Method for synthesizing lipstatin derivatives

Abstract

Methods for forming the lipstatin derivative (S)-1-((2S,3S)-3-ethyl-4-oxooxetan-2-yl)tridecan-2-yl formyl-L-alaninate are provided in accordance with embodiments of the present invention. These methods include one or more reaction steps including Mitsunobu coupling of N-formyl L-alanine in the presence of di-t-butyl azodicarboxylate (DBAD) and triphenylphosphine. Compounds formed by the methods of the present invention, compositions including compounds formed by the methods of the present invention, and methods for inhibiting lipase activity and / or treating pancreatitis using compounds formed by the methods of the present invention are also provided.

Description

[Technical Field]

[0001] [Priority statement] This application claims priority to U.S. Provisional Patent Application No. 63 / 178,728, filed April 23, 2021, which in whole constitutes a part of this specification by reference.

[0002] The present invention relates to a method for synthesizing pharmaceutical compounds. The present invention also relates to a small molecule lipase inhibitor and a method for synthesizing the same. In particular, the present invention relates to a method for synthesizing lipstatin derivatives. [Background technology]

[0003] Pancreatitis is an inflammation of the pancreas that can occur when digestive enzymes are activated while still present in the pancreas, stimulating pancreatic cells and causing inflammation. The annual incidence of acute pancreatitis is approximately 20 to 40 cases per 100,000 people, and this incidence has been increasing over the past few decades. (Yadav et al., Epidemiology of Pancreatitis, GI Epidemiology: Diseases and Clinical Methodology, 2) nd See Ed., 2014, Blackwell Publishing. Furthermore, severe acute pancreatitis has a mortality rate of approximately 29%. Munoz et al., Am Fam Physician (2000) 62:164-74. Therefore, novel methods for treating pancreatitis are being researched.

[0004] Lipase inhibitors are a class of compounds marketed as anti-obesity agents. Lipstatin, a natural product isolated from Streptomyces toxytricini, is one of the earliest known lipase inhibitors. Orlistat, a saturated derivative of lipstatin, was developed by Hoffman-La Roche and is marketed as an anti-obesity drug under the trade names Xenical® and Alli®. Lipase inhibitors can also be useful in treating other disorders such as pancreatitis.

[0005] One of the lipstatin derivatives is (S)-1-((2S,3S)-3-ethyl-4-oxooxetane-2-yl)tridecane-2-ylformyl-L-alaninate, which is also referred to as compound IA herein.

[0006] [ka]

[0007] As shown in Figure 1, current methods for synthesizing compound IA include the illustrated synthesis procedure. While these methods can produce compound 1-A, they may not be suitable for large-scale production and / or cGMP production. These methods may also exhibit chemical handling problems, including a strong sulfur / thiol odor that is difficult to contain during scale-up, and significant health hazards due to the use of corrosive hydrofluoric acid aqueous solutions. Furthermore, the yield obtained throughout the synthesis may be low to moderate, and seven chromatographic purification steps may be required. Thus, a novel method for synthesizing compound IA is desired. [Overview of the Initiative]

[0008] In one embodiment of the present invention, a compound of formula I: [ka] (In the formula, R1 is C 5~ C15 alkyl (e.g., C 11 alkyl).) A method for synthesizing is provided. In some embodiments, the method includes contacting a compound of INT 12 with N-formyl L-alanine in the presence of di-t-butyl azodicarboxylate (DBAD) and triphenylphosphine to form a compound of formula I.

[0009]

Chemical formula

[0010] In some embodiments of the present invention, a method for synthesizing a compound of INT 2 is provided. In some embodiments, these methods include contacting a compound of INT1 with a ruthenium (R)-BINAP catalyst and hydrogen gas in a reaction mixture within a pressure vessel to form a compound of INT 2.

[0011]

Chemical formula

[0012] In some embodiments, the pressure of hydrogen gas within the pressure vessel is 200 psi or less (e.g., within the range of 100 - 200 psi). In some embodiments, the volume ratio of the dead space within the pressure vessel to the reaction mixture is 3:1 or more.

[0013] Also, in one embodiment of the present invention, a method for synthesizing a compound of formula I, wherein

Chemical formula

Chemical formula

[0014] Furthermore, in one embodiment of the present invention, a compound of formula I formed by the method according to the present invention, or a pharmaceutically acceptable salt thereof, is provided. Also provided is a composition comprising the compound according to the present invention and a pharmaceutically acceptable carrier.

[0015] Furthermore, in one embodiment of the present invention, a method is provided for inhibiting lipase activity in a subject that requires such inhibition, comprising the step of administering a therapeutically effective amount of the compound according to the present invention or a pharmaceutically acceptable salt thereof and / or a composition according to the present invention to the subject, thereby inhibiting lipase activity in the subject.

[0016] Furthermore, in one embodiment of the present invention, a method is provided for treating pancreatitis in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof and / or a composition according to the present invention to the subject, thereby treating the pancreatitis in the subject.

[0017] These and other aspects of the present invention will be described in more detail in the following description of the present invention. [Brief explanation of the drawing]

[0018] [Figure 1] This is a synthesis scheme in a prior art method for producing compound IA. [Figure 2] This is the 1H NMR spectrum of INT 1. [Figure 3] This is the 1H NMR spectrum of enantiomerically enriched INT 2. [Figure 4] This is the 1H NMR spectrum of racemic INT 2. [Figure 5] The chiral HPLC data for enantiomerically enriched INT 2 are shown. [Figure 6A]Figures 6A and 6B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material (SM) and the INT 3 product under UV lamp (Figure 6A) and PMA staining (Figure 6B) using 20% ​​ethyl acetate / heptane as the eluent. [Figure 6B] Figures 6A and 6B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material (SM) and the INT 3 product under UV lamp (Figure 6A) and PMA staining (Figure 6B) using 20% ​​ethyl acetate / heptane as the eluent. [Figure 7] This is the 1H NMR spectrum of the crude reaction mixture of INT 3. [Figure 8A] Figures 8A and 8B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material (SM) and the INT 4 product in the crude mixture (IPC, Figure 8A) and the crystallized product (Figure 8B). [Figure 8B] Figures 8A and 8B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material (SM) and the INT 4 product in the crude mixture (IPC, Figure 8A) and the crystallized product (Figure 8B). [Figure 9] This is the 1H NMR spectrum of INT 4, which exhibits keto-enol tautomerism. [Figure 10] The experimental setup for the Raney Ni hydrogenation is shown. [Figure 11A] Figures 11A and 11B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material and the INT 5 product in the crude product (Figure 11A) and the crystalline product (Figure 11B). [Figure 11B] Figures 11A and 11B are photographs of thin-layer chromatography (TLC) plates showing the elution of the starting material and the INT 5 product in the crude product (Figure 11A) and the crystalline product (Figure 11B). [Figure 12] This is the 1H NMR spectrum of crystalline INT 5. [Figure 13] This is the LC-MS spectrum of INT 6. [Figure 14] This is the LC-MS spectrum of INT 7. [Figure 15] This is the LC-MS spectrum of INT 8. [Figure 16] This is the LC-MS spectrum of INT 9. [Figure 17] This is the LC-MS spectrum of INT 10. [Figure 18] This is the LC-MS spectrum of the mother liquor containing purified PEA INT 8. [Figure 19] This is the LC-MS spectrum of INT 9 converted from PEA-purified INT 8 in the mother liquor. [Figure 20] This is the LC-MS spectrum of INT 10 converted from PEA-purified INT 8 in the mother liquor. [Figure 21] This is the LC-MS spectrum of INT 10 after crystallization. [Figure 22] This is the LC-MS spectrum of INT 9 (purified). [Figure 23] This is the LC-MS spectrum of INT 11. [Figure 24] This is the LC-MS spectrum of INT 12. [Figure 25] This is the LC-MS spectrum of a crude mixture (RABI-767) containing compound IA. [Figure 26] This is the LC-MS spectrum of the crude mixture containing compound IA after treatment with TFA / DCM. [Figure 27] This is the LC-MS spectrum of a crude mixture containing compound IA after treatment with TFA / DCM in a large-scale synthesis. [Figure 28A] This is a photograph of a column packing configuration for purifying compound IA. [Figure 28B] This is a photograph of a PMA-stained TLC plate showing the elution of compound IA with various other impurities using a 50 / 50 heptane / ethyl acetate eluent. [Figure 29] This is the LC-MS spectrum of compound IA. [Figure 30]This is the 1H NMR spectrum of compound IA. [Figure 31] The HPLC readings and spectrum of compound IA are shown. [Figure 32] The HPLC data for compound IA is shown. [Figure 33] The crystal structure of compound IA is shown. [Modes for carrying out the invention]

[0019] The present invention will now be described in detail. It should be noted that the present invention may be carried out in different forms and should not be construed as being limited by the embodiments described herein. Rather, these embodiments are provided to make this disclosure complete and comprehensive, and to fully convey the scope of the invention to those skilled in the art.

[0020] In this specification, the terminology used to describe the present invention is intended solely to describe a particular embodiment and is not intended to limit the invention. Unless otherwise clearly indicated in the context, the singular forms "a," "an," and "the" used in the description of the present invention and the appended claims are intended to include the plural forms as well.

[0021] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Furthermore, terms defined in commonly used dictionaries, for example, should be interpreted as having the same meaning as they do in the context of this application and related art, and should not be interpreted in an ideal or overly formal sense unless explicitly stated herein. The terminology used in describing the present invention herein is intended solely to describe a particular embodiment and is not intended to limit the invention. All publications, patent applications, patents, and other references referenced herein, by reference, constitute part of this specification. In the event of any inconsistency in terminology, this specification shall prevail.

[0022] In this specification, “and / or” means and encompasses all possible combinations of one or more related items listed, and, where interpreted as alternative ("or"), the absence of any combination.

[0023] The various features of the present invention described herein are expressly intended to be usable in any combination unless otherwise indicated in the context. The present invention also assumes that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted. For example, where a complex is described herein as comprising components A, B, and C, it is expressly intended that any one of A, B, or C, or any combination thereof, may be omitted and discarded.

[0024] In this specification, the transitional phrase "essentially derived from" (and its grammatical variations thereof) should be interpreted as encompassing the substances or processes described and substances or processes that do not substantially affect the fundamental novel features of the claimed invention. Accordingly, the term "essentially derived from" should not be interpreted as equivalent to "include" in this specification.

[0025] In this specification, terms such as “first,” “second,” etc., may be used to describe various elements, but it should be understood that these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. Thus, without departing from the teachings of this embodiment, the “first” element may be referred to as the “second” element.

[0026] In this specification, the term “about” is used to refer to measurable values ​​such as quantities or concentrations and is intended to include variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% from a specified value, as well as the specified value. For example, “about X,” where X is a measurable value, is intended to include X and variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% from X. The ranges given herein for measurable values ​​may include any other ranges and / or individual values ​​within that range.

[0027] In this specification, "halo" means any suitable halogen including -F, -Cl, -Br, and -I.

[0028] In this specification, "hydroxy" or "hydroxyl" means an -OH group. A "free hydroxyl group" is an unprotected -OH group. A "protected hydroxyl group" is a hydroxyl group that is bonded (e.g., covalently) to a protecting group.

[0029] In this specification, “alkyl” means a linear or branched hydrocarbon containing 1 to 15 carbon atoms, used alone or as part of another group. In this specification, “lower alkyl” means a linear or branched hydrocarbon containing 1 to 4 carbon atoms, used alone or as part of another group. Representative examples of alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, tridecyl, tetradecyl, and pentadecyl. C5~C 15 Alkyls include linear and branched saturated alkyls, such as n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, tridecyl, tetradecyl, and pentadecyl.

[0030] In this specification, the term “compound” is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the illustrated structure. Unless otherwise specified, a compound identified herein by name or structure as a specific tautomer is intended to include other tautomers. Tautomers arise from the simultaneous exchange of a single bond with an adjacent double bond and the transfer of a proton. Tautomers include prototropic tautomers, which are isomer-protonated states having the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imoid acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which a proton may occupy two or more positions in the heterocyclic system. Tautomers may be in equilibrium or sterically locked into one form by appropriate substitution.

[0031] In some embodiments, the compounds described herein may contain one or more chiral centers and may result in racemates and racemic mixtures, enantiomerically concentrated mixtures, a single enantiomer, individual diastereomers and diastereomer mixtures (including, for example, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+)(dextrorotatory) forms, (-)(levorotatory) forms, racemic mixtures thereof, and other mixtures thereof). Further chiral carbon atoms may be present in substituents such as alkyl groups. Unless otherwise indicated, all these isomers of these compounds, and mixtures thereof, are expressly included herein.

[0032] The compounds described herein may further include limitations arising from the presence of bonds in which the rotation of the bond is restricted around a particular bond, such as rings or double bonds (e.g., carbon-carbon bonds or carbon-nitrogen bonds such as amide bonds). Therefore, all cis / trans and E / Z isomers, as well as rotational isomers, are included herein. Unless otherwise indicated, the chemical designation of a compound encompasses a mixture of all possible stereochemical isomers of the compound.

[0033] The preparation of compounds described herein may involve the protection and deprotection of various chemical groups. For the chemical properties of protecting groups, see, for example, TW Greene and PGMWuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999).

[0034] The product can be identified by monitoring the reaction by methods known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), mass spectrometry, and / or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). The compounds can be purified by various methods known to those skilled in the art, unless otherwise indicated.

[0035] As used herein, "treatment" means any manner of ameliorating or otherwise beneficially altering one or more symptoms of a disease or disorder. Treatment also encompasses any pharmaceutical use of the compositions described herein, such as use for treating pancreatitis. As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition means any diminution, whether permanent or temporary, persistent or transient, that results from or is associated with the administration of the composition.

[0036] According to one embodiment of the invention, Formula I:

Chemical formula

[0037] The method steps for the synthesis of the compound of formula I are shown below. The method according to the present invention may include one or more of these method steps. In some embodiments, certain method steps may be omitted, and any single step may be considered the method according to the present invention. For example, a method according to one embodiment of the present invention may include only steps 2-14, 3-14, 4-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, 13-14, and 14. Also, any combination of intermediate steps may constitute the method according to the present invention (e.g., steps 2-4, 4-6, 8-10). In some embodiments, each method step described herein is present in the method according to the present invention, for example, in the method for synthesizing compound IA.

[0038] [Method step 1] In some embodiments of the present invention, a compound of formula 1-A and a compound of formula 1-B are brought into contact to form a compound of formula INT 1, as shown below: [ka] (In the formula, X1 is a halo (e.g., Cl or Br), and R1 is C 5~ C 15 Alkyl (e.g., C5~C) 15 (Linear alkyl) Therefore, R1 is C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , or C 15 It can be alkyl, or any range defined between these. In one particular embodiment, R1 is C 11 Alkyl (e.g., C 11 H 23 )

[0039] In some embodiments, compound 1-A and compound 1-B are brought into contact in the presence of an alkali metal such as magnesium to form compound INT 1 by aliphatic acylation. In some embodiments, an alkali metal (e.g., magnesium) is first reacted with methanol to form an alkali metal methoxide solution (optionally in toluene), to which compound 1-A is added. Next, compound 1-B, dissolved in a solvent (e.g., toluene), can be added over time, for example, by an addition funnel. In some embodiments, the reaction mixture is stopped with methanol and distilled. A strong acid, such as a mineral acid, such as hydrochloric acid, sulfuric acid, and / or phosphoric acid, can then be added. The reaction mixture can then be purified by any preferred method, for example, washing with water, drying, and / or concentrating compound INT 1. Optionally, compound INT 1 may be recrystallized (e.g., with methanol) to further purify the compound.

[0040] [Method step 2] In some embodiments of the present invention, a compound of INT1 is contacted with a ruthenium R-BINAP catalyst and hydrogen gas in a stereoselective ketone reduction reaction to form a compound of INT2: [ka] (In the formula, R1 is as defined above.)

[0041] In some embodiments, when the ruthenium pre-catalyst and the R-BINAP ligand are combined under an inert atmosphere and then added to the reaction mixture, R-BINAP, i.e., (R)-(2,2'-bis(diphenylphosphin)-1,1'-binaphthyl) and the ruthenium metal form a catalyst in situ. In some embodiments, the ruthenium pre-catalyst is a ruthenium chloride compound such as [RuCl2benzene]2. In some embodiments, the ruthenium pre-catalyst and R-BINAP are combined in a degassed solvent and under an inert atmosphere and heated (e.g., to 100°C) to form an activated catalyst solution. The INT 1 compound is dissolved in a suitable solvent (in an inert environment) to form a reaction mixture, and the activated catalyst solution is added to the reaction mixture. In some embodiments, the INT 1 compound and the activated catalyst are added to an autoclave or pressure vessel, pressurized with hydrogen gas, and heated to form an activated catalyst solution. Compound 2 is formed. In some embodiments, the pressure of the hydrogen gas in the autoclave or pressure vessel is 200 psi or less (e.g., in the range of 100 psi to 200 psi). Also, dead space in the pressure vessel may affect the purity and enantioselectivity of the resulting INT 2 compound. Therefore, in some embodiments, the ratio of dead space in the pressure vessel to the reaction mixture is 3:1 or greater (e.g., 4:1, 5:1, or 6:1). The crude mixture of INT 2 can be further purified by extraction and / or washing, and in some embodiments, it can be recrystallized by dissolving it in an organic solvent and cooled.

[0042] In some embodiments, to evaluate the enantiopurity of the INT 2 compound, the compound can be derivatized to form a moscher ester. In some embodiments, the moscher ester is formed using moscher acid (α-methoxy-α-trifluoromethylphenylacetic acid (MTPA)) with a catalyst (e.g., 4-dimethylaminopyridine (DMAP)) and a dehydrating agent (e.g., dicyclohexylcarbodiimide (DCC)). The moscher ester is then formed 1 By analyzing with 1H NMR, it is possible to determine whether or not the INT 2 compound is enantiomerically enriched.

[0043] In some embodiments, to evaluate the enantiopurity of the INT 2 compound, the compound can be derivatized with benzoyl chloride in the presence of, for example, DMAP and N,N-diisopropylethylamine (DIPEA). The derivative thus formed can be analyzed by chiral HPLC to determine the enantiomeric excess (% ee). In some embodiments, the INT 2 compound has a % ee of at least 90% (90-99% ee), at least 95% (95-99% ee), or at least 97% (97-99% ee).

[0044] [Method step 3] In some embodiments of the present invention, a compound of INT2 is brought into contact with a compound of formula 3-A to form a compound of INT3: [ka] (In the formula, R1 is as defined above, and X2 and X3 are independently halos (e.g., Br or Cl).)

[0045] In some embodiments, the O-acylation reaction of the INT 2 compound is carried out under Schotten-Baumann reaction conditions (a two-phase reaction involving water and an organic solvent such as toluene) in the presence of a catalyst (e.g., DMAP) and potassium bicarbonate (KHCO3). This reaction can be carried out at low temperatures, for example, in the range of 0°C to 15°C. After the reaction is complete, the temperature can be increased and water can be added to the reaction vessel to hydrolyze any unreacted compound 3-A. In some embodiments, the INT 3 compound thus formed can be washed with water and / or the organic phase can be extracted and concentrated.

[0046] [Method step 4] In some embodiments of the present invention, the INT 3 compound is contacted with a Grignard reagent (Reformatski reaction) and cyclized to form the INT 4 compound: [ka] (In the formula, R1 and X3 are as defined above, and INT 4 exists as a tautomer.)

[0047] In some embodiments, the Grignard reagent is a tertiary Grignard reagent such as t-butyl MgBr or t-amyl MgBr. In some embodiments, the reaction mixture containing the INT 3 compound and the Grignard reagent is slowly added to the reactor over time with an organic solvent (e.g., THF) until the reaction forms the INT 4 compound. Optionally, the reaction mixture may be distilled to remove part of the organic solvent, the product may be cooled, and / or a cold citric acid solution may be added to promote precipitation. In some embodiments, the product may be washed and / or recrystallized to purify the INT 4 compound.

[0048] [Method step 5] In some embodiments of the present invention, a compound of INT4 is brought into contact with hydrogen gas in the presence of a Raney Ni catalyst to form a compound of INT5: [ka] (In the formula, R1 is as defined above.)

[0049] In some embodiments, hydrogen gas is present in the reaction vessel at a pressure ranging from 0.5 to 5 atmospheres, and in some embodiments, at approximately 1 atmosphere. In some embodiments, the Raney Ni catalyst is freshly prepared before the reaction, for example, immediately before the reaction (it is not commercially available). In some embodiments, Raney Ni (e.g., freshly prepared Raney Ni) is added to the reaction vessel under inert conditions, and then the INT 4 compound is added to the reaction vessel. Next, hydrogen gas can be passed through the reaction mixture through a gas diffusion sparger while stirring. When the reaction is complete (as determined, for example, by TLC or LCMS), the Raney Ni can be settled and the supernatant can be removed. The product can then be purified, for example, by filtration through a Celite pad and / or by recrystallization from ethyl acetate and heptane, for example.

[0050] [Method step 6] In some embodiments of the present invention, a protecting group is added to the free hydroxyl group in the INT 5 compound by contacting the INT 5 compound with a protecting agent: [ka] (In the formula, R1 is as defined above, and R2 is a protecting group (e.g., THP).)

[0051] In some embodiments, the protecting agent is 3,4-dihydro-2H-pyran (DHP), which gives rise to a tetrahydropyranyl (THP) protecting group. Other base-stability protecting groups, such as trityl or p-methoxybenzyl, which can be removed under relatively mild conditions, may be used. In some embodiments, a weakly acidic catalyst, such as p-toluenesulfonate pyridinium (PPTS), can be added to the INT 5 compound dissolved in a solvent such as THF. The protecting agent (e.g., DHP) can then be added to the reaction flask. In some embodiments, the resulting INT 6 compound can be redissolved in an organic solvent (e.g., MTBE) and washed.

[0052] [Method step 7] In some embodiments of the present invention, the compound of INT 6 is brought into contact with a hydroxide salt to open the lactone ring and form the compound of INT 7: [ka] (In the formula, R1 and R2 are as defined above.)

[0053] In some embodiments of the present invention, the hydroxide salt is NaOH or KOH. In some embodiments, the INT 6 compound is dissolved in an organic solvent (e.g., MTBE) and added to a reaction flask. Next, an aqueous solution of the hydroxide salt (e.g., 2N NaOH) is added to the reaction flask and stirred. When the reaction is complete, the aqueous base layer can be separated and the organic layer can be washed with a brine solution (e.g., 10% NaCl). Next, the INT 7 compound can be concentrated to form a crude oil. In some embodiments, the crude oil is further purified of the INT 7 compound by azeotropic distillation with an organic solvent (e.g., MTBE and THF).

[0054] [Method steps 8 and 9] In some embodiments of the present invention, a compound of INT 7 is brought into contact with a protective agent to add a protecting group to the free hydroxyl group on the compound of INT 7, thereby forming a compound of INT 8. Next, the compound of INT 8 is reacted with an acid to deprotect the protected hydroxyl group, thereby forming a free hydroxyl group, and thereby forming a compound of INT 9: [ka] (In the formula, R1 and R2 are as defined above, and R3 is an acid-stable protecting group such as benzyl or substituted benzyl.) The term "acid-stable protecting group" refers to a protecting group that is not removed by the acid added to INT 8 when forming INT 9.

[0055] In some embodiments of the present invention, the intermediate product INT 8 is not purified before forming the compound INT 9. Therefore, these two reactions can be carried out in a one-pot synthesis. In some embodiments, the protective agent is benzyl bromide, which reacts with the compound INT 7 in the O-benzylation reaction. In some embodiments, the compound INT 7 is stirred in an organic solvent (e.g., THF), a strong base (e.g., sodium tert-butoxide) is added, and then benzyl bromide is added. This reaction can be carried out at a low temperature (e.g., in the range of 5°C to 10°C). After the reaction is complete, the reaction mixture can be heated (e.g., to 50°C). In some embodiments, an aqueous solution of an acid (e.g., a mineral acid such as HCl) is added to the reaction mixture containing the compound INT 8, thereby producing the compound INT 9. In some embodiments, the organic layer and the aqueous layer are separated, the organic layer is washed, filtered, and optionally further purified. In some embodiments, the product is extracted into an organic solvent such as MTBE, and the organic layer is evaporated to obtain the crude INT 9 reaction product. In some embodiments, the crude product may then be redissolved in an organic solvent (e.g., methyl acetate), washed with a saline solution and / or dried, then filtered, and the product sent to the next step. Thus, in some embodiments, the hydroxy THP formed by deprotection in step 9 of the method is removed from the INT 9 product before proceeding to the next step.

[0056] [Method step 10] In some embodiments of the present invention, the compound of INT 9 is contacted with (S)-phenylethylamine to form the compound of INT 10: [ka] (In the formula, R1 and R3 are as defined above.)

[0057] The product can be purified using such optional steps. In some embodiments, (S)-PEA purification can be carried out in an organic solvent, such as methyl acetate. In some embodiments, this reaction is carried out by dissolving the INT 9 compound in an organic solvent such as methyl acetate. The reaction mixture can then be cooled and (S)-(-)-α-methylbenzylamine (also referred to as (S)-(-)-1-phenylethylamine) can be slowly added to the reaction flask. The resulting crystals can then be filtered and washed (for example, with cold methyl acetate).

[0058] In some embodiments, if hydroxy DHP is present in the crude mixture, the INT 10 compound may revert to the PEA salt of INT 8 (also referred to as INT 8-PEA). Therefore, it may be advantageous to remove the hydroxy THP formed in step 9 of the method. If such reversion occurs, the INT 8-PEA can be reacted with an acid to convert the INT 8-PEA compound to the INT 9 compound. The INT 9 compound can then be purified by filtration, extraction, washing, and / or recrystallization (e.g., from methyl acetate). The INT 9 compound can then be converted to the INT 10 compound using the same (S)-PEA reaction procedure as described above.

[0059] [ka]

[0060] [Method step 11] In some embodiments of the present invention, a compound of INT 10 is brought into contact with an acid to form a purified compound of INT 9: [ka] (In the formula, R1 and R3 are as defined above.)

[0061] In some embodiments, the acid is a mineral acid such as HCl (e.g., 1N HCl). In some embodiments, the compound of INT 10 is dissolved in an organic solvent such as heptane, and then an aqueous solution of the acid (e.g., 1N HCl) is added to the reaction mixture. The organic layer is then separated from the aqueous layer, washed, dried, filtered, and / or concentrated to obtain INT 9 (purified).

[0062] [Method step 12] In some embodiments of the present invention, a compound of INT 9 or INT 9 (purified) is brought into contact with a dehydrating agent to form a compound of INT 11: [ka] (In the formula, R1 and R3 are as defined above.)

[0063] In some embodiments, the dehydrating agent comprises one or more of benzenesulfonyl chloride, toluenesulfonyl chloride, or alkylsulfonyl chloride, optionally in the presence of pyridine or substituted pyridine. In some embodiments, the dehydration reaction is carried out at low temperatures (e.g., below 5°C) and / or under an inert atmosphere. In some embodiments, the resulting INT 11 product is then extracted in heptane, washed with an aqueous solution of acid and / or base, washed with a saline solution, dried, filtered, and / or concentrated to produce the INT 11 compound.

[0064] [Method step 13] In some embodiments of the present invention, the compound of INT 11 is deprotected to form a free hydroxyl group and the compound of INT 12: [ka] (In the formula, R3 is as defined above.)

[0065] In some embodiments, R3 is a benzyl group, and deprotection is achieved by a debenzylation reaction. In some embodiments, debenzylation is achieved using a Pd / C catalyst and hydrogen gas. In some embodiments, Pd / C is present in the reaction mixture, and hydrogen gas is blown into the reaction mixture under inert conditions. The resulting crude product containing the INT 12 compound is then filtered and concentrated under reduced pressure to obtain a purified compound of INT 12.

[0066] [Method step 14] In some embodiments of the present invention, a compound of formula I is formed by contacting a compound of INT 12 with N-formyl L-alanine.

[0067] [ka]

[0068] In some embodiments, the INT 12 compound is reacted with N-formyl L-alanine by a Mitsunobu coupling reaction using triphenylphosphine and an azodicarboxylate salt. Similar compounds were synthesized using diisopropyl azodicarboxylate (DIAD), but this reagent did not function well for the synthesis of the compound of formula I. DIAD-H2 was formed as the main byproduct, and the compound of formula I was eluted simultaneously with DIAD-H2, posing a purification problem. Surprisingly, the inventors found that when di-tert-butyl azodicarboxylate (DBAD) was used as the azodicarboxylate salt, the formed DBAD-H2 and the compound of formula I exhibited sufficiently different retention times and were therefore separable by chromatography. Even more surprisingly, the reaction proceeded more rapidly when DBAD was used instead of DIAD. In some embodiments, the reaction of INT 12 with N-formyl L-alanine in the presence of DBAD proceeded about 1.5 to about 2 times (or more) faster than the reaction in the presence of DIAD.

[0069] In some embodiments, the Mitsunobu reaction is carried out under an inert atmosphere. In some embodiments, the INT 12 compound, N-formyl L-alanine, and PPh3 are added to a reaction flask along with an organic solvent such as tetrahydrofuran (THF). The reaction mixture can then be homogenized and cooled to a specified temperature below room temperature (e.g., about 5-10°C). In some embodiments, DBAD is then dissolved in the same organic solvent and slowly added to the reaction flask, for example, dropwise using an addition funnel. DBAD can be added to allow the reaction to proceed at a specified temperature (e.g., about 5-10°C). When complete, the reaction product can be concentrated and resuspended in an organic solvent such as heptane. The heptane layer can then be decanted. MTBE can then be added to the mixture and filtered to obtain the crude product as an oil. In some embodiments, the crude oil is purified by column chromatography (e.g., using ethyl acetate / heptane as the eluent). The combination of fractions from the chromatography can then be concentrated to obtain the oil. In some embodiments, purified crystals of the compound of formula I are added to a crude oil as seed crystals, and product crystals are produced over time. In some embodiments of the present invention, the compound of formula I has a purity of at least 90% (for example, a purity of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, and any range defined in between).

[0070] The methods according to the present invention may further include forming pharmaceutically acceptable salts of compounds of formula I. The term “pharmaceutically acceptable salt” means a salt that retains the biological efficacy and properties of the free base or free acid and is not usually biologically or otherwise undesirable. Salts can be formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, particularly hydrochloric acid, and organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. These salts can also be prepared by adding an inorganic base or an organic base to a free acid. Examples of salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, and magnesium salts. Examples of salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyimine resins.

[0071] The method may further include forming a composition comprising the compound of formula I. In some embodiments, the compound of formula I can be formulated into suitable pharmaceutical formulations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release formulations, or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patches and dry powder inhalers. In one embodiment, the compound is formulated into a pharmaceutical composition by techniques and procedures well known in the art (see, for example, Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

[0072] In the composition, one or more compounds or pharmaceutically acceptable derivatives thereof can be mixed with a suitable pharmaceutical carrier at an effective concentration. The compounds can be derivatized prior to the formulation as corresponding salts, esters, enol ethers or enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs. The concentration of the compounds in the composition may be effective in delivering an amount that treats, prevents, and / or improves pancreatitis or inhibits lipase activity at the time of administration.

[0073] In one embodiment, the composition is formulated for single-dose administration. To formulate the composition, a weight fraction of the compound according to the present invention is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration such that the condition being treated is mitigated, prevented, or one or more symptoms are improved.

[0074] The active compound may be contained in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects on the treated subject. The therapeutically effective concentration can be empirically determined by testing the compound in vitro and in vivo, and then extrapolating this to human administration.

[0075] The concentration of the compound of formula I (also referred to as the "active compound") in a pharmaceutical composition may depend on the absorption rate, inactivation rate, and excretion rate of the active compound, the physicochemical properties of the compound, the administration schedule, and / or the amount administered, as well as other factors well known to those skilled in the art. For example, the amount delivered may be sufficient to inhibit lipase activity and / or treat pancreatitis.

[0076] Pharmaceutical compositions can be realized as unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, as well as oil-in-water emulsions, containing suitable amounts of the compound or pharmaceutically acceptable derivatives thereof for administration to humans and / or animals. In one embodiment, a pharmaceutically therapeutically active compound and its derivatives are formulated and administered in unit dosage forms or multiple dosage forms. As used herein, a unit dosage form means a physically separate unit, suitable for human and animal subjects, and individually packaged as known in the art. Each unit dosage form contains a predetermined amount of the pharmaceutically active compound sufficient to produce the desired therapeutic effect, in combination with the necessary pharmaceutically active compound, medium, or diluent. Examples of unit dosage forms include ampoules and syringes, as well as individually packaged tablets or capsules. A unit dosage form may be administered in fractions or more. Multiple dosage forms refer to multiple identical unit dosage forms packaged in a single container to be administered as isolated unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or bottles in pint or gallon units. Thus, multiple dosage forms are multiple unit dosage forms that are not isolated within the packaging.

[0077] Pharmaceutically administerable liquid compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing an active compound as defined herein and optional pharmaceutical adjuvants in a carrier such as water, saline solution, glucose solution, glycerol, glycol, or ethanol to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain trace amounts of non-toxic adjuvants, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc., such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other similar agents.

[0078] The practical methods for preparing these dosage forms will be known or obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.

[0079] In some embodiments, compositions according to the present invention may be suitable for oral administration. Pharmaceutical oral dosage forms may be solid, gel, or liquid. Solid dosage forms may be tablets, capsules, granules, and mixed powders. Types of oral tablets include compressed chewable lollipops and tablets, which may be enteric-coated, sugar-coated, or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be realized in non-foaming or effervescent forms by combinations of other components known to those skilled in the art.

[0080] In a particular embodiment, the formulation is a solid dosage form, and in one embodiment, it is a capsule or tablet. Tablets, pills, capsules, lozenges, etc., may contain one or more of the following components or compounds having similar properties: binders; lubricants; diluents; flow enhancers; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; and film coatings. Examples of binders include crystalline cellulose, tragacanth gum, glucose solution, gum arabic viscosa, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidone, sucrose, and starch paste. Examples of lubricants include talc, starch, magnesium stearate or calcium stearate, clubmoss, and stearic acid. Examples of diluents include lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Examples of flow enhancers include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Colorants include, for example, any of the approved and certified water-soluble FD dyes and C dyes, or mixtures thereof; as well as water-insoluble FD dyes and C dyes suspended on alumina hydrate. Sweeteners include artificial sweeteners such as sucrose, lactose, mannitol, and saccharin, as well as any number of spray-dried flavors. Flavorings include, but are not limited to, peppermint and methyl salicylate, natural flavors extracted from plants such as fruits, and synthetic blends of compounds that produce a pleasant sensation. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Examples of emetic coatings include fatty acids, fats, waxes, shellac, ammonia-treated shellac, and cellulose phthalate acetate.Examples of film coatings include hydroxyethylcellulose, gellan rubber, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0081] Compounds of formula I, or pharmaceutically acceptable derivatives thereof, can be realized in compositions that protect them from the acidic environment of the stomach. For example, a composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestines. Alternatively, the composition can be formulated in combination with antacids or other similar components. If the unit dosage form is a capsule, it may contain a liquid carrier such as fatty oil in addition to the above-mentioned substances. The unit dosage form may also contain various other substances that modify the physical form of the unit dosage form, such as sugar coatings and other enteric coatings. The compound can be administered as a component of elixirs, suspensions, syrups, wafers, sprinkles, chewing gums, etc. Syrups may contain, in addition to the active compound, sucrose as a sweetener, as well as certain preservatives, colorants, and flavorings.

[0082] The active substance may be mixed with other active substances that do not impair the desired effect, or with substances that supplement the desired effect, such as antacids, H2 blockers, and diuretics. The active ingredient is a compound described herein or a pharmaceutically acceptable derivative thereof. High concentrations of the active ingredient may be up to approximately 98% by weight.

[0083] In all embodiments, the tablet and capsule formulations can be coated as known to those skilled in the art to modify or prolong the solubility of the active ingredient. For example, they can be coated with common enterally digestible coatings such as phenyl salicylate, wax, and cellulose phthalate acetate.

[0084] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent formulations reconstituted from effervescent granules. Examples of aqueous solutions include elixirs and syrups. Emulsions are oil-in-water or water-in-oil.

[0085] An elixir is a clear, sweetened aqueous alcohol preparation. A pharmaceutically acceptable carrier used in an elixir is a solvent. A syrup is a concentrated aqueous solution of sugar, such as sucrose, and may contain preservatives. An emulsion is a two-phase system in which one liquid is dispersed throughout another liquid in the form of small spheres. Pharmacochemically acceptable carriers used in emulsions include non-aqueous liquids, emulsifiers, and preservatives. In suspensions, pharmaceutically acceptable suspending agents and preservatives are used.

[0086] Pharmaceutically acceptable substances used in non-foaming granules reconstituted into liquid oral dosage forms include diluents, sweeteners, and humectants. Pharmaceutically acceptable substances used in effervescent granules reconstituted into liquid oral dosage forms include organic acids and carbon dioxide sources. Colorants and flavorings are used in all of the above dosage forms. Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Preservatives include, for example, glycerin, methylparaben and propylparaben, benzoic acid, sodium benzoate, and alcohol. Non-aqueous liquids used in emulsions include, for example, mineral oil and cottonseed oil. Emulsifiers include, for example, surfactants such as gelatin, gum arabic, tragacanth, bentonite, and polyoxyethylene sorbitan monooleate. Suspensioning agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, veegum, and gum arabic. Sweeteners include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Carbon dioxide sources include sodium bicarbonate and sodium carbonate. Colorants include any approved and certified water-soluble FD dyes and C dyes, as well as mixtures thereof. Flavorings include natural flavors extracted from plants such as fruits, and synthetic blends of compounds that produce a pleasant taste. In solid dosage forms, for example, a solution or suspension in propylene carbonate, vegetable oil, or triglycerides is encapsulated in a gelatin capsule in one embodiment. In liquid dosage forms, the solution may be diluted with a sufficient amount of pharmaceutically acceptable liquid carrier, such as water, for easy measurement for administration.

[0087] Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oil, glycol, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other similar carriers, and then encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.

[0088] Other formulations include, but are not limited to, aqueous alcohol solutions containing pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Examples of acetals include, but are not limited to, di(lower alkyl) acetals of lower alkylaldehydes, such as acetaldehyde diethyl acetal.

[0089] Parenteral administration of the composition may include intravenous, subcutaneous, and intramuscular administration. Administration may be intraperitoneal, or direct to or near the organ or tissue of interest, such as the pancreas. Parenteral formulations include sterile solutions ready for injection; sterile dry soluble products, such as lyophilized powders, including tablets for subcutaneous injection, that are ready to be combined with a solvent immediately before use; sterile suspensions ready for injection; sterile dry insoluble products, that are ready to be combined with a medium immediately before use; and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0090] When administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0091] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, metal ion sequestering or chelating agents, and other pharmaceutically acceptable substances.

[0092] Examples of aqueous media include sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, and dextrose and lactated Ringer's injection. Examples of non-aqueous parenteral media include hydrogenated plant oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Parenteral formulations packaged in multi-dose containers must be supplemented with antimicrobial agents, including phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride, at bacteriostatic or fungiostatic concentrations. Examples of isotonic agents include sodium chloride and glucose. Examples of buffering agents include phosphates and citrates. Examples of antioxidants include sodium bisulfate. Examples of local anesthetics include procaine hydrochloride. Examples of suspending and dispersing agents include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Examples of emulsifiers include polysorbate 80 (TWEEN® 80). Examples of metal ion sequestering or chelating agents include EDTA. Examples of pharmaceutical carriers for water-miscible media include ethyl alcohol, polyethylene glycol, and propylene glycol; for pH adjustment, examples include sodium hydroxide, hydrochloric acid, citric acid, or lactic acid.

[0093] By suspending the compound of formula I in a micronized form or other suitable form, or by derivatization, a more soluble effective product or prodrug can be produced. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the compound in the selected carrier or medium. The effective concentration is one that is sufficient to improve the symptoms of the condition and can be determined empirically.

[0094] The present invention may be carried out using lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solid or gel formulations.

[0095] Sterile, lyophilized powders are prepared by dissolving the compounds provided herein or their pharmaceutically acceptable derivatives in a suitable solvent. The solvent may contain excipients that improve the stability of other pharmacological components of the powder or the reconstituted solution prepared from the powder. Available excipients include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may include a buffer near neutral pH, such as citrate, sodium phosphate, or potassium phosphate, or other similar buffers known to those skilled in the art. Subsequently, the desired formulation is obtained by sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art. In one embodiment, the resulting solution is partitioned into vials for lyophilization. Each vial contains one or more doses of the compound. The lyophilized powder can be stored under suitable conditions, for example, about 4°C to room temperature.

[0096] Furthermore, in one embodiment of the present invention, a compound of formula I formed by the method according to the present invention is provided. Also provided is a pharmaceutically acceptable salt of the compound of formula I formed by the method according to the present invention. Furthermore, a composition comprising the compound of formula I prepared by the method according to the present invention, and optionally a pharmaceutically acceptable carrier, is provided.

[0097] Furthermore, the compounds, pharmaceutically acceptable salts, and / or compositions according to the present invention can be used to inhibit lipase activity and / or treat pancreatitis in a subject. The term "subject" includes both humans and animals.

[0098] The present invention will be described in more detail in the following non-limiting experimental section. [Examples]

[0099] The labeling / numbering of compounds shown in the Examples section applies only to the Examples section and may not correspond to labeling / numbering shown elsewhere in this application. Therefore, the labeling / numbering of compounds in the Examples section should not be confused with the labeling / numbering of compounds elsewhere in this application (e.g., in the Summary and Detailed Description sections and in the Claims).

[0100] Abbreviations may include round-bottom flask (RBF), starting material (SM), room temperature (RT), dichloromethane (DCM, or CH2Cl2), ethyl acetate (SiO), hexane (hex), methanol (MeOH), isopropanol (IPA), diethyl ether (Et2O), acetic acid (AcOH), 1,2-dichloroethane (1,2-DCE), tetrahydrofuran (THF), dimethylformamide (DMF), cesium carbonate (Cs2CO3), sodium sulfate (Na2SO4), and silica (SiO2).

[0101] [Example 1] Preparation of INT 1

[0102] [ka]

[0103] A 30L jacketed vessel was equipped with a vacuum-rated stirrer bearing, argon gas inlet and outlet, a connected heating / cooling device, and a thermocouple / control device. The vessel was purged with argon for 15 minutes. Methanol (3.85L) and then magnesium (120g, approximately 50 mesh) were added to the vessel. The sides of the vessel were rinsed with methanol, and the vessel was heated to 55°C, then to 65°C. The reaction mixture was stirred overnight at 65°C. Toluene (11.6L) was added to the vessel, and a distillation head was attached to the vessel. The temperature of the circulating solution was increased to 100°C, and approximately 1.5L of the solvent was distilled. The temperature of the circulating solution was decreased to 45°C, and methyl acetoacetate (2.2Kg) was added to the vessel at 45°C. The temperature was increased by 10°C during the addition. The reaction mixture was stirred at 45°C for 12 hours. The temperature of the circulating solution was gradually increased to 140°C, and the solvent was distilled to approximately 4.9 L. The temperature of the condenser was set to 60°C. Lauroyl chloride (1608 g) was dissolved in toluene (1.55 L). The solution was transferred to an addition funnel and added to a 30 L container over 2-3 hours. The reaction mixture was stirred at 60°C for 12 hours.

[0104] 1. Stop the reaction Methanol (1.8 L) was added to the reaction mixture. The color of the reaction mixture changed from orange to red, and all solids dissolved. The reaction mixture was distilled to approximately 900 ml using a distillation head and vacuum pump. The reaction mixture was stirred at 75°C until most of the tricarbonyl intermediate was converted to the product on TLC. The reaction mixture was cooled to 25°C and concentrated HCl (1502 g) was added. The reaction mixture changed from orange to yellow.

[0105] 2. Workup and Isolation The reaction was stopped and the HCl layer was separated. The organic layer was washed with water (2 × 3.5 L), 2% KHCO3 (1 × 2.8 L), and water (1 × 2.8 L). The organic layer was dried over anhydrous Na2SO4 (approximately 300 g) for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain an orange oily substance (2018 g, over 100%).

[0106] 3. Crystallization The crude product was dissolved in methanol (6054 mL) and cooled in a freezer to crystallize. The crystals were filtered through a P3 frit funnel and air-dried for 2 hours. The solid was transferred to a tray and dried in an oven at room temperature until a uniform mass was reached. INT 1 was isolated as a flaky white to yellowish-white solid (1248 g, 66%). Figure 2 shows the results for INT 1. 1 The 1H NMR spectrum is shown.

[0107] [Example 2] Preparation of INT 2

[0108] [ka]

[0109] The enantioselectivity in this reaction was found to depend on several factors, including solvent, temperature, and pressure. In particular, R&D experiments confirmed that the ratio of reaction volume to reactor dead space is a major parameter in low-pressure hydrogenation (less than 1000 psi). In a 2 L reactor volume, the reaction scale was fixed at INT 1 150 g or less (at 200 psi) to avoid hydrogen deficiency conditions associated with decreased enantioselectivity.

[0110] 1. Enantioselective synthesis of INT 2 DMF and methanol were degassed with argon for 30 minutes. [RuCl2benzene]2 (1.95 g, 3.89 mmol, Sigma), R-BINAP (2.7 g, 4.33 mmol, Sigma), and degassed DMF (95 mL, Sigma) were added to a three-necked flask that had been purged three times with evacuated air and argon. The reaction mixture was heated to 100°C, stirred for 30 minutes, and then cooled to room temperature.

[0111] INT 1 (150 g, 1.1 mol) was dissolved in degassed methanol (325 mL). The mixture was transferred to a 2 L autoclave and degassed with argon for 15 minutes. The activated catalyst prepared above was transferred to the autoclave while purging with argon. The autoclave was evacuated, purged three times with hydrogen, and then filled with hydrogen (200 psi). The reaction mixture was heated to 100 °C and stirred for 24 hours.

[0112] 2. Workup and Isolation TLC and LC-MS of the reaction mixture suggested completion of the reaction. The reaction mixture was filtered through a Celite bed, and the bed was washed with ethyl acetate (100 mL). The filtrate was concentrated under reduced pressure to obtain an oily substance.

[0113] The crude oily substance was dissolved in ethyl acetate (10 vol.) and water (10 vol.) was added. The organic layer was separated and washed with saturated NaCl (2×). The organic layer was dried over anhydrous Na₂SO₄ for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain a yellowish-white solid. The reaction to prepare INT 2 was repeated through 7 iterations.

[0114] 3. Crystallization The crude product (960 g) was placed in a round-bottom flask, and heptane (5 vols) was added. The mixture was heated to 65-70°C to form a homogeneous solution. The solution was cooled to room temperature, and then to 0-5°C. The flask was kept in a refrigerator overnight to allow more crystals to form. The crystals were collected on a coarse-grained frit funnel and then air-dried and vacuum-dried for 3 hours to obtain a yellowish-white solid (870 g, 90% recovery).

[0115] [Example 3] Derivatization of INT 2

[0116] [ka]

[0117] INT 2 (10 mg), moscheric acid (9.1 mg), DMAP (0.004 mg), DCC (12 mg), DCM (1 mL), and a magnetic stirring bar were added to a 10-drum vial. The homogeneous solution was stirred overnight at room temperature. The reaction mixture was filtered and the mother liquor was evaporated. The crude product was then prepared. 1 The sample was subjected to 1H NMR.

[0118] As shown in Figure 3, the presence of two methyl singlets between 3.5 and 3.7 suggests that INT 2 is enantiomerically enriched (a racemic mixture indicates the presence of two sets of two singlets; see Figure 4). A chiral HPLC method was also developed to measure the enantiomer excess (ee).

[0119] [Example 4] Derivatization of INT 2

[0120] [ka]

[0121] INT 2 (50 mg), DMAP (2.3 mg, Aldrich), DIPEA (37.5 mg), benzoyl chloride (54.4 mg, Aldrich), DCM (0.5 mL), and a magnetic stirring bar were added to a 10-drum vial. The vial was stirred at room temperature for 16 hours. Aliquots (10 μL) were dissolved in 10% HPLC-grade isopropyl alcohol hexane solution (1 mL, isopropyl alcohol; hexane) and subjected to chiral HPLC. The chiral HPLC data (Figure 5) suggest that the batch was enantiomerically enriched with an enantiomer excess percentage (%ee) of 97%.

[0122] [Example 5] Preparation of INT 3

[0123] [ka]

[0124] A three-necked 12L RBF was equipped with a cooling bath with a coil, a thermocouple / control device, and an overhead stirrer. When INT 2 (360g, 1.39mol, 1 equivalent), DMAP (17g, 0.139mol, 10mol%), and toluene (720mL, 2 volumes) were added, endothermic reactions (T: 17℃~4.6℃) were obtained. All solids dissolved when the temperature was raised to approximately 13℃. The condenser was set to 4℃, and propylene glycol was added to the cooling bath. The bath was rapidly cooled to 10~15℃ using dry ice, and the temperature was maintained using the condenser. KHCO3 (474g, 4.74mol, 3.4 equivalents) and water (176mL, 9.75mol, 7 equivalents) were added to the RBF. When the mixture was cooled to 15-10°C, the first dose of acid bromide (255 mL, 2.09 mol, 1.5 equivalents, density: 1.88 g / mL, Oakwood) was added over 3 hours using an addition funnel. CO2 generation began when the acid bromide was added and stopped approximately 30 minutes after the addition was complete. TLC showed that the reaction was incomplete and 10-30% of the starting materials remained. The second dose of acid bromide (130 mL, 1.05 mol, 0.75 equivalents, Oakwood) was added to the addition funnel and adjusted to be slowly added dropwise overnight at 10-15°C.

[0125] 1. Workup and Isolation The following morning, approximately 10 mL of acid bromide remained in the addition funnel. The remaining reagent was released into the reaction mixture, and TLC and LC / MS (22 hours) confirmed that the reaction was complete. The cooling bath was replaced with a water bath at approximately 40°C. Water (1.8 L, 5 vols) and MTBE (1.8 L, 5 vols, Aldrich) were added to the reaction mixture, and the temperature was raised to 25-30°C. It was then stirred for 30 minutes to hydrolyze any unreacted acid bromide. When stirring was stopped and the layers separated into multiple phases, the pH was approximately 8. The water bath was removed, and the aqueous phase was siphon-aspirated from a 12 L RBF. The organic phase was washed with water (2 ×, 720 mL, 2 vols) until the pH of the washing solution was pH 7. The organic phase was transferred to a 5 L RBF (tare weight) (all residual water in the 12 L RBF was removed using a 500 mL separatory funnel) and concentrated under reduced pressure at 30–60°C. The sediment was suspended in the product and diluted with toluene (200 mL). It was polished filtered through tare weight coarse-grained frit (approximately 800 mg of wet solid remained) and placed in a 3 L round-bottom flask (tare weight). The flask and frit were then rinsed with a minimum amount of toluene. The solvent was removed again under reduced pressure at 60°C for 2–3 hours to obtain INT 3 as a dark brown oily substance (590.76 g, yield 104%). Figure 6A shows the TLC of INT 3 under a UV lamp, and Figure 6B shows the TLC under PMA staining. The eluent was 20% ethyl acetate / heptane. Figure 7 shows the crude INT 3. 1 This shows the 1H NMR spectrum.

[0126] [Example 6] Preparation of INT 4

[0127] [ka]

[0128] A four-necked 22L RBF was equipped with an overhead stirrer, a heated mantle, two 1L addition funnels, each with a gas inlet, and a thermocouple / control device. A minimum amount of THF (1000mL, 1.75 vol.) was added to the RBF so that the solvent was in contact with the thermocouple. The control device was set to 60°C and the argon flow was started. After 20-30 minutes, INT 3 (570g, 520mL, 1.40mol, 1 vol.) was heated to approximately 40°C to reduce its viscosity and added to the 1L funnel. 1M tert-butyl Grignard (4.8L, 4.9 equivalents, 3.5 equivalents) was added in 800mL bottles, by removing the Sure Seal caps and pouring them into the argon-purged addition funnel.

[0129] When the temperature reached 55-60°C, approximately 20 mL of Grignard reagent was added to the heated THF to deoxygenate and dehydrate the solvent. The reagent solution was simultaneously added to the heated THF continuously over 4 hours (800 mL of Grignard and approximately 85 mL of INT 3 were dispensed every 40 minutes), during which time isobutylene was actively formed. Once the addition was complete, the reaction mixture was stirred at 60°C. TLC (6.5 hours) showed that INT 3 had been consumed, but the reaction mixture was allowed to mature until the 8-hour mark, and the heating was turned off.

[0130] 1. Workup The following morning, LC-MS confirmed that SM had been consumed. The reflux condenser was replaced with a distillation head, and the temperature was set to 75°C. The solvent was distilled until 2.3 L (46%, 4 vols) of THF was collected, leaving approximately 2.7 L (4.7 vols) in the still pot. The heating mantle was replaced with an ice bath, and the reaction vessel was then cooled to 0-10°C. Cold (10°C) 0.24 M citric acid (9 L, 1 mol, 1.4 equivalents, 14 vols) was added over 20 minutes (T: 8-40°C), with exothermic reaction occurring upon the addition of the first 500 mL. The product began to precipitate, and after stirring the mixture for 1.5 hours, the larger particles decomposed, making the solid easier to filter. The solid was filtered through 3 L of coarse frit at tare weight and washed with water (8 × 1 L, 1.8 vols) until the washing solution was pH 6-7. The filtered cake was dried under reduced pressure for 72 hours to obtain the crude product as a water-moistened brown solid (642.92 g, yield 155%). The moistened cake was suspended in toluene (2 L, 3 volumes) in 5 L RBF, and the resulting mixture was then distilled under reduced pressure at 50–70°C. After adding toluene (300 mL), the distillation was repeated twice. A total of 180 mL of water was collected in the distillate.

[0131] 2. Crystallization The solid in the evaporating flask was transferred to a 22 L four-necked RBF equipped with a heating mantle, thermocouple / control device, reflux condenser, and overhead stirrer, using warm toluene (3 × 500 mL; total: 1.5 L, 2.7 vol). The mixture was heated to 80°C, and heptane (3 L, 5.3 vol) was added at a rate that maintained the temperature at 70–80°C. Once the addition was complete, heating was turned off, and the mixture was allowed to crystallize while cooling overnight to ambient temperature.

[0132] The following morning, the obtained slurry was filtered through 3 L of coarse frit by tare weight, washed with 30% toluene / heptane (3 × 666 mL), and dried for 5-10 minutes. It was then placed in a 55°C oven until it reached a constant mass, yielding a yellowish-white solid (269.5 g, yield 65%). Figures 8A and 8B show the TLC of INT 4 (Figure 8A - crude product (IPC checked); Figure 8B - crystallized product). The eluent was 50% ethyl acetate / heptane. PMA staining was used. Figure 9 shows the TLC of INT 4.1 It exhibits 1H NMR (keto-enol tautomerism).

[0133] [Example 7] Preparation of INT 5

[0134] [ka]

[0135] In a 50 L three-necked RBF equipped with a thermocouple, pneumatic overhead stirrer, and gas diffusion sparger, freshly prepared Raney Ni (approximately 700 g, 150 wt%) was stirred under argon in THF (12.9 L, 20 vol). See Figure 10 for the reaction configuration. INT 4 (648 g, 2.18 mol, 1 equivalent) was added, and hydrogen was passed directly through the reaction mixture via the gas diffusion sparger. The reaction mixture was then vigorously stirred overnight.

[0136] After 18 hours of stirring, the hydrogen flow was stopped, and then argon was passed through the system for 5–10 minutes before the reaction mixture was opened to the atmosphere. The sample was then removed for ion-pair chromatography (TLC and LC-MS). LC-MS did not detect the starting materials, but TLC indicated that the starting materials were present. Since the product was observed to be beginning to crystallize in the sparger, it was rinsed 3–4 times with THF to partially dissolve it and improve the gas flow. The reaction was then restarted by resuming the hydrogen flow and stirring. This was stirred again for another 24 hours. Both TLC and LC-MS showed completion at the 18-hour mark, and the reaction was considered complete.

[0137] 1. Preparation of Celite pads A 1-2 cm layer of sand was added to 2 L of coarse frit and leveled. Separately, a slurry of 100 wt% Celite (500 g, AW standard Super-Cel NF; Sigma Aldrich) in a minimum amount of THF was prepared. A sheet of filter paper was placed on top of the sand, and the slurry was added onto the filter paper to form a 1-2 cm layer of Celite. The Celite was allowed to settle, and then a light vacuum was applied to form a dense Celite pad.

[0138] 2. Ra-Ni removal and product isolation When the reaction was complete, stirring was stopped and Ra-Ni was allowed to settle at the bottom of the round-bottom flask. The supernatant was transferred to a 4 L vacuum flask via a vacuum siphon to remove the minimum amount of Ra-Ni. The flask was then poured onto a Celite pad and filtered. At all times, any remaining filtered Ra-Ni was moistened with THF. THF (4 L) was added to the reaction round-bottom flask containing the consumed Ra-Ni, then stirred and allowed to settle. This was then siphoned as before and filtered through Celite, and this process was repeated until no product was detected (TLC) in the supernatant. THF rinse (2 × 2 L). The product solution was concentrated under reduced pressure by rotovap to obtain the crude product as a white solid (769 g).

[0139] 3. Crystallization A 12L three-necked RBF was equipped with an overhead stirrer, thermocouple, and heating mantle. Solid from a 20L evaporating flask was added to the 12L RBF. Residual solid in the 20L evaporating flask was removed with methoxy (3250 mL, 5 vols) and added to the crystallization flask. Stirring was started and the mixture was heated to 75°C, and the solid was dissolved at 60-70°C. Heptane (7800 mL, 12 vols) was added in several portions to bring the temperature to 70-80°C. Heating was then stopped, and the mixture was allowed to cool to ambient temperature over the weekend.

[0140] The obtained slurry was filtered through tare weight coarse frit, the wet cake was washed with heptane (1000 mL), and then dried under reduced pressure for 15-30 minutes. It was then placed in a 35°C vacuum oven until a constant mass was obtained as INT 5 as a yellowish-white solid (453 g, yield 69%). Figures 11A and 11B show TLCs of crude product INT 5 (Figure 11A) and crystallized INT 5 (Figure 11B). Figure 12 shows the crystallized INT 5. 1 This shows the 1H NMR spectrum.

[0141] [Example 8] Preparation of INT 6

[0142] [ka]

[0143] A three-necked 12L RBF was equipped with an overhead stirrer, heating mantle, argon gas inlet and outlet, and thermocouple / control device. INT 5 (452g, 1.5mol) was added to the flask, followed by THF (4.5L, 10 volumes), and the mixture was stirred at room temperature. The solid was partially dissolved at room temperature. Pyridinium p-toluenesulfonate (5.7g, 0.015 equivalents, 0.023mol) was added to the flask. 3,4-dihydro-2H-pyran (382.2g, 3 equivalents, 4.54mol, Aldrich) was added dropwise to the reaction mixture over 1 hour. The reflux condenser was set to 8°C, and the reaction mixture was heated at 50°C for 24 hours.

[0144] 1. Workup and Isolation TLC and LC-MS confirmed the completion of the reaction (see Figure 13). The reaction mixture was transferred to a 22 L rotavap flask and concentrated under reduced pressure. The resulting crude oil was dissolved in MTBE (4 L). The organic layer was washed with water (3 × 2 L) and then with saturated NaCl (1 × 2 L). The organic layer contained trace amounts of water, which were carried over to the next step.

[0145] [Example 9] Preparation of INT 7 A three-necked 12L RBF was equipped with an overhead stirrer, a cooling bath, and a thermocouple / control device. INT 6 (579g, 1.5mol) dissolved in MTBE (4.6L, ​​8 volumes) from the previous step was added to the flask. A 2N NaOH solution was prepared using a 32% NaOH solution. 2N NaOH (1.3L, 2.25 equivalents) was added to the reaction mixture and vigorously stirred at room temperature for 20 hours.

[0146] 1. Workup and Isolation LC-MS confirmed the completion of the reaction (see Figure 14). The reaction was stopped and the 2N NaOH layer was separated. The organic layer was washed with 10% NaCl (3 × 2 L). The organic layer was dried over anhydrous Na₂SO₄ (approximately 300 g) for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain an oily substance. The crude oily substance was azeotropically distilled with MTBE (2 × 2 L) and THF (2 × 2 L). At the end of evaporation, all the oily substance was a mixture of solid lumps and powder. The crude mixture was maintained under reduced pressure overnight for further drying (694 g, over 100%).

[0147] [Example 10] Preparation of INT 9

[0148] [ka]

[0149] A three-necked 22L RBF was equipped with an overhead stirrer, cooling bath, argon inlet and outlet, and thermocouple / control device. INT 7 (639g; 1.5mol; large lumps divided into smaller pieces) and powder were added to a flask. THF (6.3L, 10 volumes) was added to the flask and vigorously stirred at room temperature. The slurry mixture was cooled to 5-10°C. Sodium tert-butoxide (290.6g, 3.0mol, 2 equivalents) was added to the reaction mixture in several portions over 1 hour. The reaction mixture was stirred at 5-10°C for 1 hour and 30 minutes. During the stirring period, the slurry became cloudy and the color of the reaction mixture changed from light orange to dark orange. Benzyl bromide (388g, 270mL, 1.5 equivalents, 2.3mol) was diluted with THF (250mL) and added to the reaction mixture over 1 hour while maintaining the temperature at 5-10°C. The ice bath was removed, and the reaction mixture was stirred at room temperature for 20 hours.

[0150] LC-MS data suggested that the reaction had stopped with 50% of the starting material remaining. A small aliquot was removed and the reaction was attempted at 50°C. After 1 hour, the trial LC-MS data suggested that the reaction was complete. The cooling bath was replaced with a heated mantle for the reaction, and the mixture was heated at 50°C over the weekend. Subsequent LC-MS data suggested that the reaction was complete. Figure 15 shows the LC-MS of INT 8.

[0151] The heating was stopped, and the reaction mixture was stirred while cooling to room temperature. 2N HCl (2.5 L, 4 volumes) was added to the pot over 1 hour, maintaining the temperature below 45°C. The slurry reaction mixture became a clear solution. The reaction mixture was heated at 50°C for 5 hours. The heating was removed, and the resulting mixture was stirred overnight and allowed to cool.

[0152] 1. Workup and Isolation LC-MS confirmed the completion of the reaction (see Figure 16). MTBE (5 L) was added to the reaction mixture and stirred for 15 minutes. Stirring was stopped to allow the layers to settle, and the 2N HCl layer was separated. The organic layer was washed with saturated NaHCO3 (4 × 4 L) to pH 8-9. The organic layer was dried over anhydrous Na₂SO₄ (approx. 300 g) for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain a thick brick-red oil (783 g, over 100%, containing trace amounts of solvent). The crude oil was redissolved in ethyl acetate (4 L). The organic layer was washed with 0.5N HCl (1 L), then water (2 × 2 L), and brine (1 × 2 L). The organic layer was dried over anhydrous Na₂SO₄ (approx. 200 g) for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain a thick brick-red oil (765 g, over 100%, containing trace amounts of solvent).

[0153] The workup procedure can be modified to avoid the reverse reaction to INT 8-PEA. When the reaction with 2N HCl is complete, the product can be extracted into MTBE, and the organic layer can be evaporated to obtain the crude product. The crude product can then be redissolved in methyl acetate, washed with water and brine, and dried over Na2SO4. The organic layer (methyl acetate) can be filtered and carried over to the next step (INT 10).

[0154] [Example 11] Preparation of INT 10

[0155] [ka]

[0156] A three-necked 12L RBF was equipped with an overhead stirrer, cooling bath, argon inlet and outlet, and thermocouple / control device. INT 9 (615g, 1.5mol) in a 22L Rotabap flask was dissolved in methyl acetate (3.7L, 6 volumes) and transferred to the 12L RBF. The reaction mixture was stirred and cooled to 5-10°C. (S)-(-)-α-methylbenzylamine ((-)PEA, 183.3g, 195mL, Chem-Impex) was added dropwise to the reaction mixture via an addition funnel, and the temperature was maintained at 5-10°C. The concentrated solid was stirred at room temperature for 16 hours.

[0157] 1. Crystallization and Isolation The obtained crystals were cooled to 5-10°C and stirred for 2 hours. The crystals were collected on a coarse-grained frit funnel, and the solid was washed with cold methyl acetate (1 L). The crystals were air-dried for 1 hour, and then vacuum-dried at room temperature until a constant weight was reached. INT 10 was isolated as a bright yellow solid (472.7 g). Figure 17 shows the LS-MS spectrum of INT 10.

[0158] LC-MS data of the mother liquor (Figure 18) showed that some of the INT 10 was converted back to the PEA salt of INT 8 (INT 8-PEA). This is likely due to the presence of DHP in the crude product. A test reaction was carried out using INT 8-PEA, as shown in the scheme below, to convert it to INT 9 and then to INT 10. The test reaction was successful, and the residue was converted from INT 8-PEA to INT 10 by this method.

[0159] The conversion from mother liquor (INT 8-PEA) to INT 10 is shown below.

[0160] [ka]

[0161] 2. From INT 8-PEA (derived from mother tincture) to INT 10 A three-necked 12L RBF was equipped with an overhead stirrer, a heating mantle, and a thermocouple / control device. INT 8-PEA (SCR410-18B, 480g, 0.7mol) in a 22L Rotabap flask was dissolved in MTBE (2.4L, 5 volumes) and transferred to the 12L RBF. 2N HCl (2.5L, 4 volumes) was added to a pot, and the reaction mixture was heated at 50°C for 16 hours. The heating was removed, and the mixture was stirred while cooling to room temperature.

[0162] LC-MS confirmed that the reaction from INT 8-PEA to INT 9 was complete (see Figure 19). Stirring was stopped and the layers were allowed to settle, and the 2N HCl layer was separated. The organic layer was washed with saturated NaHCO3 (3 × 1 L) to pH 8-9. Next, the organic layer was dried over anhydrous Na2SO4 (approx. 300 g) for 1 hour, filtered, and concentrated under reduced pressure to obtain a concentrated brick-red oil (430 g). The crude oil was redissolved in methyl acetate (1.5 L, Aldrich). The solution was washed with 0.5N HCl (1 × 500 mL, VWR), then with water (2 × 500 mL) and brine (1 × 500 mL). The organic layer was dried over anhydrous Na2SO4 (approx. 100 g) for 1 hour, then filtered, and the Na2SO4 cake was washed with methyl acetate (500 mL). The combined filtrate (INT 9) was carried over to the next step.

[0163] A three-necked 5L RBF was equipped with an overhead stirrer, cooling bath, argon inlet and outlet, and thermocouple / control device. INT 9 dissolved in methyl acetate (2L) was transferred to the 5L RBF. The reaction mixture was stirred and cooled to 5-10°C. (S)-(-)-α-methylbenzylamine (95.4g, Chem-Impex) was added dropwise to the reaction mixture using an addition funnel, while maintaining the temperature at 5-10°C. The resulting concentrated slurry was stirred at room temperature for 16 hours.

[0164] The slurry of crystals was cooled to 5 - 10 °C and stirred for 1 hour. The crystals were filtered through a coarse - grained fritted funnel, and the solid was washed with cold methyl acetate (200 mL). The crystals were air - dried for 1 hour and then vacuum - oven dried at room temperature until a constant weight was obtained. INT 10 was isolated as a bright yellow solid (196.4 g). The LS - MS spectrum is shown in Figure 20.

[0165] 3. Recrystallization of INT 10 A 5 L three - necked RBF was equipped with an overhead stirrer, a heating mantle, an argon inlet and outlet, and a thermocouple / controller. INT 10 (674 g) was transferred into the 5 L RBF. Methyl acetate (2.7 L, 5 volumes) was added to the pot and stirred at room temperature. The slurry mixture was heated to 50 °C to completely dissolve all the solids. When the mixture became homogeneous, the heating was turned off, and the mixture was stirred overnight and cooled to room temperature.

[0166] The crystals were cooled to 5 - 10 °C and stirred for 1 hour. The crystals were filtered through a coarse - grained fritted funnel, and the solid was washed with cold methyl acetate (500 mL). The crystals were air - dried for 1 hour and then vacuum - oven dried at room temperature until a constant weight was obtained. INT 10 was isolated as a pale yellow - white solid (589 g, recovery rate 87%). The LC - MS of the recrystallized INT 10 is shown in Figure 21.

[0167] [Example 12] Preparation of Purified INT 9

[0168] [Chemical formula]

[0169] A 12 L three - necked RBF was equipped with an overhead stirrer, a cooling bath, and a thermocouple / controller. INT 10 (580 g, 1.1 mol) was added to the flask, followed by heptane (5.8 L, 10 volumes), and stirred at room temperature. 1 N HCl was prepared using 2 N HCl. 1 N HCl (1160 mL, 2 volumes) was added to the reaction mixture and stirred at room temperature for 16 hours.

[0170] 1. Workup and Isolation LC-MS confirmed the completion of the reaction (see Figure 22). Stirring was stopped and the 1N HCl layer was separated. The organic layer was washed with water (3 × 1 L) and dried with anhydrous Na₂SO₄ (approximately 300 g) for 1 hour. The organic layer was filtered and concentrated under reduced pressure to obtain an oily substance (460.7 g, over 100%, containing trace amounts of solvent).

[0171] [Example 13] Preparation of INT 11

[0172] [ka]

[0173] A three-necked 12L RBF was equipped with an overhead stirrer, cooling bath, argon gas inlet and outlet, and thermocouple / control device. INT 9 (purified) (450g, 1.11mol) in a 22L Rotabap flask was dissolved in pyridine (4.5L, 10 volumes) and added to the three-necked flask. The reaction mixture was cooled to below 5°C under argon. Benzesulfonyl chloride (342g, 1.75 equivalents, 1.94mol, Aldrich) was added to a 500mL addition funnel. The reagent was added dropwise to the flask while maintaining the temperature below 5°C. The reaction mixture was then stirred overnight at room temperature for 16 hours.

[0174] 1. Workup and Isolation The completion of the reaction was confirmed by LC-MS (see Figure 23). The reaction mixture was cooled to 5-10°C. Water (4.5 L) was added to the reaction mixture while maintaining the temperature below 20°C. The reaction mixture was stirred for 30 minutes. The product was extracted in heptane (3 × 2 L). The combined organic layers were washed with 1N HCl (2 × 1.5 L), then with 5% NaHCO3 (2 × 1.5 L) and 10% NaCl (2 × 1.5 L). The organic layers were dried over anhydrous Na₂SO₄ (approximately 400 g, Aldrich) for 1 hour. The organic layers were filtered and concentrated under reduced pressure to obtain a concentrated brick-red oily substance (423.8 g, 99%).

[0175] [Example 14] Preparation of INT 12

[0176] [ka]

[0177] Using the configuration shown in Figure 10, a three-necked 12 L RBF was equipped with an overhead stirrer, cooling bath, argon gas inlet and outlet, and thermocouple / control device. INT 11 (420 g, 1.08 mol) and then THF (4.2 L, 10 vol) were added to the flask, and the slurry was stirred under argon until a homogeneous solution was obtained. Under an argon atmosphere, 10% Pd / C (42 g, 10 wt%, Aldrich) was added to the flask. The flask was evacuated and refilled three times with hydrogen. Hydrogen was blown directly into the reaction mixture using a gas diffusion sparger. The reaction mixture was then stirred overnight at room temperature.

[0178] After 18 hours of stirring, the hydrogen flow was stopped, and argon was passed through the system for 5–10 minutes before the reaction mixture was opened to the atmosphere. The sample was then removed for IPC (LCMS). The LCMS data showed completion of the reaction and formation of INT 12 (see Figure 24).

[0179] 1. Preparation of Celite pads A slurry of 100% by weight Celite (Sigma Aldrich) in a minimum amount of THF was prepared and added to 2 L of coarse frit. One sheet of filter paper was placed on top of the slurry. The Celite was allowed to settle, and a light vacuum was applied to form a dense Celite pad.

[0180] 2. Pd / C removal and product isolation Without drying the Pd / C, the reaction mixture was carefully filtered on a Celite bed. The bed was washed with THF (3 L), and the combined filtrate was concentrated under reduced pressure using a 22 L Rota vaporizer to obtain the crude product as an oily substance. After standing at room temperature, the oily substance became a yellowish-white solid (326.6 g, yield over 100%).

[0181] [Example 15] Preparation of Compound I-A

[0182] [Chemical formula]

[0183] 1. Alternative reagents In the previous batch of Compound I-A, in the final step, diisopropyl azodicarboxylate (DIAD) was used together with triphenylphosphine as a coupling reagent. The crude reaction mixture of these steps was observed to contain diisopropyl hydrazinodicarboxylate (DIAD-H2) as the main by-product. In the case of the engineering batch, subsequent chromatographic purification showed that approximately 40% of the desired product co-eluted with DIAD-H2. To avoid such mixing in future column purifications, di-tert-butyl azodicarboxylate (DBAD) was selected as an alternative reagent for the final step. The original rationale for switching to DBAD was the prediction that the by-product (DBAD-H2) could be easily removed by decomposition. The scheme of the decomposition process is shown below.

[0184] [Chemical formula]

[0185] Presumably, by treating DBAD-H2 in the reaction mixture with TFA / DCM at high temperature, the by-product should be decomposed into CO2 (gas), N2 (gas), and isobutylene (gas) that can flow out of the reaction mixture. The activity of DBAD for the Mitsunobu reaction was tested using INT 12 (300 mg), and LCMS confirmed that the reaction functioned under the modified conditions (Figure 25). From 1 g of the crude reaction mixture, 50 mg was treated with TFA / DCM (1:2) and heated at 55 °C for 1 hour. LCMS (Figure 26) showed that the DBAD-H2 peak (RT: 5.21) disappeared without affecting the stability of Compound I-A.

[0186] A similar procedure was used to carry out another 30g-scale reaction. On a larger scale, LC-MS showed that compound IA was partially decomposed during the DBAD deprotection attempt, and a new impurity was identified as a deformylated analog of compound IA (see Figure 27). Under acidic conditions and high temperatures, compound IA decomposed along with DBAD-H2. Therefore, this method was not considered feasible on a large scale.

[0187] However, another advantage of using DBAD reagent in the Mitsunobu process was that the difference in retention times between compound IA and DBAD-H2 on TLC was greater than that between compound IA and DIAD-H2. For this reason, we decided to use DBAD reagent during the large-scale Mitsunobu reaction.

[0188] 2. Toxicity test batch A three-necked 12L RBF was equipped with an overhead stirrer, a cooling bath, a 500mL addition funnel, argon gas inlet and outlet, and a thermocouple / control device. INT 12 (274g, 0.918mol, 1 equivalent), N-formyl L-alanine (139.7g, 1.193mol, 1.3 equivalents), and PPh3 (288.9g, 1.102mol, 1.2 equivalents) were added to the flask. THF (2000mL) was added to the RBF and stirred at room temperature. The reaction mixture was partially dissolved, cooled to 5-10°C, and the argon flow was started. Di-tert-butyl azodicarboxylate (DBAD, 253.7g, 1.102mol, 1.2 equivalents) was diluted with THF (700mL) and added to the 500mL addition funnel. The solution was added dropwise to the reaction mixture while maintaining the temperature at 5-10°C. The addition funnel was rinsed with THF (40 mL), and the rinse solution was added to the reaction mixture. After the addition of DBAD was complete, the reaction mixture became a clear solution. The reaction mixture was stirred under argon flow at room temperature for 12 hours.

[0189] 3. Workup and initial purification TLC and LC-MS confirmed the completion of the reaction. The reaction mixture was transferred to a 10 L Rota vapor flask and evaporated under reduced pressure to obtain a concentrated oily substance (971 g). Heptane (1000 mL) was added, and the resulting mixture was stirred at 10-15°C for 2 hours. A semi-solid was formed, and after stirring and suspension, it was allowed to settle. Heptane was decanted, and the semi-solid was triturated with heptane (1000 mL). TLC showed that the heptane layer contained low-polarity impurities and triphenylphosphine oxide. MTBE (1000 mL) was added to the semi-solid and stirred at room temperature. The semi-solid became a free-flowing solid, and the solid was collected on a medium-grain frit funnel. The solid was washed with MTBE (2 × 500 mL). TLC and LC-MS confirmed that the solid was triphenylphosphine oxide. The combined MTBE layer was evaporated under reduced pressure to obtain 894 g of crude product as an oily substance.

[0190] 4. Column purification A large glass column (see Figure 28A) was packed with silica gel (4.4 kg, 5 volumes, 60 Å, 230-400 mesh, Aldrich) and heptane. 893 g of crude compound IA was dissolved in MTBE (1000 mL) and added to 900 g of silica. The mixture was evaporated to obtain the crude compound adsorbed on the silica. Dry silica was placed on top of the packed column, 1 cm of sand was placed on top of that, and filter paper was added. A fraction of 1000 mL was collected. The compound was eluted as follows: ethyl acetate, heptane. 100% Heptane - 14L 10% EA: Heptane - 25L - Upper Impurities 15% EA: Heptane-36L-Upper Impurities + DBAD-H2 20% EA:Heptane-120L-Product 30% EA:Heptane-120L-Product + Bottom Impurities

[0191] During 20% ​​EA:heptane elution, the initial fraction contained low-polarity impurities (by-products) along with the product, while the later fraction contained pure product. Only the pure fraction was collected by TLC (see Figure 28B - mobile phase: 50:50 heptane / ethyl acetate, PMA staining) and evaporated under reduced pressure.

[0192] The combined pure fractions were evaporated under reduced pressure to obtain an oily substance. Compound IA (1.3g) was added as a seed crystal to this oily substance, and it was kept in a freezer to solidify. Over the weekend, the oily substance became a waxy, yellowish-white solid (254g, 70%) (compound IA). Subsequent ULC analysis showed that the purity of this substance was approximately 95% (AN at 205nm). Figure 29 shows the LC-MS spectrum, and Figure 30 shows... 1 The 1H NMR spectrum is shown, and the HPLC results are shown in Figures 31 and 32. The crystal structure of compound IA is shown in Figure 33.

[0193] The above is illustrative of the present invention and should not be construed as limiting it. The present invention is defined by the following claims, which include equivalents of the claims.

Claims

1. A method for synthesizing the compound of formula I, 【Chemistry 1】 (In the formula, R １ C ５～ C １５ It is alkyl.) Compound INT 12 and N-formyl L-alanine are contacted in the presence of azodicarboxylate di-tert-butyl (DBAD) and triphenylphosphine to form formula I: 【Chemistry 2】 A method comprising the step of forming the aforementioned compound.

2. R １ However, C １１ The method according to claim 1, wherein the alkyl group is alkyl.

3. The method according to claim 1 or 2, wherein the contact step is carried out in a tetrahydrofuran (THF) solvent.

4. The aforementioned contact step, (a) A step of forming a reaction mixture by combining the INT 12 compound, N-formyl L-alanine, triphenylphosphine, and a solvent; (b) A step of cooling the reaction mixture to a temperature within a range of 5°C to below room temperature; (c) A step of dissolving DBAD in a solvent; (d) Adding DBAD to the reaction mixture while maintaining the temperature within the temperature range until the compound of formula I is formed; The method according to claim 1 or 2, including the method according to claim 1 or 2.

5. A method for synthesizing the compound of formula I, 【Transformation 3】 (In the formula, R １ C ５～ C １５ It is alkyl.) (a) Contact compound 1-A with compound 1-B, INT 1: 【Chemistry 4】 (wherein X １ is a halo). A step of forming a compound; (b) The compound of INT1 is brought into contact with the ruthenium (R)-BINAP catalyst and hydrogen gas to form INT2: 【Transformation 5】 The process of forming the compound; (c) The compound of INT 2 is brought into contact with the compound of 3-A, and INT 3: 【Transformation 6】 (In the formula, X ２ and X ３ The process of forming a compound of (each being an independent halo); (d) The compound in INT 3 is brought into contact with the Grignard reagent, and INT 4: 【Transformation 7】 The process of forming the compound; (e) The compound of INT 4 is brought into contact with hydrogen gas in the presence of a Raney Ni catalyst to form INT 5: 【Transformation 8】 The process of forming the compound; (f) The compound of INT 5 is brought into contact with the first protective agent, and INT 6: 【Chemistry 9】 (In the formula, R ２ The process of forming a compound of (which is a protecting group); (g) The compound of INT 6 is brought into contact with a hydroxide to form INT 7: 【Chemistry 10】 The process of forming the compound; (h) The compound of INT 7 is reacted with the second protective agent to protect the free hydroxyl group on the compound of INT 7 and form the compound of INT 8, and then the compound of INT 8 is brought into contact with the acid to deprotect the protected hydroxyl group on the compound of INT 8 and form INT 9: 【Chemistry 11】 (In the formula, R ３ The process of forming a compound of (which is a protecting group); (i) The compound in INT 9 is dehydrated with a dehydrating agent to obtain INT 11: 【Chemistry 12】 The process of forming the compound; (j) Deprotect the compound of INT 11 to obtain INT 12: 【Chemistry 13】 The process of forming a compound; and (k) The compound of INT 12 and N-formyl L-alanine are brought into contact in the presence of azodicarboxylate di-tert-butyl (DBAD) and triphenylphosphine to obtain formula I: 【Chemistry 14】 A method comprising the step of forming a compound.

6. The method according to claim 5, wherein in step (b), the step of contacting the compound of INT1 with the ruthenium(R)-BINAP catalyst and hydrogen gas is carried out in a reaction mixture in a pressure vessel, and the ratio of the volume of dead space in the pressure vessel to the volume of the reaction mixture is 3:1 or more.

7. The method according to claim 5, wherein in step (b), the pressure of the hydrogen gas is less than 200 psi.

8. In step (c), the contact step reacts dimethylaminopyridine (DMAP) and potassium bicarbonate (KHCO2) in a two-phase reaction. ３ The method according to claim 5, carried out in the presence of an aqueous solution.

9. The method according to claim 5, wherein in step (d), the Grignard reagent comprises tert-butylMgBr.

10. The method according to claim 5, wherein in step (e), the Raney Ni catalyst is newly prepared and the pressure of the hydrogen gas is in the range of 0.5 to 2 atmospheres.

11. In step (f), the protective agent is 3,4-dihydro-2H-pyran (DHP), R ２ The method according to claim 5, wherein is a tetrahydropyran (THP) group.

12. The method according to claim 5, wherein the dehydrating agent in step (i) comprises benzenesulfonyl chloride.

13. The method according to claim 5, wherein in step (j), the deprotection step includes a debenzylation reaction.

14. The method according to claim 5, wherein in step (j), the deprotection step comprises a debenzylation reaction using Pd / C and hydrogen gas.

15. Between steps (h) and (i), the compound of INT 9 is brought into contact with (S)-(-)-1-phenylethylamine to purify the compound of INT 9 into INT 10: 【Chemistry 15】 Form a compound, crystallize and isolate the compound in INT 10, then contact the compound in INT 10 with an acid (e.g., HCl) to obtain INT 9 (purification): 【Chemistry 16】 process of forming The method according to claim 5, further comprising:

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