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 is incorporated by reference in its entirety.

[0002] The present invention relates to a method for synthesizing pharmaceutical compounds. The present invention also relates to small molecule lipase inhibitors and methods 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 become activated while still present in the pancreas, stimulating pancreatic cells and causing inflammation. The annual incidence of acute pancreatitis is approximately 20-40 cases per 100,000 people, and this incidence has been increasing over the last few decades. Yadav et al.,Epidemiology of Pancreatitis,GI Epidemiology:Diseases and Clinical Methodology,2 nd Ed., 2014, Blackwell Publishing. Moreover, severe acute pancreatitis has a mortality rate of about 29%. Munoz et al., Am Fam Physician (2000) 62:164-74. Thus, novel methods for treating pancreatitis are being investigated.

[0004] Lipase inhibitors are a class of compounds marketed as anti-obesity agents. Lipstatin, a natural product isolated from streptomyces toxytricini, is an early known lipase inhibitor. 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 may also be useful for treating other disorders such as pancreatitis.

[0005] One lipstatin derivative is (S)-1-((2S,3S)-3-ethyl-4-oxooxetan-2-yl)tridecan-2-yl formyl-L-alaninate, shown below, and also referred to herein as compound IA.

[0006] [ka]

[0007] As shown in FIG. 1, current methods for synthesizing compound IA include the illustrated synthetic steps. Although these methods can produce compound 1-A, they may not be suitable for large-scale and / or cGMP manufacturing. These methods may also exhibit chemical processing issues, including a strong sulfur / thiol odor that is difficult to contain upon scale-up, and significant health hazards due to the use of corrosive aqueous hydrofluoric acid. Furthermore, the overall synthesis may provide low to moderate yields and may require seven chromatographic purification steps. Thus, novel methods for synthesizing compound IA are desirable. Summary of the Invention

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

[0009] [ka]

[0010] In some embodiments of the present invention, methods are provided for synthesizing a compound of INT 2. In some embodiments, these methods include contacting a compound of INT 1 with a ruthenium (R)-BINAP catalyst and hydrogen gas in a reaction mixture in a pressure vessel to form a compound of INT 2.

[0011] [ka]

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

[0013] Also in one embodiment of the present invention, there is provided a method for synthesizing a compound of formula I, comprising the steps of: [ka] (Wherein, R1 is C 5~ C 15 Alkyl (e.g. C 11 alkyl). (a) contacting a compound of formula 1-A with a compound of formula 1-B, optionally in the presence of magnesium, to obtain INT 1: [ka] forming a compound of formula (II) wherein X1 is halo (e.g., Cl or Br); (b) contacting the compound of INT1 with a ruthenium(R)-BINAP catalyst and hydrogen gas to produce INT 2: [ka] forming a compound of (c) contacting the compound of INT 2 with the compound of 3-A to obtain INT 3: [ka] wherein X2 and X3 are each independently halo (e.g., Br); (d) contacting the compound of INT 3 with a Grignard reagent to give INT 4: [ka] forming a compound of (e) contacting the compound of INT 4 with hydrogen gas in the presence of a Raney Ni catalyst to produce INT 5: [ka] forming a compound of (f) contacting the compound of INT 5 with a first protecting agent to produce INT 6: [ka] forming a compound of formula (II) wherein R2 is a protecting group (e.g., THP); (g) contacting the compound of INT 6 with a hydroxide (e.g., from a hydroxide salt such as NaOH) to produce INT 7: [ka] forming a compound of (h) reacting INT 7 with a second protecting agent to protect the free hydroxy group on compound INT 7 to form compound INT 8, and then contacting INT 8 with an acid to deprotect the protected hydroxy group on compound INT 8 to form compound INT 9: [ka] forming a compound of formula (wherein R3 is a protecting group (e.g., benzyl); (i) optionally purifying the compound of INT 9 by contacting the compound of INT 9 with (S)-(−)-1-phenylethylamine to obtain compound of INT 10: [ka] and then contacting INT 10 with an acid (e.g., HCl) to form a compound of formula (I): [ka] forming a (j) Compound INT 9 or compound INT 9 (purified) is dehydrated with a dehydrating agent to obtain INT 11: [ka] forming a compound of (k) Deprotecting the compound INT 11 to give INT 12: [ka] forming a compound of (l) contacting the compound of INT 12 with N-formyl L-alanine in the presence of di-t-butyl azodicarboxylate (DBAD) and triphenylphosphine to obtain the compound of formula I [ka]

[0023] A method is provided comprising forming a compound of the formula:

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

[0015] Also provided in one embodiment of the present invention is a method of inhibiting lipase activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to the present invention or a pharma- ceutically acceptable salt thereof and / or a composition according to the present invention, thereby inhibiting lipase activity in the subject.

[0016] Also provided in one embodiment of the present invention is a method of treating pancreatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to the present invention or a pharma- ceutically acceptable salt thereof and / or a composition according to the present invention, thereby treating pancreatitis in the subject.

[0017] These and other aspects of the invention are described in further detail in the following description of the invention. [Brief description of the drawings]

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

[0019] The present invention will be described in detail below. However, the present invention may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0020] The terminology used in the description of the invention herein is for the purpose of describing a particular embodiment only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0021] Unless otherwise defined, all terms in this specification (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In addition, terms, such as those defined in commonly used dictionaries, should be interpreted as having the same meaning as in the context of this application and related art, and should not be interpreted in an ideal or overly formal sense unless expressly defined in this specification. The terminology used in the description of the present invention in this specification is intended only to describe a specific embodiment and is not intended to limit the present invention. All publications, patent applications, patents, and other references mentioned in this specification are incorporated herein by reference in their entirety. In the event of a conflict in terminology, this specification shall prevail.

[0022] As used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combination when interpreted as alternatives ("or").

[0023] It is expressly intended that the various features of the present invention described herein can be used in any combination, unless the context indicates otherwise. The present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be excluded or omitted. For example, if a complex is described herein as comprising components A, B, and C, it is expressly intended that any of A, B, or C, or any combination thereof, can be omitted and discarded.

[0024] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) should be construed to include materials or steps described, as well as materials or steps that do not materially affect the basic novel characteristics of the invention as claimed. Thus, as used herein, the term "consisting essentially of" should not be construed as the equivalent of "comprising."

[0025] In this specification, terms such as "first" and "second" 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 only to distinguish one element from another. Thus, a "first" element may be referred to as a "second" element without departing from the teachings of the present embodiment.

[0026] As used herein, the term "about" is used in reference to a measurable value, such as an amount or concentration, and is intended to include ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variation relative to the stated value, and the stated value. For example, "about X", where X is a measurable value, is intended to include X and ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variation relative to X. Ranges given herein for measurable values ​​may include any other ranges and / or individual values ​​within the range.

[0027] As used herein, "halo" means any suitable halogen including -F, -Cl, -Br, and -I.

[0028] As used herein, "hydroxy" or "hydroxyl" refers to an --OH group. A "free hydroxy group" refers to an unprotected --OH group. A "protected hydroxy group" refers to a hydroxy group that is bound (e.g., covalently bonded) to a protecting group.

[0029] As used herein, "alkyl" refers to a straight or branched chain hydrocarbon containing 1 to 15 carbon atoms, either alone or as part of another group. As used herein, "lower alkyl" refers to a straight or branched chain hydrocarbon containing 1 to 4 carbon atoms, either alone or as part of another group. Representative examples of alkyl 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, pentadecyl, and the like. C5 to C 15 Alkyl includes straight chain and branched saturated alkyls, including, for example, 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] As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. Compounds identified herein by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. Tautomeric forms result from the simultaneous exchange of a single bond with an adjacent double bond and the migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Prototropic tautomers include, for example, ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which a proton can occupy more than one position in a heterocyclic ring system. Tautomeric forms 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 asymmetric centers and may occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+) (dextrorotatory) forms, (-) (levorotatory) forms, racemic mixtures thereof, and other mixtures thereof). Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. Unless otherwise indicated, all these isomeric forms of these compounds, as well as mixtures thereof, are expressly included herein.

[0032] The compounds described herein may further include restrictions imposed by the presence of bonds, such as rings or double bonds (e.g., carbon-carbon bonds or carbon-nitrogen bonds such as amide bonds), where rotation of the bond is restricted around that particular bond. Thus, all cis / trans and E / Z isomers and rotamers are included herein. Unless otherwise indicated, chemical designations of compounds include mixtures of all possible stereochemically isomeric forms of the compound.

[0033] Preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. For the chemistry of protecting groups, see, for example, TW Greene and PG M Huts, 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., UV-Vis), mass spectrometry, and / or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by various methods known to those skilled in the art, unless otherwise indicated.

[0035] As used herein, "treatment" refers to any manner of improving or otherwise beneficially altering one or more symptoms of a disease or disorder. Treatment also includes any pharmaceutical use of the compositions described herein, such as use to treat pancreatitis. As used herein, the improvement of symptoms of a particular disorder by administering a particular compound or pharmaceutical composition refers to any attenuation, whether permanent or temporary, permanent or transient, that may result from or be associated with the administration of the composition.

[0036] According to one embodiment of the present invention, a compound of formula I: [ka] (Wherein, R1 is C 5~ C 15 Alkyl (e.g. C5-C 15 A method for synthesizing a compound of formula (I) is provided. R1 is C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , or C 15 In one particular embodiment, for example, when the compound of formula I is compound IA, R1 can be C 11 Alkyl (e.g. C 11 H 23 ).

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

[0038] [Method step 1] In some embodiments of the invention, a compound of formula 1-A is contacted with a compound of formula 1-B to form a compound of formula INT 1, as shown below: [ka] wherein X1 is halo (e.g., Cl or Br) and R1 is C 5~ C 15 Alkyl (e.g. C5-C 15 Therefore, R1 is C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , or C 15 In one particular embodiment, R1 can be C 11 Alkyl (e.g. C 11 H 23 ).

[0039] In some embodiments, the compound of 1-A is contacted with the compound of 1-B in the presence of an alkali metal, such as magnesium, to form the compound of INT 1 by aliphatic acylation. In some embodiments, the compound of 1-A is added to the compound of 1-A by first reacting an alkali metal (e.g., magnesium) with methanol to form an alkali metal methoxide solution (optionally in toluene). The compound of 1-B dissolved in a solvent (e.g., toluene) can then be added over time, for example, via an addition funnel. In some embodiments, the reaction is quenched with methanol and distilled. A strong acid, for example, a mineral acid, such as hydrochloric acid, sulfuric acid, and / or phosphoric acid, can then be added. The reaction can then be purified by any suitable method, for example, by washing with water, drying, and / or concentrating the compound of INT 1. The compound of INT 1 can then be optionally further purified by recrystallizing the compound of INT 1 (e.g., from methanol).

[0040] [Method step 2] In some embodiments of the 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 INT 2: [ka] (wherein R1 is as defined above).

[0041] In some embodiments, the ruthenium pre-catalyst and the R-BINAP ligand are combined under an inert atmosphere and then when added to the reaction mixture, R-BINAP, i.e., (R)-(2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, and ruthenium metal form the 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 active catalyst solution. A compound of INT 1 is dissolved (in an inert environment) in a suitable solvent to form a reaction mixture, and the active catalyst solution is added to the reaction mixture. In some embodiments, the compound of INT 1 and the active catalyst are added to an autoclave or pressure vessel, which is pressurized with hydrogen gas and heated to form the INT In some embodiments, the hydrogen gas pressure in the autoclave or pressure vessel is at 200 psi or less (e.g., in the range of 100 psi to 200 psi). Also, the dead space in the pressure vessel may affect the purity and enantioselectivity of the resulting INT 2 compound. Thus, in some embodiments, the ratio of dead space in the pressure vessel to the reaction mixture is 3:1 or more (e.g., 4:1, 5:1, or 6:1). The crude INT 2 mixture can be further purified by extraction and / or washing, and in some embodiments, recrystallized by dissolving in an organic solvent and cooling.

[0042] In some embodiments, to assess the enantiopurity of a compound of INT 2, the compound can be derivatized to form a Mosher ester. In some embodiments, the formation of a Mosher ester is carried out using a Mosher acid (α-methoxy-α-trifluoromethylphenylacetic acid (MTPA)) with a catalyst (e.g., 4-dimethylaminopyridine (DMAP)) and a dehydrating agent (e.g., dicyclohexylcarbodiimide (DCC)). The Mosher ester is then derivatized to form a Mosher ester. 1 Analysis by 1 H NMR can be used to determine whether the compounds of INT 2 are enantiomerically enriched.

[0043] In some embodiments, to assess the enantiopurity of a compound of INT 2, the compound can be derivatized with, for example, benzoyl chloride in the presence of 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 compound of INT 2 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 invention, a compound of INT2 is contacted with a compound of formula 3-A to form a compound of INT 3: [ka] (wherein R1 is as defined above, and X2 and X3 are each independently halo (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 including water and an organic solvent such as toluene) in the presence of a catalyst (e.g., DMAP) and potassium bicarbonate (KHCO3). The 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 invention, a compound of INT 3 is contacted with a Grignard reagent (Reformatsky reaction) to cyclize and form a compound of INT 4: [ka] (wherein 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, t-amyl MgBr, etc. In some embodiments, the reaction mixture containing the compound of INT 3 and the Grignard reagent is added slowly over time to a reactor along with an organic solvent (e.g., THF) until the reaction forms the compound of INT 4. Optionally, the reaction mixture can be distilled to remove a portion of the organic solvent, and the product can be cooled and / or a cold citric acid solution can be added to promote precipitation. In some embodiments, the product can be washed and / or recrystallized to purify the compound of INT 4.

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

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

[0050] [Method step 6] In some embodiments of the present invention, a protecting group is added on a free hydroxy group in a compound of INT 5 by contacting the compound of INT 5 with a protecting agent: [ka] (wherein 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-stable protecting groups, such as trityl or p-methoxybenzyl, which are removable under relatively mild conditions, may also be used. In some embodiments, a weak acid catalyst, such as p-pyridinium toluenesulfonate (PPTS), may be added to a compound of INT 5 dissolved in a solvent, such as THF. A protecting agent (e.g., DHP) may then be added to the reaction flask. In some embodiments, the resulting compound of INT 6 may be redissolved in an organic solvent (e.g., MTBE) and washed.

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

[0053] In some embodiments of the invention, the hydroxide salt is NaOH or KOH. In some embodiments, the compound of INT 6 is dissolved in an organic solvent (e.g., MTBE) and added to the reaction flask. An aqueous solution of the hydroxide salt (e.g., 2N NaOH) can then be 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 (e.g., 10% NaCl) solution. The compound of INT 7 can then be concentrated to form a crude oil. In some embodiments, the crude oil is azeotropically distilled with an organic solvent (e.g., MTBE and THF) to further purify the compound of INT 7.

[0054] [Method steps 8 and 9] In some embodiments of the invention, a compound of INT 7 is contacted with a protecting agent to add a protecting group on the free hydroxy on the compound of INT 7, thereby forming a compound of INT 8. The compound of INT 8 is then reacted with an acid to deprotect the protected hydroxyl group to form a free hydroxyl group, thereby forming a compound of INT 9: [ka] (wherein 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 in forming INT 9.

[0055] In some embodiments of the present invention, the intermediate product INT 8 is not purified prior to forming the compound INT 9. Thus, these two reactions can be carried out in a one-pot synthesis. In some embodiments, the protecting agent is benzyl bromide, which reacts with the compound INT 7 in an O-benzylation reaction. In some embodiments, the compound INT 7 in an organic solvent (e.g., THF) is stirred, 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 and aqueous layers are separated, and 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 reaction product INT 9. In some embodiments, the crude product can then be redissolved in an organic solvent (e.g., methyl acetate), washed with a saline solution, and / or dried, then filtered, and the product carried on to the next step. Thus, in some embodiments, the hydroxy THP formed by deprotection in process step 9 is removed from the INT 9 product before proceeding to the next step.

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

[0057] Such optional steps can be used to purify the product. In some embodiments, (S)-PEA purification can be carried out in an organic solvent, such as methyl acetate. In some embodiments, the reaction is carried out by dissolving the compound of INT 9 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 added slowly to the reaction flask. The crystals thus formed can be filtered and washed (e.g., with cold methyl acetate).

[0058] In some embodiments, if hydroxy-DHP is present in the crude mixture, the INT 10 compound may revert back to the INT 8 PEA salt (also referred to as INT 8-PEA). Therefore, it may be advantageous to remove the hydroxy-THP formed in process step 9. If such reversion occurs, the INT 8-PEA compound can be converted to the INT 9 compound by reacting the INT 8-PEA with an acid. 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 described above.

[0059] [ka]

[0060] [Method step 11] In some embodiments of the invention, a compound of INT 10 is contacted with an acid to form a purified compound of INT 9: [ka] (wherein 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 an acid (e.g., 1N HCl) is added to the reaction mixture. The organic layer can then be separated from the aqueous layer, washed, dried, filtered, and / or concentrated to provide INT 9 (purified).

[0062] [Method step 12] In some embodiments of the invention, the compound INT 9 or INT 9 (purified) is contacted with a dehydrating agent to form a compound INT 11: [ka] (wherein 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 temperature (e.g., below 5° C.) and / or under an inert atmosphere. In some embodiments, the resulting INT 11 product is then extracted into heptane, washed with an aqueous acid and / or base, washed with a brine 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] (wherein 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 bubbled into the reaction mixture under inert conditions. The resulting crude product, which contains the compound of INT 12, is then filtered and concentrated under reduced pressure to provide the purified compound of INT 12.

[0066] [Method step 14] In some embodiments of the invention, a compound of INT 12 is contacted with N-formyl L-alanine to form a compound of formula I.

[0067] [ka]

[0068] In some embodiments, the compound INT 12 reacts with N-formyl L-alanine by Mitsunobu coupling reaction using triphenylphosphine and an azodicarboxylate. A similar compound was synthesized using diisopropyl azodicarboxylate (DIAD), but this reagent did not work well for the synthesis of the compound of formula I. Purification was problematic because DIAD-H2 was formed as the main by-product, and the compound of formula I was co-eluted with DIAD-H2. Surprisingly, the inventors found that when di-tert-butyl azodicarboxylate (DBAD) was used as the azodicarboxylate, the DBAD-H2 and the compound of formula I formed showed sufficiently different retention times that they could be separated by chromatography. Even more surprisingly, the reaction proceeded more quickly 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 proceeds 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 compound of INT 12, 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 temperature within a specified temperature range 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 via an addition funnel. DBAD can be added to allow the reaction to proceed at a temperature within a specified temperature range (e.g., about 5-10° C.). Upon completion, 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 yield 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). A combination of fractions from the chromatography can then be concentrated to yield an oil. In some embodiments, the crude oil is seeded with purified crystals of the compound of formula I to produce product crystals over time. In some embodiments of the invention, the compound of formula I is at least 90% pure (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% pure, and any ranges defined therebetween).

[0070] The method of the present invention may further comprise forming a pharma- ceutically acceptable salt of the compound of formula I. The term "pharma- ceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, and is not usually biologically or otherwise undesirable. Salts can be formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, especially 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, N-acetylcysteine, and the like. These salts can also be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, and the like.

[0071] The method may further comprise forming a composition comprising the compound of formula I. In some embodiments, the compound of formula I can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release preparations, or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparations and dry powder inhalers. In one embodiment, the compound is formulated into pharmaceutical compositions 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 their pharma- ceutically acceptable derivatives can be mixed with a suitable pharmaceutical carrier at effective concentration.Compounds can be derivatized as corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs before the above formulation.The concentration of compound in the composition can be effective to deliver the amount that treats, prevents, and / or improves pancreatitis or inhibits lipase activity when administered.

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

[0074] The active compound may be included in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects on the subject being treated. The therapeutically effective concentration may be empirically determined by testing the compound in in vitro and in vivo systems and then extrapolating for administration to humans.

[0075] The concentration of the compound of formula I (also referred to as the "active compound") in the pharmaceutical composition may depend on the absorption, inactivation, and excretion rates 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] The 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, and oil-in-water emulsions, containing a suitable amount of the compound or its pharma- ceutically acceptable derivatives for administration to humans and / or animals. In one embodiment, the phy- tratically therapeutically active compounds and their derivatives are formulated and administered in unit dosage forms or multiple dosage forms. Unit dosage forms, as used herein, refer to physically discrete units suitable for human and animal subjects and individually packaged as known in the art. Each unit dosage form contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, in combination with the necessary pharmaceutical carrier, vehicle, or diluent. Unit dosage forms include, for example, ampoules and syringes, and individually packaged tablets or capsules. A unit dosage form may be administered in a fraction or multiple thereof. A multiple dosage form refers to a plurality of identical unit dosage forms packaged in a single container to be administered as segregated unit dosage forms. A multiple-dosage form includes, for example, vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, a multiple-dosage form is a multiple of unit-dosage form that is not segregated in packaging.

[0077] Pharmaceutically administrable liquid compositions can be prepared, for example, by dissolving, dispersing or otherwise mixing an active compound, as defined herein, and optional pharmaceutical excipients in a carrier, for example, water, saline, aqueous dextrose, glycerol, glycol, ethanol, and the like, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like, such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other similar agents.

[0078] Actual methods for preparing these dosage forms will be known or apparent to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975).

[0079] In some embodiments, the compositions of the present invention may be suitable for oral administration. Pharmaceutical oral dosage forms are solid, gel, or liquid. Solid dosage forms are tablets, capsules, granules, and mixed powders. Oral tablet types include compressed chewable lozenges and tablets that may be enteric, sugar-coated, or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be realized in non-effervescent or effervescent form by the combination of other ingredients known to those skilled in the art.

[0080] In a particular embodiment, the formulation is a solid dosage form, in one embodiment a capsule or tablet. Tablets, pills, capsules, lozenges, etc. may contain one or more of the following ingredients, or compounds with similar properties: binders; lubricants; diluents; glidants; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; and film coatings. Binders include, for example, microcrystalline cellulose, tragacanth gum, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidine, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants 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. Coloring agents include, for example, any of the approved and certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD 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. Flavoring agents include natural flavors extracted from plants, such as fruits, such as, but not limited to, peppermint and methyl salicylate, as well as synthetic blends of compounds producing a pleasant sensation. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates.Film coatings include hydroxyethylcellulose, gellan gum, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0081] The compound of formula I, or its pharma- ceutically acceptable derivatives, can be realized in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other similar ingredient. When the unit dosage form is a capsule, it may contain a liquid carrier, such as fatty oil, in addition to the above types of materials. The unit dosage form may also contain various other materials that modify the physical form of the unit dosage form, such as sugar coatings and other enteric agents. The compound can be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum, etc. The syrup may contain sucrose as a sweetener, as well as certain preservatives, dyes, colorings, and flavors, in addition to the active compound.

[0082] The active ingredient may be mixed with other active ingredients that do not impair the desired action, or with ingredients that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a compound as described herein or a pharma-ceutically acceptable derivative thereof. High concentrations of the active ingredient, up to about 98% by weight, may be included.

[0083] In all embodiments, 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, with conventional enterically digestible coatings such as phenylsalicylate, waxes, and cellulose acetate phthalate.

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

[0085] Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions include non-aqueous liquids, emulsifying agents, and preservatives. Suspensions use pharma-ceutically acceptable suspending agents and preservatives.

[0086] Pharmaceutically acceptable substances used in non-effervescent granules to be reconstituted into liquid oral dosage forms include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into liquid oral dosage forms include organic acids and carbon dioxide sources. Colorings and flavorings are used in all the above dosage forms. Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Preservatives include, for example, glycerin, methyl and propyl parabens, benzoic acid, sodium benzoate, and alcohol. Non-aqueous liquids utilized in emulsions include, for example, mineral oil and cottonseed oil. Emulsifying agents include, for example, gelatin, gum arabic, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, Veegum, and gum arabic. Sweeteners include sucrose, syrups, glycerin, and artificial sweeteners such as saccharin. Wetting agents 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. Coloring agents include any of the approved and certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation. In solid dosage forms, the solution or suspension, for example in propylene carbonate, vegetable oils, or triglycerides, is enclosed in a gelatin capsule in one embodiment. In liquid dosage forms, the solution may be diluted with a sufficient quantity of a pharma- ceutically acceptable liquid carrier, for example water, to be easily measured for administration.

[0087] Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other similar carriers, and 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 alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents with one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol.Acetals include, but are not limited to, the di(lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.

[0089] Parenteral administration of the composition includes intravenous administration, subcutaneous administration, and intramuscular administration.Administration can be intraperitoneal administration, or directly to or near the organ or tissue of interest, such as the pancreas.Formulations for parenteral administration include sterile solutions ready for injection; sterile dry soluble products, such as freeze-dried powders, ready to be combined with a solvent immediately before use, including tablets for subcutaneous injection; sterile suspensions ready for injection; sterile dry insoluble products, ready to be combined with a vehicle immediately before use; and sterile emulsions.Solutions can be aqueous or non-aqueous.

[0090] If administered intravenously, carriers suitably include physiological saline or phosphate buffered saline (PBS) as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0091] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances.

[0092] Aqueous vehicles include, for example, Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Non-aqueous parenteral vehicles include hydrogenated oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Parenteral preparations packaged in multi-dose containers should contain antibacterial agents in bacteriostatic or fungistatic concentrations, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0093] The compound of formula I can be suspended in micronized or other suitable form, or derivatized to produce more soluble active product or to produce prodrug.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 vehicle.The effective concentration is the concentration sufficient to improve the symptoms of the condition, and can be empirically determined.

[0094] The invention may be practiced using lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures, or they may be reconstituted and formulated as solids or gels.

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

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

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

[0098] The invention is described in further detail in the following non-limiting experimental section. EXAMPLES

[0099] The labeling / numbering of the compounds shown in the Examples section is for the Examples section only and may not correspond to the labeling / numbering shown elsewhere in this application, and therefore should not be confused with the labeling / numbering of the compounds in the Examples section with the labeling / numbering of the 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 (EtOAc), 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 30 L jacketed vessel was fitted with a vacuum rated stirrer bearing, argon gas inlet and outlet, connected heating / cooling, and thermocouple / controller. The vessel was purged with argon for 15 minutes. To the vessel was added methanol (3.85 L), followed by magnesium (120 g, approximately 50 mesh). The sides of the vessel were rinsed with methanol and the vessel was heated to 55° C., then 65° C. The reaction was stirred at 65° C. overnight. Toluene (11.6 L) 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 1.5 L of solvent was distilled. The temperature of the circulating solution was reduced to 45° C. and methyl acetoacetate (2.2 Kg) 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 cooling temperature 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 the 30 L vessel over 2-3 hours. The reaction mixture was stirred at 60°C for 12 hours.

[0104] 1. Stopping 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 about 900 ml with a distillation head and vacuum pump. The reaction mixture was stirred at 75° C. until most of the tricarbonyl intermediate was converted to product on TLC. The reaction mixture was cooled to 25° C. and concentrated HCl (1502 g) was added. The reaction mixture turned from orange to yellow.

[0105] 2. Workup and Isolation The reaction was quenched 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 h. The organic layer was filtered and concentrated under reduced pressure to give an orange oil (2018 g, >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 fritted funnel and air dried for 2 hours. The solid was transferred into a tray and drying continued in an oven at room temperature until constant weight. INT 1 was isolated as a flaky white to off-white solid (1248 g, 66%). Figure 2 shows the solubility of INT 1. 1 The H NMR spectrum is shown.

[0107] [Example 2] Preparation of INT 2

[0108] [ka]

[0109] The enantioselectivity in this reaction was found to be dependent on several factors including solvent, temperature, and pressure. In particular, R&D experiments established that the ratio of reaction volume to reactor dead space is a key parameter in low pressure hydrogenation (<1000 psi). For a 2 L reactor volume, the reaction scale was fixed at ≤150 g INT 1 (at 200 psi) to avoid hydrogen starvation conditions associated with reduced enantioselectivity.

[0110] 1.Enantioselective synthesis of INT 2 DMF and methanol were degassed with argon for 30 min. In a three-neck flask that was evacuated and purged with argon three times, [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. The reaction mixture was heated to 100° C., stirred for 30 min, 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 into a 2 L autoclave and degassed with argon for 15 min. The activated catalyst prepared above was transferred into the autoclave while purging with argon. The autoclave was evacuated, purged with hydrogen three times, and filled with hydrogen (200 psi). The reaction was heated to 100° C. and stirred for 24 h.

[0112] 2. Workup and Isolation TLC and LCMS of the reaction mixture indicated completion of the reaction. The reaction mixture was filtered through a bed of Celite and the bed was washed with ethyl acetate (100 mL). The filtrate was concentrated under reduced pressure to give an oil.

[0113] The crude oil was dissolved in ethyl acetate (10 vol) and water (10 vol) was added. The organic layer was separated and washed with saturated NaCl (2x). The organic layer was dried over anhydrous Na2SO4 for 1 h. The organic layer was filtered and concentrated under reduced pressure to give an off-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 volumes) 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 the refrigerator overnight to allow more crystals to form. The crystals were collected on a coarse fritted funnel and then air-dried and vacuum-dried for 3 hours to give an off-white solid (870 g, 90% recovery).

[0115] [Example 3] Derivatization of INT 2

[0116] [ka]

[0117] In a 10-dram vial was added INT 2 (10 mg), Mosher's acid (9.1 mg), DMAP (0.004 mg), DCC (12 mg), DCM (1 mL), and a magnetic stir bar. The homogeneous solution was stirred at room temperature overnight. The reaction mixture was filtered and the mother liquor was evaporated. The crude product was 1 H NMR was performed.

[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 (the racemic mixture shows two sets of two singlets, see Figure 4). A chiral HPLC method for measuring the enantiomeric excess (ee) was also developed.

[0119] [Example 4] Derivatization of INT 2

[0120] [ka]

[0121] In a 10-dram vial was added 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 stir bar. The vial was stirred at room temperature for 16 h. An aliquot (10 μL) was dissolved in 10% HPLC grade isopropyl alcohol in hexanes (1 mL, isopropyl alcohol; hexanes) and subjected to chiral HPLC. Chiral HPLC data (Figure 5) suggests that the batch was enantiomerically enriched with a percent enantiomeric excess (%ee) of 97%.

[0122] [Example 5] Preparation of INT 3

[0123] [ka]

[0124] A three-neck 12 L RBF was equipped with a coiled cooling bath, thermocouple / controller, and overhead stirrer. INT 2 (360 g, 1.39 mol, 1 equiv), DMAP (17 g, 0.139 mol, 10 mol%), and toluene (720 mL, 2 vol) were added resulting in an endotherm (T: 17 °C-4.6 °C). All solids dissolved when the temperature rose to approximately 13 °C. The chiller was set to 4 °C and propylene glycol was added to the cooling bath. The bath was quickly cooled to 10-15 °C using dry ice and the chiller was used to maintain that temperature. The RBF was charged with KHCO3 (474 ​​g, 4.74 mol, 3.4 equiv) and water (176 mL, 9.75 mol, 7 equiv). When the mixture was cooled to 15-10 °C, the first portion of acid bromide (255 mL, 2.09 mol, 1.5 eq., density: 1.88 g / mL, Oakwood) was added via addition funnel over 3 h. CO2 began to evolve as the acid bromide was added and stopped approximately 30 min after the addition was complete. TLC indicated the reaction was incomplete with 10-30% starting material remaining. The second portion of acid bromide (130 mL, 1.05 mol, 0.75 eq., Oakwood) was added to the addition funnel and adjusted to add slowly dropwise at 10-15 °C overnight.

[0125] 1. Workup and Isolation The next morning, approximately 10 mL of acid bromide remained in the addition funnel. The remaining reagents were released into the reaction, and TLC and LC / MS (22 h) revealed the reaction to be complete. The cooling bath was replaced with a water bath at approximately 40 °C. Water (1.8 L, 5 vol) and MTBE (1.8 L, 5 vol, Aldrich) were added to the reaction mixture to raise the temperature to 25-30 °C. It was then stirred for 30 min to hydrolyze any unreacted acid bromide. Stirring was stopped and the layers separated into multiple phases, at which point the pH was approximately 8. The water bath was removed and the aqueous phase was siphoned from the 12 L RBF. The organic phase was washed with water (2x, 720 mL, 2 vol) until the pH of the wash was pH 7. The organic phase was transferred to a tared 5 L RBF (any residual water in the 12 L RBF was removed via a 500 mL separatory funnel) and concentrated under reduced pressure at 30-60°C. The sediment was suspended in the product, which was diluted with toluene (200 mL) and polished filtered through a tared coarse frit (approximately 800 mg of wet solids remaining) into a tared 3 L round bottom flask. The flask and frit were then rinsed with a minimum amount of toluene. The solvent was again removed under reduced pressure at 60°C for 2-3 hours to give INT 3 as a dark brown oil (590.76 g, 104% yield). Figure 6A shows the TLC of INT 3 under UV lamp and Figure 6B shows the TLC under PMA stain. The eluent was 20% ethyl acetate / heptane. Figure 7 shows the crude INT 3. 1 H NMR is shown.

[0126] [Example 6] Preparation of INT 4

[0127] [ka]

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

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

[0130] 1. Work-up The next morning, it was determined by LCMS that the SM had been consumed. The reflux condenser was replaced with a distillation head and the temperature was set to 75 °C. Solvent was distilled until 2.3 L (46%, 4 vol) of THF had been collected, leaving approximately 2.7 L (4.7 vol) 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 equiv, 14 vol) was added over 20 min (T: 8-40 °C), with an exotherm occurring with the first 500 mL addition. The product began to precipitate and the mixture was stirred for 1.5 h after which larger particles had broken down and the solid was easier to filter. The solid was filtered through a tare weight coarse 3 L frit and washed with water (8 × 1 L, 1.8 vol) until the washings were pH 6-7. The filter cake was dried under vacuum for 72 h to give the crude product as a water-wet brown solid (642.92 g, 155% yield). The wet cake was suspended in toluene (2 L, 3 volumes) in a 5 L RBF, and the resulting mixture was then vacuum distilled 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 solids in the evaporator flask were transferred using warm toluene (3 x 500 mL; total: 1.5 L, 2.7 vol) to a 22 L 4-neck RBF equipped with a heating mantle, thermocouple / controller, reflux condenser, and overhead stirrer. The mixture was heated to 80 °C and heptane (3 L, 5.3 vol) was added at a rate to maintain the temperature between 70-80 °C. When the addition was complete, the heat was turned off and the mixture was allowed to crystallize while cooling to ambient temperature overnight.

[0132] The next morning, the resulting slurry was filtered through a tare weight coarse 3 L frit, washed with 30% toluene / heptane (3 x 666 mL) and dried for 5-10 min. It was placed in a 55 °C oven until constant mass yielded an off-white solid (269.5 g, 65% yield). Figures 8A and 8B show the TLC of INT 4 (Figure 8A - crude product (IPC check); Figure 8B - crystallized product). The eluent was 50% ethyl acetate / heptane. PMA staining was used. Figure 9 shows the TLC of INT 4.1 H NMR (keto-enol tautomerism) is shown.

[0133] [Example 7] Preparation of INT 5

[0134] [ka]

[0135] Freshly prepared Raney Ni (approximately 700 g, 150 wt%) was stirred in THF (12.9 L, 20 vol) under argon in a 50 L three-neck RBF equipped with a thermocouple, air-powered overhead stirrer, and gas diffusion sparger. See FIG. 10 for the reaction setup. INT 4 (648 g, 2.18 mol, 1 equiv) was added and hydrogen was passed directly into the reaction mixture through the system via the gas diffusion sparger. The reaction was then stirred vigorously overnight.

[0136] After stirring for 18 hours, the hydrogen flow was stopped and then argon was passed through the system for 5-10 minutes before the reaction was opened to atmosphere. A sample was then removed for ion pair chromatography (TLC and LCMS). LCMS did not detect starting material, but TLC indicated starting material remained. It was noticed that the product had started to crystallize in the sparger so it was rinsed 3-4 times with THF to partially dissolve it and improve gas flow. The reaction was then restarted by resuming hydrogen flow and stirring. This was again stirred for an additional 24 hours. Both TLC and LCMS showed completion at the 18 hour mark and the reaction was deemed complete.

[0137] 1. Preparation of Celite Pads A 1-2 cm layer of sand was added to a 2 L coarse frit and leveled. Separately, a slurry of 100 wt% celite (500 g, AW standard Super-Cel NF; Sigma Aldrich) in a minimal amount of THF was prepared. A sheet of filter paper was placed on top of the sand and the slurry was added on top of 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 the Ra-Ni was allowed to settle to the bottom of the round bottom flask. The supernatant was transferred to a 4 L vacuum flask via vacuum siphon so that a minimal amount of Ra-Ni was removed. The flask was then poured onto a celite pad and filtered. At all times, any residual filtered Ra-Ni was moistened with THF. THF (4 L) was added to the reaction round bottom flask containing the spent Ra-Ni, then stirred and allowed to settle. It was siphoned and filtered through celite as before, and the process was repeated until no product was detected (TLC) in the supernatant. THF rinses (2 x 2 L). The product solution was concentrated under reduced pressure via rotovap to give the crude product as a white solid (769 g).

[0139] 3. Crystallization A 12 L three-neck RBF was equipped with an overhead stirrer, thermocouple, and heating mantle. The solids from the 20 L evaporator flask were added to the 12 L RBF. Residual solids in the 20 L evaporator flask were removed with EtOAc (3250 mL, 5 vol) and added to the crystallization flask. Stirring was started and it was heated to 75 °C, and the solids were dissolved at 60-70 °C. Heptane (7800 mL, 12 vol) was added in portions so that the temperature was 70-80 °C. The heat was then turned off and the mixture was allowed to cool to ambient temperature over the weekend.

[0140] The resulting slurry was filtered through a tare weight coarse frit and the wet cake was washed with heptane (1000 mL) and then dried under vacuum for 15-30 min. It was placed in a 35° C. vacuum oven to constant mass to give INT 5 as an off-white solid (453 g, 69% yield). Figures 11A and 11B show the TLC of crude INT 5 (Figure 11A) and crystallized INT 5 (Figure 11B). Figure 12 shows the TLC of crystallized INT 5. 1 H NMR is shown.

[0141] [Example 8] Preparation of INT 6

[0142] [ka]

[0143] A three-neck 12 L RBF was equipped with an overhead stirrer, heating mantle, argon gas inlet and outlet, and a thermocouple / controller. INT 5 (452 ​​g, 1.5 mol) was added to the flask followed by THF (4.5 L, 10 vol) and stirred at room temperature. The solids were allowed to partially dissolve at room temperature. Pyridinium p-toluenesulfonate (5.7 g, 0.015 equiv, 0.023 mol) was added to the flask. 3,4-Dihydro-2H-pyran (382.2 g, 3 equiv, 4.54 mol, Aldrich) was added dropwise to the reaction mixture over 1 h. The reflux condenser was set at 8° C. and the reaction mixture was heated at 50° C. for 24 h.

[0144] 1. Workup and Isolation The reaction was determined to be complete by TLC and LCMS (see FIG. 13). The reaction mixture was transferred into 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 saturated NaCl (1×2 L). The organic layer contained traces of water and was carried on to the next step.

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

[0146] 1. Workup and Isolation The reaction was determined to be complete by LCMS (see FIG. 14). The reaction was quenched 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 h. The organic layer was filtered and concentrated under reduced pressure to give an oil. The crude oil was azeotroped with MTBE (2×2 L) and THF (2×2 L). At the end of the evaporation, all the oil became a mixture of solid chunks and powder. The crude mixture was kept under reduced pressure overnight for further drying (694 g, >100%).

[0147] [Example 10] Preparation of INT 9

[0148] [ka]

[0149] A three-neck 22 L RBF was equipped with an overhead stirrer, cooling bath, argon inlet and outlet, and thermocouple / controller. To the flask was added INT 7 (639 g; 1.5 mol; large chunks broken into small pieces) and powder. To the flask was added THF (6.3 L, 10 vol) and stirred vigorously at room temperature. The slurry mixture was cooled to 5-10 °C. To the reaction mixture was added sodium tert-butoxide (290.6 g, 3.0 mol, 2 equiv) in portions over 1 h. The reaction mixture was stirred at 5-10 °C for 1 h 30 min. During the stirring period, the slurry became cloudy and the color of the reaction mixture changed from light orange to deep orange. Benzyl bromide (388 g, 270 mL, 1.5 equiv, 2.3 mol) was diluted with THF (250 mL) and added to the reaction mixture over 1 h while maintaining the temperature at 5-10 °C. The ice bath was removed and the reaction mixture was stirred at room temperature for 20 h.

[0150] LCMS data suggested the reaction had stopped with 50% of the starting material remaining. A small aliquot was removed and the reaction run at 50° C. After 1 hour, the run LC-MS data suggested the reaction was complete. The cooling bath was replaced with a heating mantle for the reaction and heated at 50° C. over the weekend. Subsequent LCMS data suggested the reaction was complete. The LC-MS of INT 8 is shown in FIG. 15.

[0151] The heat was turned off 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 heat was removed and the resulting mixture was stirred and cooled overnight.

[0152] 1. Workup and Isolation The reaction was determined to be complete by LCMS (see FIG. 16). MTBE (5 L) was added to the reaction and stirred for 15 min. Stirring was stopped, the layers were allowed 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 with anhydrous Na2SO4 (ca. 300 g) for 1 h. The organic layer was filtered and concentrated under reduced pressure to give a thick brick red oil (783 g, >100%, contains traces 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 with anhydrous Na2SO4 (ca. 200 g) for 1 h. The organic layer was filtered and concentrated under reduced pressure to give a thick brick red oil (765 g, >100%, contains traces of solvent).

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

[0154] [Example 11] Preparation of INT 10

[0155] [ka]

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

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

[0158] LCMS 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), likely due to the presence of DHP in the crude product. As shown in the scheme below, a test reaction was performed with INT 8-PEA to convert it to INT 9 and then to INT 10. The test reaction was successful and the remaining material was converted from INT 8-PEA to INT 10 by this method.

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

[0160] [ka]

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

[0162] The reaction of INT 8-PEA to INT 9 was determined to be complete by LCMS (see FIG. 19). The stirring was stopped, 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. The organic layer was then dried with anhydrous Na2SO4 (ca. 300 g) for 1 h, filtered, and concentrated under reduced pressure to give a thick brick red oil (430 g). The crude oil was redissolved in methyl acetate (1.5 L, Aldrich). The solution was washed with 0.5 N HCl (1×500 mL, VWR), followed by water (2×500 mL) and brine (1×500 mL). The organic layer was dried with anhydrous Na2SO4 (ca. 100 g) for 1 h, then filtered, and the Na2SO4 cake was washed with methyl acetate (500 mL). The combined filtrate (INT 9) was carried on to the next step.

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

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

[0165] 3. Recrystallization of INT 10 A three-neck 5 L RBF was equipped with an overhead stirrer, heating mantle, 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 vol) was added to the pot and stirred at room temperature. The slurry mixture was heated to 50° C. to completely dissolve all solids. When the mixture was homogenous, the heat was turned off and the mixture was stirred overnight and allowed to cool to room temperature.

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

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

[0168] [ka]

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

[0170] 1. Workup and Isolation The reaction was determined to be complete by LCMS (see FIG. 22). Stirring was stopped and the 1N HCl layer was separated. The organic layer was washed with water (3×1 L) and dried over anhydrous Na2SO4 (approximately 300 g) for 1 h. The organic layer was filtered and concentrated under reduced pressure to give an oil (460.7 g, >100%, containing traces of solvent).

[0171] [Example 13] Preparation of INT 11

[0172] [ka]

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

[0174] 1. Workup and Isolation The reaction was determined to be complete by LCMS (see Figure 23). The reaction mixture was cooled to 5-10 °C. Water (4.5 L) was added to the reaction while maintaining the temperature below 20 °C. The reaction mixture was stirred for 30 min. The product was extracted into heptane (3 x 2 L). The combined organic layers were washed with 1N HCl (2 x 1.5 L), followed by 5% NaHCO3 (2 x 1.5 L) and 10% NaCl (2 x 1.5 L). The organic layer was dried over anhydrous Na2SO4 (ca. 400 g, Aldrich) for 1 h. The organic layer was filtered and concentrated under reduced pressure to give a thick brick red oil (423.8 g, 99%).

[0175] [Example 14] Preparation of INT 12

[0176] [ka]

[0177] Using the configuration shown in Figure 10, a three-neck 12 L RBF was equipped with an overhead stirrer, cooling bath, argon gas inlet and outlet, and thermocouple / controller. INT 11 (420 g, 1.08 mol) was added to the flask, followed by THF (4.2 L, 10 vol), and the slurry was stirred under argon until a homogenous 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 with hydrogen three times. Hydrogen was bubbled directly into the reaction mixture with a gas diffusion sparger. The reaction was then stirred at room temperature overnight.

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

[0179] 1. Preparation of Celite Pads A slurry of 100 wt% Celite (Sigma Aldrich) in a minimal amount of THF was prepared and added to a 2 L coarse frit. A piece 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 through a bed of Celite. The bed was washed with THF (3 L) and the combined filtrates were concentrated under reduced pressure on a 22 L rotavapor to give the crude product as an oil. After standing at room temperature, the oil became an off-white solid (326.6 g, >100% yield).

[0181] [Example 15] Preparation of compound IA

[0182] [ka]

[0183] 1. Alternative Reagents Previous batches of compound IA utilized diisopropyl azodicarboxylate (DIAD) with triphenylphosphine as the coupling reagent in the final step. The crude reaction mixtures of these steps were observed to contain diisopropyl hydrazinodicarboxylate (DIAD-H2) as the major by-product. For the engineering batch, subsequent chromatographic purification showed that approximately 40% of the desired product was 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 expectation that the by-product (DBAD-H2) could be easily removed by decomposition. Below is a scheme of the decomposition process.

[0184] [ka]

[0185] The expectation was that treatment of DBAD-H2 in the reaction mixture with TFA / DCM at high temperature would decompose the by-products to CO2 (gas), N2 (gas), and isobutylene (gas), which could escape from the reaction mixture. INT 12 (300 mg) was used to test the activity of DBAD for the Mitsunobu reaction, and LCMS confirmed that the reaction worked 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 h. LCMS (Figure 26) showed that the DBAD-H2 peak (RT: 5.21) disappeared without affecting the stability of compound IA.

[0186] Another 30 g scale reaction was carried out using a similar procedure. At larger scale, LCMS showed that compound IA was partially decomposed during the DBAD deprotection attempt, and a new impurity was identified as the deformylated analog of compound IA (see FIG. 27). Under acidic conditions and elevated temperatures, compound IA was decomposed along with DBAD-H2. Therefore, this method was not considered viable at larger scale.

[0187] However, another advantage of using DBAD reagent in the Mitsunobu step was that the difference in retention time between compound IA and DBAD-H2 on TLC was larger 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-neck 12 L RBF was equipped with an overhead stirrer, cooling bath, 500 mL addition funnel, argon gas inlet and outlet, and thermocouple / controller. The flask was charged with INT 12 (274 g, 0.918 mol, 1 equiv), N-formyl L-alanine (139.7 g, 1.193 mol, 1.3 equiv), and PPh3 (288.9 g, 1.102 mol, 1.2 equiv). The RBF was charged with THF (2000 mL) and stirred at room temperature. The reaction mixture was partially dissolved, cooled to 5-10 °C, and an argon flow was started. Di-tert-butyl azodicarboxylate (DBAD, 253.7 g, 1.102 mol, 1.2 equiv) was diluted with THF (700 mL) and added to the 500 mL addition funnel. The solution was added dropwise to the reaction mixture, maintaining the temperature at 5-10 °C. The addition funnel was rinsed with THF (40 mL) and the rinse 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 at room temperature under a stream of argon for 12 hours.

[0189] 3. Work-up and Initial Purification The reaction was determined to be complete by TLC and LCMS. The reaction mixture was transferred to a 10 L rotavapor flask and evaporated under reduced pressure to give a thick oil (971 g). Heptane (1000 mL) was added and the resulting mixture was stirred at 10-15 °C for 2 h. A semi-solid formed which was allowed to settle after stirring suspension. The heptane was decanted and the semi-solid was triturated with heptane (1000 mL). TLC showed that the heptane layer contained less polar 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 fritted funnel. The solid was washed with MTBE (2 x 500 mL). The solid was determined to be triphenylphosphine oxide by TLC and LCMS. The combined MTBE layers were evaporated under reduced pressure to give 894 g of crude product as an oil.

[0190] 4. Column Purification A large glass column (see FIG. 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 give the crude compound adsorbed on the silica. The dried silica was placed on top of the packed column, topped with 1 cm of sand, and filter paper was added. Fractions of 1000 mL were 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 + Lower Impurities

[0191] During 20% ​​EA:heptane elution, early fractions contained less polar impurities (by-products) along with the product, and later fractions contained pure product. Only pure fractions by TLC (see FIG. 28B - mobile phase: 50:50 heptane / ethyl acetate, PMA staining) were collected and evaporated under reduced pressure.

[0192] The combined pure fractions were evaporated under reduced pressure to give an oil which was seeded with compound IA (1.3 g) and kept in the freezer to solidify. The oil became a waxy off-white solid (254 g, 70%) over the weekend (compound IA). Subsequent analysis by UPLC showed the material to be approximately 95% pure (AN at 205 nm). The LC-MS spectrum is shown in Figure 29 and Figure 30. 1 The H NMR spectrum is shown in Figure 31 and the HPLC is shown in Figure 32. Figure 33 shows the crystal structure of compound IA.

[0193] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The present invention is defined by the following claims, including equivalents of the claims to be included therein.

Claims

1. A method for synthesizing a compound of formula I, comprising: 【Chemistry 1】 (In the formula, R １ is C ５～ C １５ is alkyl. The compound of formula I: 【Chemistry 2】 forming said compound of formula (1).

2. R １ But, C １１ The method of claim 1 , wherein the alkyl group is an alkyl group.

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

4. The contacting step comprises: (a) combining INT 12, N-formyl L-alanine, triphenylphosphine, and a solvent to form a reaction mixture; (b) cooling the reaction mixture to a temperature within a specified temperature range below room temperature; (c) dissolving DBAD in a solvent; (d) adding DBAD to the reaction mixture at a rate sufficient to maintain the temperature within the specified temperature range until the compound of formula I is formed; The method of claim 1 or 2, comprising:

5. A method for synthesizing a compound of INT 2, comprising the steps of: INT 2: 【Chemistry 3】 forming a compound of formula (I).

6. 6. The method of claim 5, wherein the pressure of the hydrogen gas in the pressure vessel is less than or equal to 200 psi.

7. 7. The method of claim 5 or 6, wherein the volume ratio of dead space in the pressure vessel to the reaction mixture is 3:1 or greater.

8. The contacting step comprises: (a) combining a ruthenium pre-catalyst with R-BINAP to form a ruthenium (R)-BINAP catalyst; (b) combining the INT 1 with a solvent to form the reaction mixture; (c) adding the ruthenium (R)-BINAP catalyst to the reaction mixture; and (d) heating the reaction mixture to form INT 2; The method of claim 5 or 6, comprising:

9. A method for synthesizing a compound of formula I, comprising: 【Chemistry 4】 (In the formula, R １ is C ５～ C １５ is alkyl. (a) contacting a compound of 1-A with a compound of 1-B, optionally in the presence of an alkali metal, to obtain INT 1: 【Chemistry 5】 (In the formula, X １ is halo; (b) contacting the compound of INT1 with a ruthenium (R)-BINAP catalyst and hydrogen gas to produce INT2: 【Chemistry 6】 forming a compound of (c) contacting said compound of INT 2 with a compound of 3-A to produce INT 3: 【Chemistry 7】 (In the formula, X ２ and X ３ are each independently halo; (d) contacting said compound of INT 3 with a Grignard reagent to give INT 4: 【Chemistry 8】 forming a compound of (e) contacting the compound of INT 4 with hydrogen gas in the presence of a Raney Ni catalyst to produce INT 5: 【Chemistry 9】 forming a compound of (f) contacting the compound of INT 5 with a first protecting agent to produce INT 6: 【Chemistry 10】 (In the formula, R ２ is a protecting group; (g) contacting said compound of INT 6 with a hydroxide to produce INT 7: 【Chemistry 11】 forming a compound of (h) reacting INT 7 with a second protecting agent to protect the free hydroxy group on compound INT 7 to form compound INT 8, and then contacting INT 8 with an acid to deprotect the protected hydroxy group on said compound INT 8 to form compound INT 9: 【Chemistry 12】 (In the formula, R ３ is a protecting group; (i) optionally purifying said compound of INT 9 by contacting said compound of INT 9 with (S)-(−)-1-phenylethylamine to obtain INT 10: 【Chemistry 13】 and then contacting INT 10 with an acid to give INT 9 (purified): 【Chemistry 14】 forming (j) dehydrating the compound of INT 9 or the compound of INT 9 (purified) with a dehydrating agent to obtain INT 11: 【Chemistry 15】 forming a compound of (k) deprotecting said compound of INT 11 to give INT 12: 【Chemistry 16】 forming a compound of formula (I); and (l) contacting the compound of formula I with N-formyl L-alanine in the presence of di-tert-butyl azodicarboxylate (DBAD) and triphenylphosphine to obtain a compound of formula I: 【Chemistry 17】 forming a compound of formula (I).

10. 10. The method of claim 9, wherein in step (b), the contacting of 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 greater.

11. 10. The method of claim 9, wherein in step (b), the pressure of the hydrogen gas is less than 200 psi.

12. In step (c), the contacting step comprises contacting dimethylaminopyridine (DMAP) and potassium bicarbonate (KHCO) in a two-phase reaction. ３ 10. The process of claim 9, wherein the process is carried out in the presence of an aqueous solution of

13. 10. The method of claim 9, wherein in step (d), the Grignard reagent comprises tert-butyl MgBr.

14. 10. The method of claim 9, wherein in step (e), the Raney Ni catalyst is freshly prepared and the pressure of the hydrogen gas is in the range of 0.5 to 2 atmospheres.

15. In step (f), the protecting agent is 3,4-dihydro-2H-pyran (DHP), and R ２ The method of claim 9 , wherein is a tetrahydropyran (THP) group.

16. 10. The method of claim 9, wherein in step (i), after crystallization and isolation of said compound of INT 10, said compound of INT 10 is contacted with an acid to form purified INT 9.

17. 10. The method of claim 9, wherein the dehydrating agent in step (j) comprises benzenesulfonyl chloride.

18. 10. The method of claim 9, wherein in step (k), the deprotection step comprises a debenzylation reaction optionally using Pd / C and hydrogen gas.

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