Synthesis and purification of polyketides

Abstract

Provided herein are methods for preparing and synthesizing polyketides, including Compound A, at commercially relevant scale and pharmaceutically administrable purity. Also provided herein are preparation of synthetic intermediates useful in preparing such polyketides.

Description

1959952.00068SYNTHESIS AND PURIFICATION OF POLYKETIDES RELATED APPLICATIONS

[0001] This application claims priority of U. S. Provisional Patent Application No.64 / 022,440, filed March 30, 2026, and U. S. Provisional Patent Application No. 63 / 788,455, filed April 14, 2025, the entire content of each of which is incorporated herein by reference.SUMMARY

[0002] The double-stranded RNA-specific adenosine deaminase family of enzymes is encoded by the ADAR family genes. ADAR (adenosine deaminase acting on RNA) proteins enzymatically perform base editing, e.g., by converting adenosine to inosine, that subsequently disrupts otherwise orthogonal Watson-Crick nucleobase pairing.

[0003] Unlike therapeutic use of CRISPR-Cas9, which acts at the gene editing level and is therefore a permanent or inheritable intervention, ADAR enzymes act on double-stranded RNA, which is a transient molecule. Therefore, ADAR modulation has potential as a transient and tunable, non-heritable, therapeutic intervention. ADAR enzymes have previously been referred to as double-stranded RNA adenosine deaminase (dsRAD; " Toward the therapeutic editing of mutated RNA sequences," PNAS, 1995, 92, 8298-8302). Rather than using the CRISPR-Cas9 gene editing approach, which risks permanent genetic off-target mutations, RNA editing allows changes to the coding for protein production to be modulated without permanent gene editing. Accordingly, there is a continuing and long-standing need for therapeutic intervention at the ribonucleic acid-to-protein stage of molecular biology's central dogma, including a need for modulators of the spliceosome and ADAR enzymes. The polyketides include 12-membered ring macrolides, including Compound A, Compound B, and pladienolides (including FD-895 and the pladienolides A, B, C, D, E, F, and G). These compounds modulate the spliceosome and have shown the ability to down-regulate levels of ADAR enzymes.

[0004] Thus, provided herein are commercially scalable synthetic methods and intermediates useful in the preparation of polyketides. The synthetic methods, which include purification sequences, are useful in preparing polyketides at a purity suitable for pharmaceutical administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 depicts a chemical reaction and reagents useful in preparing polyketides herein, including preparing Compound A from INT8 and INT10.1 of 601609969430.31959952.00068

[0006] FIG. 2 depicts a chemical reaction and reagents useful in preparing macrolactonized intermediates, such as preparing INT8 from INT7, that are useful in preparing polyketides herein, including Compound A.

[0007] FIG. 3 depicts a chemical reaction and reagents useful in preparing stannylated intermediates, such as preparing INT10 from INT9, that are useful in preparing polyketides herein, including Compound A.

[0008] FIG. 4 depicts chemical reactions and reagents useful in preparing alkyne intermediates, such as INT9, including preparing C5 from B4, C6 from C5, C7 from C6, C8 from C7, C26 from C8, C27 from C26, and C28 from C27, that are useful in preparing polyketides herein, including Compound A.

[0009] FIG. 5 depicts chemical reactions and reagents useful in preparing alkyne intermediates, such as INT9, including preparing B4 from (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide, B8 from C28, B9 from B8, B10 from B9, and INT9 from B10, that are useful in preparing polyketides herein, including Compound A.

[0010] FIG. 6 depicts a series of purification steps useful for preparing purified polyketides herein, including a purified solid (lyophilized or powdered) form of Compound A suitable for clinical use or administration. FP refers to Finished / Final Product, e.g., a purified polyketide, such as Compound A.

[0011] FIG. 7 depicts another series of purification steps useful for preparing purified polyketides herein, including a purified solid (lyophilized or powdered) form of Compound A suitable for clinical use or administration. FP refers to Finished / Final Product, e.g., a purified polyketide, such as Compound A.

[0012] FIG. 8 depicts an FT-IR spectrum for Compound A prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0013] FIG. 9 depicts an expanded view of an FT-IR spectrum for Compound A prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0014] FIG. 10 depicts a proton (1H) nuclear magnetic resonance (NMR) and carbon (13C) NMR spectrum for Compound A prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0015] FIG. 11 depicts an expanded view from 6.40 to 5.20 ppm of anNMR spectrum and 180 to 120 ppm of a13C NMR spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.2 of 601609969430.31959952.00068

[0016] FIG. 12 depicts an expanded view from 4.00 to 1.80 ppm of an1H NMR spectrum and 90 to 50 ppm of a13C NMR spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0017] FIG. 13 depicts an expanded view from 1.75 to 0.95 ppm of an1H NMR spectrum and 44 to 28 ppm of a13C NMR spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0018] FIG. 14 depicts an expanded view from 1.00 to 0.50 ppm of anNMR spectrum and 28 to 6 ppm of a13C NMR spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0019] FIG. 15 depicts an1H,13C HSQC spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0020] FIG. 16 depicts an1H,1H gCOSY spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0021] FIG. 17 depicts an1H,13C HMBC spectrum for Compound A in CeDe prepared as in Example 1 (e.g., Examples 1-20 and 22) and purified as described in Example 21.

[0022] FIG. 18 depicts a series of purification steps useful for preparing purified INT10.

[0023] FIG. 19 depicts chemical reactions and reagents useful in preparing macrolactonized intermediates, such as INT7, including preparing INT4 from SM, INT5 from INT4, INT6 from INT5, and INT7 from INT6, that are useful in preparing polyketides herein, including Compound A.

[0024] FIG. 20 depicts a sequence of chemical reactions useful in preparing Compound A suitable for clinical administration.

[0025] FIG. 21 depicts an isolated solid form of INT6.

[0026] FIG. 22 depicts an isolated solid form of INT7.DETAILED DESCRIPTIONDefinitions

[0027] Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0028] The articles "a" and "an" refer to one, or to more than one, of the grammatical object of the article.3 of 601609969430.31959952.00068

[0029] Numerical values relating to measurements are subject to measurement errors that place limits on their accuracy. For this reason, all numerical values provided herein, unless otherwise indicated, are to be understood as being modified by the term "about." Accordingly, the last decimal place of a numerical value provided herein indicates its degree of accuracy. Where no other error margins are given, the maximum margin is ascertained by applying the rounding-off convention to the last decimal place or last significant digit when a decimal is not present in the given numerical value.

[0030] The term "amelioration" means a lessening of severity of at least one indicator of a condition or disease, such as a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

[0031] The term "composition" refers to a mixture of at least two or more components.

[0032] The terms "effective amount" and "therapeutically effective amount" refer to an amount of therapeutic compound, such as a compound prepared as described herein, including Compound A, Compound B, FD-895, Pladienolide B, or the like, administered to a subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect. In general, the therapeutically effective amount can be estimated initially either in cell culture assays or in mammalian animal models, for example, in nonhuman primates, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in non-human subjects and human subjects.

[0033] The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid filler, solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material, involved in carrying or transporting at least one compound described herein within or to the patient such that the compound may perform its intended function. A given carrier must be "acceptable" in the sense of being compatible with the other ingredients of a particular formulation, including the compounds described herein, and not injurious to the patient. Other ingredients that may be included in the pharmaceutical compositions described herein are known in the art and described, for example, in " Remington's Pharmaceutical Sciences" (Genaro (Ed.), Mack Publishing Co., 1985), the entire content of which is incorporated herein by reference.4 of 601609969430.31959952.00068

[0034] The term "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0035] The terms "treatment" or "treating" refer to the application of one or more specific procedures used for the amelioration of a disease. A "prophylactic" treatment, refers to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.

[0036] Value ranges herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within a range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the described subject matter and does not pose a limitation on the scope of the subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to practicing the described subject matter.

[0037] Groupings of alternative elements or embodiments of this disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. Furthermore, a recited member of a group may be included in, or excluded from, another recited group for reasons of convenience or patentability. When any such inclusion or exclusion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0038] References have been made to patents and printed publications throughout this specification, each of which are individually incorporated herein by reference in their entirety.

[0039] It is to be understood that the embodiments of this disclosure are illustrative. Accordingly, the present disclosure is not limited to that precisely as shown and described.Synthetic Intermediates and Methods

[0040] Several synthetic schemes and intermediate compounds provided herein taken together are useful in improving the commercial synthetic preparation of polyketides, including Compound A as a powder. The synthetic and purification methods herein may be5 of 601609969430.31959952.00068used under GMP (Good Manufacturing Practices) conditions, e.g., to provide Compound A at a purity suitable for clinical human administration.

[0041] Synthetic preparations of Compound A have been described, including in WO 2021 / 026273 Al, US 10,675,267 B2, Chan et al. (Cell Reports Physical Science, 2020, 1, 12, 100277), and Villa et al. (Organic Letters, 2012, 14, 21, 5396-5399). Problematically for commercial manufacturing, these synthetic routes yielded the polyketides as a wax or oil. Furthermore, according to Figure 2 of Chan, a "17 g batch [of 17S-FD-895] was prepared by five full-time employees (FTEs) over [a] 3 month period; only the effort of 3 FTEs was required the entire period." Furthermore, on comparison of at least13C NMR spectra it is evident that the herein described synthetic and purification sequences provide purer polyketide, e.g., Compound A, and at a commercially relevant scale of synthesis, e.g., useful for preparation of a commercially and clinically useful pharmaceutical agent. Additionally, preparation of Compound A in a lyophilized or powdered form as described herein results in a solid form readily manipulated for preparation of non-liquid formulations.Compound A

[0042] Adenosine deaminase acting on RNA (ADAR)l is the principal enzyme for adenosine-to-inosine editing, an RNA modification avoiding cytosolic nucleic acid sensor's activation triggered by endogenous dsRNAs. Two ADAR1 isoforms exist in mammals, a longer IFN-inducible and mainly cytoplasm-localized pl50 isoform and a shorter constitutively expressed and primarily nucleus-localized pl 10 isoform. Compound A is a demonstrated potent inhibitor of ADAR1 pl50 isoform. Thus, the importance of preparing commercially relevant volumes of Compound A, at least for therapeutic applications, is apparent. The methods herein are useful at least for preparing commercially relevant amounts of Compound A, e.g., kg amounts, and in a solid or powder form easy to manipulate during drug product manufacture processes.

[0043] In some embodiments, the schemes and intermediates provided herein may be adjusted by one of skill to prepare polyketides other than Compound A, including Compound B or stereoisomers thereof, pladienolides or stereoisomers thereof, such as pladienolide B,6 of 601609969430.31959952.00068and compounds of the same class of polyketides that are capable of binding to the SF3b complex of a spliceosome, inhibiting mRNA splicing activity, or down-regulating ADAR levels.

[0044] Pladienolide B has been synthesized previously, at least, as described, for example, by Rhoades et al. (Journal of the American Chemical Society, 2021, 143, 13, 4915-4920). Problematically for commercial scale manufacture, Pladienolide A and Pladienolide B described by Rhoades were prepared on a milligram scale and isolated as oils.

[0045] Compounds described herein can also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to2H,3H,nC,13C,14C,123I,125I,15O,17O, or18O. In some embodiments, isotopically labeled compounds are useful in drug or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such asnC or15O, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds are prepared by7 of 601609969430.31959952.00068any suitable method or by processes using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.

[0046] In some embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0047] The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4th Ed., (Wiley 1992); Carey and Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green and Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified using appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

[0048] Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources or are prepared using procedures described herein.

[0049] In some embodiments, the compounds described herein may be prepared by a method of synthesis that comprises any of the synthetic steps described in the Examples or Figures herein pertinent to the compound being prepared.

[0050] In some embodiments, provided herein are compositions, comprising a compound provided herein.

[0051] In some embodiments, the compounds or compositions provided herein are for use in the preparation of a synthesized compound.

[0052] In some embodiments, provided herein are methods of preparing a synthesized compound, comprising contacting a compound provided herein with one or more reagents to form the synthesized compound.

[0053] In some embodiments, provided herein are methods of preparing a compound provided herein, comprising contacting a precursor compound with one or more reagents to form the compound provided herein. In some embodiments, the compounds so prepared may 8 of 601609969430.31959952.00068be in a composition comprising the prepared or synthesized compound, e.g., Compound A, and a carrier. In some embodiments, the composition is a pharmaceutical composition comprising the prepared or synthesized compound, e.g., Compound A, and a pharmaceutically acceptable carrier.

[0054] The compounds prepared herein are purer than previously described at the molar or mass scale described herein. Accordingly, compositions comprising the compounds prepared as described herein, e.g., Compound A, can be prepared at a level of purity heretofore unattained in multigram (e.g., hundreds of grams or kilograms) scale, and in a solid form, e.g., powder, not achieved previously. Thus, in some embodiments, the compositions herein comprise Compound A in a purity of at least 99.0 % (e.g., as determined by HPLC in %w / w or % area). In other embodiments, the compositions herein comprise Compound A in a purity of at least 99.4 % (e.g., as determined by HPLC). In still other embodiments, the compositions herein comprise Compound A in a purity of at least 99.8 % (e.g., as determined by HPLC).

[0055] Chemical structures of certain compounds herein are depicted in Table 1.9 of 601609969430.31959952.0006810 of 601609969430.31959952.00068

[0056] Thus, in some embodiments, provided herein are compositions, comprising:a polyketide as described herein, e.g., Compound A; andone or more of:a Pd, and wherein the composition comprises less than 1 ppm of the Pd,an Sn, and wherein the composition comprises less than 20 ppm of the Sn, hexafluoro-2-propanol, and wherein the composition comprises less than 500 ppm of hexafluoro-2-propanol, orINT9, and wherein the composition comprises less than 1,000 ppm of INT9.

[0057] In some embodiments of the compositions herein, the polyketide, e.g., Compound A, is in the form of a powder. In some embodiments of the compositions herein, the polyketide, e.g., Compound, A is in the form of a solid. In some embodiments of the compositions herein, the composition is in the form of a particle. In some embodiments compositions are provided11 of 601609969430.31959952.00068that include a plurality of the particles. In some embodiments, the compositions herein are prepared by the methods provided herein.

[0058] In some embodiments of the compositions herein, the compositions further comprise one or more of:more than 99% w / w (e.g., at least 99.4% w / w or at least 99.8% by area, e.g., as determined by HPLC) of a polyketide herein, e.g., Compound A;Compound A in the form of an amorphous solid form as determined by x-ray powder diffraction;a polyketide herein, e.g., Compound A, in greater than 99% (e.g., at least about 99.7% by area) chiral purity as determined by high-performance liquid chromatography; or less than 2% w / w of water.

[0059] In some embodiments of the compositions herein, the compositions comprise at least 400 g (e.g., at least 1.25 kg) of Compound A.

[0060] In some embodiments of the compositions herein, the compositions are a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0061] In some embodiments, provided herein are solid forms of certain compounds herein, including a compound selected from Compound A, INT5, INT6, INT7, or INT8. In some embodiments, the solid form is a powder. In some embodiments, the solid form is a crystalline form. In some embodiments, the solid form is obtained by precipitation from a liquid solution. In some embodiments, the solid form is obtained by crystallization from a liquid solution.

[0062] In some embodiments, a solid form of Compound A is precipitated from a liquid solution comprising a solvent, the solvent comprising isopropanol (e.g., about 40% carbon dioxide in isopropanol).

[0063] In some embodiments, a solid form of INT5 is precipitated from a liquid solution comprising a solvent, the solvent comprising ethyl acetate and heptane (e.g., about 95-98 % v / v ethyl acetate and about 2-5 % v / v heptane). In some embodiments, the INT5 in the solid form of INT5, or in a composition comprising the solid form of INT5, has a chiral purity of at least 96%. In some embodiments, a composition comprising the solid form of INT5 comprises at least 95% w / w INT5.

[0064] In some embodiments, a solid form of INT6 is precipitated from a liquid solution comprising a solvent, the solvent comprising ethyl acetate and heptane (e.g., about 80-85 % v / v ethyl acetate and about 15-20 % v / v heptane). In some embodiments, the INT6 in the 12 of 601609969430.31959952.00068solid form of INT6, or in a composition comprising the solid form of INT6, has a chiral purity of at least 96%. In some embodiments, a composition comprising the solid form of INT6 comprises at least 98 or at least 99% w / w INT6.

[0065] Challengingly for manufacturing of commercially scalable polyketide API, e.g., Compound A, INT7 and INT8 were previously isolated as an oil (see Rhoades et al.), making transfer or manipulation, and apportionment, difficult. It has been discovered that INT7 and INT8 can be isolated as a solid form.

[0066] Thus, in some embodiments, a solid form of INT7 is precipitated from a liquid solution comprising a solvent, the solvent comprising methyl-tert-butylether and dichloromethane (e.g., about 50-70 % v / v methyl-tert-butylether and about 30-50 % v / v dichloromethane). In some embodiments, the INT7 in the solid form of INT7, or in a composition comprising the solid form of INT7, has a chiral purity of at least 96%. In some embodiments, a composition comprising the solid form of INT7 comprises at least 96 or at least 97 % w / w INT7.

[0067] In some embodiments, a solid form of INT8 is precipitated from a liquid solution comprising a solvent, the solvent comprising methyl-tert-butylether and heptane (e.g., about 50-70 % v / v methyl-tert-butylether and about 30-50 % v / v heptane). In some embodiments, the INT8 in the solid form of INT8, or in a composition comprising the solid form of INT8, has a chiral purity of at least 96%. In some embodiments, a composition comprising the solid form of INT8 comprises at least 99% w / w INT8.

[0068] In some embodiments, compositions comprising a synthetic intermediate for a polyketide herein (e.g., Compound A) are provided. In some embodiments, the compositions comprise about 95%, or more, w / w of the synthetic intermediate. Thus, in some embodiments, provided herein are compositions comprising a solid form of a compound selected from INT5, INT6, INT7, or INT8, wherein the composition comprises at least 95% w / w of the solid form of the compound. In some embodiments, the composition comprises at least or about 99, 98, 97, 96, or 95 % w / w of the solid form of the compound. The compositions are useful in preparing a polyketide herein, e.g., Compound A, or compositions comprising the polyketide (e.g., Compound A).

[0069] In some embodiments, the compositions herein comprise about 100 g, about 200 g, about 300 g, about 400 g, about 500 g, about 1 kg, or more than 1 kg of the compound or synthetic intermediate (e.g., Compound A, INT5, INT6, INT7, or INT8).13 of 601609969430.31959952.00068

[0070] In some embodiments, methods of preparing the polyketides, e.g., Compound A, herein are provided.

[0071] Thus, in some embodiments provided herein, the polyketides herein, e.g., Compound A, and thereby the compositions herein, are prepared by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step (e.g., SiO2(silica) stationary phase and hexane:acetone 1:0 to 2:1 mobile phase);2) a potassium carbonate-silica gel chromatography purification step (e.g., 10% w / w K2CO3 and 90% w / w silica a stationary phase and hexane:ethyl acetate 1:1 to 0:1 mobile phase);3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step (e.g., Phenomenex Luna C18 250mm*100mm*10um; mobile phase: [water (FA)-ACN]; gradient:30%-60% B over 20 min);5) a first supercritical fluid chromatography (SFC) purification step (e.g., DAICEL CHIRALPAK IC (250mm*30mm,10pm); mobile phase: [CO2-i-PrOH]; B%:40%, isocratic elution mode; IPA (0.05%DEA) in CO2 from 5% to 40%, 100Bar back pressure);6) an optional second supercritical fluid chromatography (SFC) purification step (e.g., repeating the first SFC purification step);7) a concentration and lyophilization purification step; and8) an optional sieving step to provide a solid form (e.g., a powder), or composition, of polyketide (e.g., Compound A).

[0072] In some embodiments, the compositions herein comprise about 99.4 to about 99.8 % w / w Compound A. In some embodiments, the compositions herein comprise about 99.4 to about 99.8 % w / w Compound A, not more than 1 ppm of Pd (e.g., a positive amount of Pd the amount of Pd being not more than 1 ppm), not more than 20 ppm of Sn (e.g., a positive amount of Sn the amount of Sn being not more than 20 ppm), not more than 500 ppm of hexafluoro-2-propanol (e.g., a positive amount of hexafluoro-2-propanol the amount of hexafluoro-2-propanol being not more than 500 ppm), and not more than 1,000 ppm of INT9 (e.g., a positive amount of INT9 the amount of INT9 being not more than 1,000 ppm). In some embodiments, the compositions are prepared by a process including a method provided herein.14 of 601609969430.31959952.00068

[0073] In some embodiments provided herein is a particle comprising about 99.4 to about 99.8 % w / w Compound A. In some embodiments provided herein is a particle comprising about 99.4 to about 99.8 % w / w Compound A, not more than 1 ppm of Pd (e.g., a positive amount of Pd the amount of Pd being not more than 1 ppm), not more than 20 ppm of Sn (e.g., a positive amount of Sn the amount of Sn being not more than 20 ppm), not more than 500 ppm of hexafluoro-2-propanol (e.g., a positive amount of hexafluoro-2-propanol the amount of hexafluoro-2-propanol being not more than 500 ppm), and not more than 1,000 ppm of INT9 (e.g., a positive amount of INT9 the amount of INT9 being not more than 1,000 ppm). In some embodiments, the particle is prepared by a process including a method provided herein.

[0074] In some embodiments, provided herein is a composition comprising a plurality of particles, each particle comprising about 99.4 to about 99.8 % w / w Compound A. In some embodiments, provided herein is a composition comprising a plurality of particles, each particle comprising about 99.4 to about 99.8 % w / w Compound A, not more than 1 ppm of Pd (e.g., a positive amount of Pd the amount of Pd being not more than 1 ppm), not more than 20 ppm of Sn (e.g., a positive amount of Sn the amount of Sn being not more than 20 ppm), not more than 500 ppm of hexafluoro-2-propanol (e.g., a positive amount of hexafluoro-2-propanol the amount of hexafluoro-2-propanol being not more than 500 ppm), and not more than 1,000 ppm of INT9 (e.g., a positive amount of INT9 the amount of INT9 being not more than 1,000 ppm). In some embodiments, the compositions are prepared by a process including a method provided herein.

[0075] In some embodiments, provided herein are one or more particles, each particle comprising the composition of claim 1, the particle comprising at least 99.4% w / w Compound A, not more than 1 ppm of Pd, not more than 20 ppm of Sn, not more than 500 ppm of hexafluoro-2-propanol, and not more than 1,000 ppm of INT9.

[0076] In some embodiments, the compositions or particles herein comprise at least 99.8% w / w Compound A, not more than 1 ppm of Pd, not more than 20 ppm of Sn, not more than 500 ppm of hexafluoro-2-propanol, and not more than 1,000 ppm of INT9.

[0077] In some embodiments, the compositions or particles herein comprise at least 99.4% w / w Compound A, a positive amount of Pd the positive amount of Pd being not more than 1 ppm, a positive amount of Sn the positive amount of Sn being not more than 20 ppm, a positive amount of hexafluoro-2-propanol the positive amount of hexafluoro-2-propanol being not more than 500 ppm, and a positive amount of INT9 the positive amount of INT9 being not more than 1,000 ppm.15 of 601609969430.31959952.00068

[0078] In some embodiments, the compositions or particles herein comprise at least 99.8% w / w Compound A, a positive amount of Pd the positive amount of Pd being not more than 1 ppm, a positive amount of Sn the positive amount of Sn being not more than 20 ppm, a positive amount of hexafluoro-2-propanol the positive amount of hexafluoro-2-propanol being not more than 500 ppm, and a positive amount of INT9 the positive amount of INT9 being not more than 1,000 ppm.

[0079] In some embodiments, provided herein is a composition comprising a plurality of particles, each particle comprising about 99.4 to about 99.8 % w / w Compound A. In some embodiments, provided herein is a composition comprising a plurality of particles, each particle comprising about 99.4 to about 99.8 % w / w Compound A, not more than 1 ppm of Pd (e.g., a positive amount of Pd the amount of Pd being not more than 1 ppm), not more than 20 ppm of Sn (e.g., a positive amount of Sn the amount of Sn being not more than 20 ppm), not more than 500 ppm of hexafluoro-2-propanol (e.g., a positive amount of hexafluoro-2-propanol the amount of hexafluoro-2-propanol being not more than 500 ppm), and not more than 1,000 ppm of INT9 (e.g., a positive amount of INT9 the amount of INT9 being not more than 1,000 ppm). In some embodiments, the compositions are prepared by a process including a method provided herein.

[0080] In some embodiments of the methods herein, an intermediate compound or a polyketide (e.g., Compound A), or a composition comprising the intermediate or the polyketide (e.g., Compound A), may be prepared as described herein by a process including one or more of the following synthetic steps:a) reacting INT8 with INT10, e.g., in the presence of a catalyst (e.g., XPHOS Pd G4) and a solvent (e.g., tetrahydrofuran), to form Compound A;b) reacting INT7 with trimethylorthoacetate (aka 1,1,1-trimethoxyethane), e.g., in the presence of an acid (e.g., camphorsulphonic acid) and a solvent (e.g., dichloromethane), to form INT8;c) reacting INT9 with tributyltinhydride, e.g., in the presence of a solvent (e.g., dichloromethane), to form INT10;d) reacting B10 with B11, e.g., in the presence of a catalyst (e.g., BF3-Et2O) and a solvent (e.g., dichloromethane), to form INT9;e) reacting B9 with 2-iodoxybenzoic acid, e.g., in the presence of a solvent (e.g., ethylacetate) and elevated temperature (e.g., about 80 °C), to form B10;16 of 601609969430.31959952.00068f) reacting B8 with cumene hydroperoxide, e.g., in the presence of a catalyst (e.g., titanium (IV) isopropoxide and (-)-diethyltartrate) in a solvent (e.g., dichloromethane), to form B9;g) reacting B8 with an epoxidase, e.g., a pldD, an fddD, an epoK, a monCi, a nanO epoxidase, or EC 1.14.99.20 (phylloquinone monooxygenase), in the presence of an FAD cofactor (e.g., flavin adenine dinucleotide), an NAD cofactor, or an NADP+cofactor in a solvent, to form B9;h) reacting C28 with a carbonate, e.g., potassium carbonate in the presence of a solvent (e.g., methanol), to form B8;i) reacting C27 with a methylating agent (e.g., Me3O+BF4“), e.g., in the presence of a base (e.g., l,8-bis(dimethylamino)naphthalene) and a solvent (e.g., dichloromethane), to form C28;j) reacting C26 with an acid (e.g., hydrochloric acid), e.g., in the presence of a solvent (e.g., acetonitrile), to form C27;k) reacting C8 with a benzoylating agent (e.g., benzoyl chloride), e.g., in the presence of a base (e.g., triethylamine) and a solvent (e.g., dichloromethane), to form C26; l) reacting C7 with a hydride (e.g., diisobutylaluminum hydride), e.g., in the presence of a solvent and reduced temperature (e.g., about -78 °C), to form C8;m) reacting C6 with triethylphosphonoacetate, e.g., in the presence of a base (e.g., triethylamine), a lithium (e.g., lithium chloride), and a solvent (e.g., acetonitrile), to form C7;n) reacting C5 with a hydride (e.g., diisobutylaluminum hydride), e.g., in the presence of a solvent and reduced temperature (e.g., about -78 °C), to form C6;o) reacting B4 with triphenylsilyl chloride, e.g., in the presence of a first base (e.g., 4- dimethylaminopyridine), a second base (e.g., pyridine), and a solvent (e.g., dichloromethane), to form C5;p) reacting (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide with a ketoreductase enzyme (e.g., KRED-A6, e.g., KRED-A6-P2D5), e.g., in the presence of a cofactor (e.g., a nicotinamide adenine dinucleotide phosphate, e.g., as a sodium salt), a solvent (e.g., isopropanol or water, or both), and buffer (e.g., potassium phosphate buffered saline), to form B4;17 of 601609969430.31959952.00068q) reacting INT6 with an iodination agent (e.g., N-iodosuccinimide), e.g., in the presence of a first solvent (e.g., l,l,l,3,3,3-hexafluoro-2-propanol) and a second solvent (e.g., acetonitrile), to form INT7;r) reacting INT5 with an osmium catalyst (e.g., potassium osmate (VI)) in the presence of a chiral ligand (e.g., a quinine ligand, e.g., hydroquinidine 1,4-phthalazinediyl diether (DHQD)2PHAL)), e.g., in the presence of a carbonate (e.g., potassium carbonate), potassium hexa cyanoferrate (III), methanesulfonamide, and a solvent (e.g., water and tert-butanol), to form INT6;s) reacting INT5 with a Rieske non-heme iron oxygenase (RO) (e.g., a toluene dioxygenase (TDO), a naphthalene dioxygenase (NDO), or a phthalate dioxygenase (PDO)) to form INT6;t) reacting INT4 with a ketoreductase enzyme (e.g., KRED-A6, e.g., KRED-A6-P2D5), e.g., in the presence of a cofactor (e.g., a nicotinamide adenine dinucleotide phosphate, e.g., as a sodium salt), a solvent (e.g., isopropanol or water, or both), and buffer (e.g., potassium phosphate buffered saline), to form INT5; andu) reacting SM with heat, e.g., heat in flow in a solvent (e.g., acetonitrile), to form INT4.

[0081] In some embodiments provided herein are compositions comprising a reactant compound (e.g., a starting material compound) and a reaction product compound, wherein the composition comprises a ratio of the reactant to the sum of the reactant and the reaction product (i.e. A / (A+B)) of less than 50%, e.g., less than 30%, less than 20%, less than 10%, less than 5%, or less than 2%. In some embodiments, the ratio is determined on a mole fraction basis or by HPLC (e.g., integrating the corresponding High-Performance Liquid Chromatography peak area(s) and determining the ratio therefrom). In some embodiments, the reactant and the reaction product compounds are those described, independently, in each of the examples herein (e.g., Example 25 where the reactant is B8 and the reaction product is B9, Example 26 where the reactant is (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide and the reaction product is B4, Example 1 where the reactant is INT8 or INT 10 and the reaction product is Compound A, Example 2 where the reactant is INT7 and the reaction product is INT8, Example 3 where the reactant is INT6 and the reaction product is INT7, Example 4 where the reactant is INT5 and the reaction product is INT6, Example 5 where the reactant is INT4 and the reaction product is INT5). In some embodiments, e.g., where the reactant is INT4, (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide, B8, or INT5, the compositions further comprise an enzyme useful for enzymatic conversion of the reactant to the corresponding reaction product (e.g., INT5, B4, B9, or INT6, respectively), such as a KRED,18 of 601609969430.31959952.00068an RO, a TDO, an NDO, or a PDO herein. In some embodiments, the compositions comprise a solvent system (e.g., the solvent system comprising water or isopropanol, or water and isopropanol).

[0082] Thus, in some embodiments provided herein are compositions comprising a reactant compound and a reaction product compound, the composition comprising a ratio of the amount of the reactant to the sum of the amount of the reactant and the amount of the reaction product, the ratio being less than 2%, wherein:a) the reactant is B8 and the reaction product is B9, and wherein the composition optionally includes an epoxidase enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system;b) the reactant is INT4 and the reaction product is INT5, and wherein the composition optionally includes a KRED enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system;c) the reactant is (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide and the reaction product is B4, and wherein the composition optionally includes a KRED enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system; ord) the reactant is INT5 and the reaction product is INT6, and wherein the composition optionally includes an RO enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system.

[0083] In some embodiments, provided herein are methods of preparing the compositions, comprising combining the reactant, the enzyme, the solvent mixture, a buffer, and a cofactor thereby forming a reaction mixture, and incubating the reaction mixture such that the composition is prepared, wherein the reaction mixture comprises a ratio of the amount of the reaction product to the sum of the amount of the reactant and the amount of the reaction product, the ratio being less than 2%, e.g., less than 1%, less than 0.5%, or less than 0.1%.

[0084] Regarding the enzymatic reaction of B8 to form B9, the enzyme has epoxidase activity. An epoxidase is a type of enzyme, often flavin-dependent monooxygenases (FMOs) or cytochrome P450s, that introduce an epoxide group (oxirane) at an alkene, e.g., into a polyketide backbone. Without being bound by theory, the epoxidase enzyme's three-dimensional binding pocket ensures that an oxygen atom is delivered to one face of the olefin, resulting in a single epoxide enantiomer being produced. To reduce costs of commercial19 of 601609969430.31959952.00068synthetic procedures, a cofactor recycling system may be used in conjunction with the enzyme having epoxidase activity.

[0085] In some embodiments, the enzyme having epoxidase activity includes a pldD (pldD is the enzyme responsible for installing the epoxide in the synthesis of pladienolide B; see Smith et al., RSC Chem. Biol., 2025, 6, 1126, and Machida et al., Biosci. Biotechnol. Biochem., 72 (11), 2946-2952, 2008), an fddD (fddD is the enzyme responsible for installing the epoxide in the synthesis of FD-895; see Smith et al.), an epoK (epothilone epoxidase, e.g., the enzyme responsible for installing the epoxide in the synthesis of epothilones; see Liang et al., Microb. Cell Fact. (2020) 19:180), a monCi (monensin epoxidase, e.g., the enzyme responsible for installing the epoxide in monensin), a nanO (nanchangmycin epoxidase, the enzyme responsible for installing the epoxide in the synthesis of nanchangmycin), or EC 1.14.99.20 (phylloquinone monooxygenase (2,3-epoxidizing)).

[0086] These epoxidase enzymes operate through a coenzyme dependent mechanism. In some embodiments, the cofactor regeneration enzyme is used with a substrate, which can include FAD, NAD, or NADP+. In some embodiments, the enzymes used in the reacting of B8 to form B9 are bound to a solid support, are not bound to a solid support, or include a combination of bound and unbound enzymes. In some embodiments, isopropanol is included in the reaction mixture to convert the oxidized cofactor (FAD, NAD, or NADP+) to its reduced form (FADH2, NADH, or NADPH). In some embodiments, glucose is included, along with GDH, in the reaction mixture to convert NADP+to NADPH.

[0087] In some embodiments regarding the reacting of INT4 to form INT5, the enzyme having ketoreductase activity is a KRED or an enzymatically active variant thereof. In some embodiments, a KRED is called a carbonyl reductase. A KRED is a type of oxidoreductase enzyme that specializes in the asymmetric reduction of ketones to form chiral alcohols. KRED's are generally grouped into three structural families based on the chain length and folding: 1) Short-chain Dehydrogenases / Reductases (SDRs); Medium-chain Dehydrogenases / Reductases (MDRs); and Aldo-Keto Reductases (AKRs).

[0088] These KRED enzymes operate through a coenzyme dependent mechanism. The ketone of the substrate (e.g., the ketone of INT4) binds within the KRED enzyme's active site, which often contains a catalytic tetrad of amino acids (typically serine, tyrosine, lysine and asparagine). A hydride ion (H⁻) is transferred from a cofactor, typically NADH or NAD(P)H, to the electrophilic carbon atom of the ketone's carbonyl group. Simultaneously, a conserved tyrosine residue in the active site acts as a general acid to protonate the carbonyl oxygen atom and provide the alcohol product. The KRED enzyme's three dimensional binding pocket20 of 601609969430.31959952.00068ensures that the hydride ion is delivered to only one specific face of the ketone, resulting in a single alcohol enantiomer being produced. To reduce costs of commercial synthetic procedures, a cofactor recycling system may be used in conjunction with the enzyme having ketoreductase activity. Thus, in some embodiments, a cofactor regeneration enzyme may be used. In some embodiments, the cofactor regeneration enzyme used herein can include glucose-6-phosphate dehydrogenase (G6PD), glucose dehydrogenase (GDH), and isocitrate dehydrogenase (IDH). In some embodiments, the cofactor regeneration enzyme is used with a substrate, which includes a glucose or isocitrate. The reacting of INT4 to form INT5 may further include an additional substrate isomerization enzyme, including aconitase, which isomerizes citrate to isocitrate. In some embodiments, the enzymes used in the reacting of INT4 to form INT5 are bound to a solid support, are not bound to a solid support, or include a combination of bound and unbound enzymes. In some embodiments, isopropanol is included in the reaction mixture to convert the oxidized cofactor (NADP+) to its reduced form (NADPH). In some embodiments, glucose is included, along with GDH, in the reaction mixture to convert NADP+to NADPH.

[0089] In some embodiments, the enzyme having ketoreductase activity includes ketoreductase 1 from Oogatea glycozyma (KREDl-Pglu), alcohol dehydrogenase from Ralstonia sp. (RADH), and alcohol dehydrogenase from Lactobacillus brevis (LbADH). In some embodiments, KREDs include, for example, those classified under the EC numbers of 1.1.1. KREDs may include alcohol dehydrogenase, carbonyl reductase, lactate dehydrogenase, hydroxyacid dehydrogenase, hydroxyisocaproate dehydrogenase, 0-hydroxybutyrate dehydrogenase, steroid dehydrogenase, sorbitol dehydrogenase, or aldoreductase, or an enzymatically active variant thereof, or a combination thereof. NADPH-dependent KREDs include those classified under the EC number of 1.1.1.2. NADH-dependent KREDs include those classified under the EC number of 1.1.1.1. In some embodiments, the KRED enzyme is KRED-A6, e.g., KRED-A6-P2D5, and the enzymatic reaction takes place in the presence of NADP and a solvent, optionally in the presence of a buffer and a nonionic surfactant having a hydrophilic head and a hydrophobic / lipophilic tail and a hydrophilic-lipophilic balance (HLB) of about 10-20 (e.g., 13 to 15), such as octylphenoxypolyethoxyethanol (IGEPAL CA-630; HLB of 13.4) or a polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether such as Triton-X100 (CAS number 9002-93-1; HLB of 13.4). In some embodiments, the solvent includes isopropyl alcohol, water, or a mixture thereof.

[0090] In other embodiments, the enzyme having ketoreductase activity includes specific engineered KRED examples, which include KRED-101, KRED-P1-B12, KRED-P1-C01, KRED-132, KRED-145, KRED-101, KRED-119, KRED-130, KRED-244, or KRED-464, or a 21 of 601609969430.31959952.00068combination thereof. In some embodiments, the enzyme having ketoreductase activity includes natural source microbial KREDs, which include Lactobacillus kefir KRED (LK-ADH), Sporidiobolus salmonicolor KRED (Ssal-KRED), or Rhodococcus erythropolis (RS-1 ADH), or a combination thereof.

[0091] In some embodiments regarding the reacting of INT5 to form INT6, the transformation may be a Sharpless Asymmetric Dihydroxylation (SAD) or a variation thereof. In other embodiments, the conversion of INT5 to form INT6 may not employ osmium catalysis. In some embodiments, INT6 may be prepared from INT5 replacing osmium catalyst systems with iron catalysts, manganese catalysts, palladium / gold catalysts and other suitable metal oxidation systems.

[0092] In some embodiments regarding the reacting of INT5 to form INT6, the transformation may employ enzyme catalysis. In some embodiments, the enzyme may comprise Rieske non-heme iron oxygenases (ROs). ROs are a diverse family of enzymes, predominantly bacterial, that catalyze stereo- and regiospecific oxygenation reactions using molecular oxygen. In some embodiments, ROs function as multi-component systems involving a Rieske [2Fe-2S] cluster and a mononuclear iron center, enabling the addition of two oxygen atoms to substrate. In some embodiments, the RO may comprise an iron-sulfur [2Fe-2S] complex. In some embodiments, the RO may be a toluene dioxygenase (TDO). In other embodiments the RO may be a naphthalene dioxygenase (NDO). Other embodiments include phthalate dioxygenase (PDO).

[0093] In some embodiments, provided herein are methods for preparing Compound A, or a composition thereof, comprising a synthetic sequence as depicted in FIG. 1, FIG. 2, FIG.3, FIG. 4, FIG. 5, FIG. 19, or FIG. 20, or a combination thereof. In some embodiments, Compound A, or a composition thereof, is purified by a process as depicted in FIG. 6 or FIG.7. In some embodiments, INT10, or a composition thereof, is purified by a process as depicted in FIG. 18.

[0094] In some embodiments provided herein, the polyketides herein, e.g., Compound A, and thereby the compositions herein, are prepared by a method of synthesis that comprises, sequentially:1) reacting INT6 with NIS (N-iodosuccinimide) to form INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of CSA (camphorsulfonic acid) to form INT8;3) reacting INT8 with INT10 in the presence of a palladium catalyst to form Compound A,22 of 601609969430.31959952.00068wherein INT10 is prepared by a method of synthesis that comprises reacting INT9 with tributyltin hydride to form INT10.

[0095] In some embodiments of the methods herein, INT10 is prepared by a method comprising a method of synthesis that comprises, sequentially:1) reacting INT9 with tributyltin hydride to form INT10; and2) optionally a method of purification of INT10 that comprises, sequentially: a silica gel chromatography purification step, a supercritical fluid chromatography (SFC) purification step, and a concentration purification step.

[0096] In some embodiments provided herein, the polyketides herein, e.g., Compound A, and thereby the compositions herein, are prepared by a method of synthesis that comprises reacting INT8 with INT10 in the presence of a palladium catalyst (e.g., Xphos Pd G4, aka SP-4-3)-[Dicyclohexyl[2',4',6'-tris(l-methylethyl)[l,l'-biphenyl]-2-yl]phosphine](methanesulfonato-KO)[2'-(methylamino-KN)[l,l'-biphenyl]-2-yl- KC]palladium) in the presence of tetra hydrofuran (THF) to form Compound A, which is optionally further purified as described herein.

[0097] In some embodiments provided herein, the polyketides herein, e.g., Compound A, and thereby the compositions herein, are prepared by a method of synthesis that comprises, sequentially:1) reacting INT6 with NIS in the presence of acetonitrile and 1,1, 1,3,3, 3-hexafluoro-2- propanol to form INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of CSA and dichloromethane to form INT8;3) reacting INT8 with INT10 in the presence of a palladium catalyst (e.g., Xphos Pd G4) in the presence of tetra hydrofuran (THF) to form Compound A, which is optionally further purified as described herein.

[0098] In some embodiments provided herein, the polyketides herein, e.g., Compound A, and thereby the compositions herein, are prepared by a method of synthesis that comprises, sequentially:1) reacting INT6 with NIS in the presence of acetonitrile and 1,1, 1,3,3, 3-hexafluoro-2- propanol to form INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of CSA and dichloromethane to form INT8;23 of 601609969430.31959952.000683) reacting INT8 with INT10 in the presence of a palladium catalyst (e.g., Xphos Pd G4) in the presence of tetra hydrofuran (THF) to form Compound A, which is optionally further purified as described herein,wherein INT10 is prepared by a method of synthesis that comprises reacting INT9 with tributyltin hydride in the presence of a palladium catalyst (e.g., PdCh PhsP ) and dichloromethane to form INT10.

[0099] In some embodiments of the methods herein, Compound A is prepared by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

[0100] In some embodiments, provided herein are methods of purifying the polyketides herein, e.g., Compound A, the method comprising, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

[0101] Thus, in some embodiments of methods of purification herein, pure polyketide, e.g., Compound A, is obtained by a six-step process, including 1) silica gel column chromatography (e.g., using acetone and n-heptane mobile phase), 2) silica gel column 24 of 601609969430.31959952.00068chromatography (e.g., to remove organostannane by-products) containing potassium carbonate (e.g., using ethyl acetate and acetone mobile phase), 3) thiosilica gel chromatography (e.g., to remove metal contaminants by stirring compound A in acetonitrile with siliaMetS DMT metal scavenger (2,4,6-trimercaptotriazine-functionalized silica gel)), 4) reverse phase high-performance liquid chromatography (e.g., on Luna C18 (250 mmx80 mm ID, 10 pm) column using acetonitrile and water as the running phase), 5) a supercritical fluid (SCF) purification (e.g., using isopropanol and CO2 at 100 bar at 38 °C on a Chiral Pack IC (200mmxl00 mm ID, 10 pm) column, and 6) concentration and lyophilization. In some embodiments, the method of purification optionally includes a sieving step to provide a consistent particle sized composition of purified polyketide, e.g., Compound A. The methods of synthesis herein may be combined with the methods of purification herein.

[0102] In some embodiments, provided herein are methods of synthesizing Compound A, the method comprising, sequentially:1) reacting INT6 with NIS to form INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of CSA to form INT8; 3) reacting INT8 with INT10;to form Compound A.

[0103] In some embodiments of the methods herein, INT10 is prepared by a method comprising a method of synthesis that comprises, sequentially:1) reacting INT9 with tributyltin hydride to form INT10; and2) optionally a method of purification of INT10 that comprises, sequentially: a silica gel chromatography purification step, a supercritical fluid chromatography (SFC) purification step, and a concentration purification step.

[0104] In some embodiments of the methods herein, the polyketides herein, e.g., Compound A, are purified by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;25 of 601609969430.31959952.000686) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

[0105] In some embodiments of the methods herein, the polyketides herein, e.g., Compound A, are prepared according to the method in a single batch, e.g., without combining multiple synthetic runs of the same method, to provide at least 400 g (e.g., at least 1.25 kg) of purified Compound A.

[0106] In some embodiments of the methods herein, Compound A is prepared according to the method in a single batch, e.g., without combining multiple synthetic runs of the same method, to provide purified Compound A in the form of a powder.

[0107] In some embodiments of the methods herein, Compound A is prepared according to the method in a single batch, e.g., without combining multiple synthetic runs of the same method, to provide purified Compound A in the form of a solid.

[0108] In some embodiments of the methods herein, Compound A is prepared according to the method in a single batch, e.g., without combining multiple synthetic runs of the same method, to provide purified Compound A in the form of a solid (e.g., a powder) in a composition wherein the composition comprises one or more of: more than 99% w / w (e.g., at least 99.4% w / w) of Compound A; Compound A in the form of an amorphous solid form as determined by x-ray powder diffraction; Compound A in greater than 99% chiral purity as determined by high-performance liquid chromatography; less than 2% w / w of water; or at least 400 g (e.g., at least 1.25 kg) of Compound A.

[0109] Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses (Crews, Leslie A et al. " Reversal of malignant ADAR1 splice isoform switching with Rebecsinib." Cell stem ce / / vol. 30,3 (2023): 250-263. e6. doi: 10.1016 / j. stem.2023.01.008). However, inflammatory-microenvironment-induced ADARlpllO to pl50 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies (Crews, Leslie A et al.). The polyketides herein, e.g., Compound A, and thereby the compositions provided herein, are useful in methods for inhibiting ADARlpl50, whether in vivo or in vitro (Crews, Leslie A et al.).

[0110] In some embodiments, the compositions described herein may include one or more other pharmacologically active compounds, such as an antiproliferative agent.26 of 601609969430.31959952.00068

[0111] Thus, provided herein are methods of inhibiting ADARlpl50 in a subject in need thereof, comprising administration of an effective amount of a polyketide composition here, e.g., a composition herein comprising purified Compound A, to a subject in need thereof. In some embodiments of the methods herein, the subject suffers from cancer.

[0112] In another embodiment, provided herein are methods of treatment, comprising administering a polyketide composition prepared as described herein, e.g., a composition comprising purified Compound A, to a patient in need of such treatment in an effective amount for the prophylactic or therapeutic treatment of a disease or disorder which is mediated by the activity of ADARlpl50.

[0113] In a further embodiment, provided herein are methods of treatment, comprising administering a polyketide composition prepared as described herein, e.g., a composition comprising purified Compound A, to a patient in need of such treatment in an effective amount for the prophylactic or therapeutic treatment of a disease or disorder which is mediated by the activity of ADARlpl50, wherein the disease or disorder mediated by the activity of ADARlpl50 is a proliferative disease, such as a liquid cancer, e.g., a leukemia.

[0114] In another embodiment, provided herein are combinations comprising a composition described herein, e.g., a composition comprising purified Compound A, and one or more other pharmacologically active compounds, such as antiproliferative agents, for simultaneous, sequential or separate administration, e.g., administration in the methods herein.

[0115] Some embodiments include a method of preparing a compound (e.g., B9, BIO, INT9, INT10, e.g., a polyketide herein, e.g., Compound A or Compound B), comprising reacting a reactant having a formula,with an epoxidase enzyme to form the compound.

[0116] Some embodiments include a method of preparing a compound having a formula,comprising reacting a reactant having a formula,27 of 601609969430.31959952.00068with an epoxidase enzyme to form the compound.

[0117] Some embodiments include a method of preparing a compound,wherein J1is an alkylsilanyl or an arylakyl silanyl, comprising reacting a reactant having a formula,with a ketoreductase enzyme to form the compound.

[0118] In some embodiments, J1comprises an SiRxR2R3functional group, wherein R1, R2and R3are independently hydrogen (H), alkyl (e.g., Ci-io alkyl), alkylaryl, aryl, heteroaryl or alkylheteroaryl. Aryl and heteroaryl functional groups may include one or more substituent groups including C i-io alkyl, halogen, cyano, amino (-NR1R2), amido (-NR1R4) where R4is C(O)R2, hydroxy or ether (OR1), acetate derivative (OR4), thiol or thioether (SR1), thioacetate derivative (SR4), nitro (NO2), sulfonate (S(O)2RX), ester (CO2R1), amide (C(O)NR1R2), sulfonamide (S(O)2NR1R2), and the like. In some embodiments, R1, R2and R3are methyl (CH3). In other embodiments, R1and R2are methyl and R3is phenyl. In another embodiment, R1and R2are methyl and R3is aryl, wherein aryl includes a substituted phenyl group. In an embodiment, R1and R2are methyl and R3is alkylaryl, wherein alkylaryl includes, e.g., a substituted benzyl group. In an embodiment, R1and R2are methyl and R3is heteroaryl, wherein heteroaryl includes a substituted heteroaromatic group, comprising one or more heteroatoms including nitrogen (N), oxygen (0) or sulfur (S) atoms.28 of 601609969430.31959952.00068

[0119] In some embodiments of the methods herein, the compound formed from thereacting with the ketoreductase enzymethe reactant is

[0120] Some embodiments include a method of preparing a compound having a formula,comprising reacting a reactant having a formula,with a ketoreductase enzyme to form the compound.

[0121] In another embodiment of the methods herein, the ketoreductase enzyme and the reactant are reacted in a mixture comprising a solvent system, optionally wherein the solvent system comprises isopropanol or water, or isopropanol and water.

[0122] In a further embodiment of the methods herein, the reactant and the ketoreductase enzyme are reacted in a mixture comprising a nicotinamide adenine dinucleotide phosphate (e.g., a p-nicotinamide adenine dinucleotide phosphate sodium salt), optionally wherein the reactant and the ketoreductase enzyme are reacted at about 20 °C to about 35 °C (e.g., about 25 °C to about 30 °C) in the mixture.29 of 601609969430.31959952.00068EXAMPLESExample 1: Preparation of Compound A

[0124] Compound A is prepared from INT8 and INT10 as depicted in FIG. 1. Synthesis of compound A: INT8 (1.0 x) and INT10 (1.3 x) were dissolved in tetra hydrofuran (8.9 x) at 25 °C. Nitrogen was bubbled through the solution and XPHOS Pd G4 (0.092 x) was added, and the atmosphere of the flask was changed to N2 (pump and replace, 3x). The temperature was adjusted to 70 °C and stirred vigorously. After 14 h, the mixture was cooled to 25 °C and filtered through a pad of celite (1 x) washing the pad with tetra hydrofuran (3 x). The solvent was exchanged with dichloromethane by concentration and dilution. Pure compound A is obtained by a process, including those as described in FIG. 6, Example 21, Example 23, or Example 24.Example 2: Preparation of INT8

[0125] INT8 is prepared from INT7 as depicted in FIG. 2. Synthesis of INT8. INT7 (1 x) was dissolved in dichloromethane (6.7 x) and charged with camphor-10-sulfonic acid (0.060 x). The atmosphere of the flask was changed to N2 (pump and replace, 3 x) and the temperature was reduced to 0 °C. Trimethylorthoacetate (1,1,1-trimethoxyethane) (0.43 x)30 of 601609969430.31959952.00068in dichloromethane (6.7 x) was stirred for 1 h at 25 °C and cooled to 0 °C and then added the solution of INT7. After 2 h of stirring at 0 °C, a solution of ammonium chloride (2.5 x) in water (10 x) was added, warmed to 25 °C and stirred at 25 °C for 2 h and let stand for 1 h. The organic layer was collected, and the aqueous layer was washed twice with dichloromethane (6.7 x and 3.3 x) with stirring for 1 h at 25 °C and settling for 1 h at 25 °C. The combined organic phases were cooled to 0 °C and stirred with 30% hydrochloric acid for 8 h at 0 °C. The organic layer was collected and washed satd. sodium chloride (10 x) and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with dichloromethane (1 x). The resulting material was concentrated and solvent exchanged into methyl-tert-butylether and diluted to 1:1 ratio with n-heptane. Solid crystalline precipitate was filtered from this mixture and was dried at 35 °C to provide INT8 in 53% yield with 99.3% purity: 89.4 g.Example 3: Preparation of INT7

[0126] INT7 is prepared from INT6 as depicted in FIG. 19. Synthesis of INT7. INT6 (1 x) was dissolved in l,l,l,3,3,3-hexafluoro-2-propanol (16 x) and the temperature was adjusted to 5 °C. / V-iodosuccinimide (0.83 x) was dissolved acetonitrile (4 x) by stirring at 25 °C for 1 h. This solution was added to the solution of INT6 at 5 °C over 2 h and stirred for an additional 1 h at 5 °C. In an independent reactor, a solution of sodium thiosulfate (4.4 x) in water (10 x) prepared by stirring for 1 h at 25 °C. This solution was added to the reaction flask at 5 °C and the mixture was allowed to warm to 25 °C with stirring. Once at 25 °C, methyl-tert-butyl ether (14.8 x) was added and stirred for 1 h. After standing for 1.5 h, the layers were separated, and the aqueous phase was extracted again with methyl-tert-butyl ether (3.7 x) with stirring for 1 and standing for 1.5 h. The two organic layers were combined and was sequentially washed with water (10 x) and 20% aqueous sodium chloride (11.7 x). The organic layers were collected and dried by filtering through a pad of anhydrous sodium sulfate (1 x filter press) with washing with methyl-tert-butyl ether (1.48 x). The solvent was changed to dichloromethane by two additions of dichloromethane (4 x) and evaporation. This solution was used directly for the next step. Alternatively, the solvent is removed to provide INT7 as a solid (see FIG. 22) in 66% yield with 96.2-97.2% purity: 120 g.Example 4: Preparation of INT6

[0127] INT6 is prepared from INT5 as depicted in FIG. 19. Synthesis of INT6. Potassium hexacyanoferrate (III) (2.5 x) was dissolved in water (15 x) and tert-butanol (11.7 x). Potassium carbonate (1 x) and (DHQD)zPHAL (0.020 x) were added sequentially. The resulting solution was stirred for 1 h at 25 °C and then the temperature was adjusted to 531 of 601609969430.31959952.00068°C. Potassium osmate (VI) dihydrate (0.0092 x) and methanesulfonamide (0.71 x) were added sequentially. This solution was stirred at 5 °C for 1 h. INT5 (1 x) was added at 5 °C and the mixture was stirred at 5 °C for 18 h. After checking for reaction completion, a mixture of sodium thiosulfate (6.8 x) and water (15 x) were added. This solution was stirred for 1 h at 25 °C at which point ethyl acetate (9 x) was added. The suspension was stirred for 1 h at 25 °C. The organic phase was collected, and the aqueous phase was extracted again with ethyl acetate (4.5 x). The two organic phases were combined and washed sequentially with water (10 x) and a mixture of sodium chloride (2 x) in water (10 x). Each extraction or wash was stirred for 1 h before settling and collection. The resulting solution was dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with ethyl acetate (1.8 x). The resulting solution was concentrated to 4 x and heptane (3.4 x) was added, and the solution was stirred for 2 h resulting in precipitation. INT6 was collected by filtration and washed with a mixture of heptane (1 x) and ethyl acetate (1 x). The resulting wet cake was dried at 35 °C for 8 h to provide INT6 as a dried cake (see FIG. 21): 200.6 g.Example 5: Preparation of INT5

[0128] INT5 is prepared from INT4 as depicted in FIG. 19. Synthesis of INT5. Potassium phosphate monobasic (0.080 x) and potassium phosphate dibasic trihydrate (0.20 x) and Triton X-100 (0.60 x) was dissolved in water (30 x). The temperature was adjusted to 25 °C and the mixture was stirred for 30 min at 25 °C until all solids were dissolved, (3-nicotinamideadeninedinucleotide phosphate sodium salt (0.050 x) along with the enzyme KRED-A6-P2D5 (0.50 x) were added and stirred for 30 min at 25 °C until dissolved. In a separate vessel, INT4 (1.0 x) was added to isopropyl alcohol (1.5 x) and the temperature was adjusted to 25 °C and stirred for 30 min until all solids dissolved. The solution of INT4 was added to the enzymatic mixture over 30 min and the temperature was adjusted to 30 °C. This mixture was stirred for 24-72 h until HPLC indicated an IPC purity of [INT4] / [INT4+INT5] of <2%. When not obtained, 0-nicotinamideadeninedinucleotide phosphate sodium salt (0.020 x) along with the enzyme kRED-A6-P2D5 (0.20 x) dissolved in water (1.0 x) was added and the mixture was stirred for 24-72 h until HPLC indicated an IPC purity of [INT4] / [INT4+INT5] of <2%. Additional enzyme kRED-A6-P2D5 and |3-nicotinamideadeninedinucleotide phosphate sodium salt were added when needed to reach this IPC. Once obtained, acetonitrile (12 x) and diatomite (2 x) were added, and the temperature was adjusted to 50 °C. After stirring for 4 h at 50 °C the temperature was reduced to 25 °C and the resulting solution was filtered through a pad of diatomite (1 x) and washed with ethyl acetate (13.5 x). The resulting solution was charged with 20% sodium chloride (10 x) and stirred for 2 h at 25 °C. The layers were separated, and the aqueous layer 32 of 601609969430.31959952.00068was washed with ethyl acetate (4.5 x). The organic layers were combined and dried by filtering through a pad of anhydrous sodium sulfate (1 x filter press) with washing with ethyl acetate (2 x). The solvent was concentrated and changed to dichloromethane (6.7 x). Pure INT5 was obtained by filtering through a silica gel pad with mixture of n-heptane (1 x) and ethyl acetate (10 x) and then n-heptane (1 x) and ethyl acetate (5 x). This mixture was concentrated to 1-3 V and heptane (10 x). The mixture was warmed to 45-50 °C and stirred for 1-4 h, then gradually cooled in steps at 30-40 °C for 1-4 h, 20-30 °C for 1-4 h, 25 °C for 4-24h and -5 °C to 5°C for 4-24h and -15 °C for 4-24 h to encourage precipitation. The solids were collected by filtration and the wet cake was dried at 25 °C for 8-24 h to afford pure INT5: 285.2 g.Example 6: Preparation of INT4

[0129] INT4 is prepared from SM as depicted in FIG. 19. Synthesis of INT4. SM (1 x) was dissolved in acetonitrile (314 x) and stirred for 30 min at 0-30°C to reach a clear solution. The solution was dried with molecular sieves (10 x) by recycled flow for 1-10 h until the water level was below 200 ppm. The resulting solution was passed through a two-stage flow system where the first stage was at 120-150°C (stage 1). The second was at 0-30°C (stage 2). The flow rate was adjusted to meet the optimal conversion through test experiments and was set to this value. The dried solution was flowed from stage 1 to stage 2. The system was then charged, and the solution was passed through the system until the reaction was complete over 1-8 h. The reaction was tested at every pass. Once complete (>70% INT4 by HPLC) the resulting material was collected, and the system was washed with acetonitrile. The product and wash were combined and dried to (1-3 x). The solvent was replaced with CH2CI2 by addition of CH2CI2 (4 x) and concentrating to 2 x (repeated twice). The pure material was obtained by silica gel column chromatography using acetone and n-heptane. The pure fractions were solvent exchanged to THF and dried: 412 g. Dried material was used for the next step.Example 7: Preparation of SM

[0130] SM is prepared as previously reported (Rhoades et al. (Journal of the American Chemical Society, 2021, 143, 13, 4915-4920)).Example 8: Preparation of INT10

[0131] INT10 is prepared from INT9 as depicted in FIG. 3. Synthesis of INT10. INT9 (0.070 x) was dissolved in dichloromethane (9.3 x) and the temperature was adjusted to -10 °C. Nitrogen was bubbled into the solution for 0.5 h. Bis(triphenylphosphine)palladium(II)chloride33 of 601609969430.31959952.00068(0.093 x) was added and the atmosphere of the flask was changed to N2 (pump and replace, 3x). The temperature was adjusted to -15 °C and tri-n-butylstannane (1.8 x) was added. After stirring for 2 h at -15 °C, n-heptane (4.8 x) was added and the resulting solution was filtered through a pad of silica gel (3 x) by washing sequentially with 1:1000 w / w ratio solution of triethylamine / n-heptane (15 x), 1:200:800 w / w ratio solution of triethylamine / methyl-tert-butylether / n-heptane (30 x), 1:200:800 w / w ratio solution of triethylamine / methyl-tert-butylether / n-heptane (10 x). The three washes were collected and concentrated, and pure material was obtained by silica gel column chromatography using triethylamine, methyl-tert-butylether and n-heptane to yield INT10 in 98.3% purity.Example 9: Preparation of INT9

[0132] INT9 is prepared from B10 and Bll as depicted in FIG. 5. Synthesis of INT9: (S)-buta-l,2-dien-l-yltributylstannane (1.5 x) was dissolved dichloromethane (7 x) and atmosphere was swapped with N2 (pump and replace, 3x). The temperature was cooled to -75±5 °C and BF3 • Et20 (1.0 x) was added and stirred for 1 h at -75±5 °C. B10 (1.0 x) in dichloromethane from step 13 was added slowly to ensure the temperature was maintained at -75±5 °C. After stirring rigorously for 1 h at -75±5 °C, methanol (2 x) was added and stirred for 15 min. The pH was then adjusted to 6-8 by the addition of 6±1% aq. sodium hydrogen carbonate. The suspension was filtered and separated. The aqueous fraction was further extracted with dichloromethane (2 x). At 25 °C, the combined organic layers stirred with water (4.75 x) containing saturated sodium hydrogen carbonate (0.25 x) for 1 h and stand for 30 min. The organic layer was collected and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with dichloromethane (0.9 x). After solvent removal, pure INT9 was obtained by short pad silica gel column chromatography using n-heptane and ethyl acetate in 51% yield.Example 10: Preparation of BIO

[0133] B10 is prepared from B9 as depicted in FIG. 5. Synthesis of B10: B9 (1 x) was dissolved in ethyl acetate (11.7 x) at the temperature was adjusted to 25 °C. 2-Iodoxybenzoic acid (IBX, 2.2 x) was added and the temperature was adjusted to 73 °C. The mixture was vigorously stirred for 12 h at which point it was cooled to 25 °C and filtered. The filter cake was washed with ethyl acetate (1 x) which was dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with ethyl acetate (0.9 x). B10 was used directly for the next step without purification. The solvent was changed to dichloromethane concentration and dilution.34 of 601609969430.31959952.00068Example 11: Preparation of B9

[0134] B9 is prepared from B8 as depicted in FIG. 5. Synthesis of B9: Dichloromethane (16 x) was charged with activated heat-resistant molecular sieves (1 x). Titanium (IV) isopropoxide (0.18 x) was added followed by a solution of (-)-diethyltartrate (0.16 x) in dichloromethane (1.3 x) at 25 °C. This solution was cooled to 15 °C and cumene hydroperoxide (1.45 x) was added followed by B8 (1 x) was dissolved in dichloromethane (1 x). After 2 h at 15 °C the reaction as cooled to at 5 °C and a solution of citric acid (1.7 x) in water (6.8 x) was added and stirred for 1 h at 0 °C. The solution was filtered with diatomite (1 x) and the cake was washed with dichloromethane (2.7 x). The layers were separated, and the aqueous layer was extracted with a second portion of dichloromethane (2.7 x). The combined organic layers were washed sequentially with sodium thiosulfate (2 x) in water (8 x), sodium carbonate (0.5 x) in water (5 x) and 20% aq. sodium chloride (6 x). After solvent removal, pure B9 was obtained by short pad silica gel column chromatography using n-heptane and ethyl acetate at 93% purity in 89% yield.Example 12: Preparation of B8

[0135] B8 is prepared from C27 as depicted in FIG. 5. Synthesis of B8: Potassium carbonate (1.6 x) was added to C28 (1 x) in methanol (7.9 x) at 25 °C. After stirring for 2 h at 25 °C the mixture was filtered, and the filter cake was washed with methyl-tert-butylether. The organic phase was diluted with methyl-te / T-butyl ether (6.3 x) and water (4.0 x). The layers were separated, and the aqueous layer was extracted further with methyl-fert-butyl ether (1.5 x). The organic layers were combined and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with methyl-tert-butylether (1 x). After solvent removal, pure B9 was obtained by short pad silica gel column chromatography using n-heptane and ethyl acetate to yield 91% purity material in a 57% yield.Example 13: Preparation of C28

[0136] C28 is prepared from C27 as depicted in FIG. 4. Synthesis of C28: C27 (1 x) was diluted with dichloromethane (66 x) and adjusted to 25 °C. At 25 °C, 1,8-bis(dimethylamino)naphthalene (0.95 x) and trimethyloxonium tetrafluoroborate (0.6 x) were added. After 1 h, this addition was repeated. The reaction was filtered, and the resulting solution was stirred with solution of NaHCCh (0.5 x) in water (10 x) for 1 h and allowed stand for 1 h. The organic layer was collected and washed with citric acid (1 x) in water (10 x) and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with dichloromethane (l x). The solvent was changed to methanol concentration and dilution. The solution of C28 (1 x) in methanol (7.9 x) was used for the next step.35 of 601609969430.31959952.00068Example 14: Preparation of C27

[0137] C27 is prepared from C26 as depicted in FIG. 4. Synthesis of C27: C26 (1 x) dissolved in acetonitrile (3.6 x) was diluted a mixture of water (3.5 x) and 35% aq. HCI (0.39 x). This mixture was stirred for 12 h at 25 °C at which point NaHCCh (0.35 x) in water (5 x) was added slowly at 25 °C. After 1 h, the mixture was extracted with ethyl acetate (4.5 x). The organic layer was collected and washed with 25% aq. sodium chloride (5 x) and dried by sodium sulfate (3 x filter press) and the solvent was exchanged to n-heptane. Pure C27 was obtained by short pad silica gel column chromatography using n-heptane and ethyl acetate to yield 90% purity material in a 60% yield from C8.Example 15: Preparation of C26

[0138] C26 is prepared from C8 as depicted in FIG. 4. Synthesis of C26: This reaction was conducted using flow process. Alcohol C8 (1.00 x) was dissolved in dichloromethane (13.3 x) by stirring at 25 °C for 30 min. The reaction was cooled to 0 °C and charged with EtsN (1.3 x). After 15 min at 0 °C, benzoyl chloride (1.2 x) was added and stirred for 2 h at 0 °C. Water (10 x) was added and stirred for 1 h at 25 °C followed by standing for 2 h at 25 °C. The organic layer was collected, washed with water (10 x) and ammonium chloride (3.7 x). The organic phases were extracted with dichloromethane and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press). The crude C26 product was used immediately for the next step by a solvent exchange with acetonitrile (3.6 x) by concentration and dilution. A f C26 (1 x) dissolved in acetonitrile (3.6 x) was used for the next step.Example 16: Preparation of C8

[0139] 08 is prepared from C7 as depicted in FIG. 4. Synthesis of C8: This reaction was conducted using flow process. Ester C7 (1.00 x) was dissolved in dichloromethane (9.3 x) by stirring at 25 °C for 30 min. The reaction was conducted in flow where the solution of C7 in THF was mixed with DIBAL-H IM in n-hexane solution maintaining the temperature at -58 °C to -78 °C and then adding acetone and an aqueous solution of potassium sodium tartrate. The organic phases were extracted with dichloromethane and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press) with washing with dichloromethane (1 x). After solvent removal, 08 was obtained at 86% purity in 90% yield from this step.Example 17: Preparation of C7

[0140] C7 is prepared from C6 as depicted in FIG. 4. Synthesis of C7: Triethylphosphonoacetate (1.08 x) was added dissolved in acetonitrile (5.52 x) and the reaction temperature was adjusted to 0 °C. Anhydrous lithium chloride (0.34 x) and36 of 601609969430.31959952.00068triethylamine (0.46 x) were then added, and temperature was adjusted to 0 °C. C6 (1.0 x) dissolved in acetonitrile (2.0 x) was added and maintaining the temperature at 0 °C. After 15 min, the mixture was warmed to 25 °C where it was stirred for 24 h. Water (4 x) was added, and the organic layer was washed with ammonium chloride (1.2 x) and the product as obtained by extraction with dichloromethane (10 x). Pure material was obtained by short pad silica gel column chromatography using n-heptane and ethyl acetate to yield 90% purity material in a 97% yield from C5.Example 18: Preparation of C6

[0141] C6 is prepared from C5 as depicted in FIG. 4. Synthesis of C6: This reaction was conducted using flow process. Amide C5 (1.00 x) was dissolved in dry THF (4.5 x) by stirring at 25 °C for 30 min. The reaction was conducted in flow where the solution of C5 in THF was mixed withlM DIBAL-H in n-hexane solution maintaining the temperature at -58 °C to -78 °C and then adding acetone and an aqueous solution of potassium sodium tartrate (5 x). The organic phases were extracted with dichloromethane and dried by filtering through a pad of anhydrous sodium sulfate (3 x filter press). The crude C6 product was used immediately for the next step by a solvent exchange with acetonitrile (2.0 x). The solution of C6 (1.0 x) dissolved in acetonitrile (2.0 x) was used for the next step.Example 19: Preparation of C5

[0142] C5 is prepared from B4 as depicted in FIG. 4. Synthesis of C5: Dichloromethane (7.98 x) was added to B5 (1.00 x) and adjusted to 25 °C. 4-Dimethylaminopyridine (0.070 x) and pyridine (2.25 x). A solution of triphenylsilyl chloride (1.77 x) in dichloromethane (5.3 x) was stirred for 1 h at 25 °C added to the solution of B5 at 25 °C where it was stirred for 1.5 h. Water (10 x) was added and stirred for 1 h at 25 °C followed by standing for 2 h at 25 °C. The organic layer was collected, washed with water (10 x) and ammonium chloride (2 x) and the solvent changed to isopropanol. Pure material was obtained by precipitation from isopropanol which after drying provided a yield of 70% of C5.Example 20: Preparation of Bll

[0143] Bll is prepared as in Chan et al. (Cell Reports Physical Science, 2020, 1, 12, 100277)).Example 21: Purification of Compound A

[0144] Compound A, prepared in a single batch as in Example 1, was purified in a single batch as depicted in FIG. 6 to yield a composition comprising about 417 g Compound A having the characteristics shown in Table 2 (e.g., greater than 99.4 % w / w pure, and greater than 37 of 601609969430.31959952.0006899.7 % chiral purity by HPLC area). About 1.25 kg of purified Compound A was similarly prepared. Purified Compound A may further be sieved.

[0145] A purification according to FIG. 6 included sequential steps:1) a silica gel chromatography purification step;2) a Sn-removal purification step (e.g., a potassium carbonate-silica gel chromatography purification step);3) a Pd-removal purification step (e.g., a thiosilica gel chromatography purification step); 4) a reverse phase high-performance liquid chromatography purification step (e.g., preparative RP HPLC);5) a supercritical fluid chromatography (SFC) purification step; and6) a concentration and lyophilization purification step.

[0146] Thus, pure compound A was obtained by a six-step process. This began by column chromatography using acetone and n-heptane. Next, organostannane by-products were removed by passage through a silica gel column (3 x) containing potassium carbonate (0.3 x) using ethyl acetate and acetone. The solvent was exchanged with acetonitrile (4.4 x). This was then followed by removal of metal contaminants by stirring compound A in acetonitrile with siliaMetS DMT metal scavenger (0.1 x) for 16 h at 25 °C. This was then followed by an HPLC purification on Luna C18 (250 mmx80 mm ID, 10 pm) column using acetonitrile and water as the running phase and a supercritical fluid (SCF) purification using isopropanol and CO2 at 100 bar at 38 °C on a Chiral Pack IC (200mmxl00 mm ID, 10 pm) column. The final material was collected by concentration and lyophilization.Table 2. Characterization of purified Compound A.Test Item Criteria Result Appearance Report result White solid Identification by LC- HRMS MS: 567.3±1.0 Da, MS: 589.3 Da 584.3±1.0 Da, or 589.3±1.0DaIdentification byXHNMR Conforms to structure Conforms to structure Identification by13CNMR Conforms to structure Conforms to structure XRPD Report result Amorphous substance Assay by HPLC (% w / w) Report result 99.438 of 601609969430.31959952.00068Test Item Criteria ResultPurity by HPLC (% area) >99.0 99.8Related substances by HPLC (% area)Individual impurity Report result RRT 1.02, 0.19 Total impurities <1.0 0.19Chiral purity by HPLC (% area) >99.0 99.7Elemental Impurities by ICP-MS (ppm)Cd <0.2 <0.06Pb <0.5 < 0.15As <1.5 <0.45Hg <0.3 <0.09Co <0.5 <0.15V <1 <0.3Ni <2 <0.6Pd <1 <0.3Sn <20 <6Li <25 <7.5Cu <30 <9Ir <1 <0.3Ag <1 <0.3Ti <25 <7.5Sb <9 <2.7Elemental Impurities of Os <1 <0.3Solvents by HS-GC (ppm)2-Propanol <5000 1245 Acetonitrile <410 <198 Dichloromethane <600 <201Ethyl acetate <5000 <198Tetra hydrofuran <720 <199n-Heptane <5000 <200Acetone <5000 <199INT9 by GC (ppm) Report result <1000 Hexafluoro-2-propanol Report result <500FA by IC (ppm) <5000 < LOQ(102)39 of 601609969430.31959952.00068Test Item Criteria ResultWater content by KR (% w / w) Report result 1.6Microbial limitsTotal Aerobic Microbial Count Report result <100(TAMC) (cfu / g)Total Yeasts and Molds Count Report result <50(TYMC) (cfu / g)Specific pathogen: E. coli Report result Absent in 1g Bacterial Endotoxins Test Report result <0.0500 EU / mg

[0147] Purified Compound A is characterized by various analytical techniques including, among others, those described in Table 2.

[0148] The FT-IR analysis was performed on a NICOLET iSlO Spectrometer. The spectrum was recorded at a resolution of 4 cm1and represented the sum of 32 scans from 650~4000 cm-1, background subtracted. Testing sample preparation (Attenuated Total Reflectance Accessory): firstly, acquire the background blank; then, place some amount of sample in the sample sensor, rotate the pressure control ring until the sample and the sensor closely contact; and finally, scan the sample and collect IR spectrum.

[0149] The FT-IR spectrum in FIG. 8 and FIG. 9 shows characteristic absorption bands consistent with the functional groups present in Compound A. The assignments of the major absorption bands are presented in Table 3.Table 3. FT-IR absorption spectral assignment for purified Compound A.Wavenumber (cm-1) Assignment3447.97 O-H stretching vibrations2966.07-2827.27 C-H stretching vibrations in CH2 groups1734.17-1716.25 C=O stretching vibrationsC=C stretching vibrations1456.55-1019.88 Bending, deformation and scissoring vibrations in the CH2 and CH3 groupsC-O-C stretching vibrationsC-C stretching vibrationsCOO symmetric stretching vibrations975.91-688.40 Out of plane C-OH bending vibrations40 of 601609969430.31959952.00068

[0150] NMR analysis was conducted on Compound A as shown below using a 3.2 mg sample of GMP grade Compound A (lot #1, prepared as described herein, e.g., Examples 1-22; FIGs. 10-17) in 800 pL of C6D6includingXH-NMR,13C-NMR, H^C-HSQC,1H,1H gCOSY, and1H,13C-HMBC spectra. NMR data was collected using Jeol ECA 500 NMR spectrometer (Macintosh iMac for instrument control and data processing, Jeol 2 inverse-detect1H / 19F, broad band (15N,13C,31P) probe with autotune module).Numbered Compound A Structure

[0151] The assignments of chemical shifts (in ppm) of proton and carbon are tabulated in Table 4.13C NMR chemical shift data were obtained from the 1-D carbon NMR experiment. Centerline of the carbon resonance due to the CeDe was used as an internal reference at 128.060 ppm.1H NMR chemical shift data were obtained from the 1-D proton NMR spectrum. The residual solvent resonance of the CeDe was used as an internal reference at 7.16 ppm. Relative integrated intensities of proton resonances were measured from the 1-D proton NMR spectrum. s=singlet, d=doublet, dd=doublet of doublets, t=triplet, m=multiplet, or - = Not applicable. Spectra are depicted herein forXH NMR and13C NMR (see FIG. 10, FIG. 11, FIG.12, FIG. 13, and FIG. 14),XH-13C HSQC (see FIG. 15), -I^H COSY (see FIG. 16), and! H-13C HMBC (see FIG. 17).

[0152] The data is reported for the anhydrous drug substance GMP (lot #1) in CeDe with atom numbering and values for13C peaks relative to standard peak at 128.060 ppm andXH peaks relative to standard peak at 7.16 ppm with mult.: multiplicity (s: singlet, d: doublet, t: triplet, m: multiplet, and - not accessible). * and ** denote overlapping peaks. NMR data was tabulated fromXH and13C spectra and verified by comparison with1H,13C-HSQC correlations. An edited1H,13C-HSQC spectrum was collected where CH and CH3 signals are phased up and CH2 signals are phased down. gCOSY NMR and1H,13C-HMBC spectra were collected to confirm the assignment of each proton and carbon atom.41 of 601609969430.31959952.00068Table 4. Purified Compound A NMR assignments.Atom # Type de, ppmcdH, ppm (mult., J in Hz)b#H's 1 C 172.12 CH238.5 2.31 (dd, J = 14.8, 3.9), 2.21 (7 = 14.9, 3.0) 2H8 CH 126.4 5.84 (dd, J = 15.3, 9.7)* 1H 9 CH 140.7 5.63 (dd, J = 15.2, 10.0) 1H 10 CH 41.1 2.39 (m)*** 1H 11 CH 82.6 5.25 (d, J = 10.6)** 1H 12 C 131.313 CH 131.7 6.13 (dd, J = 10.9, 1.4) 1H 14 CH 126.3 6.27 (ddd, J = 15.3, 10.9, 1.0) 1H 15 CH 137.9 5.84 (dd, J = 15.2, 8.2)* 1H 16 CH 41.5 2.37 (m) *** 1H 17 CH 72.8 3.47 (m) 1H 17-OH OH - 1.95 (bs) 1H 18 CH 59.6 2.59 (dd, J = 3.8, 2.2) 1H 19 CH 57.7 3.04 (dd, J = 8.2, 2.3) 1H 20 CH 39.3 1.34 (ddd, J = 11.3, 4.3, 1.9) 1H 21 CH 83.7 3.15 (td, J = 6.2, 4.2) 1H 22 CH223.8 1.66 (m), 1.40 (ddd, J = 14.0, 7.6, 6.5) 2H 23 CH310.0 0.85 (t, J = 7.4) 3H 24 CH324.7 1.03 (s) 3H 25 CH316.4 0.71 (d, J = 6.8) 3H 26 CH311.9 1.59 (d, J = 1.3) 3H 27 CH317.3 1.14 (d, J = 6.9) 3H 28 CH310.8 0.89 (d, J = 7.0) 3H 29 CH357.7 3.23 (s) 3H 30 C 169.131 CH320.8 1.63 (s) 3H42 of 601609969430.31959952.00068Atom # Type de, ppmcdH, ppm (mult., J in Hz)b#H'saThe data is reported for the anhydrous drug substance GMP (lot #1) in CeDe with atom numbering.bd values: relative to standard peak at 128.060 ppm.cd values: relative to standard peak at 7.16 ppm with mult.: multiplicity (s: singlet, d: doublet, t: triplet, m: multiplet, and -: not accessible).* and ** denote overlapping peaks.Example 22: Purification of INT10

[0153] INT10, prepared as in Example 8, was purified as depicted in FIG. 18 to yield a composition comprising about 240 g INT10 at a purity of greater than 94%.

[0154] Thus, a purification of INT10 according to FIG. 18 included sequential steps:1) a silica gel chromatographic purification step;2) a supercritical fluid chromatography (SFC) purification step; and3) a concentration step.Example 23: Purification of Compound A

[0155] Compound A, prepared as in Example 1 in a single batch, is purified in a single batch as depicted in FIG. 7 to yield a composition comprising hectograms of Compound A.

[0156] A purification according to FIG. 7 includes sequential steps:1) a silica gel chromatography purification step;2) a Sn-removal purification step (e.g., a potassium carbonate-silica gel chromatography purification step);3) a Pd-removal purification step (e.g., a thiosilica gel chromatography purification step); 4) a reverse phase high-performance liquid chromatography purification step (e.g., preparative RP HPLC);5) a first supercritical fluid chromatography (SFC) purification step;6) a second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) a sieving step.43 of 601609969430.31959952.00068Example 24: Purification of Compound A

[0157] Compound A, prepared as in Example 1 in a single batch, is purified in a single batch analogous to FIG. 6 or FIG. 7 to yield a composition comprising hectograms of Compound A, the purification sequence including sequential steps:1) a silica gel chromatography purification step;2) a Sn-removal purification step (e.g., a potassium carbonate-silica gel chromatography purification step);4) a reverse phase high-performance liquid chromatography purification step (e.g., preparative RP HPLC);5) a first supercritical fluid chromatography (SFC) purification step;6) a second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) a sieving step.Example 25: Preparation of B9

[0158] B9 is prepared from B8 as depicted in FIG. 5. Potassium phosphate monobasic (0.080 x) and potassium phosphate dibasic trihydrate (0.20 x) and Triton X-100 (0.60 x) are dissolved in water (30 x). The temperature is adjusted to 25 °C and the mixture is stirred for 30 min at 25 °C until all solids are dissolved. Flavin adenine dinucleotide disodium salt (0.050 x) along with the enzyme mt-FddD (0.40 x) are added and stirred for 30 min at 25 °C until dissolved. In a separate vessel, B8 (1.0 x) is added to isopropyl alcohol (1.5 x) and the temperature is adjusted to 25 °C and stirred for 30 min until all solids dissolved. The solution of B8 is added to the enzymatic mixture over 30 min and the temperature is adjusted to 30 °C. This mixture is stirred for 24-72 h until HPLC indicates an IPC (in-process control) purity of [B8] / [B8+B9] of <2%. When not obtained, flavin adenine dinucleotide disodium salt (0.020 x) along with the mt-FddD (0.20 x) dissolved in water (1.0 x) is added to the reaction mixture and the mixture is stirred for 24-72 h until HPLC indicated an IPC purity of [[B8] / [B8+B9] of <2%. Additional enzyme mt-FddD and P flavin adenine dinucleotide disodium salt are added when needed to reach this IPC. Once obtained, acetonitrile (12 x) and diatomite (2 x) are added, and the temperature is adjusted to 50 °C. After stirring for 4 h at 50 °C the temperature is reduced to 25 °C and the resulting solution is filtered through a pad of diatomite (1 x) and washed with ethyl acetate (13.5 x). The resulting solution is charged with 20% sodium chloride (10 x) and stirred for 2 h at 25 °C. The layers are separated, and44 of 601609969430.31959952.00068the aqueous layer is washed with ethyl acetate (4.5 x). The organic layers are combined and dried by filtering through a pad of anhydrous sodium sulfate (1 x filter press) with washing with ethyl acetate (2 x). The solvent is concentrated and changed to dichloromethane (6.7 x). B9 is obtained by filtering through a silica gel pad with mixture of n-heptane (1 x) and ethyl acetate (10 x) and then n-heptane (1 x) and ethyl acetate (5 x). This mixture was concentrated to 1-3 V and heptane (10 x) to afford B9.Example 26: Preparation of B4

[0159] B4 is prepared from (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide as depicted in FIG. 5. Synthesis of B4. Potassium phosphate monobasic (0.080 x) and potassium phosphate dibasic trihydrate (0.20 x) and Triton X-100 (0.60 x) is dissolved in water (30 x). The temperature is adjusted to 25 °C and the mixture is stirred for 30 min at 25 °C until all solids are dissolved, p-nicotinamideadeninedinucleotide phosphate sodium salt (0.050 x) along with the KRED enzyme (e.g., KRED-A6, e.g., KRED-A6-P2D5) (0.50 x) are added and stirred for 30 min at 25 °C until dissolved. In a separate vessel, (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide (1.0 x) is added to isopropyl alcohol (1.5 x) and the temperature is adjusted to 25 °C and stirred for 30 min until all solids dissolve. The solution of (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide is added to the enzymatic mixture over 30 min and the temperature is adjusted to 30 °C. This mixture is stirred for 24-72 h until HPLC indicates an IPC purity of [INT4] / [INT4+INT5] of <2%. When not obtained, (3-nicotinamideadeninedinucleotide phosphate sodium salt (0.020 x) along with the KRED enzyme (0.20 x) dissolved in water (1.0 x) is added and the mixture is stirred for 24-72 h until HPLC indicates an IPC purity of [INT4] / [INT4+INT5] of <2%. Additional KRED enzyme and p-nicotinamideadeninedinucleotide phosphate sodium salt are added when needed to reach this IPC. Once obtained, acetonitrile (12 x) and diatomite (2 x) are added, and the temperature is adjusted to 50 °C. After stirring for 4 h at 50 °C the temperature is reduced to 25 °C and the resulting solution is filtered through a pad of diatomite (1 x) and washed with ethyl acetate (13.5 x). The resulting solution is charged with 20% sodium chloride (10 x) and stirred for 2 h at 25 °C. The layers are separated, and the aqueous layer is washed with ethyl acetate (4.5 x). The organic layers are combined and dried by filtering through a pad of anhydrous sodium sulfate (1 x filter press) with washing with ethyl acetate (2 x). The solvent is concentrated and changed to dichloromethane (6.7 x). B4 is obtained by filtering through a silica gel pad with mixture of n-heptane (1 x) and ethyl acetate (10 x) and then n-heptane (1 x) and ethyl acetate (5 x). Solvents are removed to provide isolated B4.45 of 601609969430.3

Claims

1959952.00068CLAIMSWe claim:

1. A method of preparing a compound,, wherein J1is an alkylsilanyl or an arylalkyl silanyl, comprising reacting a reactant having a formula,with a ketoreductase enzyme to form the compound.

2. A method of claim 1, wherein the compound is,the reactant is,46 of 601609969430.31959952.000683. The method of claim 1 or 2, wherein the reactant and the ketoreductase enzyme are reacted in a mixture comprising a solvent system, optionally wherein the solvent system comprises isopropanol or water, or isopropanol and water.

4. The method of one of claims 1-3, wherein the reactant and the ketoreductase enzyme are reacted in a mixture comprising a nicotinamide adenine dinucleotide phosphate (e.g., a p-nicotinamide adenine dinucleotide phosphate sodium salt), optionally wherein the reactant and the ketoreductase enzyme are reacted at about 20 °C to about 35 °C (e.g., about 25 °C to about 30 °C).

5. A method of preparing Compound A,(Compound A), Compound B,(Compound B), or Pladienolide B,(Pladienolide B), the method comprising the method of one of claims 1-4.47 of 601609969430.31959952.00068A composition, comprising:Compound A having a formulaone or more of:hexafluoro-2-propanol, and wherein the composition comprises less than 500 ppm of hexafluoro-2-propanol,INT9, and wherein the composition comprises less than 1,000 ppm of INT9, and INT9 having a formulaa Pd, and wherein the composition comprises less than 1 ppm of the Pd, or an Sn, and wherein the composition comprises less than 20 ppm of the Sn.

7. The composition of claim 6, wherein the composition further comprises one or more of:more than 99% w / w (e.g., at least 99.4% w / w or at least 99.8%) of Compound A;Compound A in the form of an amorphous solid form as determined by x-ray powder diffraction;Compound A in greater than 99% chiral purity as determined by high-performance liquid chromatography; orless than 2% w / w of water.

8. The composition of claim 6 or 7, wherein Compound A is in a solid form.48 of 601609969430.31959952.000689. The composition of claim 8, wherein the solid form is a powder.

10. The composition of one of claims 6-8, wherein the composition is a particle.

11. The composition of claim 10, comprising a plurality of the particles.

12. The composition of one of claims 6-8, comprising:A) at least 99.4% w / w Compound A, not more than 1 ppm of Pd, not more than 20 ppm of Sn, not more than 500 ppm of hexafluoro-2-propanol, and not more than 1,000 ppm of INT9;B) at least 99.8% w / w Compound A, not more than 1 ppm of Pd, not more than 20 ppm of Sn, not more than 500 ppm of hexafluoro-2-propanol, and not more than 1,000 ppm of INT9;C) at least 99.4% w / w Compound A, a positive amount of Pd the positive amount of Pd being not more than 1 ppm, a positive amount of Sn the positive amount of Sn being not more than 20 ppm, a positive amount of hexafluoro-2-propanol the positive amount of hexafluoro-2-propanol being not more than 500 ppm, and a positive amount of INT9 the positive amount of INT9 being not more than 1,000 ppm; orD) at least 99.8% w / w Compound A, a positive amount of Pd the positive amount of Pd being not more than 1 ppm, a positive amount of Sn the positive amount of Sn being not more than 20 ppm, a positive amount of hexafluoro-2-propanol the positive amount of hexafluoro-2-propanol being not more than 500 ppm, and a positive amount of INT9 the positive amount of INT9 being not more than 1,000 ppm;wherein the composition optionally comprises less than 0.6% w / w water.

13. The composition of claim 12, which is in the form of a solid particle or a powder.

14. A composition, comprising a plurality of solid particles, each solid particle being the composition of claim 12.49 of 601609969430.31959952.0006815. The composition of one of claims 6-14, wherein the composition comprises at least 400 g (e.g., at least 1.25 kg) of Compound A.

16. The composition of one of claims 6-15, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

17. A method of preparing the composition of one of claims 6-16, wherein Compound A is prepared by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

18. A method of preparing the composition of one of claims 6-16, wherein Compound A is prepared by a method comprising a method of synthesis that comprises, sequentially:1) reacting INT6,INT6,with N-iodosuccinimide to form INT7,50 of 601609969430.31959952.00068INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of camphorsulfonic acid to form INT8,INT8;3) reacting INT8 with INT10,INT10;in the presence of a palladium catalyst to form Compound A.

19. The method of claim 18, wherein INT10 is prepared by a method comprising a method of synthesis that comprises, sequentially:1) reacting INT9,with tributyltin hydride to form INT10; and51 of 601609969430.31959952.000682) optionally a method of purification of INT10 that comprises, sequentially: a silica gel chromatography purification step, a supercritical fluid chromatography (SFC) purification step, and a concentration purification step.

20. The method of claim 18 or 19, wherein Compound A is prepared by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

21. A method of purifying Compound A, wherein Compound A has a formulathe method comprising, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;4) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;52 of 601609969430.31959952.000687) a concentration and lyophilization purification step; and8) an optional sieving step.

22. A method of synthesizing Compound A, wherein Compound A has a formulathe method comprising, sequentially:1) reacting INT6,INT6,with N-iodosuccinimide to form INT7,INT7;2) reacting INT7 with 1,1,1-trimethoxyethane in the presence of camphorsulfonic acid to form INT8,53 of 601609969430.31959952.00068INT8;3) reacting INT8 with INT10INT10;to form Compound A.

23. The method of claim 22, wherein INT10 is prepared by a method comprising a method of synthesis that comprises, sequentially:1) reacting INT9,INT9,with tributyltin hydride to form INT10; and2) optionally a method of purification of INT10 that comprises, sequentially: a silica gel chromatography purification step, a supercritical fluid chromatography (SFC) purification step, and a concentration purification step.

24. The method of claim 23, wherein Compound A is purified by a method comprising a method of purification that comprises, sequentially:1) a silica gel chromatography purification step;2) a potassium carbonate-silica gel chromatography purification step;3) an optional thiosilica gel chromatography purification step;54 of 601609969430.31959952.000684) a reverse phase high-performance liquid chromatography purification step;5) a first supercritical fluid chromatography (SFC) purification step;6) an optional second supercritical fluid chromatography (SFC) purification step;7) a concentration and lyophilization purification step; and8) an optional sieving step.

25. A compound having a formulawherein the compound is prepared by the method of one of claims 17-24.

26. A composition, comprising the compound of claim 25, optionally including a pharmaceutically acceptable carrier.

27. A composition, comprising:Compound A having a formulaone or more of:hexafluoro-2-propanol, and wherein the composition comprises less than 500 ppm of hexafluoro-2-propanol,INT9, and wherein the composition comprises less than 1,000 ppm of INT9, and INT9 having a formula55 of 601609969430.31959952.00068a Pd, and wherein the composition comprises less than 1 ppm of the Pd, or an Sn, and wherein the composition comprises less than 20 ppm of the Sn, and wherein the composition is prepared by the method of one of claims 12-19.

28. A method, comprising administration of the composition of any one of claims 6-16, 26, or 27 inclusive, or the compound of claim 25, to a subject.

29. A method of inhibiting ADARlpl50 in a subject in need thereof, comprising administration of an effective amount of the composition of any one of claims 6-16, 26, or 27 inclusive, or the compound of claim 25, to a subject in need thereof.

30. The method of claim 28 or 29, wherein the subject suffers from a cancer.

31. A solid form of a compound, the compound selected from INT5, INT6, INT7, or INT8.

32. The solid form of claim 31, wherein:the compound is INT5, and the solid form is prepared by a method comprising precipitating INT5 from a solution, the solution comprising INT5 and a solvent system, the solvent system comprising n-heptane (e.g., at least or about 50% v / v n-heptane or at least or about 90% v / v n-heptane, e.g., about 9:1 by volume n-heptane to ethyl acetate);the compound is INT6, and the solid form is prepared by a method comprising precipitating INT6 from a solution, the solution comprising INT6 and a solvent system, the solvent system comprising n-heptane (e.g., at least or about 50% v / v n-heptane e.g., about 1:1 by volume n-heptane to ethyl acetate);the compound is INT7, and the solid form is prepared by a method comprising precipitating INT7 from a solution, the solution comprising INT7 and a solvent system (e.g., the solvent56 of 601609969430.31959952.00068system comprising an organic solvent, e.g., dichloromethane), the solution being concentrated until the INT7 precipitates; orthe compound is INT8, and the solid form is prepared by a method comprising precipitating (e.g., crystallizing) INT8 from a solution, the solution comprising INT8 and a solvent system, the solvent system comprising n-heptane (e.g., at least or about 50% v / v n-heptane, e.g., about 1:1 by volume n-heptane to methyl-tert-butyl ether).

33. The solid form of claim 31 or 32, wherein the solid form is a powder or crystalline form.

34. A composition comprising the solid form of one of claims 31-33, wherein the composition comprises at least 95% w / w of the compound.

35. A composition comprising the solid form of one of claims 31-33, wherein the composition comprises about 99%, 98%, 97%, 96%, or 95% w / w of the compound.

36. A method of preparing a compound,comprising reacting a reactant having a formula,with a ketoreductase enzyme to form the compound.

37. A method of preparing a compound,comprising reacting a reactant having a formula,57 of 601609969430.31959952.00068 / with an epoxidase enzyme to form the compound.

38. The method of claim 36 or 37, wherein the reactant and the ketoreductase enzyme, or the reactant and the epoxidase enzyme, are reacted in a mixture comprising a solvent system, optionally wherein the solvent system comprises isopropanol or water, or isopropanol and water.

39. The method of one of claims 36-38, wherein the reactant and the ketoreductase enzyme are reacted in a mixture comprising a nicotinamide adenine dinucleotide phosphate (e.g., a p-nicotinamide adenine dinucleotide phosphate sodium salt), optionally wherein the reactant and the ketoreductase enzyme are reacted at about 20 °C to about 35 °C (e.g., about 25 °C to about 30 °C).

40. A composition, comprising a reactant compound and a reaction product compound, the composition comprising a ratio of the amount of the reactant to the sum of the amount of the reactant and the amount of the reaction product, the ratio being less than 2%, wherein: the reactant is B8 and the reaction product is B9, and wherein the composition optionally includes an epoxidase enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system;the reactant is INT4 and the reaction product is INT5, and wherein the composition optionally includes a KRED enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system;the reactant is (R)-N-methoxy-N,2-dimethyl-3-oxopentanamide and the reaction product is B4, and wherein the composition optionally includes a KRED enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system; orthe reactant is INT5 and the reaction product is INT6, and wherein the composition optionally includes an RO enzyme, and optionally includes water or isopropanol or both water and isopropanol in a solvent system.58 of 601609969430.31959952.0006841. A method of preparing the composition of claim 40, comprising combining the reactant, the enzyme, the solvent mixture, a buffer, and a cofactor thereby forming a reaction mixture, and incubating the reaction mixture such that the composition is prepared,wherein the reaction mixture comprises a ratio of the amount of the reaction product to the sum of the amount of the reactant and the amount of the reaction product, the ratio being less than 2%.59 of 601609969430.3

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