments, this reaction is conducted in a solvent which may be the same or different from the first step solvent and a base is used to facilitate the reaction.

[0073] In certain embodiments, the (R,S)-Phosphonamic Acid is separated from the reaction mixture by aqueous extraction before the second step, for example, the (R,S)- Phosphonamic Acid may be extracted with an alkaline aqueous solution. In certain embodiments, the remaining phosphonodiamidate is extracted in the organic phase and the (R,S)-Phosphonamic Acid is extracted in the aqueous phase. In certain embodiments, the (R,S)-Phosphonamic Acid is extracted in the aqueous alkaline phase. In other embodiments, the (R,S)-Phosphonamic Acid is reacted with a benzyl halide without a separation step.

[0074] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in the reaction include ACN (Acetonitrile), acetic acid, acetone, dioxane, DCM (Dichloromethane), DMAc (N,N-dimethylacetamide), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, ethanol, MTBE (Methyl Tertiary Butyl Ether), NMP (N-Methyl-2 -Pyrrolidone), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent. In certain embodiments, the solvent is protic. Non-limiting examples of protic solvents for use in reaction include water, methanol, ethanol, propanol, and isopropanol. In alternative embodiments, the solvent is an aprotic solvent.

[0075] In alternative aspects, an enzyme with the opposite selectivity may be used to hydrolyze the phosphonodiamidate. After performing the second step, (S,S)-Compound I would be formed.Dibenzyl Phosphonate Ester Route

[0076] In this chiral separation, an enzymatic hydrolysis is used to selectively hydrolyze a dibenzyl phosphonate ester to the (R)-Phosphonic Acid. (R)-Phosphonic Acid can then be converted by Mitsunobu reaction which occurs with inversion of stereochemistry to produce the desired (R,S)-Compound I.Scheme 11. Stereoselective Enzymatic Hydrolysis of Dibenzyl Phosphonate Ester

[0077] In Scheme 11, in the first step of the reaction, a dibenzyl phosphonate ester is selectively hydrolyzed with an enzyme to afford (R)-Phosphonic Acid. In typical embodiments, this reaction is conducted in a solvent.

[0078] In the second step of the reaction, the (R)-Phosphonic Acid is reacted with (S)- ethyl alaninate or a salt thereof in a Mitsunobu reaction to invert the chiral center and afford (R,S)-Compound I. In typical embodiments, this reaction is conducted in a solvent which may be the same or different from the first step solvent and one or more activating reagents may also be used to facilitate the reaction, for example azodi carb oxy late and triphenyl phosphine.

[0079] In certain embodiments, the (R)-Phosphonic Acid is separated from the reaction mixture by aqueous extraction before the Mitsunobu reaction, for example, the (R)-Phosphonic Acid may be extracted with an alkaline aqueous solution. In other embodiments, the (R)- Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof without a separation step.

[0080] In certain embodiments, the reaction is conducted in an organic solvent. Nonlimiting examples of organic solvents for use in this reaction include ACN (Acetonitrile), acetic acid, acetone, dioxane, DCM (Dichloromethane), DMAc (N,N-dimethylacetamide), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (Dimethoxyethane), DMSO (Dimethyl sulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (Hexamethylphosphoramide), methanol, ethanol, MTBE (Methyl Tertiary ButylEther), NMP (N-Methyl-2 -Pyrrolidone), pentane, pyridine, toluene, and triethyl amine. In other embodiments, the reaction is conducted in water or a mixture of water and an organic solvent. In certain embodiments, the solvent is protic. Non-limiting examples of protic solvents for use in this reaction include water, methanol, ethanol, propanol, and isopropanol. In alternative embodiments, the solvent is an aprotic solvent.

[0081] In alternative aspects, an enzyme with the opposite selectivity may be used to hydrolyze the dibenzyl phosphonate ester. After performing the Mitsunobu reaction on the hydrolysis product, (S,S)-Compound I would be formed.II. Synthesis of a Mixture of (R,S)- and (S,S)-Compound IStep 1

[0082] In Step 1, compound 3-1 is combined with a base in methanol to give compound 2-6. In certain embodiments, the base is sodium methoxide. In certain embodiments, the solvent is methanol. In certain embodiments, the reagents are combined at room temperature (RT), then heated to reflux. In certain embodiments, the reagents are combined at an elevated temperature. In certain embodiments, the reaction mixture is concentrated at under vacuum. The reactionmixture may be quenched with an acid salt. In certain embodiments, the acid salt is sodium bisulfate. After quenching, in certain embodiments, the resulting mixture is stirred until a precipitated solid is observed. In certain embodiments, the resulting mixture is stirred for from about 0 hours to about 12 hours at RT. In certain embodiments, the resulting mixture is stirred from about 15 minutes to about 12 hours, from about 30 minutes to about 12 hours, from about 45 minutes to about 12 hours, about 1 hour to about 12 hours, from about 2 hours to about 12 hours, from about 3 hours to about 12 hours, from about 4 hours to about 12 hours, from about 5 hours to about 12 hours, from about 6 hours to about 12 hours, from about 7 hours to about 12 hours, from about 8 hours to about 12 hours, from about 9 hours to about 12 hours, from about 10 hours to about 12 hours, or from about 11 hours to about 12 hours. In some embodiments, the precipitated solid is filtered. In some embodiments, the precipitate is washed with a solvent such as, for example, water.Step 2Step 2Mixture of (R,S) and (S,S) Compound I

[0083] In Step 2, the product from Step 1 (compound 2-6) is reacted with benzyl alcohol, L-alanine ethyl ester or a salt thereof, and a base to give a reaction mixture. In certain embodiments, the L-alanine ethyl ester is the hydrogen chloride salt. In certain embodiments, the base is triethylamine or pyridine. In certain embodiments, two or more bases are used. In certain embodiments, both triethylamine and pyridine are used. In certain embodiments, the reaction mixture is stirred at room temperature or above, for example, from about room temperature to about 80 °C, or from about 15 °C to about 75 °C, or from about 20 °C to about 70 °C, or from about 25 °C to about 60 °C. In certain embodiments, the reaction mixture is stirred at about 60 °C.

[0084] A solution of Aldrithiol-2, triphenylphosphine, and a base is then added to the reaction mixture to afford a mixture of (R,S)-Compound I and (S,S)-Compound I. In certain embodiments, the base is pyridine. In certain embodiments, the base is added dropwise. Incertain embodiments, the base is added dropwise over a period of about 15 minutes, about 30 minutes, about 45 minutes, or about 1 hour. In certain embodiments, the base is added dropwise over about 30 minutes. In certain embodiments, the reaction mixture is then stirred at room temperature or above, for example, from about room temperature to about 80 °C, or from about room temperature to about 75 °C, or from about room temperature to about 65 °C. In certain embodiments, the reaction mixture is then stirred at about 55 °C to 65 °C. In certain embodiments, the reaction mixture is then stirred at about 60 °C. In certain embodiments, the reaction mixture is stirred for from about 0 hr to about 25 hr, from about 1 hr to about 20 hr, from about 5 hr to about 20 hr, from about 10 hr to about 20 hr, from about 15 to about 20 hr. In certain embodiments, the reaction mixture is stirred for about 18 hr. In certain embodiments, the reaction mixture is concentrated at under vacuum. In certain embodiments, following concentration under vacuum, the resulting residue is dissolved in a solvent and the resultant solution is washed. In certain embodiments, the solvent is ethyl acetate. In some embodiments, the resultant solution is washed with aqueous sodium bicarbonate solution and water. In some embodiments, the resultant solution is dried, such as dried with for example anhydrous Na2SO4. In some embodiments, the resultant solution is filtered and concentrated.

[0085] The mixture of (R, S)-Compound I and (S,S)-Compound I can then be separated using any method described herein. For example, (R,S)-Compound I may be separated from (S,S)-Compound I using the selective enzymatic reactions described herein. In the alternative, the chiral separation may be accomplished by chromatographic methods, such as chiral column chromatography.

[0086] Once separated, (R,S)-Compound I can be converted into a monofumarate salt using any of the methods described herein. For example, it may be prepared as outlined below in Section I or III or as in PCT / US2023 / 28218, which is incorporated herein by reference in its entirety.III. Fumarate Salt Formation

[0087] In certain embodiments, (R,S)-Compound I prepared by any of the processes described herein is reacted with fumaric acid in an organic solvent or mixture of organic solvents to form (R,S)-Compound I fumarate salt. In certain embodiments, the fumarate salt is thehemifumarate, monofumarate, sesquifumarate, or difumarate. In certain embodiments, the fumarate salt is the monofumarate.

[0088] Non-limiting examples of organic solvents for use in this reaction include ACN, acetic acid, acetone, dioxane, DCM (dichloromethane), DMAc (N,N-dimethylacetamide), THF (Tetrahydrofuran), DMF (N,N-dimethylformamide), benzene, 1 -butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexanes, diethyl ether, diglyme, DME (dimethoxyethane), DMSO (dimethylsulfoxide), ethyl acetate, ethylene glycol, glycerin, heptane, HMPA (hexamethylphosphoramide), methanol, ethanol, MTBE (methyl tertiary butyl ether), NMP (N-methyl-2-pyrrolidone), pentane, pyridine, and toluene.

[0089] In certain embodiments, the organic solvent for fumarate salt formation is selected from methanol, ethanol, isopropanol, n-propanol, butanol, tert-butanol, iso-butanol, pentanol, isopentanol, hexanol, and cyclohexanol. In certain embodiments, the organic solvent for fumarate salt formation is selected from pentane, cyclopentane, hexane, cyclohexane, heptane, benzene, toluene, xylene, diethyl ether, and MTBE. In certain embodiments, the mixture of organic solvents for fumarate salt formation is a mixture of an organic solvent selected from methanol, ethanol, isopropanol, n-propanol, butanol, tert-butanol, iso-butanol, pentanol, isopentanol, hexanol, and cyclohexanol in addition to an organic solvent selected from pentane, cyclopentane, hexane, cyclohexane, heptane, benzene, toluene, xylene, diethyl ether, and MTBE. In certain embodiments, the organic solvent used in this reaction is isopropanol (IP A).ADDITIONAL EMBODIMENTS1. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting a mixture of (R,S)-Compound I and (S,S)-Compound I with an enzyme to afford a mixture of an (R)-Phosphonic Acid and (R, S)-Compound I; and b) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; wherein:(R,S)-Compound l is a compound of structure:(R, St-Compound I(S,S)-Compound I is a compound of structure:(S,S)-Compound I and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid2. The process of embodiment 1, wherein the (R)-Phosphonic Acid is separated from the mixture before the reaction with (S)-ethyl alaninate or a salt thereof.3. The process of embodiment 2, wherein an extraction is used for the separation and the (R)-Phosphonic Acid is in the aqueous phase.4. The process of embodiment 1, wherein the (R)-Phosphonic Acid is reacted with (S)- ethyl alaninate or a salt thereof without being separated from the mixture.5. The process of any one of embodiments 1-4, wherein the enzyme is a pig liver enzyme.6. The process of any one of embodiments 1-5, further comprising a solvent.7. The process of embodiment 6, wherein the solvent is water.8. A process to prepare (R,S)-Compound I or a salt thereof comprising:a) reacting a Phosphonodiamidate with an enzyme to afford a mixture of an (R,S)- Phosphonamic Acid and an Phosphonodiamidate; b) reacting the (R,S)-Phosphonamic Acid with a benzyl halide to afford (R,S)- Compound I; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound I is a compound of structure:(R, SJ-Compound I the Phosphonodiamidate is a compound of structure:and the (R,S)-Phosphonamic Acid is a compound of structure:(R, S)-Phosphonamic Acid9. The process of embodiment 8, wherein the (R,S)-Phosphonamic Acid is separated from the mixture before the reaction with benzyl halide.10. The process of embodiment 9, wherein an extraction is used for the separation and the (R,S)-Phosphonamic Acid is in the aqueous phase.11. The process of embodiment 8, wherein the (R,S)-Phosphonamic Acid is reacted with the benzyl halide without being separated from the mixture.12. The process of any one of embodiments 8-11, wherein the benzyl halide is benzyl bromide.13. The process of any one of embodiments 8-12, wherein the enzyme is a pig liver enzyme.14. The process of any one of embodiments 8-13, further comprising a solvent.15. The process of embodiment 14, wherein the solvent is water.16. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting a Dibenzyl Phosphonate Ester with an enzyme to afford (R)-Phosphonic Acid; and b) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I, wherein:(R,S)-Compound l is a compound of structure:(R, S^-Compound I the Dibenzyl Phosphonate Ester is a compound of structure:and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid17. The process of embodiment 16, wherein the enzyme is a pig liver enzyme.18. The process of embodiment 16 or 17, further comprising a solvent.19. The process of embodiment 18, wherein the solvent is water.20. A process to prepare a mixture of (R,S)-Compound I and (S,S)-Compound I comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated; c) reacting compound 2-3 with a compound of Formula 2-4 in the presence of a suitable base to afford a compound of Formula 2-5, wherein:OLG^P'OCrCe alkyl the compound of Formula 2-4 is of structure OC-I-CQ alkyl .LG is a Leaving Group which is displaced by the primary hydroxy of compound 2-3; andthe compound of Formula 2-5 is of structured) hydrolyzing the compound of Formula 2-5 in the presence of a suitable base to afford a compound of structuree) reacting compound 2-6 with (S)-ethyl alaninate or a salt thereof and benzyl alcohol to afford a mixture of (R,S)-Compound T and (S,S)-Compound I; wherein:(R,S)-Compound I is a compound of structure:(R, S)-Compound I and (S,S)-Compound I is a compound of structure:(S,S)-Compound I21. The process of embodiment 20, wherein a solvent is used in step a).22. The process of embodiment 21, wherein the solvent in step a) is ethanol.23. The process of any one of embodiments 20-22, wherein the reaction is heated in step a).The process of embodiment 23, wherein the reaction is heated to from about 80 °C to about 85 °C in step a). The process of any one of embodiments 20-24, wherein an acid is used in step b). The process of embodiment 25, wherein the acid used in step b) is methane sulfonic acid. The process of any one of embodiments 20-26, wherein the reaction is heated in step b). The process of embodiment 27, wherein the reaction is heated to reflux in step b). The process of any one of embodiments 20-28, wherein the leaving group isThe process of any one of embodiments 20-28, wherein the leaving group is - OSChMe. The process of any one of embodiments 20-30, wherein Ci-Ce alkyl is ethyl. The process of any one of embodiments 20-30, wherein Ci-Ce alkyl is methyl or propyl. The process of any one of embodiments 20-30, wherein Ci-Ce alkyl is cyclopropyl. The process of any one of embodiments 20-33, wherein a base is used in step c). The process of embodiment 34, wherein the base is magnesium / c V-butoxide. The process of any one of embodiments 20-35, wherein a solvent is used in step c). The process of embodiment 36, wherein the solvent is N-methyl-2-pyrrolidone. The process of any one of embodiments 20-37, wherein the reaction is heated in step c). The process of embodiment 38, wherein the reaction is heated to about 80 °C in step c). The process of any one of embodiments 20-39, wherein an activating reagent is used in step d). The process of embodiment 40, wherein the activating reagent is bromotrimethylsilane. The process of any one of embodiments 20-41, wherein a solvent is used in step d).The process of embodiment 42 wherein the solvent is acetonitrile or water or a mixture thereof. The process of any one of embodiments 20-43, wherein an activating reagent is used in step e). The process of embodiment 44, wherein the activating reagent is aldrithiol-2. The process of embodiment 44 or embodiment 45 wherein an additional activating agent is used in step e). The process of embodiment 46, wherein the additional activating agent is triphenyl phosphine. The process of any one of embodiments 20-47, wherein a base is used in step e). The process of embodiment 48, wherein the base is triethylamine. The process of any one of embodiments 20-49, wherein a solvent is used in step e). The process of embodiment 50, wherein the solvent is dichloromethane. The process of any one of embodiments 20-51, wherein the reaction is heated in step e). The process of embodiment 52, wherein the reaction is heated to about 40 °C in step e). A compound of structuresalt thereof.A process to prepare a compound of structure:or a salt thereof, comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)fbrmamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with tri methoxym ethane and methanol to afford a compound of structurewherein the reaction is heated. The process of embodiment 55, wherein a solvent is used in step a). The process of embodiment 56, wherein the solvent in step a) is ethanol. The process of any one of embodiments 55-57, wherein the reaction is heated in step a). The process of embodiment 58, wherein the reaction is heated to from about 80 °C to about 85 °C in step a). The process of any one of embodiments 55-59, wherein an acid is used in step b). The process of embodiment 60, wherein the acid used in step b) is methane sulfonic acid. The process of any one of embodiments 55-61, wherein the reaction is heated in step b). The process of embodiment 62, wherein the reaction is heated to reflux in step b). A process to prepare (R,S)-Compound I or a salt thereof comprising: a) preparing the mixture of (R,S) and (S,S)-Compound I according to a method of any one of embodiments 20-63;b) reacting a mixture of (R,S)-Compound I and (S,S)-Compound I with an enzyme to afford a mixture of an (R)-Phosphonic Acid and (R,S)-Compound I; c) reacting (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; and d) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound I is a compound of structure:(R, Sj-Compound I(S,S)-Compound I is a compound of structure:(S,S)-Compound I and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid65. The process of embodiment 64, wherein the (R)-Phosphonic Acid is separated from the mixture before the reaction with (S)-ethyl alaninate or a salt thereof.66. The process of embodiment 65, wherein an extraction is used for the separation and the (R)-Phosphonic Acid is in the aqueous phase.67. The process of embodiment 64, wherein the (R)-Phosphonic Acid is reacted with (S)- ethyl alaninate or a salt thereof without being separated from the mixture.68. The process of any one of embodiments 64-67, wherein the enzyme is a pig liver enzyme.69. The process of any one of embodiments 64-68, further comprising a solvent in step a) and b).70. The process of embodiment 69, wherein the solvent in step a) is water.71. The process of any one of embodiments 1-53 or 64-70, further comprising reacting (R,S)-Compound I with fumaric acid to form a fumarate salt of (R,S)-Compound I.72. The process of embodiment 71, wherein about 1 equivalent of fumaric acid is used.73. The process of embodiment 71, wherein (R,S)-Compound I is reacted with fumaric acid in a mixture of organic solvents.74. The process of embodiment 73, wherein the mixture of organic solvents is isopropanol and heptane.EXAMPLESExample 1. Synthetic Preparation of (R,S)-Compound I(R,S)-Compound I (S,S)-Compound ISYNTHETIC METHOD AStep 1

[0090] A dry reaction container is charged with N-(2-amino-4,6-dichloropyrimidin-5- yl)formamide (2-1) (1 equiv.), 2-aminoethanol (.9 equiv.) and ethanol under N2 atmosphere at room temperature. The reaction mixture is then stirred and heated to a temperature between SO- 85 °C overnight. Alternatively, the reaction mixture is stirred and heated to between 75-80 °C.After bringing the reaction temperature to 20-30°C, the reaction mixture is filtered and purified to afford the desired product, N-(2-amino-4-chloro-6-((2-hydroxyethyl)amino)pyrimidin-5- yl)formamide (2-2).Step 2

[0091] A dry reaction container is charged with N-2-(2-amino-6-methoxy-9H-purin-9-yl)ethan- l-ol (2-2) (1 equiv.), trimethoxymethane (i.e., trimethyl orthoformate) (3 equiv.), methanesulfonic acid (3 equiv.) and methanol under N2 atmosphere at room temperature. The reaction mixture is then stirred until completion which is tracked by HPLC. Once the reaction is complete the reaction mixture is filtered and purified to afford the desired product, 2-(2-amino-6- methoxy-9H-purin-9-yl)ethan- 1 -ol (2-3).Alternate Step 2

[0092] Trimethoxymethane and methanesulfonic acid are added to N-(2-amino-4-chloro-6-((2- hydroxyethyl)amino)pyrimidin-5-yl)formamide (2-2) in methanol and the reaction mixture is heated at reflux temperature for 9-11 hr. The HPLC analysis of the reaction mixture at this point shows the formation of the desired product (2-3) in 80-85% and unreacted 2-2 in 2.5-2. %. After extending the reaction for another 3-5 hr under same conditions, the formation of an unknown impurity is seen up to -10%. This impurity is retained with the product after workup and isolation. The desired product (2-3) is obtained in 61% yield with 86% purity, which is then confirmed by 'H-NMR and mass spectroscopy.Step 3

[0093] A dry reaction container is charged with 2-(2-amino-6-methoxy-9H-purin-9-yl)ethan-l-ol (2-3) (1 equiv.), magnesium tertbutoxide (2.5 equiv.), and N-Methyl-2-pyrrolidone under N2 atmosphere at room temperature. The reaction mixture is stirred for thirty minutes and then (diethoxyphosphoryl)methyl 4-methylbenzenesulfonate (2-4’) (1.2 equiv.) is added and the reaction is stirred until completion which is tracked by HPLC. Once the reaction is complete, the reaction mixture is filtered and purified to afford the desired product, diethyl ((2-(2-amino-6- methoxy-9H-purin-9-yl)ethoxy)methyl)phosphonate (2-5’).Step 4

[0094] A dry reaction container is charged with diethyl ((2-(2-amino-6-methoxy-9H- purin-9-yl)ethoxy)methyl)phosphonate (2-5’) (1 equiv.), TMSBr (2.5 equiv.), and acetonitrile under N2 atmosphere at room temperature. The reaction mixture is stirred for thirty minutes and then a solution of NaHSO4 (IN) is added and the reaction is stirred until completion which is tracked by TLC or HPLC. Once the reaction is complete the reaction mixture is filtered and purified to afford the desired product, ((2-(2-amino-6-methoxy-9H-purin-9- yl)ethoxy)methyl)phosphonic acid (2-6).

[0095] Using the product from step 4, the procedure from PCT / US2023 / 28218 can be followed to convert compound (2-6) to (R,S)-Compound I monofumarate, which is provided below.Step 5

[0096] To a solution of ((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-m ethyl)-phosphonic acid (2-6) (40 g, 0.132 mol, 1 equiv.) in DCM (560 mL) at 20-30°C under stirring was charged (S)-ethyl 2-aminopropionate hydrogen chloride salt (6) (20.19 g, 0.132 mol, 1 equiv.), benzyl alcohol (7) (71.28 g, 0.66 mol, 5 equiv.) and TEA (159.98 g, 1.58 mol, 12 equiv.), and the solution was stirred for 10-30 minutes. To this was added a solution prepared from PhsP (207.5 g, 0.792 mol, 6 equiv.) and 2,2’-dithiopyridine (Aldrithiol-2) (174.24 g, 0.792 mol, 6 equiv.) in DCM (320 mL) at 20-30°C over 60 minutes. The resulting reaction mixture was stirred at 35-45°C for 15-20 hours and concentrated to remove3 / 4thof solvent under vacuum below 40°C. To the resulting residue were added MeOH (-120 mL), distilled water (-400 mL), toluene (-400 mL), and n-heptane (-400 mL) and the mixture was stirred at 20-30°C for 0.5-1 hours. After allowing the reaction mixture to stand for 0.5 to 1 hour at 20-30°C, the organic phase was separated and the aqueous phase was extracted with a mixture of toluene (-400 mL) and n-heptane (-400 ml) to remove remaining reagents and by-products. The remaining aqueous phase was then extracted with DCM (2 x 400 mL) and upon concentration of DCM under vacuum below 40°C, the crude product was purified by silica gel column chromatography with DCM to 2% MeOH in DCM as a mobile phase. The eluting fractions containing product were combined and solvent was removed under vacuum below 40°C to give the desired product as a mixture of diastereoisomers, namely, (R,S) and (S,S) (2S)-ethyl-2-((((2-(2-amino-6-methoxy- 9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (Compound I) in 45.8 % (29.74 g) isolated yield with 98.8 % purity by HPLC. 'H NMR (400 MHz, DMSO-d6): bppm 7.85 (s, 1H), 7.34 (m, 5H), 6.44 (s, 2H), 5.36 (m, 1H), 4.90 (m, 2H), 4.17 (m, 2H), 4.07 (m, 2H), 3.95 (s, 3H), 3.82 (m, 5H), 1.18-1.24 (m, 6H). LCMS (m / z): 493.3 (MH+).Step 6 - Chiral Separation

[0097] The chiral separation of (R,S)-Compound I from (S,S)-Compound I can be accomplished using the selective enzymatic reactions described herein. In the alternative, the chiral separation is accomplished by chiral column chromatography as described below.

[0098] A diastereoisomeric mixture of (S,S)-Compound I and (R,S)-Compound I (22.50 g) was subjected to a chiral chromatography separation under supercritical fluid chromatography (SFC) separation conditions as shown below to separate and obtain the 11.7 g of (R,S)- Compound I with 98.6% purity by HPLC and 9. 1 g of (5,5)-Compound I with 95.6% purity by HPLC.SFC Conditions:Column: ChiralPak AD, 250x30mm I.D., 10pm;Mobile phases: A: CO2 and B: Ethanol (0.1% NH3H2O);Gradient: B 45% isocratic;Flow rate: 200 mL / min;Wavelength: 310 nm;Cycle time: ~6 min;Back pressure: 100 bar;

[0099] Injection amount: ~lg. Characterization of (7?,5)-ethyl-2-((((2-(2-amino-6- methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate ((R,S)- Compound I) as a free base: Purity by HPLC: 98.6%; 'H NMR (DMSO-d6 8ppm, 7.82 (s, 1H),7.30 (m, 5H), 6.38 (s, 2H), 5.30 (t, 1H), 4.83 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.84 (m, 2H), 3.60 (m, 5H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m / z): 493 (MH+).

[0100] Characterization of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate ((S,S)-Compound I) as a free base: Purity by HPLC: 95.6%; ’HNMR (DMSO-d6): Sppm 7.82 (s, 1H), 7.35 (m, 5H), 6.45 (s, 2H),5.30 (t, 1H), 4.80 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.80 (m, 3H), 3.70 (m, 2H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m / z): 493 (MH+).

[0101] In certain nonlimiting embodiments, the stereoisomers are separated using HPLC or SFC with achiral or chiral stationary phases. Non limiting examples of chiral stationary phases which may be used include Chiralpak AD, Chiralpak AS, Chiralcel OG, and Chiralcel OJ.Salt Formation - Preparation of (l?,5)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)- ethoxy )-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate ((R,S)-Compound I Monofumarate))

[0102] To a solution of (7?,5)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (3 g, 6 mmol, 1 equiv.) in IPA was added a solution of fumaric acid ( 0.765 g, 6.6 mmol, 1 .1 equiv.) in IPA through a filter at 45-55°C and stirring was continued for 1-2 hours. The seeds of the crystalline compound were then added to the reaction mixture and stirring was continued for 1-2 hours at 45-55°C. After allowing the reaction mixture to settle at 20-30°C for 4-6 hours, a drop-wise addition of n-heptane (~30 mb) was performed and stirring was continued for another 8-15 hours at 20-30°C and 0-5°C for 8-15 hours. The solid observed was filtered and the wet cake was washed with a mixture of IPA / n- heptane (1 / 3, v / v, ~5 mb). The solid cake was dried at 35-45°C for 16-24 hours under vacuum to afford the desired product, (A,5)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)- methyl)-(benzyloxy)-phosphoryl)-amino)-propi onate monofumarate ((R-S)-Compound I monofumarate)) in 85 % (3.1 g) isolated yield with 98.6 % purity by HPLC. 'H NMR (DMSO- d6): 5ppm: 7.80 (s, 1H), 7.35 (m, 5H), 6.63 (s, 2H), 6.40 (s, 2H), 5.53 (t, 1H), 4.84 (d, 2H), 4.15 (t, 2H), 4.00 (m, 2H), 3.92 (s, 3H), 3.80 (m, 3H), 3.75 (m, 2H), 1.20 (d, 3H), 1.13 (t, 3H); [(R,S)- Compound I to fumaric acid ratio was found to be 1 : 1 by 'H NMR],SYNTHETIC METHOD BStep 1

[0103] Compound 2-1 (1.75 kg, 1.0 equiv.) is charged to an inert, 30-L, jacketed, bottom outlet valve reactor followed by ethanol (14 L, 8.0 vol). The resulting slurry is warmed to an internal temperature of 80 ± 5 °C over 79 min. In a separate container, ethanolamine (1.03 kg, 2.0 equiv.) is dissolved in ethanol (3.5 L, 8.0 vol) and then added to the ethanolic solution of compound 2-1 over 1 h while maintaining an internal temperature of 80 ± 5 °C. Upon complete addition, the reaction is agitated for 3 h at 80 ± 5 °C. The batch is cooled to 40 ± 5 °C and then analysis by HPLC is performed, indicating 99.67% conversion. The slurry is then cooled further to 20 ± 5 °C and agitated for 11.5 h. The slurry is filtered onto an 18”, Nutsche filter with polypropylene cloth and the wet cake washed with ethanol (14 L, 8.0 vol) and then conditioned under N2 for 21 h. The wet cake is further dried at 55 ± 5 °C under vacuum for 24 h. Upon analysis of the dried material by NMR, there is 26.7 mol % ethanolamine relative to compound 2-2.

[0104] The crude compound 2-2 (2.122 kg, 1.0 equiv.) is charged to an inert, 30-L, jacketed, bottom outlet valve reactor followed by ethanol (17.5 L, 8.2 vol). The batch is agitatedat 20 ± 5 °C for 3 h. The slurry is filtered onto an 18” Nutsche filter with polypropylene cloth and the wet cake is washed with ethanol (8.5 L, 4.0 vol) and the conditioned under N2 for 21 h. The wet cake is further dried at 55 ± 5 °C under vacuum for 17 h and continued drying under identical conditions for another 24 h. The desired compound 2-2 is obtained in 87% yield (1.708 kg) with 99.08% purity (HPLC, AUC). The potency determined byNMR is found to be 95.8 wt %.Step 2

[0105] Compound 2-2 (1.629 kg, 1.0 equiv.) is charged to an inert, 30-L, jacketed, bottom outlet valve reactor equipped with a 2 N NaOH scrubber, followed by methanol (4.9 L, 3.0 vol) and trimethoxymethane (4.9 L, 3.0 vol). The resulting slurry is cooled to 10 ± 5 °C (target 10 °C) and methanesulfonic acid (1.015 kg, 1.5 equiv.) is added to the reaction mixture over 30 min. The reaction is warmed to 50 ± 5 °C and agitated for 22.5 h. IPC analysis by HPLC is performed indicating 98.58% conversion. The slurry is cooled to 10 ± 5 °C and then quenched with 10 wt % Na2COs (1.49 kg, 2.0 equiv.; H2O, 14.9 L, 10 vol) over 2 h while maintaining an internal temperature below 25 °C. The reaction is warmed to 10 ± 5 °C and agitated for 15 h. The reaction mixture is filtered onto an 18” Nutsche filter with polypropylene cloth and the wet cake is washed with ethanol (4.9 L, 3.0 vol) and conditioned under N2 for 2 h.

[0106] The crude compound 2-3 is charged to an inert, 30-L, jacketed, bottom outlet valve reactor followed by ethanol (4.0 L, 2.5 vol) and DI water (4.0 L, 2.5 vol). The batch temperature is adjusted to 20 ± 5 °C and agitated for 1 h. The batch is filtered onto an 18” Nutsche filter with polypropylene cloth and the wet cake is washed with ethanol (4.9 L, 3.0 vol) and conditioned under N2 for 14 h. The wet cake is further dried at 55 ± 5 °C under vacuum for 24 h. Upon analysis of the dried material by KF, there is 6190 ppm of water (n = 2) (target: <7500 ppm). Compound 2-3 is isolated in 55% yield (970 g, 819 g adjusted for potency) with 96.14 % purity (HPLC, AUC). The 'H NMR potency is found to be 84.4 wt %.Step 3

[0107] A 30-L, jacket reactor purged with nitrogen is charged with compound 2-3 (0.760 kg after potency correction, 3.63 mol, 1 equiv.), magnesium tert-butoxide (0.681 kg, 4.00 mol, 1.1 equiv.), and (diethoxyphosphory)methyl-4-methylbenzenesulfonate (2-4’) (1.405 kg, 4.36mol, 1.2 equiv.), followed by l-methyl-2-pyrrolidone (NMP, 3.80 L, 5.0 vol), and toluene (2.28 L, 3.0 vol) pre-treated with 50 ppm Statsafe. An additional amount of NMP (1 .52 L, 2 vol) is charged as a line rinse resulting in a pink slurry. The reaction temperature is adjusted to 50 ± 5 °C and the reaction is agitated for at least 1 h. The reaction temperature is re-adjusted to 80 ± 5 °C and agitation continued for at least 10 h. The reaction mixture turns into a dark brown nearsolution with a small amount of undissolved solid. The reaction temperature is then lowered to 35 ± 5 °C and analyzed by HPLC indicating 90.2% conversion (specification >90.0%) from compound 2-3 to compound 2-5’.

[0108] The reaction mixture is cooled to 5 ± 5 °C and quenched with aqueous solution of 30% citric acid (1.047 kg, 5.45 mol, 1.5 equiv.) while maintaining the temperature <25 °C. After adjusting the temperature of the reaction mixture to 20 ± 5 °C, it is washed with toluene (2.28 L, 3.0 vol) twice to remove any organic byproducts / impurities by discarding the top organic layer while retaining the product in the bottom aqueous layer. The aqueous layer is adjusted to 10 ± 5 °C and then basified to pH 8-8.5 with 6.08 L of (8.0 vol) aqueous 2 N NaOH solution (0.486 kg, 12.16 mol and DI water, 5.85 L) while maintaining temperature <30 °C. After adjusting the temperature to 25 ± 5 °C, the pH is measured and is found to be 8.2 by calibrated pH meter. The resulting aqueous basic phase is extracted with DCM (3.8 L, 5 vol) five times at 30 ± 5 °C to make sure product is fully extracted out in DCM. The organic layers are combined and then washed with 2.28 L (3 vol) of 20% w / w NaCl aqueous solution (0.524 kg and DI water, 3.51 L) twice. The resulting organic solution is distilled under reduced pressure at <40 °C until the batch volume is 7.5-8 vol per input of compound 2-3. As a result, the desired product (2-5’) is isolated as a 6 L solution in a solvent mixture of DCM / NMP / t-BuOH with solution weight: 9.51 kg and water content: 2.1 wt % by KF (n = 2). The product is found to be with 86.9% purity (HPLC, AUC, at 280 nm). The product potency is found to be 12.51 wt % by q-lH NMR (n = 2) and reaction yield is 61.6% after potency correction. 'HNMR and31P NMR spectra are consistent with the chemical structure of compound 2-5’. The LC-MS spectrum confirmed the molecular weight 360.44 [M+l]+ of compound 2-5’. The resulting solution of compound 2-5’ in DCM / NMP / t-BuOH is stored at 5 ± 5 °C in the reactor under nitrogen atmosphere before telescoping it into the next step.Step 4

[0109] A 30-L, jacket reactor with the compound 2-5’ (0.800 kg after potency correction, 2.226 mol, 1 equiv.) solution in the solvent mixture of DCM / NMP / t-BuOH (from Step 3) is held under the protection of nitrogen flow (1 LPM) with agitation on at temperature of 5 ± 5 °C. The temperature of the mixture is maintained to 20 ± 5 °C prior to azeotropic distillation with di chloromethane (DCM). The reactor is then charged with DCM (8.0 L, 10.0 vol) and then the solvents are azeotropically distilled under reduced pressure until the batch volume reaches 5.5 L (6.9 vol, target end vol: 6.0-7.0 vol). The reactor is charged again with acetonitrile (ACN, 8.0 L, 10 vol) and the solvents are azeotropically distilled under reduced pressure until the volume in the reactor reaches 5.0 L (6.25 vol, target end vol: 6.0-6.5 vol). The analysis for water content is then found to be 0.64 wt %. An aliquot of the reaction solution is taken forNMR analysis which shows no residual DCM and t-BuOH present. The reactor temperature is at 20 ± 5 °C and an additional ACN (around 3.2 L) is added to bring the total batch volume up to 10 vol (i.e., 8 L). The water content of the batch is found to be 0.32 wt %.

[0110] The temperature of a reactor is adjusted to 5 ± 5 °C and charged with tri-n- butylamine (1.616 L, 12.243 mol, 5.5 equiv.). A 2 N NaOH solution scrubber is connected to the reactor. The addition of bromotrimethyl silane (TMS-Br, 2.27 kg, 12.243 mol, 5.5 equiv.) is performed slowly while maintaining the reaction temperature at 5 ± 10 °C. After the addition of TMS-Br is complete, the reaction temperature is adjusted 30 ± 5 °C and then stirred at 30 ± 5 °C for 16 h. Analysis of an aliquot from the reaction solution by HPLC indicates >99.0% conversion of compound 2-5’ to compound 2-6 with the presence of the mono-silyl intermediate in 3.4%.

[0111] After adjusting the reaction temperature to 20 ± 5 °C, ACN is added (8.0 L, 10 vol) and the solvent is distilled under reduced pressure until batch volume reaches 12.0 L (15 vol). An additional amount of ACN (8.0 L, 10 vol) is charged and the solvent is distilled under reduced pressure until batch volume reaches 12.0 L (15 vol) to co-distill the volatile reagents, byproduct, and impurities. During distillation of solvent under reduced pressure, the maximum temperature allowed is 45 °C. The reaction temperature is adjusted to 5 ± 5 °C and to it is slowly added an aqueous solution of sodium bisulfate (NaHSC ) (1.47 kg, 12.243 mol, 5.5 equiv and DI water, 4.8 L, 6 vol) while maintaining batch temperature 20 ± 10 °C. The reaction is then agitated at 30 ± 5 °C for 16 h under nitrogen, which resulted in a brown slurry. After cooling it to 5 ± 5 °C, the reaction mixture is filtered through a 15’,’ Hastelloy, Nutsche Filter withpolypropylene cloth as the media. The resulting wet cake is conditioned under nitrogen for at least 30 min and then mixed with 4.8 L (6 vol) of DI water in a 14 L HOPE container to form a slurry. The resulting slurry is then charged back into the 30-L, jacket reactor and maintained at 30 ± 5 °C with agitation for 1 h. After maintaining the slurry at 20 ± 5 °C, it is filtered through a 15”, Hastelloy, Nutsche Filter with polypropylene cloth as the media and the resulting cake is conditioned for 5 h under nitrogen. The reactor is rinsed with isopropyl alcohol (IP A) until visually clean. The conditioned cake is mixed with IPA (5.6 L, 7 vol) in the reactor, the temperature is adjusted to 30 ± 5 °C, and then agitated for 1 h. After adjusting the temperature to 20 ± 5 °C, it is filtered through a 15”, Hastelloy, Nutsche Filter with polypropylene cloth as the media. The resulting wet cake is conditioned for 5 h under nitrogen. The filter cake is dried in vacuum oven at 45 ± 5 °C to afford 763.5 g of product (compound 2-6) as a white solid with 95.5 % purity (HPLC, AUC). Product potency is 68.96 wt % by q-31P NMR (n = 2) and reaction yield is 70.6% after potency correction. The water content is found to be 3.6 wt %. 'H NMR,13C NMR,31P NMR, and LC-MS = 304.2 [M+l]+ are in agreement with the chemical structure of compound 2-6.Step 5

[0112] A 30-L, jacketed, bottom outlet valve inerted reactor is charged with L-alanine ethyl ester hydrochloride (0.203 kg, 1.0 equiv.) and compound 2-6 (0.400 kg after potency correction, 1.0 equiv.) and the components are slurried in DCM (2.4 L, 6.0 vol). The reactor with agitation 25 ± 5 °C is charged with benzyl alcohol (0.713 kg, 5.0 equiv.) followed by triethylamine (2.200 L, 12.0 equiv.) while maintaining a temperature below 30 °C. The resulting mixture is agitated at 25 ± 5 °C for 50 min.

[0113] Another inert, 10-L, jacketed, bottom outlet valve reactor is charged with triphenylphosphine (2.077 kg, 6.0 equiv.) and 2 ’2-di thiodi py dine (1.773 kg, 6.0 equiv.) followed by DCM (3.2 L, 8.0 vol). The resulting solution is agitated at 25 ± 5 °C for 36 min.

[0114] The solution in the 10-L, j acketed, bottom outlet valve reactor is transferred to the 30-L, jacketed, bottom outlet valve reactor over 30 min. The reaction temperature is adjusted to 40 ± 5 °C and agitated for 18.5 h. The reaction mixture is cooled to 20 ± 5 °C and IPC analysis performed by HPLC indicates 99.60% conversion.

[0115] The reaction mixture is concentrated from 13 L (32.5 vol) to 6.8 L (17 vol) undervacuum while maintaining an internal temperature below 40 ± 5 °C (25 inHg) over 2 h. After the addition of methanol (4.0 L, 10.0 vol), the reaction mixture is concentrated from 10.8 L (27 vol) to 6.8 L (17 vol) under vacuum while maintaining an internal temperature below 40 ± 5 °C (25 in Hg) over 2 h. After the second distillation, the DCM content is measured to be 1.2 mol % byrH NMR at 20 ± 5 °C. The reaction mixture is diluted with methanol (1.2 L, 3.0 vol), DI water (4.0 L, 10 vol), toluene (4.0 L, 10 vol, treated with 50 ppm Statsafe. Fisher, Catalog # T324-200, lot # 217797), n-heptane (4.0 L, 10 vol, treated with 50 ppm Statsafe. Pride, Catalog # 112703, lot # H5503026CAL), and agitated for 30 min. The agitation is ceased for 34 min to allow the layers separation. The top organic layer is removed. To a remaining bottom layer, the addition of toluene (4.0 L, 10 vol, treated with 50 ppm Statsafe, same supplier and lot #), n-heptane (4.0 L, 10 vol, treated with 50 ppm Statsafe Pride, same supplier and lot #) is performed and the reaction mixture is agitated at 20 ± 5 °C for 30 min. Agitation is ceased for 30 min to allow the layers to separate. The top organic layer is removed. To the bottom aqueous layer, toluene (4.0 L, 10 vol, treated with 50 ppm Statsafe, same supplier and lot #) and n-heptane (4.0 L, 10 vol, treated with 50 ppm Statsafe. Pride, same supplier and lot #) are added and then agitated at 20 ± 5 °C for 30 min. The top organic layer is removed and the bottom aqueous layer is held in the reactor overnight under an inert atmosphere.

[0116] To the bottom aqueous layer is added DCM (4.0 L, 10 vol, same supplier and lot #) and the mixture is agitated at 20 ± 5 °C for 30 min. Agitation is ceased for 36 min to allow the layers to separate. The bottom organic (DCM) layer is collected and stored. The remaining aqueous layer is again agitated with DCM (4.0 L, 10 vol) at 20 ± 5 °C for 30 min. Agitation is ceased for 34 min to allow the layers to separate. The bottom organic (DCM) layer is collected and stored. The remaining aqueous layer is again agitated a third time with DCM (4.0 L, 10 vol) at 20 ± 5 °C for 30 min. Agitation is ceased for 30 min to allow the layers to separate. The bottom organic (DCM) layer is collected and stored. The combined organic layers (DCM) are concentrated using a rotary evaporator from 12 L (30 vol) to 0.60 L (1.5 vol) under vacuum while maintaining an internal temperature below 40 ± 5 °C (28 in Hg, Tmax40 °C) over 12 h. After concentration, 902.8 g of the crude (R,S)-Compound I and (S,S)-Compound I mixture is obtained with 24.18% purity (HPLC, AUC).

[0117] The resulting crude product (as isomeric mixture of Compound I) is purified by silica gel column chromatography. A glass column (30 in * 11 in) is packed with silica gel(7.233 kg, 8 wts based on crude mass, Silicycle) and DCM (28.5 L, 4 vol in respect to silica wt). The crude (R,S)-Compound I and (S,S)-Compound I mixture is purified on the silica column eluting with 0-2% MeOH in DCM. The fractions collected are analyzed by TLC and HPLC to determine the desired fractions. Fraction 11 contained (R,S)-Compound I and (S,S)-Compound I mixture with 89.00% purity (HPLC, AUC) are combined, concentrated to dryness on the rotary evaporator, and further dried under vacuum. Fractions 12-18 containing both (R,S)-Compound I and (S,S)-Compound I with >96% purity (HPLC, AUC) are combined, concentrated to dryness on the rotary evaporator, and further dried under vacuum. Fractions 19-21 containing both (R,S)- Compound I and (S,S)-Compound I with 88-93 % purity (HPLC, AUC) are combined and concentrated to dryness on the rotary evaporator and further dried under vacuum.

[0118] The crude (R,S)-Compound I and (S,S)-Compound I from Fraction 11 is isolated in 1.71% yield (11.06 g corrected for potency) with 88.74 % purity (HPLC, AUC) as a brown oil. The ’H NMR potency is determined to be 81.3 wt %.

[0119] The crude (R,S)-Compound I and (S,S)-Compound I from Fractions 19-21 is isolated in 5.09% yield (62.6 g not adjusted for potency) with 90.86 % purity (HPLC, AUC) as a white solid.

[0120] The crude (R,S)-Compound I and (S,S)-Compound I from Fractions 12-18 is isolated in 23.32% yield (151.59 g corrected for potency) with 97.26 % purity (HPLC, AUC) as a brown oil. The 'H NMR potency is determined to be 83.8 wt %.Step 6 - Chiral Separation

[0121] Separation of (R,S)-Compound I and (S,S)-Compound I from Fractions 12-18 is conducted using a CHIRALPAK AD-10 (50 x 250 nm) column and using 0.1% DEA in 50:50 v / v n-heptane / EtOH as the mobile phase. The (R,S)-Compound I is isolated in 76.19 g (adjusted for potency) with 94.64 % purity (UPLC, AUC) having ratio of (R,S) : (S,S) isomer of 99.66:0.34.Salt Formation

[0122] (R,S)-Compound I (76.194 g, 1.0 equiv.) is dissolved in isopropanol (230 mL, 4.0 vol) and charged into an inert, 1-L, jacketed, bottom outlet valve reactor. The container containing the (R,S)-Compound I is rinsed with isopropanol (76 mL, 1.0 vol) and the rinse istransferred to the 1-L, jacketed, bottom outlet valve reactor. The brown solution is warmed to an internal temperature of 20 ± 5 °C. Fumaric acid (19.76 g, 1 .1 equiv.) is added to the batch resulting in a thick suspension. The batch is then warmed to an internal temperature of 50 ± 5 °C over 10 min at which heavy white precipitation occurred. The batch is agitated at 50 ± 5 °C for 1 h before cooling to 25 ± 5 °C over 2 h. The batch is agitated at 25 ± 5 °C for 1 h and then to it is added n-heptane (762 mL, 10 vol) treated with Statsafe (0.03 mL, 50 ppm, Alfa Aesar, Catalog # H5787.22, lot # N10H073) over 3 h. The slurry is agitated at 25 ± 5 °C for 15 h, then the batch is cooled to 0 ± 5 °C over 5.5 h and agitated at 0 ± 5 °C for 19 h. The slurry is fdtered onto an 8”, Buchner Funnel with Whatman # 4 paper and the wet cake is washed with 33% v / v isopropanol / n-heptane (137 mL, 2.0 vol) and then conditioned under N2 for 30 min. The wet cake is further dried at 40 ± 5 °C under vacuum for 52 h to obtain the desired (R,S)-Compound I monofumarate in >99% yield (94.8 g, 100%) with 98.93% purity (UPLC, AUC). TheNMR potency is determined to be 99.8 wt %.HPLC Method - For Use in Synthetic Method BColumn: Agilent Eclipse XDB C18, 3.5 pM, 4.6 x 150 mmWavelength: 280 nmFlow Rate: 0.7 mL / minInjection Volume: 3 pL Column Oven Temperature: 35 °C Elution Mode: Gradient Run Time: 35 minDiluent: ACN / water = 1 : 1; MeOH / water = 1 : 1Mobile Phase A: 0.01 Molar (1.36 g in 1 L) potassium dihydrogen phosphate in water; add 1.0 mL orthophosphoric acid and mix well.Mobile Phase B: acetonitrile.(R,S)-Compound I to (S,S)-Compound I Ratio HPLC Method - For Use in SyntheticMethod BColumn: XB ridge C18 3.5 pm (3.0 x 150) mmFlow Rate: 1.0 mL / minDetection: By UV at 280 nmInjection Volume: 3 pLColumn Oven Temperature: 25 °CElution Mode: GradientRun Time: 45 minMobile Phase A: 0.1% TFA in WaterMobile Phase B: 0.1% TFA in MeOHUPLC Purity Method - For Use in Synthetic Method BColumn: Waters ACQUITY UPLC BEH C18, 100 x 2.1 mm, 1.7 pm,PN: 186002352Flow Rate: 0.3 mL / minDetection: By UV at 210 nmInjection Volume: 2 pLColumn Oven Temperature: 25 °CSample Temperature: 5 °CElution Mode: GradientRun Time: 65 minData Collection Time: 55 minMobile Phase A: 0.1% TFA in WaterMobile Phase B: 0.1% TFA in MeOHExample 2. Synthetic Preparation of a Mixture of (R,S)- and (S,S)-Compound IStep 1Step 1

[0123] To a stirred solution of sodium methoxide (4 eq) in methanol (10 v / w) is addedcompound 3-1 (free acid; 1 eq) at RT. The reaction mixture is stirred at reflux and the reaction progress is monitored by HPLC. After satisfactory completion of the reaction (~20 hr), the reaction mixture is concentrated at 45 °C under vacuum. The resulting residue is dissolved in water (4 v / w) and to it is added aqueous solution of sodium bisulfate (4 eq in 6 v / w of water). The resulting mixture is stirred at RT for 12 hr, the precipitated solid is filtered, washed with water (2 x 5 v / w), and dried at 45 °C under vacuum to give the expected compound 2-6 as an off- white solid. The product structure is confirmed by 'H NMR and mass spectroscopy. Batch size: 100g; yield: 77g (78%); HPLC purity: 98.77%.Step 2

[0124] A solution of compound 2-6 (1 eq), benzyl alcohol (5 eq), L-alanine ethyl ester hydrogen chloride salt (2 eq), and triethylamine (12 eq) in pyridine (24 v / w) is stirred to 60 °C. To it is added a solution of Aldrithiol-2 (7 eq) and triphenylphosphine (7 eq) in pyridine drop by drop over a period of 30 min and the resulted mixture is stirred at 60±5 °C for 18 hr. After analyzing the reaction progress by HPLC, the reaction mixture is concentrated under vacuum at 45 °C and the resulting residue is dissolved in ethyl acetate. The ethyl acetate layer is washed with aqueous sodium bicarbonate solution and water, dried over anhydrous Na2SC>4, filtered, and concentrated under vacuum at 45 °C to give a crude mixture of (R,S)- and (S,S)-Compound I as a syrupy liquid. In some cases, this crude syrupy liquid is purified by column chromatography twice using EtOH in DCM to give the expected product (a mixture of (R,S)- and (S,S)- Compound I) as a gummy liquid, which is characterized by 'H NMR, mass spectroscopy, and HPLC analysis. Yield and purity of batches are provided below:Chiral Separation

[0125] Chiral Separation of the mixture of (R,S)-Compound I and (S,S)-Compound I can be performed by any methods described herein. For example, (R,S)-Compound I may beseparated from (S,S)-Compound I using the selective enzymatic reactions described herein. In the alternative, the chiral separation may be accomplished by chiral column chromatography. Salt Formation

[0126] After isolation of (R,S)-Compound I, the compound can be converted into a monofumarate salt using any of the methods described herein. For example, it can be prepared as described in Example 1 - Synthetic Method A or as in Example 1 - Synthetic Method B.

[0127] This specification has been described with reference to embodiments of the disclosure. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the disclosure as set forth in the claims below. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of disclosure.

Claims

CLAIMSWe claim:

1. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated; c) reacting compound 2-3 with a compound of Formula 2-4 in the presence of a suitable base to afford a compound of Formula 2-5, wherein:0 the compound of Formula 2-4 is of structure.LG is a Leaving Group which is displaced by the primary hydroxy of compound 2-3; and the compound of Formula 2-5 is of structured) hydrolyzing the compound of Formula 2-5 in the presence of a suitable base to afford a compound of structuree) reacting compound 2-6 with (S)-ethyl alaninate or a salt thereof and benzyl alcohol to afford a mixture of (R,S)-Compound T and (S,S)-Compound I; f) performing a chiral separation to obtain (R,S)-Compound I from the mixture of (R,S)-Compound I and (S,S)-Compound I, optionally wherein the chiral separation is performed using chiral chromatography; and g) optionally reacting the (R, S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound l is a compound of structure:(R, SJ-Compound I and (S,S)-Compound I is a compound of structure:(S,S)-Compound I2. The process of claim 1, wherein a solvent is used in step a), optionally wherein the solvent is ethanol.

3. The process of claim 1 or 2, wherein the reaction is heated in step a), optionally wherein the reaction is heated to from about 80 °C to about 85 °C).

4. The process of any one of claims 1-3, wherein an acid is used in step b), optionally wherein the acid is methane sulfonic acid.

5. The process of any one of claims 1-4, wherein the reaction is heated in step b), optionally wherein the reaction is heated to reflux.

6. The process of any one of claims 1-5, wherein the leaving group isor -OSChMe.

7. The process of any one of claims 1-6, wherein each Ci-Ce alkyl in the compounds of Formula 2-4 and 2-5 is ethyl, methyl, propyl, or cyclopropyl.

8. The process of any one of claims 1-7, wherein a base is used in step c), optionally wherein the base is magnesium / c / 7-butoxide.

9. The process of any one of claims 1-8, wherein a solvent is used in step c), optionally wherein the solvent is N-methyl-2-pyrrolidone.

10. The process of any one of claims 1-9, wherein the reaction is heated in step c), optionally wherein the reaction is heated to about 80 °C.

11. The process of any one of claims 1-10, wherein an activating reagent is used in step d), optionally wherein the activating reagent is bromotrimethylsilane.

12. The process of any one of claims 1-11, wherein a solvent is used in step d), optionally wherein the solvent is acetonitrile or water or a mixture thereof.

13. The process of any one of claims 1-12, wherein an activating reagent is used in step e), optionally wherein the activating reagent is aldrithiol-2.

14. The process of claim 13, wherein an additional activating agent is used in step e), optionally wherein the additional activating agent is triphenylphosphine.

15. The process of any one of claims 1-14, wherein a base is used in step e), optionally wherein the base is tri ethylamine.

16. The process of any one of claims 1-15, wherein a solvent is used in step e), optionally wherein the solvent is dichloromethane.

17. The process of any one of claims 1-16, wherein the reaction is heated in step e), optionally wherein the reaction is heated to about 40 °C.

18. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting a mixture of (R,S)-Compound I and (S,S)-Compound I with an enzyme to afford (R)-Phosphonic Acid and (R,S)-Compound I, optionally wherein the enzyme is a pig liver enzyme; b) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; andc) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound l is a compound of structure:(R, Sj-Compound I(S,S)-Compound I is a compound of structure:(S,S)-Compound I and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid19. The process of claim 18, wherein the (R)-Phosphonic Acid is separated from the (R,S)-Compound I before the reaction with (S)-ethyl alaninate or a salt thereof, optionally wherein an extraction is used for the separation.

20. The process of claim 18, wherein the (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof without being separated from the (R,S)-Compound I.

21. The process of any one of claims 18-20, further comprising a solvent, optionally wherein the solvent is water.

22. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting a Phosphonodiamidate with an enzyme to afford (R,S)-Phosphonamic Acid and an Phosphonodiamidate, optionally wherein the enzyme is a pig liver enzyme; b) reacting the (R,S)-Phosphonamic Acid with a benzyl halide to afford (R,S)- Compound I, optionally wherein the benzyl halide is benzyl bromide; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound I is a compound of structure:(R, S^-Compound I the Phosphonodiamidate is a compound of structure:the (R,S)-Phosphonamic Acid is a compound of structure:

23. The process of claim 22, wherein the (R,S)-Phosphonamic Acid is separated from (R,S)-Compound I before the reaction with benzyl halide, optionally wherein an extraction is used for the separation.

24. The process of claim 23, wherein the (R,S)-Phosphonamic Acid is reacted with the benzyl halide without being separated from the (R, S)-Compound I.

25. The process of any one of claims 22-24, further comprising a solvent, optionally wherein the solvent is water.

26. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) reacting a Dibenzyl Phosphonate Ester with an enzyme to afford (R)-Phosphonic Acid, optionally wherein the enzyme is a pig liver enzyme; and b) reacting the (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; and c) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound I is a compound of structure:(R, Sj-Compound I the Dibenzyl Phosphonate Ester is a compound of structure:and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid27. The process of claim 26, further comprising a solvent, optionally where the solvent is water.

28. A compound of structuresalt thereof.

29. A process to prepare a compound of structure:salt thereof, comprising: a) reacting N-(2-amino-4,6-dichloropyrimidin-5-yl)formamide with amino ethanol to afford a compound of structureb) reacting compound 2-2 with trimethoxymethane and methanol to afford a compound of structurewherein the reaction is heated.

30. The process of claim 29, wherein a solvent is used in step a), optionally wherein the solvent is ethanol.

31. The process of claim 29 or 30, wherein the reaction is heated in step a), optionally wherein the reaction is heated to from about 80 °C to about 85 °C.

32. The process of any one of claims 29-31, wherein an acid is used in step b), optionally wherein the acid is methane sulfonic acid.

33. The process of any one of claims 29-32, wherein the reaction is heated in step b), optionally wherein the reaction is heated to reflux.

34. A process to prepare (R,S)-Compound I or a salt thereof comprising: a) preparing the mixture of (R,S)-Compound I and (S,S)-Compound I according to a method of any one of claims 11-27; and b) reacting a mixture of (R,S)-Compound I and (S,S)-Compound I with an enzyme to afford (R)-Phosphonic Acid and (R,S)-Compound I, optionally wherein the enzyme is a pig liver enzyme; c) reacting (R)-Phosphonic Acid with (S)-ethyl alaninate or a salt thereof to afford (R,S)-Compound I; d) optionally reacting the (R,S)-Compound I with an acid to afford a salt of (R,S)- Compound I, optionally wherein the salt is the monofumarate salt of (R,S)- Compound I; wherein:(R,S)-Compound l is a compound of structure:(R, S^-Compound I(S,S)-Compound I is a compound of structure:(S,S)-Compound I and the (R)-Phosphonic Acid is a compound of structure:(R)-Phosphonic acid35. The process of claim 34, wherein the (R)-Phosphonic Acid is separated from the (R,S)-Compound I before the reaction with (S)-ethyl alaninate or a salt thereof, optionally wherein an extraction is used for the separation.

36. The process of claim 34, wherein the (R)-Phosphonic Acid is reacted with (S)-ethyl alaninate or a salt thereof without being separated from the (R,S)-Compound I.

37. The process of any one of claims 34-36, further comprising a solvent in step a) and b), optionally wherein the solvent in step a) is water.

38. The process of any one of claims 1-27 or 34-37, further comprising reacting (R,S)- Compound I with fumaric acid to form a fumarate salt of (R,S)-Compound I.

39. The process of claim 38, wherein about 1 equivalent of fumaric acid is used.

40. The process of claim 38, wherein (R,S)-Compound I is reacted with fumaric acid in a mixture of organic solvents, optionally wherein the mixture of organic solvents comprises isopropanol and heptane.

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