Process of making 3-(4'-aminophenyl)-2-methoxypropionic acid, and analogs and intermediates thereof

Abstract

The disclosure provides a process for the preparation of 3-(4'-aminophenyl)-2- methoxypropionic acid, and analogs and intermediates thereof, contemplated to be capable of modulating the activity of receptors, e.g., PPARs.

Description

[0001] Docket No. GIU-074WO

[0002] PROCESS OF MAKING 3-(4’-AMINOPHENYL)-2-METHOXYPROPIONIC ACID, AND ANALOGS AND INTERMEDIATES THEREOF

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 700,074 filed on September 27, 2024, the content of which is hereby incorporated by reference herein in its entirety.

[0005] BACKGROUND

[0006] Peroxisome Proliferator Activated Receptors (PPARs) are members of the nuclear hormone receptor super family, which are ligand-activated transcription factors regulating gene expression. Certain PPARs play roles in the regulation of cell differentiation, development and metabolism of higher organisms.

[0007] Three types of PPARs have been identified: alpha, expressed in the liver, kidney, heart and other tissues and organs; beta / delta expressed, for example, in the brain; and gamma, expressed in three forms: gammal, gamma2, and gamma3. PPARy receptors have been associated with a number of disease states including fibrotic diseases, dyslipidemia, hyperlipidemia, hypercholesteremia, atherosclerosis, atherogenesis, hypertriglyceridemia, heart failure, myocardial infarction, vascular diseases, cardiovascular diseases, hypertension, obesity, inflammation, arthritis, cancer, Alzheimer's disease, skin disorders, respiratory diseases, ophthalmic disorders, IBD (irritable bowel disease), ulcerative colitis and Crohn's disease.

[0008] Further, treatment of tumor cells with ligands of PPARy receptors can induce a decrease in cellular proliferation, cell differentiation and apoptosis, and therefore may be useful in preventing carcinogenesis. Intestinal anti-inflammatory activity may be dependent on binding and subsequent activation of PPARy receptors.

[0009] Accordingly, effective processes for making compounds capable of modulating the activity of PPARs are needed to address the treatment of such diseases.

[0010] IPTS / 200134844.1 Docket No. GIU-074WO

[0011] SUMMARY

[0012] The disclosure provides, for example, a process for the preparation of compounds which may be modulators of PPARs.

[0013] Also contemplated herein is a process for the preparation of analogs and intermediates thereof.

[0014] In one aspect, the present disclosure provides a process for preparing a substantially optically pure compound of Formula (II): lphos catalyst, thereby forming the compound of Formula (II), or a salt thereof.

[0015] In another aspect, the present disclosure provides a process of for preparing a substantially optically pure compound of Formula (I), salt thereof, the process comprising the process for preparing a substantially optically pure compound of Formula (II), as disclosed herein, wherein the process further comprises acylating the compound of Formula (II), or a salt thereof, thereby forming the compound of Formula (I) or a salt thereof.

[0016] IPTS / 200134844.1 Docket No. GIU-074WO

[0017] BRIEF DESCRIPTION OF THE DRAWING

[0018] FIG. 1 depicts the structures of Rh catalysts tested.

[0019] FIG. 2 depicts the structure of Co-catalyst tested.

[0020] FIG. 3 depicts the structures of Ru catalysts tested.

[0021] FIG. 4 depicts the structures of Walphos-Rh catalysts and related Rh catalysts tested.

[0022] FIG. 5A depicts the synthetic scheme including the hydrogenation step with H2 and Pd / C.

[0023] FIG. 5B depicts the synthetic scheme including the hydrogenation step with Rh-Walphos catalyst.

[0024] DETAILED DESCRIPTION

[0025] The features and other details of the disclosure will now be more particularly described. Before further description of the present disclosure, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[0026] DEFINITIONS

[0027] The term “substantially optically pure,” “substantially enantiomerically pure,” “optically pure” or “enantiomerically pure” as used herein when referring to a compound (e.g., a compound described herein) means that at least 95%, for example, at least 96%, at least 97%, or at least 98% of the compound has the desired stereogenic center in a given configuration. It will be appreciated that the percentage is expressed as a percentage of both enantiomers of the compound. For example, a compound of Formula I is substantially optically pure if, based on the total of both the levorotatory and dextrorotatory enantiomers, at least 95% is (5)-(-)-3-(4-acetamidophenyl)-2- methoxypropionic acid (the levorotatory enantiomer).

[0028] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. IPTS / 200134844.1 Docket No. GIU-074WO

[0029] The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

[0030] The term “pharmaceutical composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0031] The term “pharmaceutically acceptable salt(s)” as used herein refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, -toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0032] The compounds of the disclosure may contain one or more stereogenic centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “7?” or “5,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof.

[0033] IPTS / 200134844.1 Docket No. GIU-074WO

[0034] Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

[0035] Individual stereoisomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0036] The compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the disclosure embrace both solvated and unsolvated forms. In some embodiments, the compound is amorphous. In some embodiments, the compound is in a crystalline form.

[0037] The disclosure also embraces isotopically labeled compounds of the disclosure which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as2H, 3H,13C,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively.

[0038] Certain isotopically-labeled disclosed compounds (e.g., those labeled with3H and14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon-14 (z.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability.

[0039] IPTS / 200134844.1 Docket No. GIU-074WO

[0040] Further, substitution with heavier isotopes such as deuterium (z.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.

[0041] Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the e.g., Examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0042] Processes

[0043] In one aspect, provided herein is a process for preparing a substantially optically pure compound of Formula (II): lphos catalyst, thereby forming the compound of Formula (II), or a salt thereof.

[0044] In some embodiments, the hydrogenation further forms a compound of Formula (IV): salt thereof, wherein the ee (enantiomeric excess) of the hydrogenation is at least 60% (i.e., S:R=80%:20%), at least 70% (i.e., S:R=85%: 15%), at least 80% (S:R=90: 10), at least 90% (i.e., S:R=95%:5%), or at least 95% (i.e., S:R=97.5%:2.5%) favoring the formation of the compound of Formula (II).

[0045] Following hydrogenation, the compound of Formula (II) may optionally be isolated by

[0046] IPTS / 200134844.1 Docket No. GIU-074WO contacting the solution with an acid, for example, acetic acid or hydrochloric acid, or a mixture thereof.

[0047] In some embodiments, the process further comprises resolving the mixture of a compound of Formula (II) and a compound of Formula (IV).

[0048] In some embodiments, the hydrogenation reaction is in the presence of a solvent. In some embodiments, the solvent is an alcohol solvent. Contemplated alcohol solvents may include at least one of methanol, ethanol, isopropanol, and butanol. In some embodiments, the solvent is methanol.

[0049] In some embodiments, the hydrogenation reaction takes place at a lower temperature and with a lower pressure of hydrogen gas for a first period of time and then at a higher temperature and with a higher pressure of hydrogen gas for a second period of time.

[0050] In some embodiments, the lower temperature is from about 20 °C to about 35 °C, for example, about 20 °C, about 25 °C, about 30 °C, or about 35 °C. In some embodiments, the higher temperature is from about 50 °C to 70 °C , for example, about 50 °C, about 55 °C, about 60 °C, about 65 °C, or about 70 °C.

[0051] In some embodiments, the lower pressure is from about 10 atm to about 20 atm, for example, about 10 atm, about 15 atm, or about 20 atm. In some embodiments, the higher pressure is from about 25 atm to about 35 atm, for example, about 25 atm, about 30 atm, or about 35 atm.

[0052] In some embodiments, the first period of time is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. In some embodiments, the first period of time is from about 1 hour to about 8 hours, from about 1 hour to 7 hours, from about 1 hour to 6 hours, from about 1 hour to 5 hours, or from about 1 hour to 4 hours. In some embodiments, the second period of time is at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours or at least 24 hours. In some embodiments, the second period of time is from about 5 hours to 24 hours, from about 5 hours to 18 hours, or from about 5 hours to 12 hours.

[0053] IPTS / 200134844.1 Docket No. GIU-074WO

[0054] In some embodiments, the structure of the Rh-Walphos catalyst is:

[0055] In some embodiments, the amount of the Rh-Walphos catalysts is at least 1 % molar, at least 2% molar, at least 3% molar, at least 4% molar, at least 5% molar, at least 6% molar, at least 7% molar, at least 8% molar, at least 9% molar, at least 10% molar, at least 15% molar, at least 20% molar, from about 1% molar to about 20% molar, from about 1% molar to about 15% molar, from about 1% molar to about 10% molar, from about 1% molar to about 5% molar, or from about 5% molar to about 10% molar, each based on the substrate to be hydrogenated.

[0056] In some embodiments, the Rh-Walphos catalyst is generated in situ from a Rh agent and a Walphos agent in about a 1 to 1 ratio.

[0057] Resolving the mixture of a compound of Formula (II) and a compound of Formula (IV) to form a substantially optically pure compound of Formula (II) may include: a) resolving the mixture of a compound of Formula (II) and a compound of Formula (IV) in the presence of a chiral acid thereby forming a chiral salt of the compound of Formula (II); and b) neutralizing the chiral salt of the compound of Formula (II) thereby forming the compound of Formula (II).

[0058] In some embodiments, the chiral acid is selected from the group consisting of (5)-(+)- camphor-10-sulfonic acid, (2 / ,3 / )-(+)-tartaric acid, fS')-(-)-malic acid, (15)-(+)-3- bromocamphor-10-sulfonic acid, (5)- 1 -phenyl ethane sulphonic acid, dibenzoyl-L-tartaric acid, glutamic acid, (77?,35)-camphoric acid, (75)-camphanic acid and (7?)-(-)-mandelic acid and all other chiral acids that can lead to resolution of racemic mixture or an enantiomer thereof, e.g., (S)-(+)-camphor-10-sulfonic acid.

[0059] IPTS / 200134844.1 Docket No. GIU-074WO

[0060] In some embodiments, the chiral salt of the compound of Formula (II) is:

[0061] •(S)-(+)-camphor-10-sulfonic acid

[0062] In some embodiments, resolving may further comprise the initial maintenance of a temperature between 55 °C - 60 °C (e.g., 58 °C) while stirring, and / or may occur in the presence of acetone and water, followed by cooling the temperature to 37 °C - 42 °C.

[0063] At the end of resolving, if specifications are not met, reprocessing can occur where the solution maintained at the resolving temperature while stirring for a longer time.

[0064] Neutralizing may include contacting the chiral salt of the compound of Formula (II), with (i) an aqueous base (e.g., ammonium hydroxide); and then (ii) acidifying the solution by adding an acid (e.g., acetic acid). Neutralizing may occur in the presence of one or more solvents, e.g., in the presence of water and ethyl acetate.

[0065] Also provided herein is a process of for preparing a substantially optically pure compound of Formula (I), salt thereof, the process comprising the process as disclosed herein, wherein the process further comprises acylating the compound of Formula (II), or a salt thereof, thereby forming the compound of Formula (I) or a salt thereof.

[0066] Acylating may include contacting the compound of Formula (II) with an acylating agent (e.g., acetic anhydride) in the presence of an organic solvent selected from the group consisting of ethyl acetate, tetrahydrofuran, diethyl ether, dichloromethane, and toluene, e.g., ethyl acetate. Such acylating may occur at a temperature between 60 °C to 70 °C, for example, between 65 °C IPTS / 200134844.1 Docket No. GIU-074WO to 70 °C. Acylating may further comprise dissolving the compound isolated from the previous step in one or more solvents, e.g., water and / or ethyl acetate, to prepare a solution and contacting the solution with an acylating agent, e.g., acetic acid. Such step may occur at a temperature between 60 °C to 70 °C, for example, at a temperature between 65 °C to 70 °C.

[0067] In some embodiments, after optionally resolving the mixture of a compound of Formula (II) and a compound of Formula (IV) to form a substantially optically pure compound of Formula (II), a mother liquor derived from the resolution step may still contain the desired enantiomer (as a salt of the resolving agent) together with the undesired one. In these embodiments, resolving may optionally further comprise: a) recovering the mixture of enantiomers (as chiral salts of the resolving agent) from the mother liquor, neutralizing the chiral salts to form a mixture of a compound of Formula (II) and a compound of Formula (IV), and resolving the mixture providing additional substantially optically pure compound of Formula (II), thereby increasing the total process yield; or b) distilling part of the mother liquor and precipitating the desired enantiomer as a salt of the chiral resolving agent from the mother liquor, thereby increasing the total process yield.

[0068] Also contemplated herein is a process of racemizing the undesired enantiomer or chiral salt thereof in the presence of a base and resolving the resulting mixture of (R) and (S) enantiomers using a resolution process contemplated herein to provide the desired enantiomer.

[0069] Contemplated bases include those selected from the group consisting of hydroxides, alkoxides (e.g., methoxide), amides (e.g., lithium diisopropylamide), hydrides (e.g., NaH), organolithiums, and Grignard reagents. For bases that require a counterion, exemplary counterions contemplated herein may include alkali metals or alkaline earth metals, e.g., lithium, sodium, potassium, or calcium; or organic counterions, e.g., tetraalkyl ammoniums.

[0070] In some embodiments, the compound of Formula (I) can be produced on a multi -kilogram scale, for example, at least about 8 to 11 kg, about 13 to 15 kg, or about 130 to 150 kg is obtained. In some embodiments, at least about 130 kg of the compound of Formula (I) is IPTS / 200134844.1 Docket No. GIU-074WO obtained.

[0071] In some embodiments, the substantially optically pure compound of Formula (I) is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the desired enantiomer:

[0072] (expressed as a percentage of both enantiomers). In some embodiments, the content of (5)-(-)-3- (4-aminophenyl)-2-methoxypropionic acid may not, for example, be more than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, or 0.5% by HPLC.

[0073] In some embodiments, the salt is a pharmaceutically acceptable salt.

[0074] The disclosure provides, at least in part, compounds represented by Formula (I), Formula (II), Formula (III), and Formula (IV), as depicted above. Also contemplated herein are pharmaceutical compositions that include a compound represented by Formula (I) and a pharmaceutically acceptable excipient and / or carrier.

[0075] The contemplated pharmaceutically acceptable excipients include buffers, carriers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration, e.g., suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The use of such media and agents for pharmaceutically active substances is known in the art. The excipient(s) should be “acceptable” in the sense of being compatible with the other ingredients of the compositions and not deleterious to the recipient.

[0076] The pharmaceutical compositions of the present disclosure may be administered topically. In various embodiments, the topical compositions are in the form of a gel. Suitable carriers / excipients that may be used in the topical compositions discussed herein are known in the art and include, but are not limited to, solubilizers such as C2 to Cs straight and branched

[0077] IPTS / 200134844.1 Docket No. GIU-074WO chain alcohols, diols and triols, moisturizers and humectants such as glycerin, amino acids and amino acid derivatives, polyamino acids and derivatives, pyrrolidone carboxylic acids and its salts and derivatives, surfactants such as sodium laureth sulfate, sorbitan monolaurate, emulsifiers such as cetyl alcohol, stearyl alcohol, thickeners such as methyl cellulose, ethyl cellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyvinyl alcohol and acrylic polymers.

[0078] The pharmaceutical compositions described herein can be presented in a dosage unit form and can be prepared by any suitable method, for example, by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0079] Daily topical administration of the compounds or compositions described herein may be once per day in the morning, and / or once per day in the evening during a treatment period which may be between one week, two weeks, one month, two months, or three months and one year may be effective to prevent and / or ameliorate acne or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation. The topical composition can be administered in an amount of, for example, between about 1.0 mL / 5 cm2and 1.0 mL / 50 cm2, or between about 1.0 mL / 5 cm2and 50 mL / 50 cm2, or between about 1.0 mL / 5 cm2and 100 mL / 50 2 cm .

[0080] Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also contains an alcohol and, optionally, an oil. In embodiments, “organic macromolecules,” i.e., gelling agents, are crosslinked acrylic acid polymers such as the “carbomer” family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the CARBOPOL™ trademark. Hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin may also be included. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be

[0081] IPTS / 200134844.1 Docket No. GIU-074WO dispersed by trituration, mechanical mixing, or stirring, or combinations thereof.

[0082] The pharmaceutical compositions may also be prepared with liposomes, micelles, and microspheres. Liposomes are microscopic vesicles having a lipid wall including a lipid bilayer, and can be used as drug delivery systems herein as well. Generally, liposome compositions are poorly soluble or insoluble pharmaceutical agents. Liposomal preparations for use in the instant disclosure may include cationic (positively charged), anionic (negatively charged), and neutral preparations. Cationic liposomes are readily available. For example, N[l-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trade name LIPOFECTIN™ (ThermoFisher). Similarly, anionic and neutral liposomes are readily available as well, e.g., from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE), among others. These materials can also be mixed with DOTMA in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0083] Micelles are known in the art as included of surfactant molecules arranged so that their polar head groups form an outer spherical shell, while their hydrophobic, hydrocarbon chains are oriented towards the center of the sphere, forming a core. Micelles form in an aqueous solution containing surfactant at a high enough concentration so that micelles naturally result. Surfactants useful for forming micelles include, but are not limited to, potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, docusate sodium, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecylammonium chloride, polyoxyl 8 dodecyl ether, polyoxyl 12 dodecyl ether, nonoxynol 10, and nonoxynol 30. Micelle compositions can be used in conjunction with the present disclosure either by incorporation into the reservoir of a topical or transdermal delivery system, or into a composition to be applied to the body surface.

[0084] Microspheres, similarly, may be incorporated into the present pharmaceutical compositions and drug delivery systems. Like liposomes and micelles, microspheres essentially IPTS / 200134844.1 Docket No. GIU-074WO encapsulate a drug or drug-containing composition. Microspheres are generally, although not necessarily, formed from synthetic or naturally occurring biocompatible polymers, but may also be included of charged lipids such as phospholipids. Preparation of microspheres is well known in the art and described in the pertinent texts and literature.

[0085] Various additives, known to those skilled in the art, may be included in the topical compositions. For example, solvents, including relatively small amounts of alcohol, may be used to solubilize certain composition components. In embodiment, the composition includes a suitable enhancer, e.g., but are not limited to, ethers such as di ethylene glycol monoethyl ether (available commercially as TRANSCUTOL™) and di ethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80), and lecithin (U.S. Patent No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzyl alcohol, and the like; polyethylene glycol and esters thereof such as polyethylene glycol monolaurate (PEGML); amides and other nitrogenous compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, l-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, particularly citric acid and succinic acid. AZONE™ and sulfoxides such as DMSO and Cio MSO may also be used.

[0086] The present pharmaceutical compositions may also include conventional additives such as opacifiers, antioxidants, fragrance, colorant, gelling agents, thickening agents, stabilizers, surfactants, and the like. Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, ie., to inhibit growth of microbes such as yeasts and molds. Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of / 2-hydroxybenzoic acid (ie., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof.

[0087] The present pharmaceutical compositions may also contain irritation-mitigating additives to minimize or eliminate the possibility of skin irritation or skin damage resulting from the pharmacologically active base or other components of the composition. Suitable irritationmitigating additives include, for example: a-tocopherol; monoamine oxidase inhibitors, IPTS / 200134844.1 Docket No. GIU-074WO particularly phenyl alcohols such as 2-phenyl-l -ethanol; glycerin; salicylic acids and salicylates; ascorbic acids and ascorbates; ionophores such as monensin; amphiphilic amines; ammonium chloride; N-acetylcysteine; cis-urocanic acid; capsaicin; and chloroquine. The irritant-mitigating additive, if present, may be incorporated into the present at a concentration effective to mitigate irritation or skin damage, typically representing not more than about 20 wt. %, more typically not more than about 5 wt. %, of the composition.

[0088] Therapeutic Applications

[0089] Also provided herein are methods of using the compounds or pharmaceutical compositions described herein. In some embodiments, provided herein are methods for preventing or treating a skin disease or disorder, e.g., acne, by acting on the main pathogenetic factors of acne, for example, reduced sebum secretion (both in terms of quantity and quality of sebum), inhibition of inflammation (in both sebocytes and keratinocytes), and inhibition of hyperkeratinization (e.g., keratinocytes altered proliferation and differentiation induced by inflammatory cytokines). The methods can also include preventing a disease or a disorder characterized by alteration of sebocyte differentiation by administering the compositions disclosed herein, for example, topically to the skin (e.g., the epidermis) or other body surface, for example to a specific region or regions of the skin (e.g., the back, the face, the cheeks, the forehead, the chin, the nose, the head, the neck, the shoulders, the arms, the legs, and / or the chest) via topical administration.

[0090] In some embodiments, methods of treating or preventing acne or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation include administering the composition disclosed herein to a patient in need thereof in combination with a therapy for treating or preventing acne or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation. Therapies for treating or preventing acne or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation include, but are not limited to, light-based therapies, laser therapies, photodynamic therapy, chemical peels (e.g., chemical peels that include one or more applications of a chemical solution, such as salicylic acid, glycolic acid, or retinoic acid), direct extraction of whiteheads and blackheads, and steroid injection.

[0091] In some embodiments, the contemplated disease or the disorder to be treated with the compound or composition described herein is selected from non-inflammatory acne, IPTS / 200134844.1 Docket No. GIU-074WO inflammatory acne, acne vulgaris, acne fulminans, acne mechanica, acne conglobata, gramnegative folliculitis, pyoderma faciale, sebaceous hyperplasia, sebaceous adenitis, comedones (including whiteheads, blackheads, papules), pustules, nodules, cysts, cystic lesions, mild acne, moderate acne, severe nodulocystic acne. In some embodiments, the amount of the compound or composition administered is an amount that is effective to induce sebocyte differentiation and / or an amount that is effective to reduce insulin-induced LOX activity and / or inflammation.

[0092] The disclosed compositions may be administered to subjects (e.g., animals and / or humans) in need of prevention or treatment in dosages that will provide optimal pharmaceutical efficacy.

[0093] Generally, a therapeutically effective amount of active component will be in the range of from about 0.1 mg / kg to about 100 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 1000 mg / kg, from about 1 mg / kg to about 1000 mg / kg, from about 1 mg / kg to about 100 mg / kg, from about 1 mg / kg to 10 mg / kg, from about 10 mg / kg to about 20 mg / kg, from about 20 mg / kg to about 30 mg / kg, from about 30 mg / kg to about 40 mg / kg, from about 40 mg / kg to about 50 mg / kg, from about 50 mg / kg to about 60 mg / kg, from about 60 mg / kg to about 70 mg / kg, from about 70 mg / kg to about 80 mg / kg, from about 80 mg / kg to about 90 mg / kg, from about 90 mg / kg to about 100 mg / kg, from about 100 mg / kg to about 500 mg / kg, from about 500 mg / kg to about 1000 mg / kg, or from about 1000 mg / kg to about 2000 mg / kg. A therapeutically effective amount of active component can be in the range of about 0.5 mg / kg to about 50 mg / kg or about 0.5 mg / kg to about 25 mg / kg, or about 0.5 mg / kg to about 20 mg / kg, or about 0.5 mg / kg to about 15 mg / kg, or about 0.5 mg / kg to about 10 mg / kg.

[0094] For example, a therapeutically effective amount of active component can be about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, or about 1000 mg / kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health status of the particular patient, the relative biological efficacy of the compounds, compositions of compounds, the presence and types of excipients in the composition, and the route of administration.

[0095] IPTS / 200134844.1 Docket No. GIU-074WO

[0096] The initial dosage administered may be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage may be smaller than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg / kg to 20 mg / kg.

[0097] Dosing frequency can vary, depending on factors such as route of administration, dosage amount, and the disease condition being treated. Exemplary dosing frequencies are once per day, twice per day, three times per day, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, once every other day, once every three days, once every four days, once every five days, once every six days, once every eight days, once every nine days, once every ten days, once every eleven days, once every twelve days, once every thirteen days, and once every two weeks.

[0098] The method of delivery of the compounds or pharmaceutical compositions disclosed herein, may vary, but may involve application of a composition of the disclosure to an area of body surface affected with or at risk of being affected with acne or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation.

[0099] The dose regimen of the compositions described herein will depend on a number of factors that may readily be determined, such as severity or likely severity of the acne, or the disease, the condition, or the disorder characterized by alteration of sebocyte differentiation being treated, prevented, and / or ameliorated, and the responsiveness or likely responsiveness of the condition being treated, prevented, and / or ameliorated, but will normally be one or more doses per day, with a course of administration lasting from several days to several months. In general, the composition can be applied one to four times daily.

[0100] Sebocyte biology

[0101] The sebaceous gland and hair follicles together form the pilosebaceous unit of the skin. Sebocytes are the main cellular units of the sebaceous gland, which produces sebum - a mixture of cell debris, lipids, antimicrobial substances, free fatty acids, and matrix metalloproteinases. Sebum is a composed mainly of lipids, including: triglycerides, free fatty acids, wax esters, squalene, cholesterol esters, and cholesterol. Sebum functions to prevent excess water from entering the skin, while trapping and preventing necessary moisture from getting out.

[0102] IPTS / 200134844.1 Docket No. GIU-074WO

[0103] Additionally, sebum has antimicrobial and antioxidant delivery properties. While too little sebum can result in dry skin, excessive amounts of sebum results in clogging of the sebaceous gland and bacterial growth. The body reacts to bacterial growth caused by sebum blockage by mobilizing white blood cells. White blood cell mobilization in turn causes skin inflammation. Sebum synthesis is strongly regulated by hormone levels, in particular by androgens, which stimulate lipid production in sebocytes and sebocyte differentiation.

[0104] Acne pathology

[0105] The compositions and methods described herein are useful for preventing or treating a skin disease or disorder, e.g., acne, or a disease, a condition, or a disorder characterized by alteration of sebocyte differentiation in a patient. Acne and related conditions include, but are not limited to, non-inflammatory acne, inflammatory acne, acne vulgaris, acne fulminans, acne mechanica, acne conglobata, gram-negative folliculitis, pyoderma faciale, sebaceous hyperplasia, sebaceous adenitis, comedones (including whiteheads, blackheads, papules), pustules, nodules, cysts, cystic lesions, mild acne, moderate acne, and severe nodulocystic acne.

[0106] In general, acne occurs when hair follicles become clogged with dead skin cells and sebum, and / or as a result of excessive growth of the skin bacterium Propionib acterium acnes. Causes of acne include genetic predisposition, hormonal activity associated with puberty and female menstrual cycles (e.g., increased levels of androgens, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), growth hormone (GH), and insulin-like growth factor 1 (IGF-1)), bacterial overgrowth or infection, diet, stress, and environmental factors. Characteristics of acne include blackheads or whiteheads, pimples, oily skin, and scarring. Acne can present on areas of the skin, for example, the face, arms, shoulders, chest, head, and back.

[0107] Major pathogenic factors associated with acne are hyperkeratinization, obstruction of sebaceous follicles resulting from abnormal keratinization of the infundibular epithelium, stimulation of sebaceous gland secretion by androgens, and microbial colonization of pilosebaceous units by Propionibacterium acnes. In general, acne develops when a mixture of sebum and desquamated cells obstructs sebaceous follicles. The accumulation of sebum and cells distends the sebaceous follicle, forming a microcomedo, which expands to form an open or closed comedo. Without being bound by theory, P. acnes is believed to colonize and proliferate in the comedo, releasing inflammatory cytokines and chemotactic factors. This release of

[0108] IPTS / 200134844.1 Docket No. GIU-074WO inflammatory cytokines and chemotactic factors weakens the follicular wall, causing it to rupture, and allowing the keratin-sebum mixture to leak into the dermis. This series of events results in the inflammatory lesions (e.g., papules, pustules, nodules, and cysts) associated with acne.

[0109] The body’s inflammatory response is believed to play an important role in the pathogenesis of acne. For instance, early stage acne lesions are associated with lymphoid perivascular infiltrate, and accumulation of polymorphonuclear leukocytes continues in later stages of acne, causing distension, pustule formation, and, ultimately, lesion rupture. Additionally, P. acnes is believed to activate TLR2 in monocytes, resulting in proinflammatory cytokine release (including IL- 12 and IL-8) and expression of antimicrobial peptides (including defensin-1, defensin-2, and defensin-3). Protease release by P. acnes also activates protease- activated receptor-2 (PAR-2) on keratinocytes, which enhances transcription of proinflammatory cytokines IL-la, IL-8, and tumor necrosis factor-a (TNF-a), matrix metalloproteinases, and the cathelici din LL-37.

[0110] Diet may play an important role in acne development as well. For instance, hyperglycemic carbohydrates, milk and dairy products, and saturated fats are increased in the Western diet and contribute to acne development. Insulin / insulin-like growth factor (IGF-1) signaling, along with branched-chain amino acids (BCAAs), glutamine, and palmitate, all of which are also generally increased in individuals who partake in a Western diet and increase mammalian target of rapamycin complex 1 (mTORCl) signaling. Furthermore, increased mTORCl activity in keratinocytes stimulates keratinocyte proliferation, pro-inflammatory NFKB signaling, increased transcription of pro-inflammatory cytokines (e.g., TNFa, IL-6, IL-8 IL- 17, IL-20, IL-22 and IL-23), lipid biosynthesis, comedone development, and acne. Increased mTORCl activity also promotes T cell activation and generation of CD4+and CD8+effector T cells. IGF-1 signaling also stimulates sterol response element binding protein-1 (SREBP-1) expression and lipogenesis in sebocytes via activation of the PI3K / AKT pathway, and IGF-1 suppresses nuclear FoxOl in sebocytes, which also contributes to increased lipogenesis.

[0111] Acne evaluation

[0112] Severity of acne and related disorders can be evaluated using different methods, including counting lesions and grading based on different observational criteria. Multiple

[0113] IPTS / 200134844.1 Docket No. GIU-074WO grading systems exist, including the global acne grading system (GAGS), in which the face, chest, and back are divided into six areas and each area is assigned a factor based on its size. (See Doshi et al., (1997) Int J Dermatol 36:416-8.) Each type of lesion is given a value depending (e.g., no lesions = 0, comedones = 1, papules = 2, pustules = 3 and nodules = 4), and the score for each area is calculated by multiplying the factor for each area by a severity grade of 0-4. The global score is the sum of each area’s score. A score of 1-18 is considered mild, a score of 19-30 is considered moderate, a score of 31-38 is considered severe, and a score of greater than 39 is considered very severe. In the Investigator’s Global Assessment (IGA) of acne severity grading system, a patient is assigned a score of 0-4 based on established criteria. In particular, a score of 0 (clear) indicates residual hyperpigmentation and potentially the presence of erythema; a score of 1 (almost clear) indicates a few scattered comedones and a few small papules; a score of 2 (mild) indicates that less than half the face is involved but that some comedones, papules, and pustules are observed; a score of 3 (moderate) indicates that more than half the face is involved, many comedones, papules, and pustules are present, and one nodule may be present; and a score of 4 (severe) indicates that the subject’s entire face is involved, and is covered with comedones, numerous papules and pustules, and a few nodules and cysts.

[0114] Methods of counting lesions generally require recording the number of each type of acne lesion and determining the overall severity of the lesions. Counting lesions or evaluating acne severity using, for example, the GAGS or IGA scoring systems, can be performed in connection with the methods described herein. A patient or patients without acne can be characterized as having a score of zero with either the GAGS or IGA scoring systems.

[0115] Procedures for making compounds described herein are provided below with reference to Schemes 1-6. In the reactions described below, it may be necessary to protect reactive functional groups (such as hydroxyl, amino, or carboxyl groups) to avoid their unwanted participation in the reactions. The incorporation of such groups, and the methods required to introduce and remove them are known to those skilled in the art (for example, see Greene, Wuts, Protective Groups in Organic Synthesis. 4th Ed. (2007)). The deprotection step may be the final step in the synthesis such that the removal of protecting groups affords compounds of Formula I, as disclosed herein. Starting materials used in the following schemes can be purchased or prepared by methods described in the chemical literature, or by adaptations thereof, using methods known by those

[0116] IPTS / 200134844.1 Docket No. GIU-074WO skilled in the art. The order in which the steps are performed can vary depending on the groups introduced and the reagents used, but would be apparent to those skilled in the art.

[0117] EXAMPLES

[0118] The procedures disclosed herein can be conducted in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials.

[0119] At least some of the compounds identified as intermediates e.g., as part of a synthetic scheme or figure disclosed herein are contemplated as compounds of the disclosure.

[0120] Abbreviations:

[0121] General:

[0122] HPLC high-performance liquid chromatography

[0123] LC liquid chromatography

[0124] MHz megahertz

[0125] MS mass spectrometry

[0126] NMR nuclear magnetic resonance

[0127] Me methyl

[0128] Ph phenyl

[0129] Et ethyl

[0130] SM starting material

[0131] Sat. saturated

[0132] Anh. anhydrous

[0133] IPTS / 200134844.1 Docket No. GIU-074WO

[0134] Solvents, Reagents and Conditions

[0135] AcOH acetic acid

[0136] CSA camphorsulfonic acid

[0137] EtOAc ethyl acetate

[0138] NaOMe sodium methoxide

[0139] RT room temperature

[0140] TBME methyl tert-butyl ether

[0141] MIK methyl isobutyl ketone

[0142] General experimental:

[0143] 'll NMR

[0144] 1H NMR spectra were recorded using a Varian Mercury 400 MHz NMR-spectrometer. Chemical shifts for protons were reported as parts per million in 5 scale using solvent residual peak (DMSO-de: 2.50 ppm or CDCh: 7.26 ppm or CD3OD: 4.78 and 3.31 ppm) as an internal standard. Data are represented as follows: chemical shift (5), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qn = quintuplet, sx = sextet, sp = septuplet, m = multiplet, br = broad, dd = doublet of doublets, dt = doublet of triplets, qd = quartet of doublets, dquin = doublet of quintets), coupling constant (J, Hz) and integration (# H).

[0145] 13C NMR

[0146] 13C NMR were recorded by dissolving the sample in DMSO-de operating at 100 MHz. Fully decoupled spectra were acquired.

[0147] HPLC / MS (Method A)

[0148] Equipment: Agilent Technologies 1200 HPLC system equipped with a VWD and coupled to a 6110 Single Quadrupole.

[0149] Polarity (MS): Positive

[0150] Capillary: 4000V (+)

[0151] Drying Gas: 13 L / min

[0152] Nebulizer Pressure: 55 psi

[0153] Gas Temperature: 350°C

[0154] IPTS / 200134844.1 Docket No. GIU-074WO

[0155] Fragmentor: 100 V

[0156] Scan range: 50-1000 m / z

[0157] Column: Kinetex EVO 50 x 4,6 mm, 2.6 mm.

[0158] Gradient: NH4HCO3 pH=8.0:ACN (95:5)— 0.5 min— (95:5)— 6.5 min— (0: 100)—

[0159] 2 min— (0: 100); post run 1.5min

[0160] Flow: 1.5 mL / min

[0161] Wavelength: 210 nm

[0162] Temperature: 40°C

[0163] Injection volume: 1 mL

[0164] Sample preparation: 1 mg / mL in MeOH

[0165] HPLC (Method B) (chemical purity)

[0166] Column30: Acquity BEH C18 150x2.1 mm (1.7 mm)

[0167] Gradient: 0.1% HCIO4 in water: MeOH (99: 1) — 10 min — (1 :99) — 5 min — (1 :99)

[0168] Flow: 0.25 mL / min

[0169] Wavelength: 220 nm

[0170] Temperature: 30°C

[0171] Injection volume: 5 pL

[0172] Sample preparation: 1 mg / mL in water

[0173] HPLC Method C (chiral purity of Compound (I) and 5 -nitro Compound (III, when the Carbon-Carbon double bond is hydrogenated and before the reduction of Nitro group)

[0174] Equipment: Agilent Technologies 1260 Infinity II HPLC system equipped with a DAD.

[0175] Column: Chiralpak QN-AX 150x4.6 mm

[0176] Mobile phase: HCOONH4 (10 mM in MeOH) with 0.2% HCOOH

[0177] Gradient: isocratic

[0178] Flow: 1.0 mL / min

[0179] Wavelength: 245 nm

[0180] Temperature: 25°C

[0181] IPTS / 200134844.1 Docket No. GIU-074WO

[0182] Injection volume: 2 pL

[0183] Sample preparation: 1 mg / mL in methanol

[0184] HPLC Method D (chiral purity of compound (II))

[0185] Equipment: Agilent Technologies 1100 HPLC system equipped with a VWD.

[0186] Column: Chiralpak IC 250x4.6 mm (5 mM)

[0187] Mobile phase:

[0188] A: n-heptane

[0189] B : EtOH with 0.2% DEA

[0190] Gradient: isocratic (75% A)

[0191] Flow: 0.5 mL / min

[0192] Wavelength: 258 nm

[0193] Temperature: 25°C

[0194] Injection volume: 20 pL

[0195] Sample preparation: 1 mg / mL in methanol

[0196] IR

[0197] Equipment: Agilent Cary 630 FTIR.

[0198] Spectral width: 650-4000 cm-1.

[0199] Scans: 32

[0200] Resolution: 4

[0201] The chiral purity % is calculated from the following expression: (Ai x 100) / (Ai + Ad), where Ai = (5)-(-)-3-(4-Acetamidophenyl)-2-methoxypropionic acid peak area in the sample solution (levorotatory enantiomer) and Ad = ( ’)-(+)-3-(4-Acetamidophenyl)-2-methoxypropionic acid peak area in the sample solution (dextrorotatory enantiomer). The above methods may be used to determine optical purity or enantiomeric purity of a compound as referenced herein.

[0202] Example 1: Preparation of Compound of Formula (I)

[0203] Previously, the compound of formula (I) was prepared according to FIG. 5 A. In the

[0204] IPTS / 200134844.1 Docket No. GIU-074WO present application, the compound of formula (I) is prepared according to FIG. 5B, where an asymmetric catalyst is used in the hydrogenation step to produce the desired enantiomer stereoselectively.

[0205] As shown in FIG. 5 A, the hydrogenation reaction without an asymmetric catalyst yields a racemic mixture of the product. However, as detailed in the following Examples, it was discovered that the hydrogenation reaction can produce the desired enantiomer with a high selectivity (e.g., S:R =95:5) by using Rh-Walphos catalyst and optimized conditions.

[0206] Products used for the preparation of the ligand Rh-Walphos: a) Bis(norbornadiene)rhodium(I) tetrafluorob orate, CAS: [36620-11-8], b) (15)-l-[(lA)-l-[Bis[3,5-bis(trifluoromethyl)phenyl]phosphino]ethyl]-2-[2- (diphenylphosphino)phenyl]ferrocene (Walphos), CAS: [565184-33-0],

[0207] Rh-Walphos is obtained by mixing a) and b) in a ratio of 1 : 1.

[0208] Example 2: Catalyst Screen

[0209] To find optimal conditions for the asymmetric hydrogenation step, different catalysts containing Rh (FIG. 1) were tested in the hydrogenation reaction as shown in Scheme 1.

[0210] Scheme 1. Hydrogenation reaction

[0211] Only Rh-3 was commercially available as the catalyst formed. Rh-1, Rh-4 and Rh-5

[0212] IPTS / 200134844.1 Docket No. GIU-074WO were prepared in situ by stirring of the ligand and the metal complex.

[0213] [Rh(NBD)2][BF4], Walphos, and a solvent were loaded to a first suitable test tube under Ar and were stirred using a magnetic stir bar. The resulting solution was stirred at RT for 1 hour. The starting material Formula (III), a base, and a solvent were loaded to a second suitable test tube under Ar and were stirred using a magnetic stir bar and a suspension was obtained.

[0214] The solution of the catalyst in the first test tube was added to the suspension of the starting material Formula (III) in the second test tube and the second test tube was placed inside a suitable high pressure reactor which is purged with consecutive cycles of vacuum / N? and vacuum / H?. The reactor was then subjected to H2 at the selected pressure and the hydrogenation reaction occurred.

[0215] The results are presented in Tables 1 to 3. Under the reaction conditions adapted from the literature (entry 1), which involved the use of mild conditions of pressure (4 atm) and temperature (RT), no reaction took place. At high pressure (25 atm) and temperature (50°C), total conversion to aniline (Formula (II)+(IV)) was observed with e.e.=80% (entry 2). The reaction was repeated under the same reaction conditions and the results were comparable (entry 3). With the same solvent (CF3CH2OH) at lower pressure (15 atm) and 50°C, total conversion to 5-nitro was achieved with e.e.=65% (entry 4). In MeOH at 15 atm and 50°C, total conversion to 5-nitro with e.e=86- 87% was obtained (entry 5). The outcome of the reaction was similar using either NaOMe (entries 1 to 5) or EtsN (entries 6-7) as base.

[0216] Table 1 : Results of the asymmetric hydrogenation with Walphos-Rh (Rh-1).

[0217] IPTS / 200134844.1 Docket No. GIU-074WO dOrg. Lett. 2005, 7, 1947. >28 V.

[0218] With catalyst Rh-3, low e.e. (as 5-nitro compound) were obtained (maximum of 36%). At high pressure (30 atm) the main product obtained was 5-nitro (entry 6) with low e.e. (34%). At lower pressure (15 atm) partial conversion to 5-nitro (entries 7-10) was observed.

[0219] Table 2. Results of the asymmetric hydrogenation with (S,S)-Et-Duphos (Rh-3) dJACS 1998, 120, 4345,b20 V.

[0220] Under the best reaction conditions obtained for Walphos-Rh (Rh-1) (P=30 atm, T=50 °C, see entry 2, Table 3), both Josiphos catalysts were tested but the results were not better than Walphos-Rh. These catalysts were not further explored.

[0221] IPTS / 200134844.1 Docket No. GIU-074WO

[0222] Table 3 : Results of the asymmetric hydrogenation with Josiphos 1 (Rh-4) and Josiphos

[0223] 2 (Rh-5)

[0224] Cobalt-containing catalyst as shown in FIG. 2 was tested. This catalyst was prepared in situ as indicated in literature (JACS 2020, 142, 11, 5272). The results are shown in Table 4.

[0225] Table 4: Results of the asymmetric hydrogenation with (R,R)-PhBPE-CoCh 6H2O catalyst. aJACS 2020, 142, 77, 5272.

[0226] With this catalyst, no reaction was observed even at high pressure and temperature. This catalyst was not further studied.

[0227] Different catalysts containing Ru were also tested (FIG. 3). Ru-1, Ru-2, and Ru-3 were prepared in situ by stirring of the ligand and the complex metal. The results are shown in Table 5. With Ru-1 no conversion was observed (entries 1 to 4). With Ru-2 and 10 atm in MeOH, incomplete reaction to 5-nitro with e.e. =67% (entry 5) was observed. Under the same reaction conditions but in DCM, a lower e.e. was obtained (entry 6). With Ru-2 and high pressure (30 atm), total conversion to 5-nitro was achieved with e.e. -70% in both solvents tested (MeOH

[0228] IPTS / 200134844.1 Docket No. GIU-074WO and DCM) (entries 7-8). Different ratios of ligand to Ru were also tested with Ru-3 (entries 9-12).

[0229] Table 5 : Results of the asymmetric hydrogenation with different Ru catalyst. aOrg. Lett 2004, 6(18), 3147-3150.b40 V,c9 V.

[0230] Example 3: Optimization of the asymmetric hydrogenation

[0231] Based on the above results, Walphos-Rh (Rh-1) was selected as the asymmetric catalyst to be used in the hydrogenation step of the synthesis. Various conditions were tested to optimize the selectivity of this reaction.

[0232] IPTS / 200134844.1 Docket No. GIU-074WO

[0233] Table 6: Results of the initial optimization of the asymmetric hydrogenation with Walphos-Rh

[0234] (Rh-1) catalyst

[0235] IPTS / 200134844.1 Docket No. GIU-074WO a(S) enantiomer, e.e determined of the major compound Experiments where P, T and / or time were modified during the progress of the reaction.

[0236] Several reaction parameters were studied: the use of MeOH and CF3CH2OH, the % molar of catalyst, the stoichiometry between the ligand and the metal to form the catalyst and the temperature and pressure.

[0237] The results of the experiments showed that enatioselectivity was slightly higher in MeOH than in CF3CH2OH. If the amount of the catalyst was less than 5% molar, the e.e. became lower. The optimal stoichiometry between the ligand and the metal to form the catalyst was about a 1 to 1 ratio.

[0238] When the reaction was performed at harsh conditions of pressure and temperature (25 atm, 50 °C) from the beginning with the aim to transform the starting material Formula (III) directly into the aniline Formula (II) (carboxylate form), the results were not reproducible (entries 13- 17). To overcome this issue, the reaction was performed stepwise. In the initial step, the starting material Formula (III) was converted to the 5-nitro compound, ensuring optimal enantioselectivity. In the second step, 5-nitro compound was converted to aniline Formula (II) and Formula (IV) while maintaining the e.e. value. Experiments were carried out by controlling carefully the pressure and the temperature in each stage of the reaction (entry 22): the first step (transformation of Formula (III) into 5-nitro) at lower pressure and temperature (15 atm, RT to 30 °C, 4.5 h) and the second step (transformation of 5-nitro into aniline Formula (II) and Formula (IV)) at higher pressure and temperature (30 atm, 60 °C) (the reaction was left overnight). Under these reaction conditions, complete conversion to aniline Formula (II) and Formula (IV) was achieved with high e.e. = 90%.

[0239] IPTS / 200134844.1 Docket No. GIU-074WO

[0240] These results confirmed that reduction of the nitro group before the double bond is detrimental for the catalytic system and decreases the final enantiomeric excess. Therefore, careful control of reaction conditions at each step of the hydrogenation reaction is required.

[0241] The same reaction was repeated multiple times to confirm that the results were reproducible. The reaction was initially conducted at 15 atm and 30 °C for 4.5 h and then at 30 atm 60 °C overnight.

[0242] Table 7: Results of the asymmetric hydrogenation with Walphos-Rh (Rh-1) catalyst in the optimized reaction conditions b (S) enantiomer.

[0243] When the reaction was performed with the same reaction conditions at the same scale (50 mg), the results were comparable. The most optimal reaction conditions found in this experiment: NaOMe (1 eq), 5% molar catalyst, ligand:Rh (1 : 1), MeOH (8 V), 15 atm RT 4-5 h then 30 atm 60°C overnight.

[0244] V indicates the ratio between the volume of solvent (mL) used and the quantity of the starting material (grams). E.g., the volume of MeOH added in the first tube is 0.2 mL (0.2 / 0.05=4) and the volume of MeOH added in the second tube is 0.2 mL (0.2 / 0.05=4) and therefore 8 V in total.

[0245] [Rh(NBD)2][BF4] (4.2 mg, 0.011 mmol, 0.05 eq), Walphos (10.4 mg, 0.011 mmol, 0.05 eq), and MeOH (0.2 mL, 4 V) were added to a 10 mL test tube (the first test tube) under Ar and

[0246] IPTS / 200134844.1 Docket No. GIU-074WO were stirred with a magnetic stir bar. The resulting solution was stirred at RT for 1 hour.

[0247] The starting material Formula (III) (50 mg, 0.224 mmol, leq), NaOMe (0.051 ml, 0.224 mmol, 1 eq) and MeOH (0.2 ml, 4V) were added to a second 10 mL test tube under Ar and were stirred using a magnetic stir bar, and a suspension was formed.

[0248] The solution of the the catalyst from the first test tube was added to the suspension of the starting material Formula (III) in the second test tube and the second test tube was placed inside a Berghoff reactor which was purged with consecutive cycles of vacuum / N2 (2 atm) (3x) and vacuum / Fb (2 atm) (3x).

[0249] The mixture was subjected to 15 atm H2 at 30 °C for 4-5 h. Once the IPC confirms the complete formation of 5-nitro compound, the hydrogenation was then performed at pressure of 30 atm and a temperature of 60 °C overnight to complete the transformation of the nitro group to the amino group.

[0250] Example 4: One pot procedure with different Walphos ligands

[0251] The effect of different substituents in the Walphos ligand was also studied. Different commercially available Walphos ligands were tested (FIG. 4).

[0252] Table 8: Resultsausing different Walphos as ligands

[0253] IPTS / 200134844.1 Docket No. GIU-074WO a5% catalyst, ligand:Rh (1: 1), MeOH (8 V), NaOMe (1 eq). 15 atm Hz 30°C 4.5 h then 30 atm 60°C o / n b(S) enantiomer, complex profile, more peaks in addition to R and S enantiomers.

[0254] The results from different Walphos ligands were compared with the results obtained with Rh-1. None of the Walphos tested gave better results than Rh-1. Rh-7 (entry 3) and Rh-9 (entry 5) gave complex profile in the chiral HPLC with many other unidentified peaks apart from the expected R and S enantiomers. The reaction with Rh-6 was scaled up to about 500 mg (entry 2 vs entry 6) of starting material to prepare a sufficient amount of the product to study the chiral resolution and subsequent steps to the compound of Formula (I).

[0255] Example 5: Scale up of the hydrogenation

[0256] The hydrogenation with the optimal conditions (5% catalyst, ligand:Rh (1 : 1), MeOH (9 V), NaOMe (1 eq), 15 atm, RT, 4-5 h then 30 atm, 60°C overnight) was scaled up to about 3 g of starting material. The results are presented in Table 9.

[0257] Table 9: Results of the scale-up experiments. b calculated as sodium salt of aniline Formula (II).

[0258] The experimental procedure was adjusted slightly with the scale-up of the reaction.

[0259] IPTS / 200134844.1 Docket No. GIU-074WO

[0260] Working with -3 g of starting material, the catalyst was pre-formed in a separate reactor and transferred via cannula into the reactor containing the starting material and NaOMe in MeOH. This procedure setting a specific pressure and temperature profile in each stage of the reaction was reproducible and yielded the final aniline Formula (II) with an e.e. of -90%.

[0261] The aniline product Formula (II) obtained with e.e -90% was subjected to additional enantiomeric enrichment to reach e.e. -99.9% and the subsequent steps to the compound of Formula (I) were performed as described in International Publication No. WO 2020 / 161362.

[0262] Example 6: Additional enantiomeric enrichment by formation of camphorsulfonic salt of Formula (n)

[0263] Preliminary attempts to isolate the aniline product Formula (II) directly from the crude material of the hydrogenation step were carried out, but its isolation either by precipitation or extraction from the reaction mixture proved to be challenging.

[0264] The crude product resulting from the asymmetric hydrogenation was dried and the resulting oil was treated with water and TBME. The organic and aqueous phases were separated and the aqueous phase containing the product Formula (II) was lyophilized to remove water.

[0265] Isolation of aniline Formula (II) as a sodium salt: the lyophilized crude product was taken up in MeOH and was divided in several fractions which were treated with several antisolvents (heptane, toluene, cyclohexane, acetone, MIK and TBME) in order to achieve the precipitation of the sodium salt. Only with TBME a cloudy solution was observed (which could not be isolated), in the rest of the cases solutions were obtained.

[0266] Isolation as aniline Formula (II): the lyophilized crude product was taken up in MeOH and was acidified with AcOH. Then the resulting solution was divided in several fractions which were treated with several antisolvents (heptane, toluene, cyclohexane, acetone, MIK and TBME) to evaluate the precipitation of Formula (II). Only with MIK a sticky solid was obtained, in the rest of the cases solutions were obtained.

[0267] After these initial attempts, a different strategy for isolating Formula (II) directly from the hydrogenation crude products was sought: precipitation of the corresponding camphorsulfonic

[0268] IPTS / 200134844.1 Docket No. GIU-074WO salt (used in the previous process through racemic resolution) after the convenient solvent exchange. Since hydrogenation is carried out with 1 equivalent of MeONa and the aniline of Formula (II) is then obtained as a sodium carboxylate, the salt precipitation has to be performed using 2 equivalents of CSA.

[0269] Thus, complete enantiomeric enrichment of aniline Formula (II) (obtained with high e.e. (-90%) after the asymmetric hydrogenation) has been carried out by crystallization of the camphorsulfonic (CSA) salt, using the procedure described in WO 2020 / 161362. After evaporation of the solvent (MeOH), the resulting crude oil was dissolved in acetone / water and treated with 2 equivalents of CSA. The CSA salt of Formula (II) was crystallized as a beige solid from this acetone / water mixture and achieved excellent e.e. (99%) and purity NMR,

[0270] Preliminary results are presented in Table 10:

[0271] Table 10: Results of the CSA resolution.

[0272] “Expected weight of aniline.bsalt crystallized contaminated with excess of CSA (ratio by 'H-NMR aniline Formula (II): CSA 1:2.5, yield calculated considering the excess of CSA).

[0273] The salt is obtained with moderate yield (-50-55%) but with excellent e.e. (>99%) as a beige solid. When the ratio acetone:water was 10: 1, the salt precipitated with moderate yield

[0274] IPTS / 200134844.1 Docket No. GIU-074WO

[0275] (entry 2).

[0276] In order to improve the yield, the ratio of acetone:water was modified to 20:1 (entry 3) (decrease of the amount of water). As a result, the salt precipitated along with excess of CSA (1.5 eq of excess, as revealed by 'H-NMR). (The yield has been re-calculated considering a ratio of aniline Formula (II): CSA = 1 :2.5).

[0277] The CSA salt of Formula (II) was confirmed by 'H-NMR and HPLC / MS and purified from catalyst ligand.

[0278] Example 7: Last steps: salt cleavage and acetylation to afford N-acetyl-GED-0507-34-levo

[0279] The last steps (cleavage and acetylation) of the synthesis were carried out according to the procedures described in WO 2020 / 161362 without any further optimization.

[0280] The cleavage of the CSA salt was performed i n the presence of NH4OH and desired aniline of Formula (II) was obtained by centrifugation of the crude after treatment with

[0281] The results are presented in Table 11.

[0282] Table 11 : Results of the CSA resolution.

[0283] IPTS / 200134844.1 Docket No. GIU-074WO

[0284] Free aniline (the compound of Formula (II)) was obtained with moderate yield and the expected high e.e. (>99%).

[0285] Acetylation

[0286] The acetylation process to achieve the target compound N-acetyl-GED-0507-34-levo (the compound of Formula (I)) was carried out by treatment of Formula (II) with acetic anhydride (Scheme 4):

[0287] Scheme 4: Acetylation of Formula (II) to obtain target compound N-acetyl-GED-0507-34-levo.

[0288] The results are presented in Table 12.

[0289] Table 12: Results of the acetylation to obtain target compound N-acetyl-GED-0507-34-levo. aThe crude was evaporated to dryness. It contains 4% wt EtOAc by 'H-NMR.

[0290] Example 8: Preparation of (2-nitrophenyl)-2-methoxyacrylic acid (Formula (III))

[0291] Starting from 4-nitrobenzaldehyde, Formula (III) used in the present investigation was prepared following the method (WO2020 / 161362) (Scheme 5).

[0292] IPTS / 200134844.1 Docket No. GIU-074WO

[0293] Formula (III)

[0294] Scheme 5. Synthesis of Formula (III) following method reported in WO2020 / 161362.

[0295] The intermediate Formula (III) was obtained with moderate yield according to the described method (-55% in most of the experiments at different scales (2 g scale, 75 g scale and 30 g scale). In a 1 L 3 necked round bottomed flask equipped with magnetic stirring and internal thermometer 4-nitrobenzaldehyde (75 g, 496 mmol), methyl 2-methoxyacetate (79 mL, 793 mmol, 1.6 eq) and anhydrous THF (150 mL, 2 V) were placed under N2 and protected from light. The resulting suspension was cooled at -5 to 5°C (ice bath). In another 1 L round bottomed flask equipped with magnetic stirring and protected from light, anhydrous THF (113 mL, 1.5V) and NaOMe (45 g, 833 mmol, 1.6 eq) were placed. The second round bottomed flask was purged for 1 min. with N2 and then it was cooled to -10°C to -5°C. The first mixture was added over the second mixture at T<10°C and formed a dark solution. After the addition was complete, the empty first flask was rinsed with anhydrous THF (38 mL) and poured into the second one. The reaction was stirred at -10°C to 10°C for ca. 5 minutes, maintaining the temperature at -10°C to 10°C. Cold toluene (150 mL) was added. A dark suspension formed. AcOH (75 mL) was slowly added and the suspension was stirred for 10 min at -10°C to 10°C. A brown suspension with sluggish stirring was formed. Water was added (225 mL) and the suspension was stirred at 0 to 10°C for 10 min and for 10 min at RT. The layers were separated; the organic one was washed with sat. NaCl (2x50 mL), dried over anh. MgSCh, filtered and the solvent was removed under vacuum. *H-NMR (CDCh) conforms to structure, purification required. The solid obtained was suspended in toluene (113 mL, 1.5 V). The suspension was heated at 45°C for 30 min. The mixture was cooled to -10°C to 0°C and stirred for 1 h at this temperature. The solid was filtered, washed with toluene and dried under vacuum (65% yield). *H-NMR (CDCh) conforms to structure.

[0296] In a 1 L 3 necked round bottomed flask, equipped with magnetic stirring and internal thermometer, methyl 3-hydroxy-2-methoxy-3-(4-nitrophenyl)propanoate (82.3 g, 322 mmol, 1 IPTS / 200134844.1 Docket No. GIU-074WO eq) and anh. toluene (321 mL, 3.9 V) were placed under N2 and protected from light. The orange suspension was cooled to 0°C - 10°C. EtsN (66 mL, 473 mmol, 1.5 eq) was added.

[0297] Methanesulfonyl chloride (27.8 mL, 359 mmol, 0.96 eq) was added to the orange suspension and the reaction was stirred at 0°C -10°C for 30 min. An abundant solid precipitate formed. Water (329 mL) was added dropwise and the mixture was stirred at 55°C for 10 min. Layers were separated and the aqueous one was extracted with toluene (2x75 mL). The combined organic layers were dried over anh. MgSCh, filtered and the solvent was removed under vacuum to yield 106.7 g. MeOH (247 mL, 3 V) were added and the system was purged with N2. NaOH (353 g, 2651 mmol) was added dropwise to the orange suspension. The reaction was very exothermic, temperature controlled with an ice water bath. The mixture was stirred at RT for 2h. Water (165 mL, 2 V) was added. The mixture was stirred at 60°C for 10 min. At this temperature, 85% H3PO4 (56.9 mL, 929 mmol, 2.9 eq) was slowly added until a pH <3.0 was obtained. The mixture was mechanically stirred at 60°C for 15 min. The mixture was stirred at RT for 45 min, filtered, washed with water, dried under vacuum over P2O5 at 40°C o / n. Yield = 72.1 g (overweight). 'H-NMR (DMSO) conforms to structure. The solid was dispersed in MeOH:H2O (1 :3) (576 mL) for 30 min. The solid was then filtered, washed with water and dried under vacuum over P2O5 at 40°C o / n (80% yield). 'H-NMR (DMSO) conforms to structure.

[0298] Example 9: Asymmetric hydrogenation with Rh / Walphos. Preparation of crude acid of Formula (D).

[0299] Intermediate Formula (III) is reduced to the sodium salt of Formula (II) according to the procedure developed in the previous activity. Isolation of Formula (II) by precipitation after acidification is studied

[0300] Scheme 6: Preparation of crude Formula (II)

[0301] Hydrogenation is carried out in MeOH (8 V) with NaOMe (1 eq) using 5% molar of the catalyst Rh-1 (Figure 1) prepared in situ from (7?)-Walphos and [Rh(NBD)2]+[BF4] ligand:Rh IPTS / 200134844.1 Docket No. GIU-074WO

[0302] (1 : 1). The procedure involves a first part carried out at 15 atm. H2 pressure, at RT for 4-5 h (reduction of the double bond) and a second part at 30 atm. H2 pressure, at 60°C o / n (reduction of the NO2 group). Compound of Formula (II) is obtained with e.e. -90%.

[0303] Scheme 7: Structure of Rh catalysts tested.

[0304] In the previous activity the maximum scale for this transformation was 3 g. The present work involves an increase of the working scale; thus, the hydrogenation step has been successfully scaled up to 8-10 g of intermediate Formula (III). The best reaction conditions defined in the previous work have not been modified. The investigation has been focused on identifying a procedure (that could be further scaled-up) allowing the isolation of acid Formula (II) by precipitation from the reaction media.

[0305] The different hydrogenations carried out and the isolation procedures applied are indicated in Table 13:

[0306] Table 13: Hydrogenation experiments.

[0307] IPTS / 200134844.1 Docket No. GIU-074WO

[0308] IPTS / 200134844.1 Docket No. GIU-074WO aYield calculated as % obtained grams / theoretical grams (purity not considered).be.e. determined before the division of the crude in parts.

[0309] Each entry is commented in more detail below:

[0310] Entry 1: Starting from 3 g of intermediate Formula (III), the first hydrogenation experiment was carried out as in the previous investigation to prove that the resulting e.e. with the Walphos ligand (ca. 90 %).

[0311] Once the reaction was finished, aiming to remove color and the residual Rh of the dark crude obtained, the use of a Rh scavenger (QuadraPure TM (Aldrich 655422-5G), a thiourea resin) was tested. The methanolic crude solution was treated with the resin scavenger ((1 : 1) in weight with respect of [Rh(NBD)2]+[BF4]), o / n RT.) but crude discoloration was not observed (possible removal of Rh was not evaluated). No more attempts to remove Rh were carried out. After filtration of the crude through a Celite pad, MeOH was evaporated under vacuum. The resulting crude was dissolved in H2O and basified to pH=9, then this aqueous solution (containing Formula (II) as sodium salt) was washed with different organic solvents (MIK, Et2O, DCM). Only using MIK two phases were observed and the ligand was effectively extracted in the organic phase. The aqueous phase was then acidified to pH=4 and, after multiple attempts, acid Formula (II) successfully precipitated from a solvent mixture formed by H2O (3V) / AcOEt (3 V). A dark solid (with overweight, probably due to the presence of salts, such as NaCl), difficult to filter, was obtained. The work-up procedure applied in this experiment, involving organic washes of the basic aqueous phase, was tedious (the separation of the organic phase from the aqueous one was difficult) and no more attempts in this sense were carried out.

[0312] Entry 2: To study the isolation procedure of acid Formula (II), this investigation required an increase of the working scale of the hydrogenation. For this reason, the second hydrogenation

[0313] IPTS / 200134844.1 Docket No. GIU-074WO experiment was carried out at 15 g scale with a hydrogenation reactor of higher volume (compared to the one used in experiment described in Entry 1). In this first experiment, carried out in a different reactor, the strict anhydrous and inert reaction conditions were not preserved, due to the adaptation of the reaction set up to the scale. As a result, the e.e obtained in this experiment was very low (-10%), thus the hydrogenation process is very sensitive to the experimental factors and must be performed under strict inert conditions.

[0314] Despite this poor result, the crude reaction of this experiment was used to study the isolation procedure of acid Formula (II). After removing MeOH crude was dissolved in acetone / FEO and acidified to pH=4. By this method acid Formula (II) precipitated as a grey solid that could be filtered (Pore 3 (16-40 mm)) more easily compared to the one obtained in the previous experiment (Entry 1, which was precipitated from EEO / AcOEt). After this observation, the mixture acetone / water was considered promising to induce precipitation of acid Formula (II) and its subsequent filtration. The resulting yield was acceptable (60 %) and the catalyst’s ligand is removed as it remains in the mother liquors (according to HPLC / MS analysis).

[0315] In view of the low e.e obtained in this experiment, a different (strictly inert) set up procedure was designed in the same hydrogenation reactor to be applied in the next hydrogenation experiments.

[0316] Entry 3: With the available reactors, the most suitable scale to carry out a hydrogenation at 30 bar EE pressure, under strict anhydrous conditions, is 8-10 g of intermediate Formula (III). At this scale the strict anhydrous conditions required can be assured. Thus, the first experiment with the suitable set up was successfully carried out at 8 g scale and the resulting e.e was >90%.

[0317] This crude was divided in two parts to study its isolation to compare two possible procedures to induce the precipitation and subsequent filtration of acid Formula (II) (in this way the use of EtOAc / EEO or acetone / H2O was studied). Comparing both procedures the use of acetone / EEO was preferred in terms of yield and quality. It is important to remark that filtration of Formula (II) from acetone / EEO is faster than from EtOAc / EEO.

[0318] Entry 4: Hydrogenation was carried out at 10 g scale, good e.e. was obtained (e.e. >90%) and the crude was divided in three equal parts to modulate conditions for the isolation procedure

[0319] IPTS / 200134844.1 Docket No. GIU-074WO of acid Formula (II). The amount of water was slightly reduced (part B and part C compared to part A) but no significant changes on the results were obtained.

[0320] Entry 5: The last hydrogenation was carried at 10 g scale, good e.e. was obtained and the whole crude was treated according to the best identified method. MeOH was evaporated, acetone (4.2V) and H2O (0.2 V) were added and the mixture was acidified with 37% HC1 to pH=3.9. The resulting solid was stirred at RT for 1 h and then filtered and washed with acetone. 8.3 g (86% yield) of acid Formula (II) as a dark solid were obtained (HPLC=87% (a / a), KF=2.6%).

[0321] After these experiments at larger scale than in the previous work, a procedure to isolate acid Formula (II) by precipitation has been defined with good results. Acid Formula (II) is obtained with acceptable mass yield (60-65%), good e.e. (>90%) and moderate chemical quality (crude). Considering the chemical HPLC quality, yield is ca 45% in the different experiments. To reach the final required quality this crude requires further chemical and enantiomeric purification.

[0322] Example 10: Asymmetric hydrogenation with Rh / Walphos Preparation of the compound of Formula (II) procedure.

[0323] 100 mL round bottomed flask, provided with magnetic stirring and under Ar. Place [Rh(NBD)2]+[BF4]-17 (838 mg, 2.24 mmol, 0.05 eq), (R)-Walphos (562 mg, 0.604 mmol, 0.05 eq) and methanol (45 mL, 4.5 V). The orange solution was stirred at RT for 1 h. In a Berghof high pressure reactor provided with magnetic stirring, compound of Formula (III) (10 g, 44.8 mmol, 1 eq), NaOMe (10.2 mL, 44.8 mmol, 1 eq) and MeOH (45 mL, 4.5 V) were placed, under Ar to form a suspension. The solution was added into the Berghof with a cannula. The Berghof was purged with cycles vacuum / N? (3x) and vacuum / H? (3x), charged with 15 atm H2 at 25-30°C (external temperature) for 4-5 h. e.e. =88%, then charged with 30 atm H2 and heat at 58-63°C (external temperature) for 16-18 h. e.e=96%. The crude was transferred into a round bottomed flask and evaporated to dryness under vacuum to form a dark oil. Acetone (42 mL, 4.2 V) and water (2 mL, 0.2 V) were added at pH=9. pH was adjusted to 3.8-4.2 with 37% HC1, forming a solid in suspension. The suspension was stirred for 15 min at RT, then confirmed pH=3.8-4.2. Then stirred at 1 h at RT. Filter and wash with acetone (2 x 0.5 V). 8.3 g, (86% yield), grey solid.

[0324] IPTS / 200134844.1 Docket No. GIU-074WO

[0325] Example 11: Additional enantiomeric enrichment by formation of camphorsulfonic salt

[0326] The different batches of Formula (II) were submitted to further purification and additional enantiomeric enrichment by formation of the camphor sulfonic salt (Scheme 8).

[0327] Scheme 8: Preparation of CSA salt.

[0328] Results obtained for the different batches are indicated in Table 14:

[0329] Table 14. Additional enantiomeric enrichment by formation of the CSA salt experiments.

[0330] IPTS / 200134844.1 Docket No. GIU-074WO

[0331] The different batches of Formula (II) were treated with ca. 1 equivalent of (S)- camphorsulfonic acid (CSA) in acetone / water (according to the method described in WO2020161362A1) and the CSA salt crystallized as a solid from this media reaching excellent e.e. (99%) and good quality (as revealed by 'H-NMR and HPLC / MS).

[0332] In Entry 1 residual salts might contaminate CSA salt resulting in overweight.

[0333] In Entry 2 the yield is low since the starting acid Formula (II) is practically racemic (as commented in Entry 2, Table 13).

[0334] In Entries 3, 4 and 5, at 8-10 g working scale, the overall yield (from 2 to 4 calculated as mass yield) has been 40-45%.

[0335] Example 12: Cleavage of the CSA salt

[0336] The cleavage of the CSA salt was performed in presence of NFUOH and desired Formula (II) was obtained by centrifugation of the crude after treatment with AcOH (Scheme 9).

[0337] IPTS / 200134844.1 Docket No. GIU-074WO

[0338] Scheme 9: Cleavage of CSA salt to generate free compound of Formula (II).

[0339] The initial reaction conditions described in WO2020161362A1, involving the use of

[0340] EtOAc, were improved using acetone. Results are gathered in Table 15:

[0341] Table 15: Results of the cleavage of the CSA salt.

[0342] IPTS / 200134844.1 Docket No. GIU-074WO

[0343] The isolation procedure of enantiomerically pure compound of Formula (II) has also been studied.

[0344] Most experiments have been carried out in acetone / water media and acid Formula (II) is obtained with good mass yield (ca 70%) and high quality (HPLC purity -99% a / a).

[0345] The reaction conditions have been slightly modulated aiming to increase the yield. The amount of water used in the crystallization media has been reduced (Entry 4, (part B)) but no significant changes have been observed.

[0346] At this point, the overall yield of the process from CSA salt to enantiomerically pure Formula (II) is 30-35% and resulting compound Formula (II) has a high chemical and optical purity

[0347] Example 13: Procedure for Cleavage of the CSA salt

[0348] In a 50 mL round bottomed flask, provided with magnetic stirring, CSA salt (9.3 g, 21.8 mmol, 1 eq) and water (18.6 mL, 2 V) were placed. The resulting suspension was heated at 20- IPTS / 200134844.1 Docket No. GIU-074WO

[0349] 40°C until nearly complete dissolution. Water (1.9 mL, 0.2 V) and acetone (9.3 mL; 1 V) were added. The resulting solution was flushed with N2, heated at 55-60°C, NH4OH (1.9, 29.5 mmol, 1.3 eq) was added and the solution was stirred for 15 min at 55-60°C. AcOH (1.1 mL, 15.5 mmol, 0.7 eq) were added, the solution was stirred at RT for 0.5 h and then cooled to 2-7°C and the mixture was stirred for 1 h to form a solid precipitate. The solid was centrifuged and washed with water (6.5 mL, 0.7 V) and acetone (6.5 mL, 0.7 V). 2.9 g (70% yield) off-white solid.1H- NMR (DMSO) conforms to structure. It contains residual CSA. KF=1.6%.

[0350] Example 14: Acetylation to afford N-acetyl-GED-0507-34-levo

[0351] The acetylation process to achieve the target compound N-acetyl-GED-0507-34-levo was carried out by treatment of the compound of Formula (II) with acetic anhydride (Scheme 10) as described in W02020161362A1.

[0352] Scheme 10: Acetylation of Formula (II) to obtain target compound N-acetyl-GED-0507-34-levo (Formula (I)).

[0353] Results are gathered in Table 16:

[0354] Table 16: Results of the acetylation to obtain target compound N-acetyl-GED-0507-34-levo.

[0355] IPTS / 200134844.1 Docket No. GIU-074WO

[0356] The process was carried out according to the reported method and the product precipitated out as described. Final characterization by LCMS, 'H-NMR,13C-NMR and IR of N- acetyl-GED-0507-34-levo has been carried out, and product complies with the required specifications (99% e.e. and NLT 99.0% a / a by HPLC). For all experiments, after seeding, the crystallization of final product is observed. Moderate yield is due to the difficult handling at the working scale with the available set up of the centrifugation system.

[0357] Example 14: Scale-up hydrogenation

[0358] The last hydrogenation crude (Entry 5 (Table 13)) obtained has been transformed into purified Formula (II) without divisions to confirm the overall yield of this asymmetric hydrogenation and the subsequent purification. The complete procedure starting from 10 g of intermediate Formula (III) to purified compound Formula (II) is depicted in Scheme 10 and compared to the process described in the patent:

[0359] IPTS / 200134844.1 Docket No. GIU-074WO

[0360] Scheme 10: Results of the improved process from compound Formula (III) to purified acid of Formula (II) (overall yield refers to reactions from intermediate Formula (III) to compound Formula (II))

[0361] Purification methods are based on crystallization strategies suitable for further scale-up. Overall yield has been -10-15% increased with respect to the racemic route. To note that the working scale has been very low compared to that described in patent WO2020161362A1.

[0362] Example 15: Procedure for acetylation of compound Formula (II) to obtain target N-acetyl-

[0363] GED-0507-34-levo (Formula (I))

[0364] In a 25 mL round bottomed flask, provided with magnetic stirring and a reflux condenser compound of Formula (II) (2.8 g, 14.2 mmol) and EtOAc (4,2 mL, 1.5 V) were placed under N2. The suspension was heated at 65-70°C and acetic anhydride (1.7 mL; 17.7 mmol; 1.2 eq) was added dropwise. The solution was heated at 60-70°C for 1.5 h. The suspension was cooled at 10- 20°C, stirred for 0.5 h, seeded, stirred for 1 h and centrifuged. The solid was washed with EtOAc IPTS / 200134844.1 Docket No. GIU-074WO

[0365] (1.4 mL; 0.5 V) and H2O (1.5 V). (54% yield). 'H-NMR,13C-NMR (DMSO) conforms to structure.

[0366] Example 16: Comparison of the key steps of the enantioselective vs racemic resolution routes

[0367] An initial comparison of the yields of the key steps (hydrogenation+resolution) of both routes (current route (Scheme 11) vs asymmetric route (Scheme 12)) is presented in this section.

[0368] Resulting yields are compared at the stage of the camphorsulfonate salt of Formula (II), a common intermediate of both routes.

[0369] Scheme 11 : Results of the key steps (racemic hydrogenation+resolution) of current route.

[0370] Scheme 12: Results of the key steps (asymmetric hydrogenation+resolution) of the asymmetric route. S

[0371] The initial results indicate an increase of yield of -10-15% in the asymmetric route with respect to the current route.

[0372] IPTS / 200134844.1 Docket No. GIU-074WO

[0373] The asymmetric reduction under the previously developed reaction conditions (NaOMe (1 eq), 5% molar catalyst, ligand:Rh (1 : 1), MeOH (8 V), 15 atm H2 RT 4-5 h then 30 atm H2 60°C o / n) has been successfully scaled up to 8-10 g scale.

[0374] Conditions to isolate intermediate of Formula (II) by precipitation just after the hydrogenation have been defined (acetone (4.2V)-H2O (0.2V)) as well as the subsequent purification and additional enantiomeric enrichment resolution by formation of the camphorsulfonate salt. Also, conditions for its posterior cleavage have been modulated.

[0375] The last step (acetylation) of the synthesis has been carried out according to the described procedure without any further optimization. Target compound N-acetyl-GED-0507-34- levo has been isolated by precipitation with the required specifications.

[0376] The complete procedure has been established displaying a good potential scalability.

[0377] From intermediate of Formula (III) to intermediate of Formula (II), results show an increase of yield of -10-15% for the asymmetric route with respect to the current route (involving a racemic resolution).

[0378] The results show that the asymmetric route of synthesis has a high potential to provide a process for the manufacturing of N-Acetyl-GED-0507-34-Levo which may be competitive in comparison with the process through racemic resolution described in WO2020161362A1.

[0379] INCORPORATION BY REFERENCE

[0380] All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0381] EQUIVALENTS

[0382] While specific embodiments of the subject disclosure have been discussed, the above

[0383] IPTS / 200134844.1 Docket No. GIU-074WO specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0384] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0385] IPTS / 200134844.1

Claims

Attorney Docket No. GIU-074WOCLAIMSWhat is claimed is:

1. A process for preparing a substantially optically pure compound of Formula (II):lphos catalyst, thereby forming the compound of Formula (II), or a salt thereof.

2. The process of claim 1, wherein the hydrogenation further forms a compound of Formula (IV):salt thereof, wherein the ee (enantiomeric excess) of the hydrogenation is at least 60% (i.e. S:R=80%:20%), at least 70% (S:R=85%: 15%), at least 80% (S:R=90: 10), or at least 90% (S:R=95%:5%), favoring the formation of the compound ofFormula (II).

3. The process of claim 2, wherein the process further comprises resolving the mixture of a compound of Formula (II) and a compound of Formula (IV).

4. The process of any one of claims 1-3, wherein the hydrogenation reaction is in the presence of a solvent.IPTS / 200134844.1Attorney Docket No. GIU-074WO5. The process of claim 4, wherein the solvent is methanol.

6. The process of any one of claims 1-5, wherein the hydrogenation reaction takes place at a lower temperature and with a lower pressure of hydrogen gas for a first period of time and then at a higher temperature and with a higher pressure of hydrogen gas for a second period of time.

7. The process of claim 6, wherein the lower temperature is about room temperature or about 308. The process of claim 6 or 7, wherein the higher temperature is about 60 °C.

9. The process of any one of claims 6-8, wherein the lower pressure is about 15 atm.

10. The process of any one of claims 6-9, wherein the higher pressure is about 30 atm.

11. The process of any one of claim 6-10, wherein the first period of time is at least 4 hours.

12. The process of any one of claims 6-11, wherein the second period of time is at least 5 hours, at least 10 hours, at least 12 hours, or at least 18 hours.

13. The process of any one of claims 1-12, wherein the structure of the Rh-Walphos catalyst is:

14. The process of any one of claims 1-13, wherein the amount of the Rh-Walphos catalysts is at least 5% molar.

15. The process of any one of claims 1-14, wherein the Rh-Walphos catalyst is generated in situ from a Rh agent and a Walphos agent in about 1 to 1 ratio.

16. A process of for preparing a substantially optically pure compound of Formula (I),IPTS / 200134844.1Attorney Docket No. GIU-074WOsalt thereof, the process comprising the process of any one of claims 1-16, wherein the process further comprises acylating the compound of Formula (II), or a salt thereof, thereby forming the compound of Formula (I) or a salt thereof.

17. The process of any one of claims 1-16, wherein the salt is a pharmaceutically acceptable salt.58IPTS / 200134844.1

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