Processes for preparing a KRAS g12c inhibitor

Abstract

Provided are processes suitable for preparing the KRAS G12C inhibitor, (5aSa,17aRa)-20-Chloro-2-[(2S,5R)-2,5-dimethyl-4-(prop-2-enoyl)piperazin-1-yl]-14,17-difluoro-6-(propan-2-yl)-11,12-dihydro-4H-1,18-(ethanediylidene)pyrido[4,3-e]pyrimido[1,6-g][1,4,7,9]benzodioxadiazacyclododecin-4-one, (the compound of Formula (I)), to provide sufficient quantities of drug substance to support clinical trials and commercial operations. Also provided are certain synthetic intermediates useful in preparing the compound of Formula (I). The present disclosure also provides crystalline polymorphic forms of the compound of Formula (I) as well as processes for their preparation.

Description

PROCESSES FOR PREPARING A KRAS G12C INHIBITORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 683,017, filed August 14, 2024, the disclosure of which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on July 31, 2025, is named 26037-WO-PCT_SL and is 3,154 bytes in size.FIELD

[0003] This disclosure relates generally to processes suitable for preparing the KRAS G12C inhibitor, (5aSa,17aRa)-20-Chloro-2-[(25,5J?)-2,5-dimethyl-4-(prop-2-enoyl)piperazin-l-yl]- 14,17-difluoro-6-(propan-2-yl)-l l,12-dihydro-477-l,18-(ethanediylidene)pyrido[4,3- e]pyrimido[l,6-g][l,4,7,9]benzodioxadiazacyclododecin-4-one, hereinafter the compound of Formula (I), to provide sufficient quantities of drug substance to support clinical trials and commercial operations.BACKGROUND

[0004] International Publication No. WO2021 / 086833 discloses the compound of Formula (I) and its potent inhibition of guanine nucleotide exchange in the SOS-catalyzed exchange assay. The compound also demonstrated potent inhibition in a cellular phosphor-ERK assay. The compound of Formula (I) possesses excellent bi oavai 1 ability which allows it to be administered orally to human patients.

[0005] The compound of Formula (I) is currently in Phase I clinical studies evaluating its safety, pharmacokinetics, and efficacy alone or as part of various combination therapies in patients with advanced solid tumor which have identified as having KRAS G12C mutation. ClinicalTrials.gov ID No. NCT05067283. Early results from this study indicate that the compound of Formula (I) as monotherapy and in combination with pembrolizumab showed manageable safety and preliminary antitumor activity in study subjects with previously treated solid malignancies and previously untreated non-small cell lung cancer (NSCLC) with a KRAS G12C mutation. Patients are currently enrolling in a Phase 3 study evaluating the efficacy and safety of the compound of Formula (I) in combination with pembrolizumab as first-line treatment in patients with metastatic NSCLC which have a KRAS G12C mutation and programmed cell death ligand 1 tumor proportion score >50%. ClinicalTrials.gov ID No. NCT06345729.

[0006] The compound of Formula (I) is a single atropisomer and the synthetic route disclosed in WO2021 / 086833 includes a supercritical fluid chromatographic separation step of an early synthetic intermediate to provide the desired atropisomer. The disclosed synthesis was suitable for providing enough drug substance to support early preclinical assays but is less suitable for providing larger quantities of the drug substance. To provide sufficient drug substance to supply such clinical studies and to support commercial operations in the future, new scalable and efficient processes were desired.SUMMARY

[0007] In one aspect, the present disclosure provides a process for preparing a compound ofFormula (I)

[0008] In another aspect, the present disclosure provides certain synthetic intermediates useful in preparing the compound of Formula (I) and salts thereof.

[0009] In yet another aspect, the present disclosure provides crystalline polymorphic forms 1 and 2 of the compound of Formula (I) as well as methods for their preparation.

[0010] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows the X-ray powder diffraction (XRPD) pattern of form 1 of the compound of Formula (I).

[0012] FIG. 2 shows the X-ray powder diffraction (XRPD) pattern of form 2 of the compound of Formula (I).DETAILED DESCRIPTION

[0013] The present disclosure provides a large-scale, high-yielding and convergent process for preparing the compound of Formula (I). The disclosed process minimizes use of hazardous reagents and transition metal catalysts. In addition, the disclosed process eliminates chromatographic separations which create excessive waste, are expensive and inconvenient for preparing commercial scale quantities of the drug substance.

[0014] In one embodiment, the present disclosure provides a process for preparing a compound of Formula (I), comprising(a) providing an atropisomeric mixture of Int-A(b) combining the atropisomeric mixture of Int-A with an optically active acid in a solvent to provide Int-A- 1: optically active acid salt;(c) converting the Int-A- 1: optically active acid salt to the compound of Formula (I).

[0015] In some embodiments of the process, the optically active acid in step (b) is selected from the group consisting of (+) dibenzoyl tartaric acid. (-) ditoluoyl tartaric acid, and (-) diacetyl tartaric acid. In one specific embodiment, the optically active acid in step (b) is (-) ditoluoyl tartaric acid. In another specific embodiment, the optically active acid in step (b) is (+) dibenzoyl tartaric acid.

[0016] In some embodiments of the process, the solvent in step (b) is toluene, dimethoxy ethane, tetrahydrofuran, or ethyl acetate. In certain embodiments of the process, the solvent in step (b) is dimethoxyethane, tetrahydrofuran, or ethyl acetate. In a specific embodiment, the solvent in step (b) is dimethoxy ethane. In another specific embodiment, the solvent is toluene. In select embodiments, the solvent is a mixture of toluene and tetrahydrofuran.

[0017] In certain embodiments of the process, the atropisomeric mixture of Int-A, optically active acid, and solvent are combined with an anti-solvent in step (b). The anti-solvent in step (b) can be a C5-C8 alkyd or C5-C8 cycloalkyl solvent. In a specific embodiment, the anti-solvent in step (b) is heptane.

[0018] In some embodiments of the process, step (b) is performed at a temperature of 40 °C to 85 °C. In certain embodiments of the process, step (b) is performed at a temperature of 40 °C to 70 °C. In a specific embodiment, step (b) is performed at a temperature of 50 °C to 60 °C. In other embodiments, step (b) is performed at a temperature of 70 °C to 85 °C, e.g., 80 °C.26037

[0019] In certain embodiments of the process, step (b) is conducted in a reaction vessel.

[0020] In one embodiment, the present disclosure provides the process wherein step (c) comprises:(cl) treating the Int-A-1 : optically active salt with a carbonic acid derivative selected from the group consisting of a di C1-C3 alkylcarbonate, phosgene, diphosgene, tnphosgene, carbonyldiimidazole and disuccinimidyl carbonate and a base in an organic solvent to formconverting Int-B to the compound of Formula (I).

[0021] In some embodiments of the process, the carbonic acid derivative is the di C1-C3 alkylcarbonate, for example, diethyl carbonate.

[0022] In certain embodiments of the process, the base is selected from the group consisting of alkali or alkaline earth metal C1-C6 alkoxides, bis(tnmethylsilyl)amide and diisopropyl amide salt. For instance, the base can be potassium bis(trimethylsilyl)amide, sodium t-butoxide, potassium t-butoxide.

[0023] In some embodiments, the process further comprises isolating Int-B. In specific embodiments, the isolation of Int-B comprises: quenching the reaction mixture with acid; adding acetonitrile or a mixture of 2-methyltetrahydrofuran and heptane; and recovering Int-B.

[0024] In other embodiments, the isolation of Int-B comprises: quenching the reaction mixture with acid; adding a mixture of acetonitrile and heptane; and recovering Int-B.

[0025] In some embodiments, step (cl) is performed in the same reaction vessel as step (b).

[0026] In one embodiment, the present disclosure further includes:(c2) treating Int-B with an electrophilic chlorinating agent and a base in an organic solvent to form a reaction mixture Int-B Rxl; andaddingthe reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx2; andconverting Int-C to form the compound of Formula (I).

[0027] In some embodiments of the process, the electrophilic chlorinating agent in step (c2) isPOCI3.

[0028] In certain embodiments of the process, the base in step (c2) is N,N-diisopropylethyl amine.

[0029] In certain embodiments, the acid addition salt form of Int-W3 is prepared by: benzylatingtreating Int-W2a with HC1 to provide the acid addition salt form of Int-W3. In specific embodiments, the HC1 can be generated in situ, e.g., from the reaction of acety l chloride and ethanol.

[0030] In other embodiments, the acid addition salt form of Int-W3 is prepared by:(a) contacting a l-benzyl-2,5-dimethylpyrazin-l-ium salt with a PyRED enzyme and NADPH to provide Int-W3; and(b) treating Int-W3 with an acid to provide the acid addition salt form of Int-W3.

[0031] In some embodiments, the PyRed enzyme in (a) comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2.

[0032] In certain embodiments, the contacting in (a) further comprises a glucose and a glucose dehydrogenase enzyme.

[0033] In certain embodiments, step (c) further comprises:(c3) treating Int-C with acryloyl chloride, an iodide salt and a base to provide the compound of Formula (I).

[0034] In some embodiments of the process, step (c) comprises:(c4) treating Int-B with an electrophilic chlorinating agent and a base in an organic solvent to form a reaction mixture Int-B Rxl; and addinglnt-W4 to the reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx3 and separating the compound of Formula (I) from the reaction mixture Int-B Rx3.

[0035] In specific embodiments, the electrophilic chlorinating agent in step (c4) is POCI3.

[0036] In specific embodiments, the base in step (c4) is diisopropylethyl amine.

[0037] In some embodiments, the solvent in step (c4) is THF. The reaction mixture can also comprise acetonitrile.

[0038] The present disclosure also provides a method for preparing the atropisomeric mixture of Int-A by cyclizing

[0039] In certain embodiments, the cyclization of Int-D comprises:(al) treating Int-D with a dehydrating agent and a base in an organic solvent to form a reaction mixture Int-D Rxl;(a2) adding an alkali or alkaline earth metal alkoxide to the reaction mixture Int-D Rxl to form a reaction mixture Int-D Rx2;(a3) adding an alkali or alkaline earth metal hydroxide to the reaction mixture Int-Dx2 to form a reaction mixture Int-D Rx3; and(a4) isolating the atropisomeric mixture of Int-A from the reaction mixture Int-D Rx3.

[0040] In some embodiments of the process, the dehydrating agent in step (al) of the cyclization of Int-D is diethylchlorophosphate.

[0041] In some embodiments of the process, the solvent in the cyclization of Int-D is THF.

[0042] In some embodiments of the process, the alkali or alkaline earth metal alkoxide in the cyclization of Int-D is sodium t-butoxide. In other embodiments, alkali or alkaline earth metal alkoxide is potassium hydroxide.

[0043] In specific embodiments, steps (al)-(a3) are performed in the same reaction vessel.

[0044] In some embodiments of the process, Int-D is prepared by treatingembodiments, the base is potassium t-butoxide.

[0045] The present disclosure provides an alternative process for preparing the atropisomeric mixture of Int-A. by:Mitsunobu conditions to form a reaction mixture Int-G Rxl;26037(a7) combining Int-G with an alkali or alkaline earth metal alkoxide in an organic solvent to form a reaction mixture Int-G Rx2;(a8) adding an alkali or alkaline earth metal hydroxide to the reaction mixture Int-G Rx2 to form a reaction mixture Int-G Rx3; and(a9) isolating the atropisomeric mixture of Int-A from the reaction mixture Int-G Rx3.

[0046] In certain embodiments of this alternative process, the Mitsunobu conditions comprise treatment of Int-E2 and Int-F with diisopropylazodicarboxylate and triphenyl phosphine in an organic solvent.

[0047] In another aspect, the present disclosure provides certain synthetic intermediates useful for preparing the compound of Formula (I). In one embodiment, the present disclosure provides a compound

[0048] In another embodiment, the present disclosure provides a compound

[0049] In another embodiment, the present disclosure provides a compound26037salt thereof.

[0050] In another aspect, the present disclosure provides certain crystalline polymorphs of the compound of Formula (I).

[0051] In one embodiment, the present disclosure provides a crystalline polymorphic form of a compound of Formula (I), which is designated Form 1, wherein the cry stalline polymorphic form designated Form 1 has d-spacings determined by powder diffraction, Cu Ka, of about 11.50, 9.75, 9.10, 8.14, 6.44, 5.78, 5.38, and 5.06 angstroms. In specific embodiments, Form 1 has at least two additional d-spacings determined by x-ray powder diffraction, Cu Ka, selected from about 7.77, 7.40, 5.63, 5.50, and 5.16 angstroms. The crystalline polymorph form 1 has a Differential Scanning Calorimetry (DSC) extrapolated onset melting temperature of about 292.9 °C. The crystalline polymorph form 1 has a Differential Scanning Calorimetry (DSC) peak melting temperature of about 295. 1 °C.

[0052] In certain embodiments, the cry stalline polymorph form 1 can be prepared by a process comprising:(a) contacting the compound of Formula (I) with a solvent pair selected from the group consisting of:(i) acetonitrile and water,(ii) methanol and water,(iii) acetone and water, and(iv) ethyl acetate and heptane;(b) allowing the crystalline polymorphic form to precipitate; and(c) isolating the precipitated crystalline polymorphic form.

[0053] In one embodiment, the present disclosure provides a cry stalline polymorphic form of a compound of Formula (I), which is designated Form 2, wherein the crystalline polymorphic form designated Form 2 has d-spacings determined by powder diffraction, Cu Ka. of about 9.27, 7.29, 6.52, 5.25, 5.12, and 4.31 angstroms. In specific embodiments, Form 2 has at least two additional d-spacings determined by x-ray powder diffraction, Cu Ka, selected from about 8.24, 7.82, 5.69, 5.63, 4.71, 4.63 angstroms. The crystalline polymorph form 2 has a Differential Scanning Calorimetry (DSC) extrapolated onset melting temperature of about 298.8 °C. The crystalline26037 polymorph form 2 has a Differential Scanning Calorimetry (DSC) peak melting temperature of about 300.4 °C.

[0054] In certain embodiments, the crystalline polymorph form 2 can be prepared by a process comprising:(a) contacting the compound of Formula (I) with a solvent or solvent pair selected from the group consisting of:(i) ethyl acetate and methyl tert-butyl ether,(ii) ethyl acetate and hexanes,(iii) xylenes and methyl tert-butyl ether, and(iv) cyclopentyl methyl ether(b) allowing the crystalline polymorphic form to precipitate; and(c) isolating the precipitated crystalline polymorphic form.Definitions

[0055] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0057] As used herein, the articles “a” and ‘"an” refer to one or to more than one (i. e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0058] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0059] As used herein, the term “atropisomeric mixture” refers to a mixture of atropisomers.

[0060] As used herein, the term “dehydrating agent” refers to substances that have the ability to expunge one or more water molecules from a compound. Examples of dehydrating agents include diethylchlorophosphate, diphenyl chlorophosphate, and alkyl tosylates.

[0061] As used herein, the term “electrophilic chlorinating agent” refers to substances that react with nucleophiles such as keto, enol, or amide substrates to provide the chloro-substituted26037 products. Examples of electrophilic chlorinating agents are phosphorus oxychloride, phosphorus trichloride, and thionyl chloride.

[0062] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0063] As used herein, the term “optically active acid” refers to chiral acids which may be used for the resolution of racemic bases. Examples of optically active acids are dibenzoyl tartaric acid, ditoluoyl tartaric acid, diacetyl tartaric acid, tartaric acid, malic acid, mandelic acid, and camphor- 10-sulfonic acid.

[0064] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0065] As used herein, the term “salt thereof’ when used in the context of a synthetic intermediate refers to salt forms that are suitable for the preparation of drug substances subject to regulatory review by health authorities. Salts can be obtained from synthetic intermediates used to prepare the compound of Formula (I) by customary' methods which are known to the person skilled in the art, for example, by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts. Suitable salts include, for example, those derived from mineral acids such as chlorides, nitrates, phosphates, sulfates, borates, fluorides, bromides, perchlorates and sulfonates.

[0066] “Percentage of sequence identity ,” “percent identity',” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, w herein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the26037 number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0067] Some of the synthetic intermediates described herein may exist as tautomers which have different points of attachment of hydrogen accompanied by one or more double bond shifts. For example, a ketone and its enol form are keto-enol tautomers. The individual tautomers as well as mixtures thereof are encompassed with synthetic intermediates provided by the present disclosure.

[0068] Some of the synthetic intermediates described herein may exist as atropisomers when the rotational energy' barrier around a single bond is sufficiently high to prevent free rotation at a given temperature, thus allowing isolation of individual conformers with distinct properties. Unless specifically indicated by way of structural formula or chemical name, the individual atropisomers as well as mixtures thereof are encompassed by the synthetic intermediates provided by the present disclosure. When resolved, individual atropisomers can be designated by established conventions such as those specified by the International Union of Pure Applied Chemistry (IUPAC) 2013 Recommendations.

[0069] In the compound of Formula (I) and the synthetic intermediates disclosed herein, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present disclosure as described and claimed herein is meant to include all suitable isotopic variations of the compounds of Formula (I) and the synthetic intermediates disclosed herein. For example, different isotopic forms of hydrogen (H) include protium (1H ) and deuterium (2H, also denoted herein as D). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds of Formula (I) and the synthetic intermediates, can be prepared without undue experimentation by conventional26037 techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates.Processes for Preparing the Compound of Formula (I)

[0070] A process for preparing the compound of Formula (I) of the present disclosure is described in the following Schemes and Examples. Starting materials and intermediates are purchased, made from known procedures, or as otherwise illustrated.Scheme 1

[0071] As shown above in Scheme 1, a mixture of the atropisomers of Int-A is resolved by a crystallization-induced diastereomeric transformation (CIDT). The combination of atropisomeric mixture of Int-A with an optically active acid in a solvent provides a slurry / suspension of the salts of Int-A with the optically active acid, wherein the desired diastereomeric salt precipitates. The optically active acid can be selected from the group consisting of (+) dibenzoyl tartaric acid, (-) ditoluoyl tartaric acid (z.e., di-p-toluoyl-L-tartaric acid), and (-) diacetyl tartaric acid. The transformation may be conducted using a number of different organic solvents including dimethoxyethane, toluene, tetrahydrofuran, or ethyl acetate.

[0072] The combination with the optically active salt is performed at a temperature of 40 °C to 85 °C. Preferably, the temperature is from 70 °C to 85 °C, e.g., 80 °C to provide the Int-A: optically active acid salt with high diastereoselectivity.

[0073] An anti-solvent can optionally be added to the reaction mixture which improves the yield of Int-A: optically active acid. The anti-solvent added can be a C5-C8 alkyl, e.g., heptane or C5-C8 cycloalkyl solvent.26037

[0074] To form Int-B, the Int-A- 1 : optically active salt is treated with a carbonic acid derivative selected from the group consisting of a di C1-C3 alkylcarbonate, phosgene, diphosgene, triphosgene, carbonyldiimidazole and disuccinimidyl carbonate and a base in an organic solvent. The carbonic acid derivative can be in certain embodiments, a di C1-C3 alkylcarbonate, such as diethyl carbonate. The base can be selected from alkali or alkaline earth metal C1-C6 alkoxide (e.g, sodium tert-butoxide), bis(trimethylsilyl)amide (e.g., KHMDS) and diisopropyl amide salt. The solvent can be an ether solvent, such as THF or dimethoxy ethane or heptane.

[0075] In specific embodiments, Int-B can be isolated by quenching the reaction mixture with acid, adding acetonitrile and heptane and recovering Int-B. In other specific embodiments, Int-B can be isolated by quenching the reaction mixture with acid, adding 2-methyltetrahydrofuran and heptane and recovering Int-B.

[0076] Preferably, the cyclization of the Int-A: optically active salt with the carbonic acid derivative occurs immediately after the CIDT transformation of the atropisomeric mixture of Int- A. The Applicant has also found that performing the cyclization step immediately after the CIDT transformation provides Int-B with a higher enantiomeric excess and higher isolated yields. In some embodiments, the treatment of the Int-A:optically active acid is performed in the same reaction vessel as the CIDT transformation of the atropisomeric mixture of Int-A. In addition, the Applicant has found that performing the cyclization step at -10 °C to 0 °C, e.g.. -5 °C, provides Int-B in high enantiomeric excess.26037Scheme 2

[0077] In one embodiment, Int-B can be converted to the compound of Formula (I) using the reaction sequence provided in Scheme 2. Int-B can be treated by an electrophilic chlorinating agent, e.g, phosphorus oxychloride (POCI3), and a base, e.g, N,N-diisopropylethylamine in an organic solvent, e.g., THF or acetonitrile, and optionally 1,2,4-triazole to form a reaction mixture Int-B Rxl. Int-W3 is added to the reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx2. Typically, the Int-W3 is added to the reaction mixture Int-B Rxl in an acid addition salt form, such as a hydrochloride salt. The intermediate dichloropyridopyrimidone (or chlorotriazolopyrimidone) is typically not isolated but is treated directly with Int-W3 to provide Int-C, which can be purified, for example, by crystallization.

[0078] Int-C is treated with acryloyl chloride, an iodide salt, to provide the compound of Formula (I). The treatment is typically performed in an organic solvent such as acetonitrile. In specific embodiments, the iodide salt is an alkali metal iodide, such as sodium iodide.26037und of Formula (I)

[0079] The compound of Formula (I) can also be prepared by the alternative reaction scheme shown in Scheme 3. Int-B can be treated by an electrophilic chlorinating agent, e.g., POCI3, and abase, e.g., N,N, diisopropylethylamine in an organic solvent, e.g., THF, to form a reaction mixture Int-B Rxl. Int-W4 is added to the reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx3. Reaction mixture Int-B Rx3 can further contain acetonitrile. Typically, Int-W4 is added to the reaction mixture Int-B Rxl in an acid addition salt form, such as a methanesulfonate or tetrafluoroborate salt. The compound of Formula (I) is isolated from the reaction mixture Int-B Rx3. The intermediate dichloropyridopyrimidone is typically not isolated.26037Scheme 4

[0080] In some embodiments, the atropisomeric mixture of Int-A can be synthesized using the reaction sequence shown in Scheme 4. Int-El is treated with Int-F and a base, e.g., potassium tert-butoxide, to form Int-D. Int-D can be cyclized by treating Int-D with a dehydrating agent (e.g., diethylchlorophosphate) and a base, in an organic solvent, e.g., THF, to form a reaction mixture Int-D Rxl. The phosphorylated intermediate G2 is formed from reaction mixture Int-D Rxl.

[0081] An alkali or alkaline earth metal alkoxide, e.g., sodium tert-butoxide, is added to the reaction mixture Int-D Rxl to form a reaction mixture Int-D Rx2. The chloro cyano-substituted pyridyl intermediate D2 is formed from reaction mixture Int-D Rx2. An alkali or alkaline earth metal hydroxide is added to the reaction mixture Int-D Rx2 to form a reaction mixture Int-D Rx3. Finally, the atropisomeric mixture of Int-A is isolated from the reaction mixture Int-D Rx3. In specific embodiments, it is convenient to perform the treatment of Int-D, and reagent additions to the reaction mixtures Int-D Rxl and Int-D Rx2 in the same reaction vessel, without isolations of Int-G2 and Int-Gl .26037Scheme 5

[0082] In other embodiments, the atropisomeric mixture of Int-A can be synthesized using the reaction sequence shown in Scheme 5. Int-E2 and Int-F are combined under Mitsunobo conditions to form a reaction mixture Int-G Rxl. In specific embodiments, the Mitsunobo conditions comprise treatment of Int-E2 and Int-F with diisopropylazodicarboxylate and triphenyl phosphine in an organic solvent. Int-G is isolated from the reaction mixture Int-G Rxl.

[0083] Int-G is combined with an alkali or alkaline earth metal alkoxide, e.g., sodium tert- butoxide. to form a reaction mixture Int-G Rx2. Int-Gl is formed from reaction mixture Int-G Rx2, but isolation of this intermediate is not necessary. An alkali or alkaline earth metal hydroxide is added to the reaction mixture Int-G Rx2 to form a reaction mixture Int-G Rx3. Finally, the atropisomeric mixture of Int-A is isolated from the reaction mixture Int-G Rx3.26037Scheme 6Pyrazinium halide lnt-W3

[0084] Scheme 6 provides two alternative routes for preparing Int-W3. In one approach, the Boc-protected piperazine is treated with a benzylating agent, e.g, benzyl bromide, in the presence of a base to provide the benzyl- and Boc-protected piperazine intermediate Int-W2a. The Boc protecting group is removed using an acid, e.g., HC1, to provide Int-W3 which may be conveniently isolated as a salt.

[0085] In another approach, a l-benzyl-2,5-dimethylpyrazin-l-ium halide can be contacted with PyRED enzyme with NADPH serving as a reductant to provide Int-W3. Typically, NADPH is provided in a cascade with co-factor-recycling enzy me, <?.g, glucose dehydrogenase.EXAMPLES

[0086] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0087] Throughout the synthetic schemes and examples, abbreviations and acronyms may be used with the following meanings unless otherwise indicated:

[0088] AcCl = acetyl chloride; AcOH = acetic acid; AY = assay yield; aq. = aqueous; BHT = butylated hydroxytoluene; BSA = bis(timethylsilyl)acetamide; CIDT = cry stallization-induced diastereomeric transformation; DCM = di chloromethane; DIEA / DIPEA / Huenig’s base = N,N- diisopropylethylamine; DIAD = diisopropylazodicarboxylate; DME = 1,2-dimethoxy ethane;DMF = N,N -dimethylformamide; DMF-DMA = N,N-dimethylformamide dimethyl acetal; dppf = l,l'-bis(diphenylphosphino)ferrocene; DMAP = 4-dimethylaminopyridine; DMS = dimethylsulfate; EtOAc = ethyl acetate; EtOH = ethanol; h / hr = hour(s); GDH = glucose26037 dehydrogenase enzyme; g = gram; HO At = l-hydroxy-7-azabenzotriazole; IPA = isopropanol; IP AC = isopropyl acetate; IY = isolated yield; kg= kilogram; L = liter; MeCN = acetonitrile; MeOH = methanol; MHz = megahertz; MeTHF = 2-methyl tetrahydrofuran; MsOH = methanesulfonic acid; MTBE = methyl te / 7-butyl ether; min = minutes; pL = microliter; pmol = micromole; mg = milligram; mL = milliliter; mmol = millimole; M = molar; mol = mole; MOPS = (3-(N-morpholino)propanesulfonic acid) buffer; MPa = megapascal; nBu = n-butyl; NMI = N- methylimidazole; NMP = N-methyl-2-pyrrolidone; Pd / C = palladium on carbon; ppm = parts per million; PyRED = pyrazinium reductase enzyme; rt = room temperature; TBAHS = tetrabutylammonium hydrogen sulfate; tBu = terf-butyl; THF = tetrahydrofuran; tol = toluene; UHPLC = ultra-high-performance liquid chromatography; V = volume, wt.% = weight percentage.

[0089] The compounds and crystalline forms thereof described herein can be prepared according to the procedures of the following schemes and examples, using appropriate materials and are further exemplified by the following specific examples. The examples further illustrate details for the preparation of the compounds and crystalline forms of the present disclosure. Those skilled in the art will readily understand that know n variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds and their crystalline forms. These examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosure.

[0090] Concentration refers to the removal of the volatile components at reduced pressure (<?.g., by rotary evaporation or distillation) unless otherwise noted. All temperatures are in degrees Celsius unless otherwise noted. Mass spectra (MS) were measured by electrospray ion-mass spectroscopy (ESI) in positive ion detection mode and m / z refers to the [M+H]+ion unless otherwise noted. 'H NMR spectra w ere recorded at 400-500 MHz at ambient temperature unless otherwise noted. Protons reported as 0.5 H are due to rotameric signals.

[0091] Powder X-ray diffraction data were acquired on a Bruker D8 Advance system equipped with a Lynxeye XE-T detector. The instrument was configured in the Bragg-Brentano configuration and equipped with a sealed tube Cu radiation source. X-ray monochromatization was achieved by detector energy' discrimination. A fixed slit optical configuration was employed. Data was acquired between 2 and 40° 20. Samples were prepared by gently pressing pow dered samples onto a shallow cavity zero background silicon holders. XRD sample stage rotate speed at 15 degree per minutes. X-ray was run at a voltage of 40.0 kV and 40 mA current. Tables 1 and 2 below list the XRPD peak locations for the isolated crystalline forms. The tables provide the peak positions (expressed in degrees 20) D-Spacings (expressed in angstroms), and peak intensities.26037

[0092] Differential scanning calorimetry (DSC) for the crystalline forms of the compound of Formula (I) were determined using a TA Instruments Q200 or Discovery calorimeter at a heating rate of 10 °C / min under a nitrogen atmosphere in an aluminum pan with pin holes.

[0093] The extrapolated onset and peak melting temperatures and enthalpy of each thermal event were determined for each form.Example 1: Preparation of 5-chloro-2-oxo-6-(2,3,6-trifluorophenyl)-l,2-dihydropyridine-3- carbonitrile (Int-El)Synthesis of l-(2.3.6-trifluorophenyl)ethan-l-one

[0094] To a solution of 1,2,4-tiifluorobenzene (2.32 g, 1.835 mL, 1 eq, 17.56 mmol) in THF (20 mL) at -60 °C was added n-hexyllithium solution (7.64 mL, 1 eq, 17.56 mmol), while maintaining the internal temperature below -55 °C. Upon complete addition, the reaction was aged for 1 hour at -60 °C. This was followed by the dropwise addition of zinc chloride solution in THF (35.1 mL. 1 eq. 17.56 mmol), maintaining the internal temperature below -55 °C. The resulting solution was warmed up to -10 °C, followed by the addition of copper(I) chloride (0.174 g, 0.1 eq, 1.756 mmol) and acety l chloride (1.374 mL, 1.1 eq, 19.32 mmol). The reaction was then allowed to warm up to room temperature then was aged overnight. The following day, the reaction was quenched with IN HC1 (15 mL) and diluted with isopropyl acetate (15 mL). The aqueous layer was removed, and the organic layer was washed again with IN HC1 (15 mL) followed by IN NH4OH (2 x 15 mL) and water (15 mL). The product was obtained as an oil, 1- (2,3,6-trifluorophenyl)ethan-l-one (1.98 g, 9.88 mmol, 56.2% IY). 'H NMR (500 MHz, CDC13) 5 7.25 (qd, J= 9.3. 5.2 Hz. 1H), 6.95 - 6.90 (m, 1H), 2.61 (s, 3H).13C NMR (126 MHz. CDCh) 5 193.50, 155.20 (ddd, J= 250.7, 5.2, 3.0 Hz), 147.91 (ddd, J = 257.0, 15.1, 7.9 Hz), 147.18 (ddd, J = 247.2, 13.3, 3.9 Hz), 119.94 - 119.60 (m), 119.41 (ddd, J= 19.6, 10.3, 1.8 Hz), 111.8626037(ddd, J= 25.1, 6.1, 4.3 Hz), 32.29;19F NMR (471 MHz, CDCh) 8 -117.28 (d, J= 15.7 Hz), - 135.99 (d, J= 21.2 Hz), -141.00 (dd, J= 21.2, 15.8 Hz).Synthesis of (E)-3-(dimethylamino)-l-(2.3.6-trifluorophenyl)prop-2-en-l-one

[0095] To a flask containing l-(2,3,6-trifluorophenyl)ethan-l-one (100 g, 1 eq, 574 mmol) was added 1,1 -dimethoxy -JVJV-dimethylmethanamine (137 g, 153 mL, 2 eq, 1.15 mol). The mixture was then stirred at 100 °C for 6 hours. After completion of the reaction (monitored by UHPLC), the mixture was quenched with water (5 V, 500 mL) and extracted with ethyl acetate (12 V total, 600 mL x 2) then died over anhy drous Na2SC>4. The combined organic phases were concentrated under reduced pressure. A small amount of MTBE was added (0.2 V. 20 mL), followed by hexane (5 V, 500 mL) as antisolvent. The solid was filtered to give (E)-3-(dimethylamino)-l- (2,3,6-trifluorophenyl)prop-2-en-l-one (112 g, 489 mmol, 85.1% IY).1H NMR (500 MHz, CDCh) 3 7.84 (br s, 1H), 7.10 (dt, J= 13.9, 6.9 Hz, 1H), 6.84 (t, J= 7.9 Hz, 1H), 5.32 (s, 1H), 3.12 (s. 3H), 2.89 (s, 3H).13C NMR (151 MHz, DMSO, 383.0 K) 6 179.06, 155.01, 154.35 - 152.74 (m), 146.80 - 144.90 (m), 121.26, 116.39 (dd. J = 19.7, 9.8 Hz), 111.15 (dt, J= 25.5, 5.3 Hz), 97.29, 39.60;19F NMR (471 MHz, CDCh) 8 -118.54 (d, J= 15.5 Hz), -136.64, -142.08.Synthesis of Sodium (lZ.3E’)-5-amino-4-cvano-5-oxo-l-(2.3.6-trifluorophenyl)penta-L3- dien-1 -plateProcedure A

[0096] A flask was charged with 2-cyanoacetamide (3.71 g, 1.01 eq, 44.1 mmol) and EtOH (100.0 mL, 10 V). At this point, sodium ethoxide (15.6 g, 17.9 mL, 21 vvt.%, 1.1 eq, 48.0 mmol) was added, and the reaction mixture was stirred for 10 mins. This resulted in a heterogeneous mixture. Then. (E)-3-(dimethylamino)-l-(2.3.6-trifluorophenyl)prop-2-en-l-one (10.0 g. 1 eq.43.6 mmol) was added as a solid in one portion. The reaction was allowed to stir overnight. Upon completion, the ethanol volume was reduced to 1 V then MTBE (10 V) was added, and the26037 mixture was stirred for 1 hr. The resulting solid was collected via filtration then washed with MTBE to give sodium (lZ,3E')-5-amino-4-cyano-5-oxo-l-(2,3,6-trifluorophenyl)penta-l,3-dien- 1-olate (12.54 g, 41.29 mmol, 94.6% IY, 95.55 wt.%). ’H NMR (500 MHz, DMSO) 5 8.08 (d, J = 13.3 Hz, 0.29 H), 7.50 - 7.35 (m, 1H), 7.24 (d, J= 14.3 Hz, 0.67H), 7. 15 - 7.04 (m, 1H), 6.21 (d, J = 73.9 Hz, 2H), 5.48 (d, J= 14.3 Hz, 0.63H), 5.31 (d, J= 13.3 Hz, 0.29H).19F NMR (471 MHz, CDCh) 5 -118.80 (d, J= 15.7 Hz), -119.13 (d, J= 15.8 Hz), -138.59 (d, J= 24.6 Hz), - 138.67 (d, J= 24.3 Hz), -142.81 (dt, J= 24.4, 16.1 Hz).Synthesis of 2-oxo-6-(2.3.6-trifluorophenyl)-1.2-dihydropyridine-3-carbonitrile

[0097] To a flask was added (lZ,3E')-5-amino-4-cyano-5-oxo-l-(2,3,6-trifluorophenyl)penta- 1,3-dien-l-olate (15.00 g, 92.03 wt.%, 1 eq, 47.57 mmol) followed by IPA:H2O (8:2, 150 mL, 10 V total). Then, acetic acid was added (5.713 g, 5.447 mL. 2 eq, 95. 14 mmol). The pH was measured at this point and was determined to be ~ 4.8. The reaction was then aged at 80 °C overnight. The resulting solid was filtered and washed with water and MTBE to give of 2-oxo-6- (2,3,6-trifluorophenyl)-l,2-dihydropyridine-3-carbonitrile as a solid (9 g, 47.57 mmol, 73% IY, 96.25 wt.%). ‘H NMR (500 MHz, DMSO) 5 13.06 (s. 1H), 8.29 (d. J = 7.4 Hz. 1H), 7.74 (qd, J = 9.5, 5.1 Hz, 1H). 7.35 (tt, J= 6.0, 2.8 Hz, 1H), 6.67 (s, 1H).19F NMR (471 MHz. DMSO) 8 - 116.82, -135.33, -141.80 (dd, J= 22.5, 15.4 Hz).Synthesis of 2-oxo-6-(2.3.6-trifluorophenyl)-1.2-dihydropyridine-3-carbonitrileProcedure B

[0098] A flask was charged with w-propanol (10.0 V, 100.0 mL) then 2-cyanoacetamide (3.82 g, 1.04 eq, 45.38 mmol) and sodium / e / 7-butoxide (4.61 g, 1.1 eq. 47.99 mmol). To this flask was then added DMAc (1.0 V, 10.0 mL). The reaction mixture was stirred for 30 mins at 25 °C. This resulted in a white heterogeneous mixture. At this point, ( / 'j-3-(di methyl amino)- 1 -(2,3,6-26037 trifluorophenyl)prop-2-en-l-one (10.0 g, 1.0 eq, 43.63 mmol) was added as a solid in one portion. The reaction was allowed to stir for at least 1 hours at 25 °C. Upon completion, the reaction mixture was concentrated at < 45 °C internal temperature to ~5.0 V (- 50.0 mL). Then, w-propanol (5.0 V, 50.0 mL) was added to reach a total volume of -10.0 V (-100 mL). Then, propionic acid was added (3.23 g, 3.25 mL, 1.0 eq, 43.63 mmol). The reaction was then aged at 92 °C for at least 20 hours. This resulted in a heterogenous mixture. The reaction was then cooled to 25 °C. Then, propionic acid (3.23 g, 3.25 mL, 1.0 eq, 43.63 mmol) was added. The resulting heterogeneous mixture was aged at 25 °C for 3 hours. The solids were filtered and then two slurry washes were performed, each with 15 mL of 1 : 1 n-propanol: water. Then, two displacement washes were performed, each with 15 mL water, to give of 2-oxo-6-(2,3,6- trifluorophenyl)-l,2-dihydropyridine-3-carbonitrile as a solid (10.02 g, 39.67 mmol, 91% IY, 99.06 wt.%).JH NMR (500 MHz, DMSO) 5 13.06 (s, 1H), 8.28 (d, J= 7.4 Hz, 1H), 7.73 (qd, J =9.5, 5.1 Hz, 1H), 7.35 (tdd, J= 9.3, 3.5, 2.1 Hz, 1H), 6.67 (br s. 1H).19F NMR (471 MHz, DMSO) 6 -116.81, -135.32, -141.80 (dd, J= 22.6, 15.3 Hz).13C NMR (126 MHz, DMSO) 5160.5, 154.6 (d, J = 247.7 Hz), 148.6, 147.7 (ddd, J= 252.5, 15.4, 6.8 Hz), 145.7 (ddd, J= 243.1, 12.5 Hz, 3.3 Hz), 140.7, 119.9 (dd, J= 19.2, 10.1 Hz), 116.0, 112.6, 112.3 (ddd.24.2. 6.8, 4.0 Hz), 109.0. 103.5.Synthesis of 5-chloro-2-oxo-6-(2.3.6-trifluorophenyl)-L2-dihvdropyridine-3-carbonitrileProcedure A

[0099] A vial was charged with methanol (5.0 mL) and 10 M sodium hydroxide (0.4 mL, 4.0 mmol, 1 eq.). To this solution was added 2-oxo-6-(2,3,6-trifluorophenyl)-l,2-dihydropyridine-3- carbonitrile (1.0 g, 4.0 mmol, 1 eq.) followed by l,3-dichloro-5,5-dimethylimidazolidine-2,4- dione (0.83 g, 4.2 mmol. 1.05 eq.). The mixture was stirred at room temperature for 2 hours after which IM HC1 (3.5 mL) was added dropwise. The mixture was allowed to age for 5-10 minutes and additional IM HC1 (1.5 mL) was added. The suspension was aged for 30 min-1 hour and the solids were collected via filtration. The filter cake was washed with 2 volumes (1 : 1 Methanol: Water) twice. The material was dried under vacuum with a flow of nitrogen to give a solid (Int- El, 0.908 g, 80% IY).Synthesis of 5-chloro-2-oxo-6-(2.3.6-trifluorophenyl)-L2-dihvdropyridine-3-carbonitrileProcedure B

[0100] A reaction vessel was charged with Isopropyl Acetate (500.0 mL) and 2-oxo-6-(2,3,6- trifluorophenyl)- 1 ,2-dihydropyridine-3 -carbonitrile (50.0 g, 195.9 mmol, 1 eq.). The mixture was cooled to 10°C and then NaOCl (11.9 wt%, 206.0 mL, 367.0 mmol, 1.87 eq) was added dropwise over an hour. The mixture was allowed to age overnight and the temperature was adjusted to 25°C. Ascorbic acid (IM, 200.0 mL, 200 mmol, 1.02 eq) was added and the solution was stirred 30 min. Hydrochloric Acid (37 wt%, 20.0 mL) was added over 5 minutes and the biphasic mixture was stirred 10 minutes. The aqueous layer was discarded, and the organic layer was washed with water (100.0 mL, twice). The organic layer was distilled (55°C, 160 mmHg) to reach a final volume of 250.0 mL. The solution was seeded with Int-El (500 mg, 2.55 mmol) keeping a temperature of 55°C. n-Heptane (750.0 mL) was added over 12 hours at 55°C. After addition, temperature was ramped down to 0°C over 12 hours. The white slurry was then filtered and washed with 50.0 mL of Isopropyl Acetatevi-heptane (1:3 v:v) then 50.0 mL of w-heptane. A white solid was obtained after drying under vacuum / nitrogen blanket. (Int-El, 44.9 g, 80% IY).

[0101] ' H NMR (500 MHz, CD3OD) 5 (ppm): 8.32 (s 1H), 7.52 (qd. J = 9.4 Hz. J = 5.0 Hz, 1H), 7.19 (m, 1H).13C NMR (151 MHz, CD3OD) 5 (ppm): 161.99 (CO), 156.38 (dm, J = 248.5 Hz, CF), 148.88 (s, CH), 148.81 (ddd, J = 253.6 Hz, J = 15.4 Hz, J = 6.9 Hz, CF), 148.43 (ddd, J = 245.5 Hz, J = 12.4 Hz, 4.0 Hz, CF), 142.66 (Cq), 121.23 (dd, J= 19.5 Hz, J = 9.9 Hz, ArF CH), 117.61 (Cq), 115.08 (Cq), 113.36 (m, CN), 113.11 (ddd, J = 23.9 Hz. J = 6.7 Hz, J = 4.2 Hz, ArF CH). ^FfH} NMR (470.5 MHz. CD3OD) 6 (ppm): -118.13 (s, J = 15.6 Hz), -136.46 (br). - 143.67 (app t, .7 = 17.4 Hz).Example 2: Preparation of 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (Int-F) - Procedure ASynthesis of 2-isopropyl-4-methoxynicotinonitrileethyl isobutyrate 4-Methyl-3-oxopentanenitrile

[0102] Ethyl isobutyrate (11.85 g, 13.98 mL, 102 mmol), THF (100 mL), and potassium tert- butoxide (22.89 g, 204 mmol) were combined. The reaction mixture was stirred and placed in a water bath at room temperature. To the stirring solution was added acetonitrile (4. 187 g, 5.33 mL, 102 mmol). After 30 min the reaction mixture was quenched by adding water (200 mL) followed by EtOAc (lOOmL). To the stirring biphasic mixture was added cone. HC1 (16mL). The mixture was stirred for 2 minutes and the stirring was stopped to allow the mixture to settle. The organic phase was collected, dried over MgSO-i. filtered, and concentrated to provide crude 4- methyl-3-oxopentanenitrile (10.4 g, 65 wt%). The crude material could be used in further transformations without purification.Synthesis of 2-isopropyl-4-methoxynicotinonitrile trimethyl orthoacetate, acetic anhydrideDMF-DMA ammonium acetate

[0103] 4-Methyl-3-oxopentanenitrile (5.0 g, 86.5 wt.%, 38.9 mmol), trimethyl orthoacetate (14.9 mL, 117 mmol), and acetic anhydride (1 1.0 mL, 117 mmol) were combined. The stirred batch was heated to 60 °C for 16-20 h. The batch was cooled to 22 °C then charged with DMF- DMA (12.9 mL, 97.2 mmol). The batch was stirred at 22 °C for 4-6 h then charged with ammonium acetate (12.0 g, 156 mmol) and methanol (5.0 mL). The stirred batch w as heated to 45 °C for 16-20 h. The batch was cooled to 22 °C to form a slurry then was charged with water (62.5 mL) over 4 h. The batch was stirred at 22 °C for 12 h then was cooled to 0-5 °C and stirred at this temperature for 2 h. Solids were filtered, washed with methanol / w ater (1 :2, 120 mL), then dried under vacuum at 20-25 °C to afford 2-isopropyl-4-methoxynicotinonitrile (6.85 g, 83% yield). 'H NMR (DMSO-d6. 600 MHz) 5 8.62 (d, J = 5.9 Hz, 1H), 7.15 (d, J = 5.9 Hz, 1H), 3.98 (s, 3H), 3.31 (hept, J = 6.8 Hz, 1H), 1.23 (d, J = 6.8 Hz, 6H).13C NMR (DMSO-d6, 151 MHz) 5 170.3, 167.2, 154.0, 114.3, 105.5, 96.4, 56.8, 34.1, 21.5.26037Synthesis of 4-(2-hydroxyethoxy)-2-isopropylnicotinonitrile

[0104] Potassium tert-butoxide (653 mg), ethylene glycol (15 mL) and acetonitrile (10 mL) were combined. The stirred batch was allowed to stir at ambient temperature and then charged with 2-isopropyl-4-methoxynicotinonitrile (5.00 g, 28.5 mmol) in one portion at ambient temperature. The reaction was allowed to stir at 80 °C for 18 h. After 18 h, the reaction was cooled to room temperature, then low pressure vacuum was applied to the reaction vessel, then the batch was further allowed to stir for an additional 18 h. The batch was cooled to 40 °C and seeded with 1 wt % of 4-(2-hydroxyethoxy)-2-isopropylnicotinonitrile (50 mg), forming a slurry. The batch was allowed to age at 40 °C for 1.5 h, after which the batch was slowly cooled to ambient temperature for one hour and then cooled to 0 °C. The slurry was charged with water (60 mL) at ambient temperature over 4 h after which the reaction was allowed to stir at ambient temperature for 18 h. Solids were filtered, washed with water (10 mL) and then dried under vacuum at ambient temperature to afford 4-(2-hydroxyethoxy)-2-isopropylnicotinonitrile (4.6 g, 79% yield). The conversion may also be performed in the absence of acetonitrile. 1H NMR (500 MHz, CDC13) 5 8.59 (1H, d, J = 5.9 Hz), 6.79 (1H, d, J = 5.9 Hz), 4.34 - 4.18 (2H, m), 4.07 (2H, q, J = 5.1 Hz), 3.49 (1H, p, J = 6.8 Hz), 2.65 (1H, t, J = 6.2 Hz), 1.34 (6H, d, J = 6.8 Hz). 13C NMR (CDC13, 125 MHz) 5: 172.1, 166.7, 153.7, 114.3, 104.9, 98.1, 70.7, 60.6, 34.7, 21.6.Synthesis of 4-(2-hydroxyethoxy)-2-isopropylnicotinamide

[0105] In a 100 mL reaction vessel was added 4-(2-hydroxyethoxy)-2-isopropylnicotinonitrile (5.0 g, 24 mmol), water (25 mL) and sulfolane (5.0 mL). Sodium hydroxide (4.8 mL, 10.0M, 48mmole) was added to the mixture. While stirring at 15-25 °C under a nitrogen sweep was added a solution of hydrogen peroxide (20 mL, 15wt.%. 97mmole) over a period of 2 hours. The resulting mixture was stirred at 15-25 °C for another 18 hours. A sodium thiosulfate solution (9.7 mL, 5M, 48mmole) was added to the reaction mixture and was stirred for 1 hour. To the resulting suspension was added sodium chloride (6.5 g) and the suspension was stirred for 2 hours. The26037 mixture was cooled to 10 °C, filtered and the solid was washed with 10 mL water twice. The product was dried in a vacuum oven at 30 °C for 20 hours to give of 4-(2-hydroxyethoxy)-2- isopropylnicotinamide (4.2 g, 78% yield).JH NMR (400 MHz, DMSO-d6) 5 8.35 (d, J = 5.8 Hz, 1H), 7.70 (s, 1H), 7.45 (s, 1H), 6.93 (d, J = 5.8 Hz, 1H). 4.81 (t. J = 5.3 Hz. 1H), 4.08 (t, J = 5.2 Hz, 2H), 3.69 (q, J = 5.2 Hz, 2H), 3.31 (s, 3H), 3.09 (p, J = 6.7 Hz, 1H), 1.17 (d, J = 6.7 Hz, 6H). 13C NMR (400 MHz, DMSO-d6) 5 168.10, 163.44, 161.47, 150.53, 122.67, 106.19, 70.36, 59.68, 32.58, 22.89.Synthesis of 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (Int-F)

[0106] In a 40 mL reaction vessel was added 4-(2-hydroxyethoxy)-2-isopropylnicotinamide (1.0 g, 4.4 mmol), water (5.0 mL) and sulfolane (0.5 mL). Sodium hydroxide (0.87 mL, 10M, 8.8 mmol) was added to the mixture. Sodium hypochlorite (4.3 mL, 12.5wt.%, 8.8mmole) was added and the reaction mixture was heated to 55-65 °C. After 3 hours, extra sodium hypochlorite (0.9 mL) was added and the reaction mixture was heated to 55-65 °C for another 16 hours. The reaction mixture was cooled to 15-25 °C and the pH was adjusted to 7 with hydrochloric acid (5M). Sodium chloride (0.5 g) was added to the reaction mixture and the product was extracted with THF. The organic solution was concentrated and the product was purified by column chromatography on silica gel (elution EtOH:EtOAc 1 :3) to give Int-F (0.42 g, 50% yield). 1H NMR (400 MHz, DMSO-d6) 5 7.70 (d, J = 5.3 Hz, 1H), 6.67 (d, J = 5.3 Hz, 1H). 4.96 (d, J = 5.6 Hz, 1H), 4.64 (s, 2H), 4.02 - 3.90 (m, 2H), 3.73 (d, J = 4.4 Hz, 2H), 3.31 (s, 1H), 3.15 (p, J = 6.7 Hz, 1H), 1.14 (d, J = 6.7 Hz, 6H). 13C NMR (400 MHz, DMSO-d6) 5 151.24, 150.04, 138.10, 130.41. 105.07, 70.21, 60.02, 29.01, 21.69.Alternate Procedure for Synthesis of 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (Int-F)

[0107] A vessel was sequentially charged with water (45 L), 4-(2-hydroxyethoxy)-2- isopropylnicotinamide (1.0 kg, 4.46 mol, 1.0 equiv.), and 30 wt% aq. sodium hydroxide (1.8 kg, 13.50 mol, 3.0 equiv.). The batch temperature was adjusted to 10 °C and then sodium hypochlorite (10 wt% in water, 3.8 kg, 5.10 mol, 1.14 equiv.) was added dropwise over 1 h. The reaction was warmed to room temperature and aged for 4 h. The reaction mixture was further26037 warmed to 60 °C and aged for an additional 16 h. The reaction mixture was cooled to 20 °C, quenched with sodium £-ascorbate (25 wt% in water, 1.1 kg, 1.39 mol, 0.31 equiv.) and then aged for 4 h. HC1 (16 wt% in water, 2.8 kg, 12.29 mol, 2.76 equiv.) was added to reach a target pH of 7 and the mixture was aged for an additional 1 h. Additional HC1 (16 wt% in water, 1.2 kg, 5.27 mol, 1.18 equiv.) was added to reach a target pH of 3-5 and then the mixture was aged for an additional 4 h. Sodium chloride (2.8 kg, 47.91 mol, 10.7 equiv.) was added and then the mixture w as filtered to remove residual solids. Additional sodium chloride (15 wt% in water, 0.2 kg, 0.51 mol. 0.12 equiv.) was added and then sodium hydroxide (30 wt% in water, 0.37 kg, 2.78 mol, 0.62 equiv.) was added over 1-2 h to precipitate the product from the solution. This slurry was aged for 2 h before adjusting the pH to 11 by slowly adding sodium hydroxide (30 wt% in water, 0.4 kg, 10.0 mol, 2.24 equiv.) over 2 h. The slurry was aged for an additional 2 h and then cooled to 5 °C, where it was aged for an additional 16 h. The solids w ere filtered off and sequentially washed with pre-cooled 15 wt% sodium chloride (1 kg) followed by pre-cooled water (1.5 L x 2). The solids were dried under vacuum at 50 °C and then the product was isolated as a white solid (707 g, 3.60 mol, 80.8%).Example 3: Preparation of 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (Int-F) - Procedure BSynthesis of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-chloro-3-nitroDyridine

[0108] DMF (1060 kg), 2,4-dichloro-3-nitropyridine (141 kg, 730.6 mol), 2-((tert- butyldimethylsilyl)oxy)ethan-l-ol (142 kg, 805.3 mol), and K3PO4 (310 kg) were combined, the temperature was adjusted to 55-65 °C, and the mixture was stirred for 22 hours. The temperature of the reaction mixture was adjusted to 0-10 °C and the reaction was quenched by the addition of a AcOH / EtOAc solution (347 kg; 2.0 equiv AcOH in 6 vol EtOAc), to pH = 7. The mixture was dilute further by the addition of EtOAc (445 kg) and w ater (1409 kg). The organic phase w as separated, and the temperature was adjusted to 20-30 °C. The organic layer was washed 6 times with 20% Na2S0 aqueous (2528 kg total). The organic phase was concentrated to 380 L below2603735 °C. The temperature was then adjusted to 20-30 °C and n-heptane (481 kg) was added slowly. The mixture was concentrated to 410 L below 40 °C, the temperature was adjusted to 20-30 °C and 422 g product seed was added. The mixture was aged for 2 hours and then cooled slowly to - 10-0 °C and aged at this temperature for 5 hours. The mixture was filtered, and the filter cake was washed twice with n-heptane (139 kg + 134 kg). The wet cake was dried at 35 -45 °C for 20 hours and the title compound (182.73 kg) was isolated in 75% yield.NMR (400 MHz, CDC13- ) 5 ppm 0.00 (s, 6 H) 0.82 (s, 9 H) 3.91 - 3.93 (m, 2 H) 4.21 - 4.24 (m, 2 H) 7.02 (d, J=5.88 Hz, 1 H) 8.27 (d, J=5.88 Hz,l H).Synthesis of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-3-nitro-2-(r) -l-en-2-vDpyridinePd(dppf)CI2

[0109] Water (222 kg) and DMF (805 kg) were combined and the temperature was adjusted to 20-40 °C. To the solution was added K3PO4 (238 kg). 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)- 2-chloro-3-nitropyridine (170 kg, 510.7 mol), and isopropenylboromc acid pinacol ester (102 kg, 1.19 eq, 607.0 mol) and the resulting mixture was sparged with argon for 3 hours. To the reaction mixture was added Pd(dppf)Ch (1.90 kg, 0.005 equiv), the sides of the vessel were rinsed with DMF (35kg), and the resulting mixture was sparged with argon for an additional 2 h. The reaction temperature was then adjusted to 55-65 °C and allowed to react for 6 hours, at which point the mixture was cooled to 20-30 °C. To the mixture was added water (850 kg) and MTBE (1007 kg) and the mixture was stirred for an hour and then allowed to stand for an hour after halting the stirring. The bottom two aqueous phases of the resulting three phase mixture were removed and the organic phase was washed 3X with 10% Na2SO4. The resulting organic phase was discolored with CUNO (6%) at 20-30 °C for 6 hours. The CUNO was removed via fdtration and the filter cake was washed twice with MTBE (259 kg + 256 kg). The solvent was then switched from MTBE to ethanol keeping the temperature below 45 °C. The desired product was obtained as a solution in ethanol in 98.0% yield. 'H NMR (400 MHz. CDC13- ) 5 ppm 0.00 (s, 6 H) 0.81 (s, 9 H) 2.12 (s, 3 H) 3.90 (t, J=4.88Hz, 2 H) 4.16 (t, J=4.88 Hz,2 H) 5.19(s,lH) 5.27 (s, 1 H) 6.89 (d, J=5.75 Hz, 1 H) 8.42 (d, J=5.75, 1 H).26037Synthesis of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-2-isopropylpyridin-3-amine

[0110] The previously prepared ethanol solution of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)- 3-nitro-2-(prop-l-en-2-yl)pyridine (169 kg; 846.2 kg x 20.0 wt.%) was combine with Pd / C (2.54 kg) and additional ethanol (95 kg). The reaction mixture yvas then placed under a hydrogen atmosphere with the pressure adjusted to 1. 1-1.3 MPa and the reaction temperature set to 50-60 °C. After 20 hours the reaction mixture was cooled to 20-40 °C and the solution was filtered, the cake washed with additional ethanol, and the desired product obtained as a solution in ethanol (150.74 kg; 97.2% isolated yield). 'H NMR (400 MHz, DMSO-6) 5 ppm 0.06 (s, 6 H) 0.87 (s, 9 H) 1.14 (d, J=6.75, 6 H) 3.14-3.17 (m, 1H), 3.93-3.95 (m, 2H), 4.06-4.08 (m, 2 H) 4.44 (s, 2 H) 6.72 (d, J=5.38Hz. 1 H) 7.73 (d, J=5.25. 1 H).Synthesis of 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol[OHl] The previously prepared ethanol solution of 4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)- 2-isopropylpyridin-3-amine was combined with 4M HCl / MeOH (271 kg) and the mixture was stirred for 1 hour at 20-30 °C. The reaction mixture was concentrated to 280 L while keeping the temperature below 50 C. Water (750 kg) was added and the resulting mixture was concentrated to 300 L while maintaining the temperature below 65 °C. The temperature of the mixture was adjusted to 15-25 °C and 7.5% NaCl and 14.2% aqueous NaOH (221 kg) were added dropwise. Product seed (0.45 kg) was added and additional 7.5% NaCl and 14.2% aqueous NaOH (140 kg) were added, after which the reaction mixture was stirred for 5 hours with the temperature being controlled to 15-25 C and the pH = 14. The slurry was then filtered and the wet cake was washed with water (289 kg) and n-heptane (402 kg). The resulting wet cake was dried for 5 hours at SOO °C and then an additional 16 hours at 70-80 °C. Int-F was isolated as a white cry stalline solid (87.48 kg, 92% isolated yield).

[0112] XH NMR (400 MHz, DMSO- <7s):5 ppm 1.14 (br d. J=6.38 Hz. 5 H) 0.91 - 1.35 (m. 1 H) 2.88 - 3.25 (m, 1 H) 3.08 - 3.22 (m, 1 H) 3.73 (br d, J=3.25 Hz, 1 H) 3.65 - 3.82 (m, 1 H) 3.89 - 4.08 (m, 1 H) 3.97 (br s, 1 H) 4.65 (br s, 1 H) 4.56 - 4.75 (m, 1 H) 5.00 (br t, J 5.69 Hz, 1 H) 4.93 - 5.06 (m, 1 H) 6.66 (br d, J-4.63 Hz, 1 H) 6.58 - 6.73 (m, 1 H) 7.71 (br d, .7-4.63 Hz, 1 H)260377.63 - 7.80 (m, 1 H).13C NMR (101 MHz, DMSO-rfo) 8 ppm 21.68, 29.01, 60.02, 70.19, 105.04, 130.40, 138.09, 150.01, 151.23. HRMS (ESI) m / z calculated for CioHi7N202+[M+H]+197.12845 found 197.12813.Example 4: Preparation of 23-chloro-l3,l6-difluoro-42-isopropyl-5,8-dioxa-3-aza-2(2,6), 4(3,4)-dipyridina-l(l,2)-benzenacyclooctaphane-25-carboxamide (Int-A) - Procedure ASynthesis of 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3.6-difluorophenyl)-5-chloro-2-oxo-1.2-dihvdropyridine-3-carbonitrile (Int-D)Procedure A

[0113] Potassium tert-butoxide (280 mg, 2.70 mmol) was added to an ice-cold mixture of 5- chloro-2-oxo-6-(2,3,6-trifluorophenyl)-l,2-dihydropyridine-3-carbonitrile (300 mg, 94.8% wt.%, 999 pmol) and 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (235 mg. 1.20 mmol) in THF (3 mL). The reaction was then stirred at room temperature for 18 h. Acetic acid (114 pL. 2.00 mmol) was added to neutralize the reaction followed by aqueous 20 wt.% sodium chloride solution (3-5 mL). The mixture was then extracted with ethyl acetate, the combined organic layers were dried over magnesium sulfate and concentrated. The obtained crude product was purified by column chromatography using dichloromethane / methanol to obtain the desired product as a solid (415 mg, 86% yield). 'H NMR (600 MHz, DMSO) 5 (ppm): 8. 18 (s 1H), 7.72 (d, J = 5.4 Hz, 1H), 7.53 (ddd, J= 11.1 Hz, J = 9.3 Hz, J= 5.2 Hz, 1H), 7.17 (td, J= 8.9 Hz, J = 3.4 Hz, 1H), 6.66 (d, J= 5.4 Hz, 1H), 4.45 (dt, J = 11.5 Hz, J = 4.2 Hz, 1H), 4.39 (dt, J = 11.5 Hz, J = 4.2 Hz, 1H), 4. 18 (t, J = 4.4 Hz, 2H), 3. 17 (hept, J = 6.8 Hz, 1H), 1.16 (d, J = 6.7 Hz. 3H), 1.15 (d. J = 6.7 Hz, 3H).13C NMR (151 MHz, DMSO) 5 (ppm): 162.02 (CO), 154.7426037(d, J = 244.1 Hz, CF), 151.14 (d, J = 242.0 Hz, CF), 150.66 (Cq), 149.33 (Cq), 146.06 (CH), 145.51 (Cq), 144.09 (dd, J = 13.1 Hz, J = 5.6 Hz, Cq), 137.29 (CH), 129.83 (Cq), 119.13 (CH), 117.79 (Cq), 115.99 (Cq), 112.81 (CN), 111.03 (dd, J = 24.2 Hz, J = 8.2 Hz, CH), 104.77 (CH), 101.05 (Cq), 72.52 (d, J = 5.5 Hz, CH2), 66.98 (CH2), 28.52 (CH), 21.11 (CHs). 21.06 (CHs).19F NMR (471 MHz, DMSO) 5 (ppm): -1 17.81 (d, J = 15.6 Hz), -133.42 (d, J = 15.8 Hz).Synthesis of 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3.6-difluorophenyl)-5-chloro-2-oxo-1.2-dihydropyridine-3-carbonitrile (Int-D)Procedure B

[0114] 5 -chloro-2-oxo-6-(2,3,6-trifluorophenyl)-l,2-dihydropyridine-3-carbonitrile (60.0 g, 209 mmol, 1 eq) and 2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethan-l-ol (50.0 g, 251 mmol, 1.2 eq) were dissolved in THF (180.0 mL) and heated up to 40°C. Potassium tert-butoxide (IM, 440.0 mL, 440 mmol. 2. 1 eq) was added dropwise over four hours at 40°C and the reaction was aged for 16 h at this temperature. Acetic acid (1.79 mL, 31.4 mmol, 0.15 eq) was added and reaction temperature was adjusted to 35°C. The yellow solution was concentrated via distillation to 240 mL. The temperature was adjusted to 25°C and water (420.0 mL) was added dropwise over 15 minutes. Acetic acid (0.5M, 90.0 mL) was added over 10 minutes. The resulting clear yellow solution was seeded with Int-D (600.0 mg, 1.30 mmol, 0.006 eq) and aged for two hours. Acetic acid (0.5M, 390.0 mL) was added over 20 hours. The slurry' was then heat to 45°C and aged for 2 hours before cooling back down to 25°C over 2 hours and then aging at this temperature for an additional 16 hours. The white solid was filtered and sequentially washed with THF:Water 1 :5 v:v (2 x 240.0 mL) and then with water (240.0 mL). The solid was then dned under vacuum / a blanket of nitrogen to obtain Int-D as a hydrate (91.9 g, 94.9 wt%, 90.5% yield). 'H NMR (600 MHz, DMSO) 5 (ppm): 8.18 (s 1H), 7.72 (d, J - 5.4 Hz, 1H), 7.53 (ddd, J = 11.1 Hz, J = 9.3 Hz, J = 5.2 Hz, 1H), 7.17 (td, J = 8.9 Hz, J= 3.4 Hz, 1H), 6.66 (d, J= 5.4 Hz, 1H), 4.45 (dt, J = 11.5 Hz, J = 4.2 Hz, 1H). 4.39 (dt, J = 11.5 Hz, J = 4.2 Hz. 1H), 4. 18 (t, J = 4.4 Hz. 2H), 3.17 (hept, J = 6.8 Hz, 1H), 1.16 (d, J = 6.7 Hz, 3H), 1.15 (d, J = 6.7 Hz, 3H).13C NMR (151 MHz, DMSO) 5 (ppm): 162.02 (CO), 154.74 (d, J = 244.1 Hz, CF), 151.14 (d, J = 242.0 Hz, CF), 150.66 (Cq), 149.33 (Cq), 146.06 (CH), 145.51 (Cq), 144.09 (dd, J = 13.1 Hz, J = 5.6 Hz, Cq), 137.29 (CH),26037129.83 (Cq), 119.13 (CH), 1 17.79 (Cq), 115.99 (Cq), 112.81 (CN), 1 11.03 (dd, J = 24.2 Hz, J = 8.2 Hz, CH), 104.77 (CH), 101.05 (Cq), 72.52 (d, J = 5.5 Hz, CH2), 66.98 (CH2), 28.52 (CH), 21.11 (CH3), 21.06 (CH3).19F NMR (471 MHz, DMSO) 5 (ppm): —117.81 (d, J = 15.6 Hz), -133.42 (d, J = 15.8 Hz).Synthesis of 23-chloro-l3.l6-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)- dipyridina- 1(1 ,2)-benzenacy clooctaphane-25-carboxamide (Int-A)

[0115] 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3,6-difluorophenyl)-5-chloro-2- oxo-l,2-dihydropyridine-3-carbonitrile (350.0 mg, 89.6% wt, 680.5 pmol) and 4- dimethylaminopyridine (4.2 mg, 34 p ol) were dissolved in THF (3.5 mL) and the solution was cooled to 10 °C. N,N-diisopropylethylamine (261 pL. 1.50 mmol) was added followed by diethyl phosphorochlondate (197 pL, 1.36 mmol) and the reaction was stirred at 20 °C for 35 min. The mixture was then cooled to 10 °C and a solution of sodium tert-butoxide in THF (1.7 mL, 2.0 M, 3.40 mmol) was added. After 45 min, the reaction was allowed to warm to room temperature. THF (1.0 mL) and aqueous potassium hydroxide (681 pL, 4 M, 2.72 mmol) were added the mixture was heated at 60 °C for 12 h. The mixture was cooled to room temperature to precipitate the product which was isolated as a solid (204 mg, 65% yield) via filtration and washing with THF / H2O 1 / 1.Synthesis of 23-chloro-l3.l6-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)- dipyridina-l(l,2)-benzenacvclooctaphane-25-carboxamide (Int-A)26037Procedure B

[0116] Flask A was charged with THF (10.0 V, 100.0 rnL), tri-w-propylamine (4. 13 mL, 3.11 g, 21.70 mmol, 1.0 eq), and 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3,6- difluorophenyl)-5-chloro-2-oxo-l,2-dihydropyridine-3-carbonitrile (10.0 g, 21.70 mmol, 1.0 eq). The solution was then distilled under reduced vacuum, maintaining an initial volume of -10.0 V or -100 mL, while continuously charging THF (12.0 V, 120.0 mL). The internal temperature of the stream was maintained between 40 and 50 °C during the distillation. Once complete, the temperature of the mixture was adjusted to 0 °C. Then, tri-n-propylamine (2.06 mL, 1.56 g, 10.85 mmol, 0.50 eq) was added followed by 1 -methylimidazole (NMI) (0.087 mL, 89.08 mg, 1.08 mmol, 0.05 eq). At this point, diethyl chlorophosphate (4.08 mL, 4.87 g, 28.21 mmol, 1.3 eq) was added slowly over one hour maintaining the temperature at 0 °C. The reaction mixture was then aged for 30 mins at 0 °C. To flask B was then added sodium tert-butoxide (2M in THF, 86.792 mmol, 43.40 mL. 4.0 eq) at 25 °C. The phosphate stream from flask A was slowly charged over 2 hours to flask B, maintaining the temperature at 25 °C. To flask A was charged THF (4.0 V, 40.0 mL) at 0 °C as a rinse, and this solution was then transferred to flask B at 25 °C. The mixture in flask B was then aged for 30 mins. Then, to flask B was added tetrabutylammonium hydrogen sulfate (TBAHS) solution, 55% in water (1.91 mL, 2.01 g, 3.25 mmol). This was followed by the addition of potassium hydroxide (86.792 mmol, 2.278 M, 38.10 mL, 4.0 eq) and water (1.0 V, 10.0 mL). This resulted in a biphasic mixture. The reaction was then aged at 60 °C for 20 hours then cooled to 50 °C, whereby a phase cut was performed to retain the top organic layer. The organic layer was cooled to 40 °C, and then acetic acid (1.86 mL, 1.95 g, 32.55 mmol. 1.5 eq) was added. The resulting solution was cooled to 25 °C, and product Int-A seed (1 wt%, 0. 10 g) was added. The mixture was then stirred for 30 mins at 25 °C,at which point the volume of the mixture was reduced to 10.0 V or -100.0 mL via distillation. The internal temperature of the stream was maintained between 20 and 30 °C during the distillation. Once complete, the solution temperature was adjusted to 25 °C, and the heterogeneous mixture was aged at this temperature for 3 hours. Then, water (12.0 V, 120.0 mL) was added slowly over 10 hours to form a yellow slurry that was aged for 2 additional hours. The slurry was filtered. Then, a displacement wash was performed with 20 mL 1 : 1 THF: water followed by a slurry wash with 20 mL 1: 1 THF: water. Then, a final displacement wash was performed with 20 mL 1: 1 THF: water. The resulting wet cake was dried at 40 °C to give the final product as a THF solvate (17.00 mmol, 9. 1 g, 85.70 wt%. 81% yield).

[0117] XH NMR (500 MHz, DMSO) 5 10.36 (s, 1H), 8.41 (br s, 1H), 8.35 (s, 1H), 8.20 (d, J =5.6 Hz, 1H), 7.83 (br s, 1H), 7.47 - 7.36 (m, 1H), 7.07 (td, J= 8.9 Hz, J =3.6 Hz, 1H), 7.00 (d, J = 5.7 Hz, 1H), 4.66 (d, J= 13.9 Hz, 1H), 4.60 (d, J= 10.8 Hz, 1H), 4.42 (dd, J= 13.2 Hz, J = 10.5 Hz, 1H), 3.94 (t, J= 10.8 Hz. 1H), 3.35 - 3.24 (m, 1H), 1.21 (d, J = 6.8 Hz, 3H), 1.11 (d, J =6.7 Hz, 3H).19F NMR (471 MHz, DMSO) 5 -117.49 (d, J= 15.9 Hz), -133.12 (d, J= 15.9 Hz).13C NMR (126 MHz, DMSO) 8 168.71, 161.64, 157.44, 154.60 (d, J = 244.7 Hz), 152.97, 153.03 (d, J = 242.3 Hz), 148.64, 146.65, 145.09 - 144.92 (m), 137.59, 121.72, 120.91 (d, J = 23.6 Hz), 118.07. 118.07 - 117.82 (m), 110.82 (dd, J= 24.8 Hz, J= 8.0 Hz), 110.50, 106.26. 71.37 (d. J=5.8 Hz), 65.50. 30.15, 21.78. 21.29.Example 5: Preparation of 23-chloro-l3,l6-difluoro-42-isopropyl-5,8-dioxa-3-aza-2(2,6), 4(3,4)-dipyridina-l(l,2)-benzenacyclooctaphane-25-carboxamide (Int-A) - Procedure BSynthesis of 1,4-difluoro-anisole

[0118] 1,4-difluorophenol (139 kg, 1068.5 mol) was combined with MeTHF (1,210 kg) and potassium carbonate (179 kg, 1295.2 mol). The internal temperature was adjusted to 45-55 °C.To the mixture was charged DMS (143 kg, 1133.8 mol) over a 4 hour period and the reaction mixture was stirred for 25 hours. The reaction mixture was quenched by the addition of water (700 kg) and the mixture was allowed to stir at the reaction temperature for 5 hours. The reaction mixture was cooled to 20-30 °C. The organic layer was separated and washed twice more with water (840 kg total). The organics were concentrated, and the solvent was switched to THF via distillation and addition of THF to a final volume of (564.4 kg). The title product was prepared as a 25.2 wt.% solution in THF (92% yield) and used in the next step without further manipulation.

[0119] Isolation of the product provided a sample for spectral analysis. 'H NMR (400 MHz, DMSO-O 5 ppm 3.84 (s, 3 H) 6.73 (ft, J=8.52. 3.17 Hz, 1 H) 7.10 (ddd, J=10.35, 7.16, 3.00 Hz, 1 H) 7.23 (ddd, J=\ 1. 19, 9.01, 5.44 Hz, 1 H)Synthesis of 3.6-difluoro-2-methoxyphenyl)zinc(II) chloride

[0120] The previously prepared THF solution of 1,4-difluoro-anisole was placed under an argon atmosphere and cooled to -80 - -65 °C. n-BuLi in hexane (283 kg) was charged and the mixture was stirred at -80 - -65 °C for two hours. To the stirred mixture was added a solution of ZnCh in THF (1.0M, 1136 kg) and the internal temperature was then adjusted between 0 and 25 °C. The prepared Aryl zinc reagent was used without further purification.Synthesis of 2,5-dichloro-6-(3,6-difluoro-2-methoxyphenvl)nicotinonitrile

[0121] 2,5,6 -trichloronicotinonitrile (134 kg, 646.0 mol) was combined with THF (587 kg) and the temperature of the reaction mixture was adjusted to 20-30 °C. The mixture was placed under an argon atmosphere by sparging the solution for 2 hours. To the inert mixture were added PPhs (10.2 kg. 38.9 mol), and Pd(OAc)2 (4.25 kg. 18.9 mol). The reaction mixture was sparged with26037 argon for an additional 3 hours and the temperature was adjusted to 35-45 °C. To the reaction mixture was added dropwise the previously prepared solution of the aryl zinc intermediate (1.4 equiv) over 5 hours and the reaction mixture was stirred for an additional 10 hours after which the internal temperature was adjusted 20-30 °C. The reaction mixture was quenched by the addition of 2N HC1 (402 kg) followed by toluene (592 kg) and the mixture was aged for 2 hours. The aqueous phase was separated and the organic phase was washed once more with 2N HC1 (402 kg). After agitation the aqueous phases were combined and then basified with 7% NaHCO3 (399 kg) to pH 7. The suspension was then filtered and the filter cake was washed with toluene (123 kg). The combined mother liquors were allowed to stand and the organic phase was separated. The organics were washed with 10% Na2SOq (404 kg) and the mixture was agitated. The organic was separated, decolorized with CUNO (9.6 kg), filtered, and the cake was washed with toluene (362 kg). The remaining THF was removed by distillation and the product was crystallized by the addition of n-heptane. The mixture was cooled to 0-10 °C and aged for 5 hours. The solids were filtered and the filter cake w as washed with cooled toluene / n-heptane (1 / 12, v / v; 274 kg total). The filter cake was dried under vacuum at 40-50 °C for 15 hours. 2,5- dichloro-6-(3,6-difluoro-2-methoxyphenyl)nicotinonitrile w as obtained in 65. 1 % isolated yield.

[0122] 'H NMR (400 MHz, DMSO-cfe) 6 ppm 3.83 (d, J=2.38 Hz, 3 H) 7.20 (td, J=8.97Hz, 1 H) 7.58 (ddd, J=11.57 Hz, 1 H) 8.99 (s, 1 H);13C NMR (101 MHz, DMSO- e) 5 ppm 62.39, 111.13, 111.21,111.36, 111.83, 114.30, 118.94, 119.16, 119.94, 120.04, 120.16, 120.26, 131.59, 145.22. 145.59, 145.72, 145.78, 149.39, 150.24. 152.65, 152.67, 153.90, 156.35; HRMS (ESI) m / z calculated for Ci3H7Cl2F2N2O+[M+H]+314.9898 found 314.99048.Synthesis of 2.5-dichloro-6-(3.6-difluoro-2-hvdroxyphenyl)nicotinonitrile (Int-E2)int-E2

[0123] DCM (1303 kg) and 2,5-dichloro-6-(3,6-difluoro-2-methoxyphenyl)nicotinonitrile (125 kg. 396.8 mol) were combined and placed under an nitrogen atmosphere. The mixture was stirred until a clear solution was formed. In a separate vessel AlCh (212 kg, 1590.0 mol) was combined with DCM (1301 kg) and the reaction temperature was adjusted to 15-25 °C. The solution of 2,5- dichloro-6-(3,6-difluoro-2-methoxyphenyl)nicotinonitrile was added dropwise to the AlCh solution and the mixture was stirred for 3 hours between 20-30 °C. The reaction mixture was26037 cooled to 0-10 °C and was quenched by the addition of water (1252 kg) after which the temperature was adjusted to 20-30 °C. The mixture was then diluted with DCM (1150 kg) and the organic phase was separated. The organic phase was then washed with twice with water (680 kg + 682 kg, total: 1362 kg). The organic phase was then decolorized by treatment with CUNO (8%) at 20-30 °C for 4 hours. The mixture was filtered, and the filter cake was washed twice with THF (twice (247 kg + 246 kg, total 493 kg). The resulting solution was used without further purification.

[0124] Isolation of the product provided a sample for spectral analysis. 'H NMR (400 MHz, DMSO-O 5 ppm 6.85 (td. J=9.04, 3.56 Hz. 1 H) 7.42 (ddd, J=10.54, 9.22. 5.38 Hz, 1 H) 10.66 (s, 1 H);13C NMR (101 MHz, DMSO-O 5 ppm 105.89, 105.96, 106.13, 106.20, 111.38, 114.37, 114.73, 114.90, 118.09, 118.19, 118.29, 118.40, 132.03, 144.01, 144.07, 144.17, 144.24, 145.02, 149.28. 149.73, 153.45, 153.47, 154.33, 156.72; HRMS (ESI) m / z calculated for C12H5CI2F2N2CF [M+H]+300.97415 found 300.9751.Svnthesis of 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3.6-difluorophenyl)-2.5-dichloronicotinonitrile (Int-G)

[0125] The previously prepared THF solution of 2,5-dichloro-6-(3,6-difluoro-2- hydroxyphenyl)nicotinonitrile, Int-E2, was concentrated to 900 L while maintaining the temperature below 35 °C. To this solution was added Int-F (85.6 kg, 436.2 mol) and the mixture was stirred while adjusting the temperature between 20-30 °C. The water content of the reaction mixture was controlled by azeotropically distilling water through addition and removal of THF (596 kg and 532 kg). The reaction mixture was cooled to 10-20 °C, triphenyl phosphine (114 kg) was added, and the mixture was stirred for 30 min. To the mixture was added DIAD (89.2 kg) dropwise over a 5 hour period and the mixture was stirred for 1 hour after complete addition. The reaction mixture was quenched by the addition of water (24 kg) and the mixture was concentrated to 5 volumes. The temperature was adjusted to 15-25 °C, methanol (766 kg) was added slowly, the batch was concentrated to 8 volumes and the mixture was stirred for 3 hours. The slurry was filtered and the wet cake was washed with IPA (272 kg). The wet cake was dried26037 under vacuum at 50-60 °C for 10 hours followed by 20 hours at 75-85 °C. The title compound (169.35 kg) was isolated as a solid in 87% yield.

[0126] ‘H NMR (400 MHz, CDC13-tf) 5 ppm 1.24 - 1.46 (m, 1 H) 1.34 (dd, J=8.57, 6.82 Hz, 5 H) 3.03 - 3. 16 (m, 1 H) 3.62 (br s, 2 H) 4.08 - 4.23 (m, 2 H) 4.46 - 4.60 (m. 2 H) 6.50 (d. J=5.38 Hz, 1 H) 6.88 - 6.99 (m, 1 H) 7.20 - 7.43 (m, 2 H) 7.92 (s, 1 H) 7.96 (d, J=5.50 Hz, 1 H).13C NMR (101 MHz, CDC13-d) 8 ppm 21.28, 30.08, 67.61, 73.40, 73.46, 105.33, 111.86, 112.09, 112.17, 112.31, 114.48, 118.17, 119.00, 119.91, 120.02, 120.24, 130.11, 132.27, 139.54, 144.43, 145.15. 150.14, 151.42, 151.52, 151.69, 153.48. 153.86, 154.85, 157.26. HRMS (ESI) m / z calculated for C22H19CI2F2N4O21[M+H]1479.08476 found 479.08456.Synthesis of 23-chloro-l3.l6-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)- dipyridina-l(1.2)-benzenacyclooctaphane-25-carbonitrile

[0127] 6-(2-(2-((3-amino-2-isopropylpyridin-4-yl)oxy)ethoxy)-3,6-difluorophenyl)-2,5- dichloronicotinonitrile (166.6 kg, 347.6 mol) was combined with THF (1396 kg), the temperature was adjusted to 40-50 °C, and the water content was controlled by azeotropically drying the mixture through addition and removal of THF. To the dried mixture was added NaOtBu (97.6 kg) while maintaining the temperature between 0-30 °C. Upon complete addition the reaction mixture was warmed to 20-30 °C and allowed to stir for 2 hours to give the intermediate macrocyclized nitrile product.Synthesis of 23-chloro-l3.l6-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)- dipyridina- 1(1 ,2)-benzenacy cl ooctaphane-25-carboxamide (Int- A)26037

[0128] The mixture was then charged with THF (700 kg) and 12.5% aqueous KOH (580 kg), the temperature was adjusted to 60 -70 °C, and the reaction mixture was stirred for 35 hours. The reaction mixture was cooled to 45-55 °C and the aqueous phase was separated. The organic phase was combined with N-acetyl-L-cysteine solution (24.4 kg in 44 kg water with pH adjusted to 7 with 9.3 kg KOH). The temperature was adjusted to 20-30 °C. The mixture was combined with product seed (0.15 kg) and water (1452 kg), the mixture was concentrated to 15 X, and was allowed to stir for 1 hour. The suspension was filtered and the cake was washed with THF / H2O (V7V: 2 / 3; 754 kg) and water (246 kg). The wet cake was dried at 60 C for 20 hours. The desired compound (Int-A) (163.4 kg) was obtained as a yellow solid in 90% isolated yield. 'H NMR (500 MHz, DMSO) 8 10.36 (s, 1H), 8.41 (br s, 1H), 8.35 (s, 1H), 8.20 (d, J= 5.6 Hz, 1H), 7.83 (br s, 1H), 7.47 - 7.36 (m, 1H), 7.07 (td, J= 8.9 Hz, J =3.6 Hz, 1H), 7.00 (d, J= 5.7 Hz, 1H), 4.66 (d, J = 13.9 Hz, 1H), 4.60 (d, J= 10.8 Hz, 1H), 4.42 (dd, J= 13.2 Hz, J= 10.5 Hz, 1H), 3.94 (t, J = 10.8 Hz, 1H), 3.35 - 3.24 (m, 1H), 1.21 (d, J = 6.8 Hz, 3H). 1.11 (d, J = 6.7 Hz, 3H).19F NMR (471 MHz, DMSO) 6 -117.49 (d, J= 15.9 Hz), -133.12 (d, J= 15.9 Hz).13C NMR (126 MHz, DMSO) 8 168.71, 161.64, 157.44, 154.60 (d, J= 244.7 Hz), 152.97, 153.03 (d, J= 242.3 Hz), 148.64, 146.65, 145.09 - 144.92 (m), 137.59, 121.72, 120.91 (d, J = 23.6 Hz), 118.07, 118.07 - 117.82 (m), 110.82 (dd, J= 24.8 Hz, J= 8.0 Hz), 110.50, 106.26. 71.37 (d. J= 5.8 Hz), 65.50. 30.15, 21.78. 21.29.Example 6: Preparation of26-chloro-33,36-difluoro-12-isopropyl-21,22,23,24-tetrahydro- 4,7-dioxa-2(l,7)-pyrido[2,3-d]pyrimidina-l(3,4)-pyridina-3(l,2)-benzenacycloheptaphane- 22, 24-dione (Int-B)Procedure A26037Synthesis of 23-chloro-13.16-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)- dipyridina-l(1.2)-benzenacvclooctaphane-25-carboxamide (2S.3S)-2.3- bis(benzoyloxy)succinate (Int-A: (2S.3S)-2,3-bis(benzoyloxy)succinate)

[0129] Dibenzoyl-D-tartaric acid (124.6 kg, 346.15 mol) was combined with DME (1190 kg) and the resulting solution was stirred at 60 °C. CIDT substrate, Int-A, (139 kg, 301 mol) was then added in 10 portions, and stirred at 60 °C for 1 h after adding each portion. The resulting slurry' was stirred at 60 °C for 5 hours, and n-heptane (1340 kg) was added at 60 °C over 8 h. The slurry was stirred at 60 °C for 6.5 h, and subsequently was cooled to -5 °C.Synthesis of 26-chloro-33.36-difluoro-12-isopropyl-21.22.23.24-tetrahydro-4.7-dioxa-2(1.7)-pyrido[2.3-d1pyrimidina-l(3.4)-pyridina-3(1.2)-benzenacycloheptaphane-22.24-

[0130] To the stirred slurry' of Int-A:optically active acid from the previous step were added pre-cooled solution of THF (1437 kg) at -10 °C. and diethyl carbonate (75 kg, 632.1 mol) at -5 °C. A solution of KHMDS in THF (1184 kg, 1309.35 mol) was added into the mixture at -5 °C for 4 h. The mixture was stirred at -5 °C for 2 h. The mixture was quenched with 9.3 wt.% aq. H3PO4 (1250 kg) and heated to 40 °C. The organic was separated, and the solvent was switched to 2 to 3 vol. with 2-Me-THF (1290 kg). 2-MeTHF (1807 kg) and water (556 kg) were added. The organic was separated and switched to 2 to 3 vol. with 2-Me-THF (2590 kg). The mixture was heated at 50 °C and then n-heptane (758 kg) was added at 50 °C over 8 h. The mixture was cooled to 5 °C over 3 h and then stirred at 5 °C for 8 h. The solids were filtered. The cake was subjected to displacement wash with 2-Me-THF:heptane (1:8, 660 kg). The cake was re-slurried at 5 °C for 6 h with 2-Me-THF (174 kg) and n-heptane (378 kg). The solids were filtered, and the26037 cake was subjected to displacement wash with 2-Me-THF:heptane (1:8, 660 kg). The wet cake was dried at 50 °C for 20 h to give the title compound (146.85 kg, 75.8% yield, 98.5% chiral purity). ’H NMR (500 MHz, DMSO) 5 12.26 (s, 1H), 8.56 (s, 1H), 8.44 (<7, J = 5.7 Hz, 1H), 7.49 (ddd, J= 11.0, 9.5. 5.2 Hz, 1H), 7.18 - 7.08 (m. 1H), 7.14 (d, J= 5.7 Hz, 1H), 4.74 (d, J= 14.1 Hz, 1H), 4.58 (d, 7= 10.8 Hz, 1H), 4.46 - 4.35 (m, 1H), 3.99 (t, J= 11.2 Hz, 1H), 3.09 (hept, 7 = 6.6 Hz, 1H), 1.22 (d, J = 6.6 Hz, 3H), 0.88 (d, 7= 6.7 Hz, 3H).19F NMR (471 MHz, DMSO) 3 - 117.05 (d, J= 16.0 Hz), -132.48 (d, 7= 16.1 Hz).13C NMR (126 MHz, DMSO) 6 165.65, 160.14. 158.12, 154.41 (d, J = 243.9 Hz), 152.87 (d, J = 243.2 Hz), 151.86, 150.35, 149.42, 149.06. 145.71 - 144.03 (m), 137.36, 126.00, 119.63 (d, J= 20.6 Hz), 118.96 (dd, J = 21.2 Hz, J = 10.3 Hz), 118.44, 112. 19, 110.97 (dd, J = 24. 1 Hz, 7= 7.9 Hz), 107.20, 70.50 (d, J = 6.2 Hz), 66.18, 29.79, 23.04.Procedure BStep 1 - Synthesis of 23-chloro-13,16-difluoro-42-isopropyl-5.8-dioxa-3-aza-2(2.6).4(3.4)-dipyridina-l(1.2)-benzenacyclooctaphane-25-carboxamide (2R,3R)-2,3- bis((4-methylbenzoyl)oxy)succinate

[0131] To a 100 L vessel equipped with a reflux condenser, was added Toluene (15 L) and Int- A (1.500 kg, 2.781 mol). While stirring the resulting slurry at 80 °C, a solution of Di-p-toluoyl- L-tartaric acid monohydrate (1.238 kg, 3.060 mol) in Toluene (10.8 L) and THF (1.2 L) was added over 5 hours.Step 2 - Synthesis of 26-chloro-33.36-difluoro-12-isopropyl-21.22.23.24-tetrahvdro-4.7- dioxa-2(1.7)-pyrido[2.3-d1pyrimidina-l(3.4)-pyridina-3(1.2)-benzenacycloheptaphane-22,24-dione26037

[0132] To the stirred slurry from Step 1, was sequentially added diethyl carbonate (1.70 L, 13.91 mol) followed by 1 M potassium tert-butoxide in THF (17.21 L, 16.69 mol) at -5 °C over 1 h. The mixture was heated to 20 °C and quenched with 1 M aq. H3PO4 (8.34 L). The separated organic layer was washed with water (8.30 L). The organic solution was concentrated under vacuum to 10 V (15 L). To the homogeneous solution at 20 °C was added Acetonitrile (3.75 L) over 1 h. After stirring the resulting slurry at 25 °C for 3 h, n-Heptane (26.3 L) was added to the slurry over 5 hours. The solids were filtered at 5 °C. The cake was subject to two displacement washes with 4: 1 :7 mixture of Toluene: Acetonitrile: n-Heptane (4.5 L). The wet cake was dried at ambient temperature for 24 h to give the title compound as a toluene solvate (1.54 kg, 83.9 wt%. 95.4% yield, 99.5% chiral purity).

[0133] 'H NMR (500 MHz, DMSO) 5 12.29 (s, 1H), 8.56 (s, 1H), 8.45 (d, J= 5.7 Hz, 1H), 7.46 (ddd, J= 11.0, 9.4, 5.2 Hz, 1H), 7.23 (app. t, J= 7.5 Hz, 2H), 7.18 - 7.06 (m, 5H), 4.75 (app. d, J= 14.0 Hz, 1H), 4.65 - 4.53 (m, 1H), 4.47 - 4.34 (m, 1H), 4.01 (app. t, J= 11.2 Hz, 1H), 3.10 (hept, J= 6.6 Hz, 1H), 2.28 (s, 3H), 1.23 (d, J= 6.6 Hz, 3H), 0.88 (d, J= 6.7 Hz, 3H).19F NMR (471 MHz, DMSO) 5 -116.98 (d, J= 15.9 Hz), -132.48 (d, J= 15.9 Hz).13C NMR (126 MHz, DMSO) 5 165.68, 160.15, 158.15, 154.43 (app. d, J= 244.1 Hz), 151.92 (dd, J= 243.2, 2.9 Hz), 151.91 (d, J= 1.9 Hz), 150.38, 149.44, 149.07. 144.90 (dd, J= 14.3, 7.2 Hz), 137.37, 137.31. 128.86, 128.16, 126.05, 125.27. 119.64 (dd, J = 20.6. 3.4 Hz), 118.92 (dd, J = 21.3, 10.4 Hz), 1 18.46, 112.18, 1 10.95 (dd, J= 24.0, 7.9 Hz), 107.21 , 70.50 (d, J= 6.2 Hz), 66.20, 29.80, 23.01, 21.00, 20.86.Example 7: Preparation of (2R,5S)-l-benzyl-2,5-dimethyl piperazine (Int-W3)

[0134] Three different procedures were used to prepare Int-W3.Procedure A26037Synthesis of 7'c77-butv-4-benzyl-(2R.5S)-2.5-dimethyl piperazinel-1 -carboxylate (Int-W2a)lnt-W2a

[0135] A I L round bottom flask equipped with a stir bar was charged with tert-butyl (2S,5 )- 2,5-dimethylpiperazine-l -carboxylate (Int-W2c, 100 g, 467 mmol, 1.0 equiv) and DCE (500 mL). N-ethyl-N-isopropylpropan-2-amine (DIPEA) (122 mL, 700 mmol, 1.5 equiv) was added followed by benzyl bromide (55.5 mL, 467 mmol, 1.5 equiv) and the reaction was stirred at 50 °C and 600 rpm for 24 hours.

[0136] The reaction was washed with water (250 mL), dried over MgSCL and concentrated and the crude product was purified by column chromatography (silica) using a gradient of ethyl acetate / hexanes from 95-5 to 8-2 which resulted in tert-butyl (2.S'.5 / ?)-4-benzyl-2.5- dimethylpiperazine- 1 -carboxylate as an oil (Int-W2a, 105 g, 345 mmol, 73.9 % yield).JH NMR (400 MHz. CD2CI2) 5 7.38-7.36 (m. 2H), 7.32-7.28 (m. 2H), 7.24-7.21 (m. 1H), 4.19-4.13 (m. 1H), 3.66 (d, J= 12.6 Hz, 1H), 3.60 (d, J= 13.6 Hz, 1H), 3.47 (d, J= 13.6 Hz, 1H), 3.28 (d, J = 12.6 Hz, 1H), 2.95-2.89 (m, 1H), 2.70 (dd, J= 11.7 Hz, J= 4.1 Hz, 1H), 2.18 (d, J= 11.7 Hz, 1H), 1.44 (s, 9H), 1.21 (d, .7= 6.7 Hz, 3H), 0.98 (d, J= 6.5 Hz, 3H).nC NMR (101 MHz, CD2CI2) 5 155.58 (CO), 140.05, 128.91. 128.55, 127.19, 79.24. 59.24, 52.64. 49.31, 47.38, 44.79, 28.59, 16.23, 7.69.Synthesis of (2R.5S)-1 -benzy l -2.5-dimethyl piperazine dihydrochloride (Int-W3 2HC1) acetyl chloride ethanol2-Me-THF40 °C, 4 h

[0137] A 2 L round bottom flask equipped with an overhead stirrer was charged with tert-buty l (2S,5A)-4-benzyl-2,5-dimethylpiperazine-l -carboxylate (165 g. 542 mmol, 1.0 equiv) and 2- MeTHF (1000 mL, contains 150-400 ppm BHT as stabilizer) to form a first solution. In a separate flask, acetyl chloride (270 mL, 3794 mmol, 7 equiv) was added to an ice-cooled solution of ethanol (222 mL, 3794 mmol, 7.0 equiv) and 2-MeTHF (100 mL) to generate a fresh solution26037 of HC1 in 2-MeTHF. The HC1 solution was then added to the first solution and the mixture was heated to 40°C for 4 h.

[0138] The reaction was cooled to room temperature and the slurry was filtered. The solids were washed with 2-MeTHF (2 x 10 mL) and then dried under vacuum and air flow to yield (27?,5S)-l-benzyl-2,5-dimethylpiperazine dihydrochloride as a solid (Int-W3-2HC1, 145 g, 523 mmol, 97 % yield). 'H NMR (500 MHz, CD3CN+D2O 3 / 1) 5 7.56-7.54 (m, 2H), 7.52 - 7.45 (m, 3H), 4.81 (d, J= 13.2 Hz, 1H), 4.13 (d, J= 13.2 Hz, 1H), 3.84-3.78 (m, 1H), 3.73 - 3.61 (m, 2H), 3.67 (dd, J= 14.2 Hz, J = 3.3 Hz, 1H), 3.43 (dd, J= 14.3 Hz, J= 12.2 Hz, 1H), 3.25 (dd, J= 13.7 Hz. J= 3.3 Hz. 1H), 3.13 (dd, J= 13.6 Hz, J= 12.2 Hz. 1H), 1.62 (d. J= 6.5 Hz. 3H), 1.26 (d. J = 6.6 Hz, 3H).13C NMR (126 MHz, CD3CN+D2O 3 / 1) 8 132.45, 131.22, 130.39, 129.00, 57.58, 57.12, 52.53, 49.93, 46.69, 15.83, 14.75.Procedure BPyrazinium bromide Bn piperazine Bn piperazine bis-HCI

[0139] To a solution of D-glucose (4.52 kg, 25.1 mol) in 0.1 M MOPS pH 6.8 buffer (20.0 L) under nitrogen was added l-benzyl-2,5-dimethylpyrazin-l-ium bromide (2.00 kg, 7.17 mol), PyRED enzyme lyophilized cell-free extract (100 g. SEQ ID NO:1), GDH lyophilized cell-free extract (10.0 g. Prozomix) and NADP+disodium salt (113 g, 0. 143 mol). The reaction was stirred at 30 °C for 28 h with the pH maintained at pH 6.25 by the addition of 2 N NaOH (aq.) solution. Sodium sulfate (4.00 kg) was added and the pH adjusted to pH 12.0 by the addition of 10 N NaOH (aq). solution. 2-MeTHF (16.0 L) was added and the mixture stirred at 40 °C for 1 h. The mixture was cooled to 25 °C and filtered through SOLKA-FLOK (powdered cellulose filter aid) followed by a filter flush with additional 2-MeTHF (8.00 L). The organic layer was separated and washed with 15 wt% NaCl (aq.) solution (10.0 L). The organic layer was separated and concentrated to 7.20 L with additional 2-MeTHF constant volume distillation until KF <2000 ppm. MeOH (4.80 L) was added to generate a (2 .5S)- 1 -benzyl-2,5-dimethylpi perazine freebase solution in 2-MeTHF / MeOH.

[0140] In a separate vessel, acetyl chloride (1.52 L, 21.5 mol) was added slowly to a mixture of MeOH (2.90 L) and 2-MeTHF (4.40 L) at 0-15 °C. The solution was warmed to 20 °C and 0.37 L of the (2 / ?.55')-l -benzyl-2.5-dimethylpiperazine freebase solution in 2-MeTHF / MeOH was26037 added. The solution was seeded with (27?,5S)-l-benzyl-2,5-dimethylpiperazine bis-HCl (14.0 g) and stirred for 1 h. The remaining (2 ?,55)-l-benzyl-2,5-dimethylpiperazine freebase solution in 2-MeTHF / MeOH was then added over 4 h and then aged overnight. The solid was fdtered and washed with 1:2 MeOH:2-MeTHF (5.90 L) and 2-MeTHF (5.90 L + 2.65 L) and dried under nitrogen at room temperature overnight to afford (2 .5S)- 1 -benzy 1-2,5-dimethy 1 piperazine bis- HCl as an off-white crystalline solid (1.654 kg, 5.97 mol, 83.2%), >99% ee, >99% de.

[0141] The transformation can also be performed using PyRED enzyme having SEQ ID NO:2.Procedure C

[0142] (Tert-butoxycarbonyl)-L- Alanine (20.00g, 105.7 mmol, 1 eq) and N-Benzyl-D-AlanineMethyl Ester (90 wt%, 24.97 g, 116.3 mmol, 1.1 eq) were dissolved in 200 mL of DCM. 1-Hydroxy-7-azabenzotriazole (1.44 g, 10.57 mmol, 0.1 eq) and EDC HC1 (22.29 g, 116.3 mmol,1.1 eq) were added as powders via a funnel. The funnel was rinsed with 100 mL of DCM and the mixture was stirred at room temperature overnight. The mixture was then stirred with 200 mL of water for 10 minutes and then the aqueous layer was discarded. The organic layer was further washed with HC1 (0.1M, 200 mL x 2) followed by brine (200 mL). The organic layer was then dried over magnesium sulfate and concentrated under vacuum to give a pale yellow oil (34 g, 88% yield, uncorrected for wt%) as the di-amino-acid.

[0143] Acetyl chloride (16.22 g, 206.7 mmol, 7 eq) was dissolved in 54 mL of Ethyl Acetate.Methanol (6.81 g, 212.6 mmol, 7.2 eq) was added dropwise over an hour with caution (strong exotherm). The freshly prepared solution of HC1 in Ethyl acetate was added dropwise to a solution of the crude di-amino-acid (10.76 g, 29.52 mmol, 1 eq) in 108 mL of Ethyl Acetate over 30 minutes. After two hours stirring at room temperature, a saturated solution of potassium carbonate (108 mL) was added dropwise over an hour. The aqueous layer was separated and re-26037 extracted with ethyl acetate (2 x 54 mL). The combined organic layers were dried over magnesium sulfate and concentrated under vacuum. (3S,6R)-l-benzyl-3,6-dimethylpiperazine- 2, 5-dione (4.9 g, 71%) was obtained as a colorless oil, that spontaneously cry stallized over 48 hours on standing.

[0144] Crude (3S,6R)-l-benzyl-3,6-dimethylpiperazine-2, 5-dione (1.00 g, 3.83 mmol, 1 eq.) w as dissolved in 10 mL of toluene in a 40 mL scintillation vial. Tetramethyldisiloxane (4. 10 mL, 23.0 mmol, 6 eq.) was added followed by zinc (II) triflate (278 mg, 0.765 mmol, 20 mol%). A stir bar was added and the vial w as sealed with a pressure relief cap. The reaction mixture was then heat at 100°C for 3 hours. The reaction was brought to room temperature, depressurized, and poured into a 200 mL Erlenmeyer. With strong stirring, KOH (20 mL, 25wt% in MeOH) was added and the mixture was stirred at room temperature overnight. Water was added (50 mL) and the mixture was extracted into EtOAc (3 x 50 mL). The organic phase was separated, dried over MgSO4 and concentrated. The thick slightly yellow oil was dissolved in heptane (10 mL) and HC1 (7.7 mL, 2M in Et20, 4 eq.) was added. Upon addition of the HCL a w hite solid precipitated from the solution. It w as filtered and washed successively with MTBE (2 x 5 mL) and heptane (5 mL). The white solid was dried under vacuum, dissolved in MeOH (10 mL) and layered with MTBE (15 mL). The Bn piperazine bis-HCl was obtained as white crystals (585 mg, 2.43 mmol, 99 wt%) in 63% yield.Example 8: Preparation of (2S,5R)-4-acryloyl-2,5-dimethylpiperazin- 1-ium Acid Addition Salts (Int-W4 Acid Addition Salt)Preparation of (2S.5R)-4-acryloyl-2.5-dimethylpiperazin-l-ium methanesulfonate (Int- W4 CHqSOqH. Procedure A26037Synthesis of tert-butyl (2S.5R)-4-acryloyl-2.5-dimethylpiperazine-l -carboxylate

[0145] IP AC (486 kg) was combined with the Boc-protected piperazine (56.6 kg. 264. 1 mol) followed by BHT (0.085 kg. 0.386 mol) and NaHCO? (33.3 kg. 396.4 mol) and the temperature was adjusted to 0-10 °C. To the mixture was added acry loyl chloride (26.4 kg, 291.7 mol) dropwise and the mixture was stirred for 24 hours. The mixture was quenched by the addition of water (285 kg) maintaining the temperature below' 25 °C. The mixture was agitated for 1 hour, the phases were allowed to separate, and the aqueous phase was removed. The organic phase was w ashed three times with water (170 kg x3). The organic phase was switched to acetonitrile by addition of acetonitrile and distillation of IP AC. The resulting solution of the title compound in acetonitrile w as used in the next step w ithout further purification.

[0146] Isolation of the product provided a sample for spectral analysis. 'H NMR (400 MHz, DMSO-O 5 ppm 0.98 - 1.18 (m, 6 H) 1.41 (br s, 9 H) 2.94 - 3.28 (m, 2 H) 3.37 - 3.77 (m. 2 H) 3.98 - 4.42 (m, 2 H) 5.69 (ddd, 7=10.35, 6.03, 2.25 Hz, 1 H) 6.13 (dd, 7=16.63, 1.88 Hz, 1 H) 6.66 - 6.88 (m, 1 H); HRMS (ESI) m / z calculated for C10H17N2O.C [M+H]+213. 12337 found 213.12319.Synthesis of (2S.5R)-4-acryloyl-2.5-dimethylpiperazin-l-ium methanesulfonate (Int-W4CH SO3H)Boc lnt-W4’CH3SO3H

[0147] Methanesulfonic acid (47.8 kg, 497.3 mol) was combined with acetonitrile (393 kg) and the temperature was adjusted to 20-30 °C. To the mixture was added the previously prepared solution of tert-butyl (2S,5R)-4-acryloyl-2,5-dimethylpiperazine-l -carboxylate (330.4 kg x 20.3 wt.%, 250.0 mol) and the mixture was stirred for 16 hours. The desired product, Int-W426037CH3SO3H, was obtained as a solution in acetonitrile in 100% assay yield. The solution was used without further purification.

[0148] Isolation of the product provided a sample for spectral analysis.JH NMR (400 MHz, DMSO-O 5 ppm 1.12 - 1.32 (d. 6 H) 2.99 (dd, 1 H) 3.15 - 3.49 (m, 2 H) 3.62 (br s, 1 H) 3.99 (br d, 1 H) 4.60 (br s, 1 H) 5.64 - 5.78 (m, 1 H) 6.12 (dd, 1 H) 6.74 (dd, 1 H) 8.18 (br s, 3 H) 8.80 - 9.14 (m, 2 H); HRMS (ESI) m / z calculated for C9H17N2O [M+H]+169.13354 found 169.13383.Preparation of (2S.5R)-4-acryloyl-2.5-dimethylpiperazin-l-ium tetrafluoroborate (Int-W4HBFzt. Procedure B)lnt-W4*BF4

[0149] Tert-butyl (2S,5R)-4-Benzyl-2,5-dimethylpiperazine-l-carboxylate (1.0 g, 3.3 mmol), IP AC (10.0 mL). and Pd / C (0.35 g, 0.33 mmol) were combined. The vessel was evacuated and back-filled three times with H2. The batch was stirred at 20-25 °C for 16 h. The vessel was purged with N2 then filtered and rinsed with IP Ac (5.0 mL). Sodium bicarbonate (0.44 g, 5.3 mmol) was charged and the batch was cooled to 0-5 °C. Acryloyl chloride (0.29 mL. 3.6 mmol) was charged. The batch was stirred at 0-5 °C for 2.5 h then the temperature was adjusted to 20-25 °C and the batch w as washed three times with w ater (15 mL). The batch was concentrated to 1 / 3 of the original volume. Tetrafluoroboric acid (48% aq. solution) (0.38 mL, 2.4 mmol) was charged. The batch was stirred at 20-25 °C to form a slurry. Solids were filtered, washed with IP Ac, then dried under vacuum at 20-25 °C to afford Int-W4 HBF4 (332 mg. 62% yield). 'H NMR (DMSO-d6, 600 MHz) 5 8.9-8.6 (m, 2H), 6.8 (dd, J = 16.6, 10.5 Hz, 1H), 6.2 (dd, J = 16.6,2.2 Hz, 1H), 5.8 (dd, J = 10.5, 2.2 Hz, 1H), 4.6 (br s, 1H), 4. 1-3.9 (m. 1H), 3.6 (br s, 1H), 3.5 -3.2 (m. 2H), 3.0 (dd, J = 13.3, 2.4 Hz, 1H), 1.24 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 6.8 Hz, 3H).13C NMR (DMSO-d6, 151 MHz) 5 165.2, 128.4, 127.7, 47.4, 43.8 (br), 40.9, 15.5 (br), 13.2.26037Example 9: Preparation of (5aSa,17aRa)-20-ChIoro-2-[(2S,5R)-2,5-dimethyl-4-(prop-2- enoyl)piperazin-l-yl]-14,17-difluoro-6-(propan-2-yl)-ll,12-dihydro-4H-l,18-(ethanediylidene)pyrido[4,3-e]pyrimido[l,6-g][l,4,7,9]benzodioxadiazacyclododecin-4-one(compound of Formula (I), Procedure A)Synthesis of 24-((2S.5R)-4-benzyl-2.5-dimethylpiperazin-l-yl)-26-chloro-33.36-difluoro-12-isopropyl-21.22-dihydro-4.7-dioxa-2(1.7)-pyrido[2.3-d1pyrimidina-l(3.4)-pyridina-3(1.2)-benzenacycloheptaphan-22-one (Int-C )

[0150] 26-chloro-33.36-difluoro-12-isopropyl-2L22,23,24-tetrahydro-4,7-dioxa-2(L7)- pyrido[2,3-d]pyrimidina-l(3,4)-pyridina-3(l,2)-benzenacycloheptaphane-22, 24-dione (Int-B, 15 g, 75.7 wt.%, 23.3 mmol), THF (113 mL), and DIPEA (6.93 m , 39.6 mmol) were combined. POCh was charged and the stirred batch was heated to 30-40 °C for 3 h. The batch was cooled to 20-25 °C. H lung's base (16.3 mL, 93.0 mmol) and (2R.5S)-l-benzyl-2.5 -dimethylpiperazine hydrochloride (Int-W3, 7.06 g, 87.5 wt.%, 25.7 mmol) were charged. The batch was stirred at 20- 25 °C for 16-20 h then charged with EtOAc (150 mL) and 5 wt.% aq. K3PO4. The phases were separated then the organic layer was washed twice with water (300 mL). The batch was charged26037 wi th activated carbon (3 g) and stirred for 20 min at 20-25 °C. The batch was filtered through CELITE and concentrated under reduced pressure. THF was charged to bring the total volume to 70 mL. The batch was heated to 45 °C and was charged with seed crystals to make a slurry. The slurry was charged with heptanes (60 mL) over 4 h. The slurry was stirred for 1 h at 45 °C then cooled to 22 °C over 4 h and stirred at this temperature for 12 h. The solids were filtered, washed with THF / heptanes (2: 1), then dried under vacuum at 20-25 °C to afford (Int-C, 15.7 g. 77% yield). NMR (CD3CN, 600 MHz) 5 8.40 (d, J = 5.7 Hz, 1H), 8.22 (s, 1H), 7.47 (ddd, J = 10.9, 9.5, 5.2 Hz, 1H), 7.41 (d, J = 7.4 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H). 7.14 - 7.09 (m, 2H), 4.71 (d, J = 13.9 Hz, 1H), 4.65 - 4.55 (m. 3H), 4.37 - 4.27 (m. 1H), 4.01 (t, J =11.2 Hz, 1H), 3.70 (d, J = 13.6 Hz, 1H), 3.65 - 3.57 (m, 1H), 3.52 (d, J = 13.6 Hz, 1H), 3.21 (s, 1H), 3.01 (hept, J = 6.7 Hz, 1H), 2.81 (d, J = 9.5 Hz, 1H), 2.33 (d, J = 11.8 Hz, 1H), 1.61 (d, J =5.2 Hz, 3H), 1. 17 (d, J = 6.7 Hz, 6H), 0.97 (d, J = 6.7 Hz, 3H).Alternate Synthesis of 24-((2S,5R)-4-benzyl-2.5-dimethylpiperazin-l-yl)-26-chloro-33.36-difluoro-12-isopropyl-21.22-dihvdro-4.7-dioxa-2(1.7)-pyridor2.3-d1nyrimidina- l(3.4)-pyridina-3(1.2)-benzenacvcloheptaphan-22-one (Int-C)Int-BH (1 .1 equiv)DIPEA (4.0 equiv)MeCN (05 V) 25 °C, 22 hWorkup:1 ) H2O (0.5 V), 1h2) H2O (7V) isolation26037

[0151] To a 100 mL reaction vessel under nitrogen was charged Int-B (Toluene solvate, 6.00 g, 81.6 wt%, 1.0 equiv. 10.1 mmol), 1 ,2,4-triazole (3.72 g, 5.3 equiv. 53.3 mmol) and acetonitrile (39 mL, 6.5V). Diisopropylethylamine (10.9 mL, 6.3 equiv. 63.4 mmol) was charged to the vessel and the reactor was cooled to 5 °C. POCh (1.31 mL 1.4 equiv. 14.1 mmol) was added dropwise over 45 min and then the vessel was warmed to 22 °C and aged for an additional Ihr. Diisopropylethylamine (6.94 mL 4.0 equiv. 40.2 mmol)) was added to the reaction mixture followed by Benzy l Piperazine (3.09 g, 1,11 eq, 11.2 mmol). The reaction mixture was aged for 16h before quenching by the dropwise addition of H2O (3.0 mL) and aging for an additional 2h. H2O (13.8 mL) was charged to the reaction vessel and then the mixture was seeded with Int-C Seed (102 mg) and aged for three hours. H2O (28.2 mL) was charged dropwise over 8h. Upon completion of water addition, the slurry was aged for an additional 8 h. The slurry7was cooled to 15 °C over 0.5 h and then aged for additional 1 h. The mixture was filtered, and the cake was washed with 60% H2O in acetonitrile (2 x 18 mL). The cake was dried under vacuum with nitrogen sweep at room temp for 0.5 h and then at 40 °C over night to afford Int-C (6.36 g, 97.34 wt%, 91.4% yield) as a yellow solid.

[0152] 'H NMR (500 MHz, CDCh) 5 8.48 (d, J= 5.7 Hz, 1H), 8.13 (s, 1H), 7.45 - 7.38 (m, 2H), 7.38 - 7.32 (m, 2H), 7.32 - 7.26 (m, 1H), 7.17 - 7.09 (m, 1H), 6.82 (td. J= 8.7. 3.5 Hz, 1H), 6.64 (d, .7= 5.7 Hz, 1H), 4.79 - 4.55 (m, 3H), 4.48 (d, J= 14.0 Hz, 1H), 4.37 - 4.29 (m, 1H), 4.15 (t, J= 10.9 Hz, 1H), 3.82 - 3.66 (m, 2H), 3.56 (d, J = 13.4 Hz, 1H), 3.34 - 3.24 (m, 1H), 3.06 (hept, J = 6.7 Hz, 1H), 2.88 (dd. J = 12.0, 3.6 Hz, 1H), 2.43 (d, .7= 11.8 Hz, 1H), 1.68 (d, .7= 6.5 Hz, 3H), 1.34 (d, .7= 6.7 Hz, 3H), 1.27 (d, .7= 5.6 Hz, 3H), 1.11 (d, .7= 6.8 Hz, 3H).13C NMR (126 MHz, CDCL) 6 166.17, 161.96, 158.85, 156.26 (d, J = 2.0 Hz), 155.25, 154.31 (d, JC-F = 2.0 Hz), 153.46 (d, JC-F = 3. 1 Hz), 151.98 (d, JC-F = 2.1 Hz), 151.76, 151.52 (d, JC-F = 3.2 Hz) , 150.12, 145.52 (dd, JC- = 14.4, 7.1 Hz), 138.69, 135.29, 128.71, 128.53, 127.37, 124.32. 121.08, 120.67 (dd, JC-F = 20.4, 3.3 Hz), 118.01 (dd, JC-F = 21.1, 10.2 Hz), 110.63 (dd, JC-F = 24.2. 7.7 Hz), 106.03. 105.76. 70.05 (d. JC-F = 6.3 Hz). 66.08, 58.54. 53.24, 52.28. 49.79, 47.94, 30.69, 22.52, 21.38, 16.19, 7.87.19F NMR (471 MHz, CDCL) 5 -117.94 (dJ = 15.5 Hz), - 133.40 (d, J= 15.6 Hz).Synthesis of (5aSa.l7aRa)-20-Chloro-2-r(2S,5R)-2.5-dimethyl-4-(prop-2- enovDpiperazin- 1 -yl] - 14.17 -difluoro-6-(propan-2-yl)- 1 L 12-dihvdro-4H- 1.18-26037(ethanediylidene)pyrido[4.3-e1pyrimido g1[1.4.7.91benzodioxadiazacvclododecin-4- one (compound of Formula (I))

[0153] Acetonitrile (15 mL) was added to a flask containing benzy l amine (5.00 g, 7.15 mmol) and sodium iodide (2.36 g. 15.7 mmol). The mixture was stirred at 20-25 °C to give a solution. Acryloyl chloride (1.16 mL. 14.3 mmol) was added dropwise and the reaction mixture was stirred at 20-25C overnight. Pyridine (1.16 mL, 14.3 mmol) was added and the reaction mixture was stirred for three hours. Then, to the reaction mixture was added EtOAc (50 mL, 10 vol) followed by 20 mL 5 wt.% sodium bicarbonate. The mixture was agitated and the aqueous phase (bottom) was removed. To the organic phase was added 20 mL 5 wt.% sodium thiosulfate (sodium ascorbate may also be used in place of sodium thiosulfate). The mixture was agitated and the aqueous phase (bottom) was removed. The resulting organic phase was washed twice more with 20mL water and the organic phase was collected. The solvent of the organic solution was swapped from EtOAc to MeCN via distillation to a final volume of 25mL (5vol). The resulting solution was diluted with 5vol water and the mixture was used in the next step.

[0154] To a separate vessel containing 75 mL water was added 5 wt.% seed (250 mg). The mixture was stirred at 45 °C and the above reaction mixture was added dropwise over a 12 hour period. The resulting white suspension was cooled to 20 °C over a three hour period and then allowed to age for an additional 2 hours. The suspension was filtered and the collected solids were washed with twice with 10 mL 1 :4 MeCN: Water followed by washing with 10 mL water. The cake w as dried under the flow7of nitrogen with vacuum suction overnight to afford the compound of Formula (I) (86% yield). 'HNMR (CDCh, 500 MHz): 3 7.45-7.32 (m, 5 H), 6.55 (d, J = 8.2 Hz, 1H), 5.05 (d, .7= 11.6 Hz, 1H). 4.90 (d, J= 11.6 Hz. 1H), 4.02 (m, 2H). 3.90 (m, 2H), 3.30 (m, 1H), 2.99 (dt, 11.7, 1.1 Hz, 1H), 2.86 (m, 2 H), 2.64 (d, J= 11.7 Hz, 1H), 2.3726037(dd, J= 14.6, 6.9 Hz, 1H), 2.04-1.82 (m, 4H), 1.58 (m, 1H), 1.45 (s, 9H), 1.30 (m, 2H);13C NMR (CDCls, 125 MHz) 5: 168.3, 167.5, 154.7, 135.6, 129.2 (2C), 128.8, 128.6 (2C), 79.7, 78.3, 60.4, 57.8, 47.5, 46.8, 42.5 (br, 2C), 32.0, 31.7, 28.4 (3C), 20.8, 17.2.Example 10: Preparation of (5aSa,17aRa)-20-Chloro-2-[(2S,5R)-2,5-dimethyl-4-(prop-2- enoyl)piperazin-l-yl]-14,17-difluoro-6-(propan-2-yl)-ll,12-dihydro-4H-l,18- (ethanediylidene)pyrido[4,3-e]pyrimido[l,6-g][l,4,7,9]benzodioxadiazacyclododecin-4-one (compound of Formula (I), Procedure B)

[0155] THF (100 IL) was combined with DIPEA (49.6 kg) and the temperature was adjusted to 20-30 °C. To this mixture was added dropwise POCI3 (55.8 kg) followed by the addition of Int-B (110 kg, 225.9 mol) in 10 portions. The temperature was then adjusted to 30-40 °C and the mixture was stirred for 17 h.Synthesis of (5aSa,17aRa)-20-Chloro-2-[(2S,5R)-2.5-dimethyl-4-(prop-2- enoyl)piperazin- 1 -yl] -14,17 -difluoro-6-(propan-2-yl)- 11 , 12-dihy dro-4H- 1,18- (ethanediylidene)pyrido[4.3- benzodioxadiazacyclododecin-4-one (compound of Formula (I))26037

[0156] The reaction temperature containing the dichloro pyridopyrimidone above was adjusted to -5 °C and DIPEA (117 kg) was added. To the stirring mixture was added dropwise Int- W4 CH3SO3H (38.0 kg, 253.0 mol) as a solution in acetonitrile and the reaction was aged for 3 hours. The temperature was then adjusted to 15-25 °C and the mixture was concentrated to -540 L maintaining the temperature below 50 °C. To the mixture were charged EtOAc (1100 L) and water (220 kg) and the resulting solution was adjusted to pH 7.0 by adding 488 kg 10% Na2CC>3 aq. The temperature was adjusted to 20-30 °C and the mixture was stirred for 1.5 hours. The phases were allowed to separate and the aqueous phase was removed. Additional water (550 kg) was combined with the retained organic phase and the mixture was agitated for an hour. The phases were then allowed to separate and the aqueous phase was removed. The organic phase was then treated with 14% CUNO (3 M) for 8 hours and the batch was filtered. The resulting filter cake was washed three times with water saturated EtOAc (550 kg; 3.5% H2O) for three hours each wash. The resulting solution was then concentrated to -340L maintaining the temperature below 40 °C and the solvent was switched to THF by distillation. The temperature of the resulting mixture was adjusted to 40-50 °C and API seed (20 g) was added. To the stirring mixture was added n-heptane (300 kg) over 8 hours and the resulting suspension was aged for 5 additional hours. The suspension was filtered and the cake was washed with THF / n-heptane (349 kg; 1 : 1 v: v) twice. The resulting wet cake was combined with EtOAc (1652 kg) and water (550 kg). The temperature was adjusted to 35-45 °C and stirred for 1 hour. The stirring was stopped, the phases were allowed to separate, and the aqueous phase was removed. The temperature was adjusted to 20-30 °C and the retained organics were treated with 14% CUNO (3M). After treatment the CUNO was removed via filtration and the filter cake washed with water-saturated EtOAc (3.5 % H2O). The resulting solution was concentrated to -330 L maintaining the temperature below 40 °C and the temperature was then adjusted to 45-55 °C. To the mixture was added seeds of the title compound (0.017 kg) and the mixture was stirred for 3 hours. The mixture was combined with EtOAc (739 L) and the mixture was concentrated to -440 E while maintaining the batch below 40 °C. The solvent was then completely switched to EtOAc by adding EtOAc and removal of the solvent by distillation to a final volume of -440 L. The temperature was then adjusted to 45-55 °C, aged for 3 hours, MTBE (900 kg) was added dropwise over an 8 hour period, and the resulting mixture was stirred for 2 hours at 40-50 °C. The batch was cooled to 20-30 °C, stirred for 4 hours, filtered, and the filter cake was washed wi th EtOAc / MTBE (240 kg; 1:2 v / v) twice. The wet cake was dried at 40-50 °C for 20 hours to give the title compound (105.3 kg) as a white solid in 73.1% isolated yield.26037

[0157] The compound of Formula (I) can be prepared using a similar method by substituting 2S,5R)-4-acryloyl-2,5-dimethylpiperazin-l-ium tetrafluoroborate (Int-W4 HBF4) for (2S,5R)-4- acryloyl-2,5-dimethylpiperazin-l-ium methanesulfonate (Int-W4 CH3SO3H) The synthesis using Int-W4 HBF4 is described below.Step 1 : 24.26-dichloro-33.36-difluoro-12-isopropyl-21.22-dihydro-4.7-dioxa-2(T.7)- pyrido[2.3-d1pyrimidina-l(3.4)-pyridina-3(1.2)-benzenacvcloheptaphan-22-one

[0158] DIPEA (4.64 ml, 26.4 mol) and POCh (2.319 mL, 24.88 mmol) were combined with THF (100 ml). To the resulting solution, was added Int-B (10 g, 15.55 mmol), and the mixture was stirred at 35 °C for 24 h.Step 2: Compound of Formula (I)

[0159] To the mixture from Step 1 cooled to 0 °C, was added DIPEA (10.92 ml, 62.2 mmol).To the resulting mixture, was added the piperazine acrylamide (4.68 g, 17.57 mmol) at 0 °C, and then was stirred at 0 °C for 2 h. The solvent was concentrated to 5 vol. below 50 °C and ethyl acetate (100 mL) was added. The resulting mixture washed with 6 wt% aq. Na2COs (50 mL) and water (50 mL). To the organic solution, was added AQUAGUARD (10 g) and then the resulting slurry was stirred at the ambient temperature for 18 h. The solids were removed by filtration. The filtrate was concentrated to 5V of THF (50 mL) below 40 °C. After stirring the solution at 45 °C for 3 h. n-heptane (40 mL) was added to the solution at 45 °C over 4 h. After stirring the mixture26037 at 45 °C for 8 h, the mixture was cooled to 25 °C over 1 h. The solids were filtered. The cake was subjected to displacement wash with THF:heptane (1:1, 40 mL). The wet cake was dried at the ambient temperature for 20 h to give the compound of Formula (I) (8.97 g, 85.0% yield).Final Purification of the Compound of Formula (I)

[0160] The compound of Formula (I) as synthesized above (103.7 kg) was combined with MeOH (505 kg). The temperature was adjusted to 20-30 °C, and the mixture was treated with 10% CUNO (3M) for 10 hours. The batch was filtered and the cake was washed with MeOH (602 kg). The resulting solution was filtered, the filtrate was concentrated to 6 volumes maintaining the batch below 40 °C, and the temperature was adjusted to 45-55 °C. To the warm solution was added filtered water (416 kg) dropwise over 8 hours, then seed crystals of the compound of Formula (I) (0.230 kg) were added and the resulting mixture was stirred for 4 hours. Additional filtered water (206 kg) was then added over 5 hours, the temperature was adjusted to 20-30 °C, and the mixture was allowed to stand for 5 hours at this temperature. The slurry was filtered and the cake was washed with MeOH / Water (368 kg; 1: 1 v / v) twice. The cake was dried at 35-45 °C for 58 hours. The purified compound of Formula (I) was obtained as a solid (92.75 kg, 89% isolated yield).Example 11 : Preparation of (5aSa,17aRa)-20-Chloro-2-[(2S,5R)-2,5-dimethyI-4-(prop-2- enoyl)piperazin-l-yl]-14,17-difluoro-6-(propan-2-yl)-ll,12-dihydro-4H-l,18- (ethanediylidene)pyrido[4,3-e]pyrimido[l,6-g][l,4,7,9]benzodioxadiazacyclododecin-4-one (compound of Formula (I), Procedure C)Step 1 : Preparation oftert-butyl (2R.5S)-4-(26-chloro-33.36-difluoro-12-isopropyl-22- oxo-21.22-dihvdro-4.7-dioxa-2(1.7)-pyrido[2.3-d1pyrimidina-l(3.4)-pyridina-3(1.2)- benzenacvcloheptaphane-24-yl)-2.5-dimethylpiperazine-l -carboxylate (Int-H)Boc

[0161] Phosphoryl trichloride (33.3 mL) and DIPEA (62.3 mL) and THF (1000 mL) were combined. The stirred batch was allowed to stir at ambient temperature and then charged with 26-chloro-33,36-difluoro-12-isopropyl-21,22,23,24-tetrahydro-4,7-dioxa-2(l,7)-pyrido[2,3- d]pyrimidina-l(3,4)-pyridina-3(l,2)-benzenacycloheptaphane-22, 24-dione (Int-B) (170 g, 264 mmol) in one portion, followed by a 100 mL THF rinse and then allowed to stir at ambient temperature for 23 hours. The reaction mixture was then analyzed using UPLC, which indicated full conversion of Int-B. The batch was cooled to 4 °C in an ice bath then DIPEA (139 mL) and (2A,55)-tert-butyl 2,5-dimethylpiperazine-l -carboxylate (68.0 g, 317 mmol) were then added to the mixture and reaction allowed to age in the ice bath and allow to slowly warm to ambient temperature for 4 hours. The reaction mixture was then analyzed using UPLC, which indicated full conversion to Int-H. The reaction was concentrated directly and purified using automated column chromatography (330 g column. AcOEt / MeOH from 100 / 0 to 80 / 20) to afford Int-H (121 g, 67.0 % yield).Step 2: Preparation of Int-I

[0162] Int-H (300 mg, 0.439 mmol), acetonitrile (2.7 mL), bis(timethylsilyl)acetamide (107 mL) were combined. Methanesulfonic acid (71.3 mL) as a solution in acetonitrile (300 mL) was added to the reaction slowly over 10 minutes at ambient temperature. The batch was then allowed to stir at 80 °C for 18 h. The batch was cooled to ambient temperature and seeded with 1 \\ t % of Int-I (3 mg), forming a slurry. The batch was allowed to age at ambient temp for 10 minutes, after which it was charged with 1 M H3PO4 (3.3 mL). The organic and aqueous layers were separated, and the organic layer was distilled at a constant volume until a KF of 300 ppm was attained. Methanesulfonic acid (25.2 mL) was added to the reaction flask and the mixture was allowed to stir at ambient temperature. The reaction mixture was seeded again with 1 wt % of Int-I (3 mg), forming a slurry. The solids were then filtered, and washed with acetonitrile (1.5 mL), then dried under vacuum at ambient temperature to afford Int-I (170 mg, 57% yield). 'H NMR (500 MHz, MeOD) 5 8.45 (1H, d. J= 5.8 Hz), 8.41 (1H, s), 7.32 (1H, td. J= 10.8. 5. 1 Hz),7.09 (Ih, d, J= 5.9 Hz), 6.97 (1H, td, J= 9.0, 3.3 Hz), 4.67 (1H, d, J= 11.4 Hz), 4.60 (1H, d, J = 14.5 Hz), 4.35 (IH, t, J= 10.8 Hz), 4.14 (IH, t, J= 11.2 Hz), 3.98 (IH, d, J= 14.7 Hz), 3.90 (IH, m), 3.65 (IH, dd, J= 13.4, 3.9 Hz), 3.40 (IH, dd, J= 13.3, 3.3 Hz), 3.13 (IH, p, J= 6.7 Hz), 2.72 (5H, s), 1.79 (3H, d, J= 6.9 Hz). 1.57 (3H, d, J= 6.7 Hz), 1.32 (3H, d, J= 6.8 Hz), 1.07 (3H, d, J = 6.8 Hz).13C NMR (125 MHz, MeOD) 5: 165.4, 163.4, 159.6, 155.8, 153.0, 151.2, 149.8, 135.9, 125.5, 120.7, 106.8, 106.4, 70.35 (d, J= 6.2 Hz), 66.5, 50.3, 49.2, 48.1, 42.9, 38.1, 30.1, 21.3, 19.9, 14.5, 13.4.Step 2: Preparation of Compound of Formula (I)

[0163] To a solution of Int-I (5.0 g, 8.1 mmol) in 100 mL of EtOAc was added 25.0 mL of 10 wt. % Na2COs (aq). To the stirring biphasic mixture was added acryloyl chloride (0.84 g, 0.75 mL, 9.3 mmol) over 30 minutes via syringe pump. The resulting biphasic mixture was stirred for 2 hours and then the layers were separated. The organic layer was washed with 20 mL of water and the layers were separated. The resulting organic layer was concentrated in vacuo and diluted with 25 mL of THF. The solution was heated to 45 °C, and 20 mL of n-heptane was added over a period of 4 hours. The resulting slurry w as aged at 45 °C for 1 hour and then cooled to 20 °C over 4 hours. The slurry was held for 20 hours and then filtered and washed with 1 : 1 THF:n-heptane (2 x 10 mL). The solid was dried under a nitrogen sweep to provide the compound of Fomula (I) as a THF solvate (4.47 g, 81% yield).Example 11: Preparation of Form 1 of the Compound of Formula (I)Preparation of Form 1 from an Ethyl Acetate: Heptane Mixture

[0164] To a 4 mL vial, 101.07 mg of compound of Formula (I) (amorphous form) was added followed by the addition of 2 mL of ethyl acetate-heptane (1: 10, v / v). Mixture was stirred and aged at 50 °C for at least 115 hours. Solids were isolated and dried in the vacuum oven for 16.5 h to provide Form 1 of the compound of Formula (I)26037

[0165] Fig. 1 show the X-ray powder diffraction (PXRD) pattern of form 1 of compound of Formula (I). The 29 positions and d-spacings are provided in Table 1 below.Table 1X-Ray Powder Diffraction 20 Positions D-Spacing and Relative Intensities for Crystalline PhaseForm 126037Differential Scanning Calorimetry

[0166] The DSC of Form 1 shows an extrapolated melting onset of 292.9 °C and a peak melting temperature of 295. 1 °C.Example 12: Preparation of Form 2 of the Compound of Formula (I)Preparation of Form 1 from a Xylenes-MTBE Mixture (Procedure A)

[0167] To a 1.2 mL vial, 250 pl acetonitrile solution was added the compound of Formula (I) (with a concentration of 40 mg / mL). The solution is evaporated under reduced pressure, followed by the addition 1 mL of xylenes-MTBE solvent mixture (1: 1, v / v). The vial is then sealed and subjected to acoustic mixing (with the use of a Resodyn LabRam) at 10% Intensity for 5 days. A solid was isolated to provide Form 2.26037Preparation of Form 2 from Cvclopentyl Methyl Ether (Procedure B)

[0168] To a 1.2 mL vial, 250 pl acetonitrile solution was added the compound of Formula (I) (with a concentration of 40 mg / mL). The solution is evaporated under reduced pressure, followed by the addition 1 mL of cyclopentyl methyl ether. The vial is then sealed and subjected to acoustic mixing (with the use of a Resodyn LabRam) at 10% Intensity for 5 days. A solid was isolated to obtain Form 2.

[0169] Fig. 2 show the X-ray pow der diffraction (PXRD) pattern of form 2 of compound of Formula (I). The 20 positions and d-spacings are provided in Table 2 below-.Table 2X-Ray Powder Diffraction 20 Positions D-Spacing and Relative Intensities for Crystalline Phase Form 226037Differential Scanning Calorimetry

[0170] The DSC of Form 2 shows an extrapolated melting onset of 298.8 °C and a peak melting temperature of 300.4 °C.

[0171] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to26037 those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0172] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety' for all purposes. Other embodiments are within the follow ing claims.SEQUENCE LISTING

[0173] SEQ ID NO: !:MTVSVLGLGPMGQALAGALVTAKYPTTVWNRTPGKADALLARGARWADTPAAACAA SQLILVNVVDQAAALAVLDAAGDAIAGRVVVGLSSDIPAGAHRTEELVIARGGRYLDGA IMSPTWDIGTPSASILFAGPQDLFDAHRSVFDALATVNWVGPDVGRAAAYDMALLDIM WTSFSGFVHALAMAKAHNIAPRELLPHASNIAAILPPIFVEIAERVEADNHGNAQATVSS VAASVRHLIAASHSAGFDAGALEAFKGYVDNAVAAGHGADMISRIRPAAFSGHTPGSHHHHHH

[0174] SEQ ID NO: 2:MTVSVLGLGPMGQALAGALVTAKYPTTVWNRTPGKADALLARGARWADTPAAACAA SQLILVNVVDQAAALAVLDAAGDAIAGRVVVGLSSDIPAGAHRTEELVIARGGRYLDGA IMSPTWDIGTPSASILFAGPQDLFDAHRSVFDALATVNWVGPDVGRAAAYDMALLDIM WTSFSGFVHALAMAKAHNIAPRELLPHASNIAAILPPIFVEIAERVEADNHGNAQASVSS VAASVRHLIAASHSAGFDAGALEAFKGYVDNAVAAGHGADMISRIRPAAFSGHTPGSH HHHHH

Claims

WHAT IS CLAIMED IS:

1. A process for preparing a compound of Formula (I)(a) providing an atropisomeric mixture of Int-A(b) combining the atropisomeric mixture of Int-A with an optically active acid in a solvent to provide Int-A- 1: optically active acid salt;2. The process of claim 1, wherein the optically active acid in step (b) is selected from the group consisting of (+) dibenzoyl tartaric acid, (-) ditoluoyl tartaric acid, and (-) diacetyl tartaric acid.

3. The process of claim 1, wherein the atropisomeric mixture of Int-A, optically active acid, and solvent in step (b) are combined with an anti-solvent.

4. The process of claim 1, wherein step (b) is performed at a temperature of 40 °C to 85 °C.

5. The process of claim 1, wherein step (c) comprises:(cl) treating the Int-A-1 : optically active salt with a carbonic acid derivative selected from the group consisting of a di C1-C3 alkylcarbonate, phosgene, diphosgene, triphosgene, carbonyldiimidazole and disuccinimidyl carbonate and a base in an organic solvent to formconverting Int-B to the compound of Formula (I).

6. The process of claim 5, wherein the carbonic acid derivative is the di C1-C3 alkydcarbonate.

7. The process of claim 5, wherein the base is selected from the group consisting of alkali or alkaline earth metal C Cg alkoxides, bis(trimethylsilyl)amide and diisopropyl amide salt.

8. The process of claim 5, wherein step (c) further comprises:(c2) treating Int-B with an electrophilic chlorinating agent and a base in an organic solvent to form a reaction mixture Int-B Rxl; andthe reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx2; andconverting Int-C to form the compound of Formula (I).

9. The process of claim 8, wherein Int-W3 is added in an acid addition salt form.

10. The process of claim 9, wherein the acid addition salt form of Int-W3 is prepared by: benzylatingtreating Int-W2a with HC1 to provide the acid addition salt form of Int-W3.

11. The process of claim 9, wherein the acid addition salt form of Int-W3 is prepared by:(a) contacting a l-benzyl-2,5-dimethylpyrazin-l-ium salt with a PyRED enzyme and NADPH to provide Int-W3; and(b) treating Int-W3 with an acid to provide the acid addition salt form of Int-W3.

12. The process of claim 11, wherein the PyRed enzyme in (a) comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO:2.

13. The process of claim 1 1, wherein the contacting in (a) further comprises a glucose and a glucose dehydrogenase enzyme.

14. The process of claim 9, wherein step (c) further comprises:(c3) treating Int-C with acryloyl chloride, an iodide salt and a base to provide the compound of Formula (I).

15. The process of claim 5, wherein step (c) comprises:(c4) treating Int-B with an electrophilic chlorinating agent and a base in an organic solvent to form a reaction mixture Int-B Rxl; and addingthe reaction mixture Int-B Rxl to form a reaction mixture Int-B Rx3: and separating the compound of Formula (I) from the reaction mixture Int-B Rx3.

16. The process of claim 15, wherein the electrophilic chlorinating agent is POCI3.

17. The process of claim 1, wherein the atropisomeric mixture of Int-A is prepared by cyclizing18. The process of claim 17, wherein the cyclization of Int-D comprises:(al) treating Int-D with a dehydrating agent and a base in an organic solvent to form a reaction mixture Int-D Rxl;(a2) adding an alkali or alkaline earth metal alkoxide to the reaction mixture Int-D Rxl to form a reaction mixture Int-D Rx2;(a3) adding an alkali or alkaline earth metal hydroxide to the reaction mixture Int-Dx2 to form a reaction mixture Int-D Rx3; and(a4) isolating the atropisomeric mixture of Int-A from the reaction mixture Int-D Rx3.2603719. The process of claim 18, wherein Int-D is prepared by treatingbase to form Int-D.

20. The process of claim 1, wherein the atropisomeric mixture of Int-A is prepared by(a5) combiningMitsunobu conditions to form a reaction mixture Int-G Rxl; and(a7) combining Int-G with an alkali or alkaline earth metal alkoxide in an organic solvent to form a reaction mixture Int-G Rx2;(a8) adding an alkali or alkaline earth metal hydroxide to the reaction mixture Int-G Rx2 to form a reaction mixture Int-G Rx3; and(a9) isolating the atropisomeric mixture of Int-A from the reaction mixture Int-G Rx3.

21. A compoundsalt thereof.

22. A compoundsalt thereof.

23. A compoundsalt thereof.

24. A crystalline polymorphic form of a compound of Formula (I)which is designated Form 1, wherein the crystalline polymorphic form designated Form 1 has d- spacings determined by powder diffraction, Cu Ka, of about 11.50, 9.75, 9.10, 8.14, 6.44, 5.78, 5.38, and 5.06 angstroms.

25. A crystalline polymorphic form of a compound of Formula (I)which is designated Form 2, wherein the crystalline polymorphic form designated Form I has d- spacings determined by powder diffraction, Cu Ka, of about 9.27, 7.29, 6.

52. 5.

25.

5.

12. and 4.31 angstroms.

Images