Processes for preparing MDM2 inhibitor

Abstract

To provide a process for preparing MDM2 inhibitors useful for treating solid tumors such as breast, colon, lung, and prostate tumors, as well as liquid tumors such as lymphomas and leukemias.SOLUTION: Provided are commercial processes for preparing 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid and intermediates thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention provides a process for preparing 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid ("Compound A") and intermediates thereto. [Background technology]

[0002] p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which rarely have p53 activation, tumor cells are constantly under cellular stress from a variety of insults, including hypoxia and proapoptotic oncogene activation. Therefore, it has been proposed that there is a strong selective advantage for inactivating the p53 pathway in tumors, and that elimination of p53 function may be a prerequisite for tumor survival. In support of this concept, three groups of researchers have used mouse models to demonstrate that the absence of p53 function is a continuous requirement for the maintenance of established tumors. When researchers restored p53 function to tumors with inactivated p53, the tumors regressed.

[0003] p53 is inactivated by mutation and / or loss in 50% of solid tumors and 10% of liquid tumors. Other key members of the p53 pathway are also genetically or epigenetically altered in cancer. The oncoprotein MDM2 inhibits p53 function and is activated by gene amplification in incidences reported as high as 10%. MDM2 is in turn inhibited by another tumor suppressor, p14ARF. Downstream alterations of p53 can lead to p53 WT It has been suggested that this may be responsible for at least partial inactivation of the p53 pathway in tumors. WTTumors appear to exhibit a reduced apoptotic capacity, while their ability to undergo cell cycle arrest remains intact. One cancer treatment protocol involves the use of small molecules that bind to MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation, 2) binding to and blocking the p53 transcriptional activation domain, and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms are blocked by neutralizing the MDM2-p53 interaction. In particular, this therapeutic protocol inhibits p53 WT MDM2 inhibition may be applicable to tumors with p53-inactivated tumours, and studies with small molecule MDM2 inhibitors have produced promising reductions in tumor growth both in vitro and in vivo. Furthermore, in patients with p53-inactivated tumours, stabilisation of wild-type p53 in normal tissues by MDM2 inhibition may allow selective protection of normal tissues from mitotic toxins. Summary of the Invention

[0004] The present invention relates to compounds that can inhibit the interaction between p53 and MDM2 and activate p53 downstream effector genes. Therefore, the compounds of the present invention are useful in treating cancer, bacterial infections, viral infections, ulcers, and inflammation. In particular, the compounds of the present invention are useful for treating solid tumors such as breast, colon, lung, and prostate tumors, and liquid tumors such as lymphoma and leukemia. As used herein, MDM2 refers to human MDM2 protein, and p53 refers to human p53 protein. Human MDM2 can also be referred to as HDM2 or hMDM2.

[0005] The compound, 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (sometimes referred to herein as Compound A), is an MDM2 inhibitor and has the following chemical structure: Compound A is disclosed in PCT Application Publication No.

[0006] [ka] It has been disclosed in WO2011 / 153509 (Example 362) and has been studied in human clinical trials for the treatment of various cancers. The present invention provides improved methods for preparing Compound A, as well as intermediate compounds thereof.

[0007] In one embodiment, the present invention provides the following compound (DHO):

[0008] [ka] A method for preparing a compound (ABA)

[0009] [ka] with methoxymethylene-N,N-dimethyliminium methyl sulfate. In one embodiment, the reaction is carried out in the presence of a base. In certain embodiments, the base is an alkali metal salt or an alkaline earth metal salt, such as KOAc, NaOAc, LiOAc, CaCO3, and K2CO3. In one embodiment, the reaction is carried out in a solvent. In certain embodiments, the solvent is benzene, toluene, o-xylene, m-xylene, p-xylene, hexane, tetrahydrofuran, ethyl acetate, HMPA, HMPT, DMSO, ethylene glycol, DME, DMF, diethyl ether, acetonitrile, methanol, ethanol, acetone, or a mixture thereof.

[0010] In one embodiment, the present invention provides the compound (SUL)

[0011] [ka] A method for preparing the compound

[0012] [ka] with an isopropylating agent, such as, but not limited to, isopropylsulfinate zinc chloride. In one embodiment, the reaction is carried out in the presence of an alkaline earth metal salt. In certain embodiments, the alkaline earth metal salt is a magnesium salt, such as, but not limited to, MgBr or MgCl. In certain embodiments, the isopropylating agent is generated in situ from isopropyl magnesium chloride. In one embodiment, the reaction is carried out at a temperature of 100° C. to 200° C., such as, for example, 100° C. to 150° C., such as, for example, 120° C., or 150° C. to 200° C., such as, for example, 180° C.

[0013] In one embodiment, the invention provides a crystalline form of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO), characterized by a reflection X-ray powder diffraction pattern comprising peaks at 7.3°±0.2° 2θ, 14.5°±0.2° 2θ, 15.8°±0.2° 2θ, 15.9°±0.2° 2θ, and 23.1°±0.2° 2θ. In one embodiment, the reflection X-ray powder diffraction pattern of the DHO crystalline further comprises peaks at 8.5°±0.2° 2θ, 10.0°±0.2° 2θ, 11.0°±0.2° 2θ, 13.4°±0.2° 2θ, 18.8°±0.2° 2θ, and 22.0°±0.2° 2θ. In one embodiment, the reflection X-ray powder diffraction pattern of the DHO crystalline further comprises peaks at 6.3°±0.2° 2θ, 10.5°±0.2° 2θ, 11.5°±0.2° 2θ, 12.8°±0.2° 2θ, 14.8°±0.2° 2θ, 15.2°±0.2° 2θ, 17.0°±0.2° 2θ, and 17.5°±0.2° 2θ. 2° 2θ, 17.8° ± 0.2° 2θ, 18.4° ± 0.2° 2θ, 19.0° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, 20.7° ± 0.2° 2θ, 21.2° ± 0.2° 2θ, 21.3° ± 0.2° 2θ, 22.4° ± 0.2° 2θ, 23. 6°±0.2° 2θ, 24.2°±0.2° 2θ, 24.9°±0.2° 2θ, 25.7°±0.2° 2θ, 26.3°±0.2° 2θ, 27.0°±0.2° 2θ, 28.3°±0.2° 2θ, 28.7°±0.2° 2θ, 29.3°±0.2° 2θ, 29.7°±0.2°

[0033] The crystalline form of DHO further comprises one or more peaks at: 30.8°±0.2° 2θ, 31.4°±0.2° 2θ, 31.8°±0.2° 2θ, 33.0°±0.2° 2θ, 34.2°±0.2° 2θ, 35.8°±0.2° 2θ, 37.0°±0.2° 2θ, and 37.5°±0.2° 2θ. In one embodiment, the crystalline form of DHO is a crystalline anhydrate. In one embodiment, reflection X-ray powder diffraction of the crystalline DHO is performed using Cu-Kα radiation.

[0014] The following figures depict specific embodiments of the described invention and are not intended to limit the invention in any way. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the rate of conversion of (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (DLAC) to (S)-2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1-hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide (ABA) over time at 60°C. [Figure 2] 1 shows the rate of conversion of DLAC to ABA over time at 115° C. [Figure 3] Figure 1 shows the solubility of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO) during the crystallization process at 25°C. [Figure 4] Yield of (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one (SUL) over time using isopropylsulfinate magnesium chloride at 120 °C (14 mol% water in the reaction mixture vs. (3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium naphthalene-1-sulfonate, hemitoluene solvate (OXOS)). [Figure 5] Figure 1 shows the yield of SUL over time using isopropylsulfinate magnesium chloride at 180°C (11 mol% water (vs. OXOS) in the reaction mixture). [Figure 6] 1H NMR analysis of different isopropyl sulfinate species in THF-d8. [Figure 7] Figure 1 shows the yield of SUL over time using Mg sulfinate-ZnCl2 at 120 °C (17 mol% water (vs. OXOS) in the reaction mixture). [Figure 8] Figure 1 shows the yield of SUL over time using Mg sulfinate-ZnCl2 at 180 °C (17 mol% water (vs. OXOS) in the reaction mixture). [Figure 9] Figure 1 shows the LC area % of (3R,5R,6S)-3-((1,2,4-trioxolan-3-yl)methyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one (OZO) compared to the weight % of water in the reaction mixture at 20°C. [Figure 10] 1 shows a schematic of an apparatus for continuous mode ozonolysis and Pinnic oxidation. [Figure 11] A photograph of the continuous ozone decomposition treatment device is shown. [Figure 12] 1 shows a schematic of an apparatus for semi-batch mode ozonolysis and Pinnic oxidation. [Figure 13] 1 shows the consumption rate of SUL for semi-batch mode ozonolysis. [Figure 14] The evolution of spargers for ozonolysis production development is shown. [Figure 15] 2 shows the solubility of 232-DAB during the crystallization process. [Figure 16] 1 shows the solubility of Compound A during the crystallization process. [Figure 17] 1 shows the powder X-ray diffraction (PXRD) pattern of crystalline DHO measured in reflectance mode. [Figure 18] 1 shows the powder X-ray diffraction (PXRD) pattern of crystalline DHO measured in reflectance mode, with sticks indicating peak positions. [Figure 19] 1 shows a thermogram from a differential scanning calorimetry (DSC) analysis of crystalline DHO. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides methods for preparing 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid ("Compound A"), as well as intermediates thereto and methods for preparing these intermediates.

[0017] In one aspect, the present invention provides a method for producing highly pure Compound A.

[0018] In another aspect, the present invention employs the bench-stable Vilsmeier reagent, methoxymethylene-N,N-dimethyliminium methylsulfate (Corbett, MT; Caille, S., Synlett 2007, 28, 2845), to achieve selective in situ activation of a primary alcohol intermediate in the preparation of compound A.

[0019] In another aspect, the present disclosure employs a bench-stable, crystalline isopropyl calcium sulfinate to achieve a high-yield preparation of the sulfone intermediate in the preparation of Compound A.

[0020] In another aspect, the present disclosure employs a safe ozonolysis reaction carried out in an aqueous solvent mixture in either a batch or continuous manufacturing mode in a method for preparing Compound A.

[0021] In another aspect, the present invention provides a crystalline form of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO), characterized by a reflection X-ray powder diffraction pattern comprising peaks at 7.3°±0.2° 2θ, 14.5°±0.2° 2θ, 15.8°±0.2° 2θ, 15.9°±0.2° 2θ, and 23.1°±0.2° 2θ. In one embodiment, the reflection X-ray powder diffraction pattern of the DHO crystalline further comprises peaks at 8.5°±0.2° 2θ, 10.0°±0.2° 2θ, 11.0°±0.2° 2θ, 13.4°±0.2° 2θ, 18.8°±0.2° 2θ, and 22.0°±0.2° 2θ. In one embodiment, the reflection X-ray powder diffraction pattern of the DHO crystalline further comprises peaks at 6.3°±0.2° 2θ, 10.5°±0.2° 2θ, 11.5°±0.2° 2θ, 12.8°±0.2° 2θ, 14.8°±0.2° 2θ, 15.2°±0.2° 2θ, 17.0°±0.2° 2θ, and 17.5°±0.2° 2θ. 2° 2θ, 17.8° ± 0.2° 2θ, 18.4° ± 0.2° 2θ, 19.0° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, 20.7° ± 0.2° 2θ, 21.2° ± 0.2° 2θ, 21.3° ± 0.2° 2θ, 22.4° ± 0.2° 2θ, 23. 6°±0.2° 2θ, 24.2°±0.2° 2θ, 24.9°±0.2° 2θ, 25.7°±0.2° 2θ, 26.3°±0.2° 2θ, 27.0°±0.2° 2θ, 28.3°±0.2° 2θ, 28.7°±0.2° 2θ, 29.3°±0.2° 2θ, 29.7°±0.2°

[0033] The crystalline form of DHO further comprises one or more peaks at: 30.8°±0.2° 2θ, 31.4°±0.2° 2θ, 31.8°±0.2° 2θ, 33.0°±0.2° 2θ, 34.2°±0.2° 2θ, 35.8°±0.2° 2θ, 37.0°±0.2° 2θ, and 37.5°±0.2° 2θ. In one embodiment, the crystalline form of DHO is a crystalline anhydrate. In one embodiment, reflection X-ray powder diffraction of the crystalline DHO is performed using Cu-Kα radiation.

[0022] In another aspect, the present disclosure provides control of the purity of Compound A by crystallization of the 1,4-diazabicyclo[2.2.2]octane (DABCO) salt of Compound A, which can be effectively purified.

[0023] In one embodiment, the present invention provides a method suitable for scale-up to prepare compound A from the starting material DLAC in 49.8% overall yield (99.9 LC area %).

[0024] The term "comprising" is intended to be open-ended, including the indicated component but not excluding other elements.

[0025] The term "therapeutically effective amount" refers to an amount of a compound or combination of therapeutically active compounds that ameliorates, attenuates, or eliminates one or more symptoms of a particular disease or condition, or prevents or delays the onset of one of more symptoms of a particular disease or condition.

[0026] The terms "patient" and "subject" are sometimes used interchangeably and refer to animals, such as dogs, cats, cows, horses, sheep, and humans. Particular patients are mammals. The term patient includes men (male) and women (female).

[0027] The term "pharmaceutically acceptable" means that the referenced substance, e.g., a compound of the invention, or a salt of said compound, or a formulation containing said compound, or a particular excipient, is suitable for administration to a patient.

[0028] Terms such as "treating," "treat," or "treatment" include preventative (eg, prophylactic) and palliative treatment.

[0029] The term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), that is typically included in a formulation and / or for administration to a patient.

[0030] The compounds of the present invention can be administered to patients in a therapeutically effective amount. The compounds can be administered alone or as part of a pharmaceutically acceptable composition or formulation. Additionally, the compounds or compositions can be administered multiple times, such as by bolus injection, all at once, by a series of tablets, or delivered substantially uniformly over a period of time, such as by transdermal delivery. It is also noted that the dose of the compound can be varied over time.

[0031] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may also be administered in combination with one or more additional pharmaceutically active compounds / agents. It is noted that the additional pharmaceutically active compounds / agents may be traditional small organic chemical molecules, or may be macromolecules, such as proteins, antibodies, peptibodies, DNA, RNA, or fragments of such macromolecules.

[0032] When a patient is given or has been given multiple pharmaceutically active compounds, the compounds can be administered simultaneously or sequentially.For example, in the case of tablets, the active compounds can be found in one tablet or in separate tablets, and the separate tablets can be administered at once or sequentially in any order.In addition, it should be recognized that the composition can be in different forms.For example, one or more compounds can be delivered via tablets, and another can be administered via injection or orally as syrup.All combinations, delivery methods and administration sequences are envisioned.

[0033] The term "cancer" refers to the physiological condition in mammals that is characterized by unregulated cell growth. General classes of cancer include carcinoma, lymphoma, sarcoma, and blastoma.

[0034] The compounds of the present invention can be used to treat cancer. The method of treating cancer comprises administering to a patient in need thereof a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt thereof.

[0035] The compounds of the present invention can be used to treat tumors. The method of treating tumors comprises administering a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

[0036] The invention also relates to the use of a compound of the invention in the manufacture of a medicament for the treatment of a condition such as cancer.

[0037] Cancers that may be treated using the compounds of the invention include carcinomas, such as cancers of the bladder, breast, colon, rectum, kidney, liver, lung (small cell lung cancer and non-small cell lung cancer), esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage (leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and leukemia); The present invention relates to a compound that can be used to treat various cancers, including, but not limited to, lymphoma, hairy cell lymphoma, and Burkett's lymphoma; hematopoietic tumors of myeloid lineage (including acute and chronic myeloid leukemia, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma, and other sarcomas, such as soft tissue and bone); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma, and schwannoma); and other tumors (including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, follicular thyroid carcinoma, and Kaposi's sarcoma). Other cancers that can be treated using the compounds of the present invention include endometrial cancer, head and neck cancer, glioblastoma, malignant ascites, and hematopoietic cancer.

[0038] Particular cancers that may be treated with the compounds of the invention include soft tissue sarcomas, bone cancers such as osteosarcoma, breast tumors, bladder cancer, Li-Fraumeni syndrome, brain tumors, rhabdomyosarcoma, adrenocortical carcinoma, colorectal cancer, non-small cell lung cancer, and acute myeloid leukemia (AML).

[0039] In certain embodiments of the invention relating to the treatment of cancer, the cancer is p53 wild type (p53 WT In another particular embodiment, the cancer is identified as p53 WT and CDKN2A mutants. In another aspect, the present invention provides diagnostics for determining which patients should be administered the compounds of the present invention. For example, a sample of the patient's cancer cells may be obtained and analyzed to determine the status of the cancer cells with respect to p53 and / or CDKN2A. In one aspect, WT Patients with cancers that are p53 are selected for treatment over patients with cancers that are mutated with respect to p53. WT and patients with cancers that have mutant CDNK2A proteins are selected over patients that do not have these characteristics. The collection of cancer cells for analysis is well known to those skilled in the art. WT The term "CDNK2A mutant" refers to the protein encoded by genomic DNA SEQ ID NO: NC_000017 version 9 (7512445..7531642) (GenBank); the protein encoded by cDNA SEQ ID NO: NM_000546 (GenBank); or the protein having GenBank SEQ ID NO: NP_000537.3. The term "CDNK2A mutant" means a CDNK2A protein that is not wild-type. The term "CDNK2A wild-type" refers to the protein encoded by genomic DNA SEQ ID NO: 9:21957751-21984490 (Ensembl ID); the protein encoded by cDNA SEQ ID NO: NM_000077 (GenBank) or NM_058195 (GenBank); or the protein having GenBank SEQ ID NO: NP_000068 or NP_478102.

[0040] In another aspect, the present invention relates to the use of compounds of the present invention in combination with one or more pharmaceutical agents that are inhibitors of proteins in the phosphatidylinositol 3-kinase (PI3K) pathway. Combination of compounds of the present invention with inhibitors of proteins in the PI3K pathway has shown synergistic effects in cancer cell proliferation assays, including enhanced apoptosis and cell killing. Examples of proteins in the PI3K pathway include PI3K, mTOR, and PKB (also known as Akt). PI3K proteins exist in several isoforms, including α, β, δ, and γ. It is envisioned that PI3K inhibitors that can be used in combination with compounds of the present invention may be selective for one or more isoforms. By selective, we mean that a compound inhibits one or more isoforms over other isoforms. Selectivity is a concept well known to those skilled in the art and can be measured using known activities in in vitro or cell-based assays. Preferred selectivity includes greater than 2-fold, preferably greater than 10-fold, or more preferably greater than 100-fold selectivity for one or more isoforms over other isoforms. In one embodiment, the PI3K inhibitor that may be used in combination with the compounds of the invention is a PI3K alpha selective inhibitor, hi another embodiment, the compound is a PI3K delta selective inhibitor.

[0041] Examples of PI3K inhibitors that can be used in combination with the compounds of the present invention include those described in, for example, WO2010 / 151791, WO2010 / 151737, WO2010 / 151735, WO2010 / 151740, WO2008 / 118455, WO2008 / 118454, WO2008 / 118468, US20100331 293, US20100331306, US20090023761, US20090030002, US20090137581, US20090054405, US20090163489, US20100273764, US20110092504, or those disclosed in WO2010 / 108074.

[0042] Compounds that inhibit both PI3K and mTOR (dual inhibitors) are known. In yet another aspect, the present invention provides the use of a dual PI3K and mTOR inhibitor for use in combination with a compound of the present invention.

[0043] mTOR is a protein in PI3K pathway.The use of mTOR inhibitor in combination with the compound of the present invention is another aspect of the present invention.Suitable mTOR inhibitor that can be used in combination with the compound of the present invention includes, for example, those disclosed in WO2010 / 132598 and WO2010 / 096314.

[0044] PKB (Akt) is also a protein in PI3K pathway.The use of mTOR inhibitor in combination with the compound of the present invention is another aspect of the present invention.The PKB inhibitor that can be used in combination with the compound of the present invention includes, for example, those disclosed in US7,354,944, US7,700,636, US7,919,514, US7,514,566, US20090270445A1, US7,919,504, US7,897,619 and WO2010 / 083246.

[0045] The combinations of the present invention may also be used in combination with radiation therapy, hormone therapy, surgery and immunotherapy, which are well known to those skilled in the art.

[0046] Since one embodiment of the present invention contemplates the treatment of diseases / conditions using a combination of pharmaceutically active compounds that may be administered separately, the present invention further relates to combining separate pharmaceutical compositions in the form of a kit. The kit comprises two separate pharmaceutical compositions: a compound of the present invention and a second pharmaceutical compound. The kit includes containers for containing the separate compositions, such as divided bottles or divided foil packets. Additional examples of containers include syringes, boxes, and bags. Typically, the kit includes instructions for use of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician or veterinarian.

[0047] An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms (such as tablets and capsules). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. The tablets or capsules are then placed in the recesses, and the sheet of relatively stiff material is sealed to the plastic foil on the side of the foil opposite to the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably, the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure to the recesses, thereby forming openings in the sheet at the locations of the recesses. The tablets or capsules can then be removed through said openings.

[0048] It may be desirable to provide a memory aid on the kit, for example, in the form of numbers next to the tablets or capsules, whereby the numbers correspond to the days of the regimen on which the designated tablets or capsules should be taken. Another example of such a memory aid is a calendar printed on a card, for example, "Week 1, Monday, Tuesday, ... etc., Week 2, Monday, Tuesday, ...", etc. Other variations of memory aids will be readily apparent. A "daily dose" may be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a compound of the present invention may consist of one tablet or capsule, while a daily dose of a second compound may consist of several tablets or capsules, or vice versa. The memory aid should reflect this and assist in the correct administration of the active agents.

[0049] In another specific embodiment of the present invention, a dispenser is provided that is designed to dispense the daily doses one by one in the order of their intended use. Preferably, the dispenser is equipped with a memory aid to further encourage compliance with the regimen. An example of such a memory aid is a mechanical counter that indicates the number of daily doses that have been dispensed. Another example of such a memory aid is a battery-powered microchip memory coupled with a liquid crystal readout or an audible reminder signal, which, for example, reads the date and time the last daily dose was taken and / or reminds the user when the next dose should be taken.

[0050] The compounds of the present invention, and if desired, other pharmaceutically active compounds, can be administered to a patient orally, rectally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously), intravesically, vaginally, intraperitoneally, intravesically, topically (e.g., as a powder, ointment, or drops), or as a buccal or nasal spray. All methods used by those skilled in the art to administer pharmaceutically active agents are contemplated.

[0051] Suitable compositions for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, and glycerol), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0052] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Microbial contamination can be prevented by adding various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0053] Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (a) dissolution inhibitors. (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin and bentonite; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, dosage forms may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols, etc.

[0054] Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other well-known in the art.They can also contain opacifiers, and can be of a composition that releases one or more active compounds in a certain part of the intestinal tract in a delayed manner.Examples of embedding compositions that can be used are polymeric substances and waxes.Active compounds can also be in the form of microencapsulation with one or more of the above-mentioned excipients, if appropriate.

[0055] The liquid dosage form for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.In addition to active compound, liquid dosage form can contain the inert diluent commonly used in this field, such as water or other solvent, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame seed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, or the mixture of these substances.

[0056] Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents. Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances.

[0057] Compositions for rectal administration are preferably suppositories, which can be prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at ordinary room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active ingredient.

[0058] The dosage forms for external administration of the compounds of the present invention include ointments, powders, sprays and inhalants.One or more active compounds are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants that may be required.Ophthalmic preparations, eye ointments, powders and solutions are also contemplated within the scope of the present invention.

[0059] The compounds of the present invention may be administered to patients at dosage levels ranging from about 0.1 to about 3,000 mg per day. For a normal human adult weighing about 70 kg, a dosage ranging from about 0.01 to about 100 mg per kilogram of body weight is typically sufficient. The specific dosage and dosage range that can be used will depend on many factors, including the patient's needs, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. Determination of dosage ranges and optimal dosages for a particular patient is within the skill of one of ordinary skill in the art.

[0060] The compounds of the present invention can be administered as pharmaceutically acceptable salts, esters, amides, or prodrugs. The term "salt" refers to inorganic and organic salts of the compounds of the present invention. Salts can be prepared in situ during the final isolation and purification of the compounds, or by reacting the purified compound in its free base or acid form with a suitable organic or inorganic base or acid and isolating the salt thus formed.

[0061] Examples of pharmaceutically acceptable esters of the compounds of the present invention include C1-C8 alkyl esters.Acceptable esters also include C5-C7 cycloalkyl esters, as well as aryl alkyl esters, such as benzyl.C1-C4 alkyl esters are commonly used.Esters of the compounds of the present invention may be prepared according to methods well known in the art.

[0062] Examples of pharmaceutically acceptable amides of the compounds of the present invention include amides derived from ammonia, primary C1-C8 alkylamines, and secondary C1-C8 dialkylamines. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycloalkyl group containing at least one nitrogen atom. Amides derived from ammonia, C1-C3 primary alkylamines, and C1-C2 dialkyl secondary amines are commonly used. Amides of the compounds of the present invention may be prepared according to methods well known to those skilled in the art.

[0063] The term "prodrug" refers to a compound that is converted in vivo to provide a compound of the invention. The conversion may occur by various mechanisms, such as through hydrolysis in blood. A discussion of the use of prodrugs is provided in T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0064] Illustratively, since the compounds of the present invention contain a carboxylic acid functional group, prodrugs may be prepared by substituting a C1-C8 alkyl, (C2-C 12 ) alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 4 to 10 carbon atoms These may include esters formed by substitution with groups such as 1-(N-(alkoxycarbonyl)aminomethyl, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolactone-4-yl, di-N,N-(C1-C2)alkylamino(C2-C3)alkyl (e.g., β-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl, and piperidino, pyrrolidino, or morpholino(C2-3)alkyl having the atom.

[0065] The compounds of the present invention may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms. All stereoisomeric forms of the compounds, as well as mixtures thereof, including racemic mixtures, are contemplated as part of the present invention. Additionally, the present invention contemplates all geometric and positional isomers. For example, if a compound contains a double bond, both cis and trans forms (designated Z and E, respectively) as well as mixtures thereof are contemplated.

[0066] Mixtures of stereoisomers, such as diastereomeric mixtures, can be separated into their individual stereochemical components on the basis of their physical chemical differences by known methods, such as chromatography and / or fractional crystallization. Enantiomers can also be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (e.g., alcohol), separating the resulting diastereomers, and then converting the individual diastereomers to the corresponding pure enantiomers (e.g., by hydrolysis).

[0067] The compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water (hydrates), ethanol, etc. The present invention contemplates and encompasses both the solvated and unsolvated forms as described herein.

[0068] It is also possible that compounds of the present invention may exist in different tautomeric forms. All tautomeric forms of the compounds of the present invention are contemplated. For example, all tautomeric forms of the tetrazole moiety are included in the present invention. Also, for example, all keto-enol or imine-enamine forms of the compounds are included in the present invention.

[0069] Those skilled in the art will understand that the names and structures of compounds contained herein may be based on a particular tautomer of the compound. Although a name or structure for only a particular tautomer may be used, it is intended that all tautomers are encompassed by the present invention unless otherwise stated.

[0070] The present invention is also intended to encompass compounds synthesized in vitro using laboratory techniques such as those well known to synthetic chemists, or compounds synthesized using in vivo techniques, such as through metabolism, fermentation, digestion, etc. It is also envisioned that the compounds of the present invention may be synthesized using a combination of in vitro and in vivo techniques.

[0071] The present invention also includes isotopically labeled compounds that are identical to the compounds described herein except for the fact that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 16 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, and 36 In one aspect, the present invention provides a compound in which one or more hydrogen atoms are replaced with deuterium ( 2 H) atoms.

[0072] Compounds of the present invention that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, e.g., 3 H and 14 Those in which radioactive isotopes such as C are incorporated are useful in drug and / or substrate tissue distribution assays. 3 H, and carbon-14, i.e., 14 C isotopes are particularly preferred for their ease of preparation and detectability. Additionally, deuterium, i.e., 2Substitution with heavier isotopes such as H can offer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds of the invention can generally be prepared by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0073] The compounds of the present invention may exist in various solid states, including crystalline and amorphous states. Different crystalline states (also called polymorphs) and amorphous states of the compounds of the present invention are contemplated as part of the present invention as described herein.

[0074] In synthesizing the compounds of the present invention, it may be desirable to use certain leaving groups. The term "leaving group" ("LG") generally refers to a group displaceable by a nucleophile. Such leaving groups are known in the art. Examples of leaving groups include, but are not limited to, halides (e.g., I, Br, F, Cl), sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH), N-hydroxysuccinimide, N-hydroxybenzotriazole, and the like. Examples of nucleophiles include, but are not limited to, amines, thiols, alcohols, Grignard reagents, and anionic species (e.g., alkoxides, amides, carbanions), and the like.

[0075] All patents, published patent applications and other publications referenced herein are hereby incorporated by reference.

[0076] The specific experimental examples presented in this application illustrate specific embodiments of the present invention. These examples are intended to be representative and are not intended to limit the scope of the claims in any way.

[0077] 1 H-NMR spectra were typically recorded on a 500.13 MHz Bruker 5 mm PABBI probe with a z-axis gradient.1 A Bruker Avance III 500 spectrometer system (Bruker, Billerica, MA) operating at H frequency or 400.23 MHz with a Bruker 5 mm PABBO probe with z-axis gradient was used. 1 NMR spectra were acquired on a Bruker Avance II or Avance III 400 spectrometer operating at H frequencies. Samples were typically dissolved in 500 μL of DMSO-d or CD 0D for NMR analysis. 1 H chemical shifts are referenced to residual solvent signals from DMSO-d6 at δ 2.50 and CD3OD at δ 3.30.

[0078] Significant peaks are listed, typically including the number of protons, multiplicity (s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet; br s, broad singlet), and coupling constants in Hertz (Hz). Electron ionization (EI) mass spectra were typically recorded on an Agilent Technologies 6140 Quadrupole LC / MS mass spectrometer (Agilent Technologies, Englewood, CO). Mass spectrometry results are reported as the ratio of mass to charge, optionally followed by the relative abundance of each ion (in parentheses). Starting materials in the following examples are typically available from commercial sources such as Sigma-Aldrich, St. Louis, MO, or via published literature procedures.

[0079] X-ray powder diffraction data (XRPD) were obtained using a Bruker D8 Discover X-ray diffraction system (Bruker, Billerica, MA) equipped with a Brownian detector and a Cu-Kα radiation source operating in Bragg-Brentano reflection geometry. 2θ values ​​are generally accurate to within ±0.2°. Samples were generally prepared without any special treatment other than the application of slight pressure to obtain a flat surface. Samples were measured uncovered unless otherwise noted. Operating conditions included a tube voltage of 40 kV and a current of 40 mA. A variable divergence slit was used with a 3° window. The step size was 0.019° 2θ with a step time of 35.2 s, and the scan range was 3–40.4°.

[0080] Differential scanning calorimetry (DSC) was performed using a Perkin Elmer DSC-7 or a TA Instruments Q2000 instrument. Samples were prepared in closed gold sample pans at a temperature ramp rate of 5°C / min from 20°C to approximately 350°C. The DSC thermogram of crystalline DHO is shown in Figure 19 with a melting point at 73.86°C. [Example]

[0081] [Example 1] Methods for Preparing Selected Intermediates

[0082] [ka]

[0083] Step A. 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone [ka]

[0084] Sodium bis(trimethylsilyl)amide (117 mL of 1 M in tetrahydrofuran) was added slowly over 1 h to a −78° C. solution of 2-(3-chlorophenyl)acetic acid (10 g, 58.6 mmol) in tetrahydrofuran (58 mL). After stirring for 40 min at −78° C., a solution of methyl 4-chlorobenzoate (10 g, 58.6 mmol) in tetrahydrofuran (35 mL) was added over a 10 min period. The reaction was stirred at −78° C. for 3 h and then warmed to 25° C. After 2 h at 25° C., the reaction was quenched with saturated aqueous ammonium chloride solution, and most of the tetrahydrofuran was removed under reduced pressure. The residue was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with saturated sodium chloride solution, dried over sodium sulfate, filtered, and the filtrate was concentrated. The product was recrystallized from ether / pentane to give 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone as a white solid.

[0085] Alternative Procedure for Preparing 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone To a mixture of chlorobenzene (170 L, 1684 mol), 3-chlorophenylacetic acid (50 kg, 293 mol), and dimethylformamide (0.7 L, 9 mol) at 0°C was added thionyl chloride (39.1 kg, 329 mol) over the course of 30 minutes. The mixture was warmed to 15°C and stirred for 6 hours. The mixture was cooled to 0°C, and aluminum chloride (43 kg, 322 mol) was added over the course of 1.5 hours. The mixture was warmed to 20°C and stirred for 15 hours. Water (200 L) and ethanol (200 L) were added to the mixture, and the biphasic mixture was stirred for 2 hours. The phases were separated, and the organic phase was washed twice with aqueous ethylenediaminetetraacetic acid tetrasodium salt (3 wt%, 200 L) and once with water (200 L). Heptane (1600 L) was added to the organic phase over the course of 15 minutes. The suspension was stirred for 30 minutes, cooled to -5°C, and filtered. The filtered material was dried at 40°C for 20 hours. 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone was isolated in 83.6% yield (67.4 Kg). 1H NMR(500MHz,DMSO-d6,δ ppm):8.05(m,2H),7.62(m,2H),7.33(m,3H),7.21(br d,J=7.3Hz,1H),4.45(s,2H).MS(ESI)=265.1[M+H] + . Step B: Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

[0086] [ka]

[0087] Methyl methacrylate (12.65 mL, 119 mmol) was added to a solution of 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone (30 g, 113 mmol) (from Step A) in tetrahydrofuran (283 mL). Potassium tert-butoxide (1.27 g, 11.3 mmol) was then added, and the reaction was stirred at room temperature for 2 days. The solvent was then removed in vacuo and replaced with 300 mL of ethyl acetate. The organic phase was washed with brine (50 mL), water (3 × 50 mL), and brine (50 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo to give methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate as a ca. 1:1 mixture of diastereomers. 1 H NMR (400MHz, CDCl3, δ ppm): 7.87 (m, 2H), 7.38 (m, 2H), 7.27-7.14 (series of m,4H), 4.61(m,1H), 3.69(s,1.5H), 3.60(s,1.5H), 2.45(m,1H), 2.34(m,1H), 2.10(ddd,J=13.9,9.4,5.5Hz,0.5 H), 1.96(ddd,J=13.7,9.0,4.3Hz,0.5H), 1.22(d,J=7.0Hz,1.5H), 1.16(d,J=7.0,1.5H).MS(ESI)=387.0[M+23] + . <Step C: (3S,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one and (3R,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one>

[0088] [ka]

[0089] Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate (40 g, 104.0 mmol) (from Step B) was dissolved in 200 mL of anhydrous toluene and concentrated under vacuum. The residue was placed under high vacuum for 2 hours before use. The compound was divided into 2 x 20 g batches and treated as follows: Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate (20 g, 52.0 mmol) in anhydrous 2-propanol (104 mL) was treated with potassium tert-butoxide (2.33 g, 20.8 mmol) in a 250 mL glass hydrogenation vessel. RuCl2(S-xylbinap) (S-DAIPEN) (0.191 g, 0.156 mmol, Strem Chemicals, Inc., Newburyport, MA) in 3.8 mL of toluene was added. After 1.5 hours, the vessel was pressurized to 50 psi (344.7 kPa), purged with hydrogen five times, and stirred at room temperature. The reaction was refilled with additional hydrogen as needed. After 3 days, the reactions were combined and partitioned between 50% saturated ammonium chloride solution and ethyl acetate. The aqueous layer was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over magnesium sulfate, filtered, and concentrated.

[0090] The crude product (primarily (4R,5R)-isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate) was dissolved in tetrahydrofuran (450 mL) and methanol (150 mL). Lithium hydroxide (1.4 M, 149 mL, 208 mmol) was added, and the solution was stirred at room temperature for 24 hours. The mixture was concentrated in vacuo, and the residue was redissolved in ethyl acetate. 1N aqueous hydrochloric acid was added with stirring until the aqueous layer had a pH of approximately 1. The layers were separated, and the organic phase was washed with brine, dried over magnesium sulfate, filtered, and concentrated. The material was then dissolved in 200 mL of anhydrous toluene and treated with pyridinium p-toluenesulfonate (PPTS, 0.784 g, 3.12 mmol). The reaction was heated to reflux under Dean-Stark conditions until the seco acid was consumed (approximately 2 hours). The reaction mixture was cooled to room temperature and washed with saturated sodium bicarbonate (50 mL) and brine (50 mL). The solution was dried over sodium sulfate, filtered, and concentrated. The crude material was purified by flash chromatography on silica gel (120 g column; elution with 100% dichloromethane). (3S,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one and (3R,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one were obtained as white solids with an enantiomeric ratio of approximately 94:6 and a 7:3 mixture of methyl diastereomers. 11H NMR (400 MHz, CDCl3, δ ppm): 7.22 - 6.98 (series of m, 5H), 6.91 (dt, J = 7.4, 1.2 Hz, 0.3H), 6.81 (m, 2H), 6.73 (dt, J = 7.6, 1.4 Hz, 0.7H), 5.76 (d, J = 4.1 Hz, 0.3H), 5.69 (d, J = 4.7 Hz, 0.7H), 3.67 (dt, J = 6.6, 4.3 Hz, 0.3H), 3.55 (td, J = 7.8, 4.7 Hz, 0.7H), 2.96 (d of quintets, J = 13.5, 6.7 Hz, 0.7H), 2.81 (m, 0.3H), 2.56 (dt, J = 14.3, 8.0 Hz, 0.7H), 2.32 (dt, J = 13.69, 7.0 Hz, 0.3H), 2.06 (ddd, J = 13.7, 8.4, 4.1, 0.3H), 1.85 (ddd, J = 14.1, 12.5, 7.4, 0.7H), 1.42 (d, J = 7.0 Hz, 0.9H), 1.41 (d, J = 6.7 Hz, 2.1H). MS (ESI) = 357.0 [M + 23] + . [α]< D (22 °C, c = 1.0, CH2Cl2) = -31.9°; m.p. 98 - 99 °C. <Step D. (3S,5R,6R)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one>

[0091]

Chem.

[0092] A solution of (3S,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one and (3R,5S,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (4.5 g, 13.4 mmol) (from Step C) and allyl bromide (3.48 mL, 40.3 mmol) in tetrahydrofuran (22 mL) at −35° C. (acetonitrile / dry ice bath) was treated with a solution of lithium bis(trimethylsilyl)amide (1.0 M, 17.45 mL, 17.45 mmol) in tetrahydrofuran. The reaction was warmed to −5° C. over 1 hour and then quenched with 50% saturated ammonium chloride. The reaction was diluted with 100 mL of ethyl acetate, and the layers were separated. The organic phase was washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum to give the title compound as a white solid upon standing under vacuum. Using Chiral SFC (92% CO, 8% methanol (20 mM ammonia), 5 mL / min, Phenomenex Lux-2 column (Phenomenex, Torrance, CA), 100 bar (10,000 kPa), 40 °C, 5 min method), the compound was determined to have an enantiomeric ratio of 96:4. (Major enantiomer: title compound, retention time = 2.45 min, 96%; minor enantiomer (structure not shown, retention time = 2.12 min, 4%). (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one was recrystallized by addition to refluxing heptane (slurry of 4.7 g in 40 mL) followed by dropwise addition of 1.5 mL of toluene to solubilize. The solution was cooled to 0°C. The resulting white solid was filtered and rinsed with 20 mL of cold heptane to give a white powder. Chiral SFC (92% CO2, 8% methanol, Phenomenex Lux-2 column, same method as above) indicated an enantiomeric ratio of 99.2:0.8 (major enantiomer, 2.45 min, 99.2%; minor enantiomer: 2.12 min, 0.8%). 1H NMR(400MHz,CDCl3,δ ppm):7.24(ddd,J=8.0,2.0,1.2Hz,1H), 7.20-7.15(series of m,3H), 6.91(t,J=2.0Hz,1H), 6.78(br d,J=7.6Hz,1H), 6.60(m,2H), 5.84(ddt,J=17.6,10.2,7.4Hz,1H), 5.70(d,J=5.3Hz,1H), 5.21-5.13(series of m,2H), 3.82(dt,J=11.7,4.5Hz,1H), 2.62( A BXJ AB =13.7Hz,J AX =7.6Hz,1H), 2.53(A B X,J AB =13.9Hz,J BX =7.2Hz,1H).1.99(dd,J=14.1,11.9Hz,1H),1.92(ddd,J=13.9,3.9,1.2Hz,1H). 13 C NMR (CDCl3, 100MHz, δ ppm):175.9, 140.2, 134.5, 134.3, 134.0, 132.2, 129.8, 128.6, 128.0, 127.9, 127.8, 126.4, 119.9, 83.9, 44.5, 42.4, 40.7, 31.8, 26.1.MS(ESI)=375.2[M+H] + .IR=1730cm -1 .[α] D (24℃,c=1.0,CH2Cl2)=-191°.mp111-114℃. Alternative Procedure for Preparing (3S,5R,6R)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

[0093] [ka]

[0094] Step 1: Isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

[0095]

change

[0096] A solution of 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone (Step A) (67.4 kg, 255 mol) in THF (325 L) was azeotropically dried to achieve a water content of 0.05 wt% by Karl Fischer. Methyl methacrylate (25.8 kg, 257 mol) was added to the solution, and the mixture was heated to 45°C. A solution of potassium tert-butoxide (20 wt% in THF, 14.3 kg, 25 mol) was added over the course of 30 minutes, and the mixture was stirred for 6 hours. The mixture was then cooled to 10°C, and an aqueous solution of citric acid monohydrate (20 wt%, 35 L) was added in less than 5 minutes. Isopropyl acetate (400 L) and aqueous sodium chloride solution (20 wt%, 300 L) were added. The mixture was stirred for 15 minutes, and the phases were allowed to separate. The organic phase was distilled under reduced pressure to produce a distillate volume of 560 L, while isopropanol (350 L) was added to produce a solution of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate in isopropanol (54 wt %, 140 kg total solution mass). The solution had a water content of 0.01 wt % by Karl Fischer. Additional isopropanol (420 L) and sulfuric acid (53 kg, 535 mol) were added to the solution. The mixture was warmed to reflux and stirred for 12 h, during which time 200 L of solvent was distilled and 200 L of fresh isopropanol was added to the mixture. The mixture was then cooled to 20 °C, and water (180 L) was added over the course of 30 minutes. Isopropyl acetate (270 L) was added, and the mixture was stirred for 30 minutes. The phases were separated, and the aqueous phase was extracted with isopropyl acetate (100 L). The combined organic phases were washed four times with water (200 L). The organic phase was distilled under reduced pressure to produce a distillate volume of 500 L, while isopropanol (50 L) was added to obtain a solution of isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate in isopropanol (60 wt %, 134 kg total solution mass). The solution had a water content of 0.02 wt % by Karl Fischer.Isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate was obtained as an approximately 1:1 mixture of diastereoisomers in 81% overall yield. 1 H NMR (400MHz, CDCl3, δ ppm): 7.70-7.80 (m, 2H), 7.22-7.28 (m, 2H), 7.00-7.18 (series of m,4H), 4.78-4.96(m,1H), 4.42-4.50(m,1H), 2.02-2.30(m,2H), 1.80-1.95(m,1H), 0.99-1.19(m,15H). Step 2: (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

[0097] [ka]

[0098] To a degassed solution of isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate (from Step 1) in isopropanol (60 wt %, 252 kg total solution mass, 151 kg isopropyl ester starting material, 385 mol) was added degassed isopropanol (900 L) and potassium tert-butoxide (13 kg, 116 mol). (S)-RUCY(R)-XylBINAP (also known as RuCl[(S)-diapena][(S)-xylbinap] (230 g, 0.2 mol, catalyst, Takasago International) in isopropanol (25 L) A separately prepared degassed solution of 1,2-dichloroethane (1,2-dichloroethane, ... %, 700 kg). The organic phase was distilled at atmospheric pressure and 100°C while simultaneously adding toluene (800 L) to produce a distillate volume of 2700 L. Less than 0.05 wt% isopropanol or water (by Karl Fischer) remained in the mixture after this solvent exchange. Carbonyldiimidazole (59 kg, 365 mol) was added to the toluene solution over the course of 2 h, and the mixture was stirred at 20°C for a further 2 h. The mixture was then cooled to 10°C, and a solution of orthophosphoric acid (72 kg, 545 mol) in water (400 L) was added over the course of 1 h, maintaining the temperature of the mixture below 20°C. The mixture was stirred for 30 min, the phases were allowed to separate, and the organic layer was washed with an aqueous solution of sodium chloride (25 wt%, 484 kg). Toluene (400 L) was distilled at atmospheric pressure and 110°C.After cooling the solution to 20°C, tetrahydrofuran (500 L) was added, and the water content was determined to be 0.03 wt% by Karl Fischer. The product solution was cooled to -10°C, and a solution of allyl bromide (66.8 kg, 552 mol) in tetrahydrofuran (50 L) was added. A solution of lithium hexamethyldisilazide (255 kg, 26 wt%, 492 mol) in toluene was added over the course of 6 h, and the mixture was stirred at -10°C for 1 h. The mixture was warmed to 0°C, and an aqueous solution of orthophosphoric acid (40 wt%, 400 mol) was added over the course of 3 h. The mixture was warmed to 20°C. Water (200 L) and dichloromethane (400 L) were added. The mixture was stirred for 15 minutes, and the phases were allowed to separate. The solution was distilled at atmospheric pressure and 100°C to produce a distillate volume of 1350 L, and the residual toluene in the mixture was determined to be 9.8 wt%. The mixture was cooled to 70°C. Diisopropyl ether (85 L), water (26 L), and isopropanol (65 L) were added. The mixture was cooled to 35°C, stirred for 9 h, cooled to 30°C, and filtered. The filtered material was washed three times with heptane (80 L). The solid was dried at 55°C for 48 h to give 90.1 kg of (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one in an overall yield of 63%. Chiral HPLC indicated an enantiomeric ratio of 99.95:0.05.

[0099] [Example 2] <Differences between the first-in-human method and commercial method for producing Compound A> The gram-scale synthesis of DLAC has been reported previously (see Sun et al., J. Med. Chem. 2014, 57, 1454). Based on this work, compound A was prepared by the first-in-human (FIH) synthesis method shown in Scheme 1. The intermediate OXOS was used as the regulatory starting material for this method. Ring-opening of DLAC using excess L-valinol (3 equivalents) at elevated temperature afforded the amide ABA, which was extracted into dichloromethane. Excess L-valinol was removed using an aqueous hydrochloric acid wash, and the product solution was used in the subsequent step without purification. Reducing the amount of L-valinol used in this transformation was identified as a future development goal given the high cost of this raw material. Notably, preparation of the ABA analogs ABA1 and ABA2 from the corresponding DLAC analogs DLAC1 and DLAC2, which have side chains containing groups in the same oxidation state as the carboxylic acid of compound A, was unsuccessful due to the formation of the undesired succinimides SUC1 and SUC2. Given the similar rates observed for the formation of the desired products ABA1-ABA2 and their conversion to the by-products SUC1-SUC2, it was not possible to isolate the amides ABA1-ABA2 in acceptable yields.

[0100] Scheme 1. First-in-human method for preparing compound A

[0101] [ka]

[0102] Scheme 2. Attempted preparation of ABA analogs (k1 ≒ k2)

[0103] [ka]

[0104] The oxoiminium salt OXOS was prepared from ABA by double activation with two equivalents of toluenesulfonic anhydride and 2,6-lutidine at elevated temperatures. The cation thus prepared was isolated as the 2-naphthylsulfonate salt, which provided satisfactory impurity removal properties. Identifying an alternative reagent to toluenesulfonic anhydride (TsO2) was envisioned for several reasons, including the need to eliminate the long-lasting tosylate intermediate DHO-OTs, which undergoes slow conversion to OXOS at elevated temperatures (120 °C). This intermediate (DHO-OTs) is an alkylating agent and, therefore, a potentially mutagenic impurity. One possible option considered was to isolate the crystalline intermediate DHO (Scheme 3) to increase control over the efficient removal of impurities for the synthesis sequence. However, this required the use of a reagent capable of selective chemoselective activation of the primary alcohol of ABA in the presence of secondary benzyl alcohol. Sulfonic anhydride reagents did not offer this advantage.

[0105] Scheme 3. Intermediates in the preparation of OXOS from ABA using TsO

[0106] [ka]

[0107] The preparation of SUL from OXOS was carried out by treating OXOS with isopropylsulfinic acid in the presence of sodium t-butoxide. This transformation proceeds via the reversible formation of a diastereomeric pair of sulfinate intermediates (SULFI) and their subsequent thermodynamic rearrangement to the product SUL, which is crystallized from acetonitrile and water (Scheme 4). The ALC by-product was irreversibly formed under these conditions in the presence of water. Isopropylsulfinic acid was prepared as an oil from isopropylmagnesium chloride at 20 °C and isolated after aqueous workup. To avoid the large amount of undesired ALC by-product, azeotropic drying of this reagent was required before its use in the formation of SUL. However, isopropylsulfinate was observed to decompose via disproportionation upon drying, thus avoiding this unit operation. Consequently, we sought to discover a stable crystalline salt of isopropylsulfinic acid that was stable under dry conditions and could be designated as a commercial regulatory starting material. Alternatively, a method for the in situ preparation of sulfinates from isopropylmagnesium chloride without aqueous workup and further reaction with OXOS was investigated.

[0108] Scheme 4. Intermediates and by-products produced in the preparation of SUL from OXOS

[0109] [ka]

[0110] Oxidation of the alkene group of SUL was carried out via treatment with catalytic ruthenium chloride (2 mol%) and excess sodium periodate (5 equivalents). The crude product was isolated as a crystalline ethanol solvate. Several undesirable features of this step were observed. First, the heavy metals used in this step needed to be removed, which was achieved using DARCO-G resin for first-in-human delivery. Additionally, multiple equivalents of sodium periodate were required to perform this process, and reagents had to be replenished in small batches to minimize impurity formation. Complex downstream processing protocols (extraction and filtration) were required to remove the large amounts of salt utilized for the conversion. Furthermore, multiple dimeric impurities were generated in this conversion step, making controlling the purity of the drug substance challenging. The use of the ethanol solvate of Compound A as a crystallization control point was problematic and only moderately effective in removing impurities present in the mixture. Additionally, the crystallization process had to be carried out as an evaporative process due to the low ethanol concentration (5% v / v) required to mitigate high mother liquor losses during filtration. The use of ethanol in the crystallization process was also observed to reduce the robustness of the process due to the undesired formation of the corresponding ethyl ester at temperatures above 30°C and the difficulty experienced in removing the ethyl ester from the desired ethanol solvate. Similarly, when methanol was used in the crystallization of Compound A, the formation of the corresponding methyl ester at high temperatures, which was also difficult to isolate and remove from the drug substance, significantly reduced the viability of this route for crystallization, especially for multigram-scale operations. Therefore, in addition to developing a more consistent and environmentally friendly oxidation process for the preparation of Compound A from SUL, the creation of a robust procedure for isolating the drug substance that exhibits effective control of essential attributes was required as part of a commercially viable process.

[0111] [Table 1]

[0112] [Example 3] Development of a commercial method for preparing the intermediate OXOS The thermal amidation of DLAC to ABA with L-valinol proceeded via a multi-step mechanism via the intermediate ester EST (Scheme 5). The initial transesterification of DLAC to EST was a reversible process (k > k with 2 equivalents of L-valinol) that led to the accumulation of EST before rearrangement to the amide product ABA. -1 ) was determined. When the reaction was carried out at 60°C, rapid conversion of DLAC to EST was observed at the beginning of the reaction, followed by slower conversion of EST to ABA over the course of several days (k1 > k2) (Figure 1). At higher temperatures (115°C) (Figure 2), the transformation of EST to the more stable ABA was faster, resulting in an increase in the overall rate of the reaction with increasing EST concentration.

[0113] Scheme 5. Kinetics of thermal amidation of DLAC to ABA

[0114] [ka]

[0115] The FIH method utilized a thermal melt using 3 equivalents of L-valinol to ensure rapid conversion of DLAC to ABA at 110 °C. Consistent with our mechanistic understanding of this transformation, the conversion of DLAC to EST (k1) directly affects the relative concentration of EST, so a reduction in L-valinol loading (from 3 equivalents to 2 equivalents) resulted in an overall slowdown in the reaction. When 2 equivalents of L-valinol were used, the reaction was observed to require 72 hours to reach conversion at 115 °C, using toluene (1 volume) to ensure reaction homogeneity. This longer processing time, however, can be considered justifiable based on the significant cost reduction. Removal of excess L-valinol was achieved by the addition of toluene (4 volumes) and subsequent washing of the organic mixture with aqueous hydrochloric acid solution. The resulting organic solution was azeotropically dried and polish filtered to give ABA as a 28 wt% solution in toluene containing 2.7 LC area % DHO, 1.0 LC area % starting material DLAC, and 1.0 LC area % EST in 91% assay yield. Pyrolysis of ABA to directly prepare DHO at higher temperatures led to a complex mixture of products.

[0116] The isolation of the intermediate DHO provided an additional opportunity to remove impurities from the process stream and strengthen the overall control procedure for delivering the drug substance for market application. Of paramount importance to this procedure was the identification of conditions for decoupling the dehydrative double cyclization of ABA to OXOS into two separate single cyclization reactions through the development of chemoselective activation of the primary alcohol of ABA (Condition A), which allowed for the isolation of DHO in crystalline form (Scheme 6).

[0117] Scheme 6. Sequential dehydration of ABA to OXOS

[0118] [ka]

[0119] Sulfonyl chloride and sulfonic anhydride reagents were found to be unselective in discriminating between the primary and secondary alcohols of ABA and were difficult to procure as anhydrous reagents. Furthermore, the use of acid catalysts also resulted in complex mixtures of products. However, the Vilsmeier salt reagent, methoxymethylene-N,N-dimethyliminium methyl sulfate, successfully achieved the desired selectivity. This reagent is easily prepared without special precautions to exclude moisture and can be stored at 20 °C for several months without loss of potency. Additionally, it exhibits milder reactivity compared to the common halide-derived Vilsmeier salt chloromethylene-N,N-dimethyliminium chloride, improving chemoselectivity and avoiding the formation of alkyl halide by-products. The formation of DHO from ABA using methoxymethylene-N,N-dimethyliminium methyl sulfate in toluene was evaluated at 25 °C in the presence of various mild inorganic bases, and the conversion to DHO was recorded (Scheme 7). The reaction was observed to work best with KOAc, but NaOAc was preferred due to its low hygroscopicity and cost.

[0120] Scheme 7. Evaluation of various bases in the monocyclization of ABA to DHO

[0121] [ka]

[0122] [Table 2]

[0123] The desired chemoselectivity of this transformation is achieved through the unique ability of methoxymethylene-N,N-dimethyliminium methylsulfate to undergo dynamic transesterification with alcohols through the generation of an unstable imidate intermediate. This reversibility was investigated in the activation of 4-chlorobenzyl alcohol (CHA) with the deuterated reagent DEU to generate the imidate IMI, which was found to equilibrate at a CHA / IMI ratio of 2.5 / 1 (Scheme 8). Based on this observation, it is proposed that IMABA, present at low concentrations during the reaction, undergoes rapid intramolecular substitution with the pendant amide to generate the oxazoline DHO (Scheme 8). Any imidate formed by derivatization of the secondary alcohol group of the ABA group does not undergo further cyclization to OXOS at the operating reaction temperature (30 °C).

[0124] Scheme 8. Reversible activation of alcohols using Vilsmeier reagents

[0125] [ka]

[0126] Equilibrium solubility measurements were collected for DHO in various solvents. With the exception of the antisolvent selected, water (<0.1 mg / mL), all values ​​obtained were observed to be greater than 20 mg / mL (including heptane) at 20 °C. Acetonitrile was selected as the solvent for crystallization because it results in facile removal of impurities when combined with water. A curve showing solubility values ​​at different points in the crystallization process is presented in Figure 3. Using this protocol, crystalline DHO was isolated from DLAC in 88% yield with an LC area percentage of >98% (Scheme 9).

[0127] A Bruker D8 powder X-ray diffractometer equipped with a Braun detector and a Cu-Kα radiation source operating in Bragg-Brentano reflection geometry was used to obtain the reflection PXRD pattern (Figure 17) of crystalline DHO. The obtained two-theta (2θ) values ​​were generally accurate to within an error of ±0.2°. Samples were generally prepared without any special treatment other than the application of slight pressure to achieve a flat surface. Samples were measured uncovered unless otherwise noted. Operating conditions included a tube voltage of 40 kV and a current of 40 mA. A variable divergence slit was used with a 3° window. The step size was 0.019° 2θ with a step time of 35.2 seconds. The sample was static between measurements.

[0128] The peaks listed in Table 2 were identified in the PXRD pattern of crystalline DHO.

[0129] [Table 3] TIFF2025172780000026.tif180170

[0130] Scheme 9. Commercial method for preparing DHO from DLAC

[0131] [ka]

[0132] Although the double dehydration cyclization of ABA to OXOS was successfully completed, the development of a method for converting DHO to OXOS was necessary. Methanesulfonic anhydride (MsO) was found to provide a faster conversion to OXOS compared to TsO, as the potentially mutagenic mesylate intermediate DHO-OMs was completely consumed within 10 h at 75 °C. This improvement may be due to the reduced steric hindrance experienced in the transition state leading from DHO-OMs to OXOS compared to that involved in the cyclization of DHO-OTs to OXOS. The conversion was found to proceed well in toluene using 2,6-lutidine as a base. Nucleophilic organic and inorganic bases resulted in undesirably complex mixtures of products. The OXOS mesylate salt generated during the conversion is poorly soluble in toluene and forms a separate liquid layer as the reaction proceeds. To enable further processing, it is necessary to dilute the reaction mixture with dichloromethane (8V) before removing the mesylate salt using an aqueous sulfuric acid wash. Salt metathesis with aqueous sodium 1-naphthalenesulfonate followed by distillation of dichloromethane led to the crystallization of the 1-naphthalenesulfonate toluene hemisolvate OXOS salt from DHO in 90% yield, 99.5 LC area %, and 99.7 wt % (Scheme 10).

[0133] Scheme 10. Commercial method for preparing OXOS from DHO

[0134] [ka]

[0135] The following experimental procedure describes the preparation of OXOS.

[0136] [ka]

[0137] N,N-Dimethylformamide dimethyl sulfate adduct: A 500 mL Atlas reactor equipped with a reflux condenser and an overhead stirring shaft was charged with dimethyl sulfate (200.0 mL, 2.11 mol, 1.0 equiv.) under a nitrogen atmosphere. The contents of the reactor were warmed to 60°C. DMF (200.0 mL, 2.56 mol, 1.2 equiv.) was added dropwise over 60 minutes (3.3 mL / min). Upon completion of the addition, the reaction was stirred at 60°C for 2 hours. Upon completion of the reaction, the reaction was cooled to room temperature to afford N,N-dimethylformamide dimethyl sulfate adduct as a solution in the remaining DMF (402.6 g, 2.02 mol, 95.8% assay yield, 82.8 wt % in DMF).

[0138] [ka]

[0139] (S)-2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1-hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide (ABA): A 5 L ChemGlass reactor equipped with a reflux condenser and an overhead stirring shaft was charged with (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one (DLAC) (201.8 g, 0.53 mol, 98.6 wt%, 1.0 equiv.), L-valinol (110.8 g, 1.06 mol, 2.0 equiv.), and toluene (205 mL, 1 mL / g) under a nitrogen atmosphere. The contents of the reactor were heated under reflux (115° C.) with continuous stirring for 72 hours. Upon completion of the reaction, the reaction was cooled to room temperature and diluted with toluene (800 mL, 5 mL / g). The reaction was quenched by the addition of 1 N HCl (1000 mL, 5 mL / g) in small portions. The phases were separated, followed by washing the organic layer twice with brine (2 x 400 mL, 2 mL / g). The organic phase was dried over magnesium sulfate, filtered through a polishing filter (coarse porosity) rinsing with toluene, and concentrated to a volume of approximately 800 mL to give ABA as a solution in toluene (229.4 g, 0.48 mol, 90.5% assay yield, 27.9 wt % in toluene). 11H NMR (400 MHz, CHLOROFORM-d) δ ppm: 7.05 - 7.19 (m, 5H), 6.95 (d, J = 8.50 Hz, 2H), 6.84 (d, J = 7.67 Hz, 1H), 5.85 (d, J = 8.09 Hz, 1H), 5.57 (ddt, J = 17.13, 9.98, 7.28, 7.28 Hz, 1H), 4.91 - 5.03 (m, 2H), 4.71 (d, J = 4.77 Hz, 1H), 3.66 (br s, 1H), 3.57 - 3.63 (m, 1H), 3.51 - 3.53 (m, 1H), 3.42 - 3.46 (m, 1H), 3.19 (br s, 1H), 2, or 97 (dt, J = 7.93, 4.95 Hz, 1H), 2.36 (dd, J = 13.89, 7.05 Hz, 1H), 2.13 (dd, J = 14.62, 4.87 Hz, 1H), 1.96 - 2.01 (m, 1H), 1.87 - 1.92 (m, or 1H), 1.71 - 1.82 (m, 1H), 1.10 (s, 3H), 0.88 (d, J = 7.05 Hz, 3H), 0.86 (d, J = 7.05 Hz, 3H). 13 13C NMR (101 MHz, CHLOROFORM-d) δ ppm: 177.47, 142.83, 140.46, 133.79, 133.67, 133.00, 129.49, 129.12, 127.96, 127.93, 127.68, 126.88, 118.64, 75.91, 63.44, 56.94, 49.51, 45.17, 42.13, 39.59, 29.06, 24.07, 19.40, 18.72.

[0140] [Chemical formula]

[0141] (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO): A 5 L ChemGlass reactor equipped with a reflux condenser and overhead stirring shaft was charged under a nitrogen atmosphere with (S)-2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1-hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide (ABA) (229.4 g, 0.48 mol, 27.9 wt% in toluene, 1.0 equiv.) and toluene (1145 mL, 5 mL / g). (Note: ABA was obtained as a stock solution in toluene containing 685 mL of residual toluene, so the amount of additional toluene required was 460 mL.) The contents of the reactor were warmed to 30 °C. NaOAc (48.3 g, 0.59 mol, 1.2 equiv.) and N,N-dimethylformamide dimethyl sulfate adduct (174.1 g, 0.72 mol, 82.8 wt. %, 1.5 equiv.) were added sequentially to the reaction. After stirring at 30 °C for 2 h, the reaction was cooled to room temperature. The reaction was quenched with saturated aqueous NH4Cl (750 mL, 3 mL / g) and HO (500 mL, 2 mL / g). The phases were separated, followed by washing the organic layer twice with brine (2 × 750 mL, 3 mL / g). The organic phase was dried over magnesium sulfate, filtered through a polishing filter (coarse porosity), rinsing with toluene, and concentrated in vacuo. The crude residue was recrystallized from MeCN:H2O (50:50) to give DHO as a white crystalline solid (206.5 g, 0.45 mol, 87.7% yield over two steps corrected for wt%). 11H NMR (400 MHz, CHLOROFORM-d) δ ppm: 7.07 - 7.21 (m, 5H), 6.99 (d, J = 8.29 Hz, 2H), 6.88 (d, J = 7.10 Hz, 1H), 5.44 - 5.55 (m, 1H), 4.83 - 4.97 (m, 2H), 4.73 (d, J = 5.60 Hz, 1H), 4.42 (br s, 1H), 4.03 (dd, J = 8.91, 7.67 Hz, 1H), 3.63 - 3.76 (m, 2H), 3.15 - 3.21 (m, 1H), 2.35 (dd, J = 13.89, 7.26 Hz, 1H), 2.13 - 2.18 (m, 1H), 2.07 - 2.12 (m, 1H), 1.84 (dd, J = 14.72, 8.09 Hz, 1H), 1.48 - 1.60 (m, 1H), 1.09 (s, 3H), 0.94 (d, J = 6.63 Hz, 3H), 0.82 (d, J = 6.63 Hz, 3H). 13 13C NMR (101 MHz, CHLOROFORM-d) δ ppm: 171.99, 143.48, 140.41, 133.74, 133.35, 132.92, 129.55, 129.09, 128.24, 127.84, 127.75, 126.75, 118.33, 76.63, 71.80, 69.84, 49.36, 42.13, 39.72, 38.61, 32.48, 24.20, 19.10, 18.26.

[0142]

Chem.

[0143] ​(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium naphthalene-1-sulfonate toluene hemisolvate (OXOS): A 5 L ChemGlass reactor equipped with a reflux condenser and an overhead stirring shaft was charged with (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO) (199.3 g, 0.40 mol, 93.5 wt%, 1.0 equiv) and toluene (1000 mL, 5 mL / g) under a nitrogen atmosphere. Methanesulfonic anhydride (88.2 g, 0.49 mol, 1.2 equiv.) and 2,6-lutidine (95.0 mL, 0.82 mol, 2.0 equiv.) were added sequentially to the reaction mixture. The contents of the reactor were heated to 75 °C with continuous stirring for 16 hours. Upon completion of the reaction, the reaction mixture was cooled to room temperature and diluted with dichloromethane (1600 mL, 8 mL / g). The reaction was quenched with a solution of concentrated HSO (45.0 mL, 0.82 mol, 2.0 equiv.) in HO (955 mL, 5 mL / g). The phases were separated, and the organic layer was then washed twice with an aqueous solution of sodium 1-naphthalenesulfonate (2 × 72.5 g, 0.31 mol, 0.75 equiv.) in HO (2 × 800 mL, 4 mL / g). The organic phase was dried over sodium 1-naphthalenesulfonate (10.0 g, 0.04 mol, 0.1 equiv.), filtered through a polishing filter (coarse porosity) rinsing with dichloromethane, and concentrated in vacuo. The crude residue was recrystallized from toluene to give OXOS as an off-white crystalline solid (260.1 g, 0.37 mol, 90.0% yield corrected by weight). 1H NMR(400MHz,CHLOROFORM-d)δ ppm:9.14(d,J=8.50Hz,1H), 8.35(dd,J=7.26,1.24Hz,1H), 7.86(t,J=8.71Hz,2H), 7.57(t,J=7.70Hz,1H), 7.43-7.50(m,2H), 7 .13-7.39(m,7.5H), 7.03-7.10(m,3H), 6.07(d,J=11.20Hz,1H), 5.80(ddt,J=17.00,9.90,7.39,7.39Hz,1H), 5.51(t,J=9.74Hz, 1H), 5.26-5.34(m,2H), 4.76(ddd,J=10.37,4.66,2.18Hz,1H), 4.62(dd,J=9.12,4.77Hz,1H), 3.51-3.60(m,1H), 2.86(t,J=13. 68Hz,1H), 2.65-2.71(m,1H), 2.55-2.60(m,1H), 2.35(s,1.5H), 1.95(dd,J=13.89,3.52Hz,1H), 1.52(s,3H), 0.54-0.67(m,7H). 13 C NMR(101MHz,CHLOROFORM-d)δ ppm:183.28, 142.16, 140.01, 137.71, 135.89, 134.15, 134.12, 133.28, 132.15, 13 0.38, 130.30, 129.95, 129.62, 129.43, 129.06, 128.90, 128.34, 128.09, 127.92, 12 7.66, 127.41, 127.18, 126.44, 125.88, 125.63, 125.48, 125.16, 124.28, 121.20, 73.14, 67.27, 67.06, 43.64, 43.01, 38.67, 38.56, 26.64, 22.13, 21.32, 18.08, 13.74. Alternatively, the intermediate DHO-OMs can be isolated and purified before conversion to OXOS.

[0144] [ka]

[0145] DHO-OMs 11H NMR (400 MHz, CHLOROFORM-d) δ ppm: 7.31 (d, J = 8.4 Hz, 2H), 7.24 - 7.18 (m, 2H), 7.16 (s, 1H), 7.08 (d, J = 8.2 Hz, 2H), 7.07 - 7.01 (m, 1H), 5.59 - 5.43 (m, 2H), 5.01 - 4.83 (m, 2H), 3.84 (dd, J = 8.1, 9.5 Hz, 1H), 3.55 - 3.45 (m, 1H), 3.42 - 3.34 (m, 1H), 3.24 - 3.13 (m, 1H), 2.46 (s, 3H), 2.39 - 2.28 (m, 1H), 2.28 - 2.14 (m, 1H), 1.98 (br dd, J = 7.8, 13.6 Hz, 1H), 1.72 (dd, J = 2.4, 14.3 Hz, 1H), 1.26 (br s, 1H), 1.06 (s, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.71 (d, J = 6.7 Hz, 3H). 13 13C NMR (101 MHz, CHLOROFORM-d) δ ppm: 169.69, 141.54, 135.61, 134.98, 133.92, 133.43, 129.82, 129.30, 128.81, 128.47, 127.82, 127.34, 118.21, 87.02, 77.22, 69.78, 47.99, 44.57, 39.96, 39.31, 38.46, 32.80, 21.85, 19.40, 18.26.

[0146] [Example 4] [Development of a Commercial Method for Preparing the Second-to-Last Intermediate SUL] Treatment of OXOS with isopropylsulfinate at elevated temperatures leads to the formation of a diastereomeric pair of sulfinate esters (SULFI), which rearrange to the thermodynamically more stable product SUL (Scheme 4). Previous studies have described this type of sulfinate-sulfone rearrangement as occurring via a benzhydrylsulfinate ester ion pairing and rearrangement mechanism. However, in the present method, the results of the crossover experiment shown in Scheme 11 reveal that the rearrangement involves dissociated ions. Considering that OXOS reacts quantitatively with water at temperatures above 70 °C, there is ample opportunity for the alcohol ALC to be produced. If OXOS cannot be regenerated from ALC, water must be strictly excluded from the reaction mixture. One route to this end is to prepare a salt of isopropylsulfinic acid that is stable to azeotropic drying conditions, can be isolated in high purity, and reacts efficiently with OXOS to produce SUL.

[0147] Scheme 11. Intermediates and by-products produced in the preparation of SUL from OXOS

[0148] [ka]

[0149] After evaluating several isopropyl sulfinate salt candidates for this purpose, including lithium, sodium, potassium, magnesium, and ammonium salts, calcium sulfinate dihydrate salt emerged as a stable, crystalline species. This salt was prepared from the reaction of isopropylmagnesium chloride with sulfur dioxide (Scheme 12), leading to the formation of isopropyl sulfinic acid after an aqueous hydrochloric acid quench. Treatment of this material with calcium acetate allowed for the isolation of calcium isopropyl sulfinate dihydrate (CALID) via reactive crystallization. Given the water sensitivity of the above process, the hydrate is not an ideal species for use in the preparation of SUL. However, the calcium salt was chemically stable upon azeotropic drying in toluene at elevated temperatures (up to 110 °C) (i.e., no disproportionation products were observed within 48 h). Therefore, drying of the reagent suspension can be included as part of the method prior to the addition of OXOS and the preparation of SUL. Using X-ray powder diffraction analysis of oven-dehydrated samples, CALID was observed to undergo a polymorphic change upon complete drying (<100 ppm water) at <15% RH. However, CALID is stable as a dehydrated material, converting back to its original dihydrate form upon water resorption at >20% relative humidity. A method for producing SUL involves drying a toluene suspension of CALID and OXOS by azeotropic distillation under reduced pressure to produce a mixture containing less than 100 ppm water. The dried suspensions are then combined and a solvent exchange to dimethylacetamide is performed (Scheme 13). The resulting solution is heated to 120°C and stirred for up to 20 hours. During this time, the sulfinate ester generated within the first hour at 120°C rearranges to form SUL. The typical level of the ALC impurity formed under these conditions is 3 LC area %. After aqueous workup and crystallization from acetonitrile and water (up to a 23.3 kg scale), SUL is isolated in 82% yield and >99.5 LC area %.

[0150] Scheme 12. Preparation of calcium isopropylsulfinate dihydrate (CALID)

[0151] [ka]

[0152] Scheme 13. Preparation of SUL using CALID

[0153] [ka]

[0154] Isopropyl magnesium chloride was prepared in situ as a dry reagent and used directly in the preparation of SUL, which provided an alternative procedure for the preparation of SUL from OXOS. As shown in Scheme 14, a solution of isopropyl magnesium chloride in tetrahydrofuran was treated with sulfur dioxide to prepare isopropyl sulfinate magnesium chloride. In situ FTIR (1325 cm) -1The complete consumption of sulfur dioxide was verified using a phenanthroline test to confirm the absence of alkyl Grignard compounds before further processing. Solvent exchange to N-methylpyrrolidinone (NMP) followed by the addition of OXOS afforded SUL at 120 °C or 180 °C (Scheme 14). Elimination of unwanted water and / or magnesium hydroxide chloride from the reaction mixture thus prepared presents a challenge. For example, when 3 equivalents of isopropylsulfinate magnesium chloride relative to OXOS are used, approximately 5 mol% of magnesium hydroxide chloride (relative to OXOS) is present in the starting Grignard solution. NMP (5 volumes) and OXOS incorporate 5–10 mol% water (relative to OXOS). Therefore, it is difficult to avoid the minimum formation of 15 mol% ALC using this reagent. Interestingly, the level of ALC observed during the formation of SUL under these conditions exceeds the measured amount of water or hydroxide contained in the reaction mixture, to a limit that depends on the operating temperature. In addition to the direct ring-opening of OXOS with water or hydroxide salts (Scheme 4), a second mechanism must be induced to afford the formation of ALC. This putative mechanism involves the reaction of SULFI with isopropylsulfinate magnesium chloride to give ALC (Scheme 15).

[0155] Although there is a plausible mechanism that explains the conversion of ALC to SUL at high temperatures (e.g., 180 °C), it does not occur at a commercially suitable rate at 120 °C (Figure 4). Treatment of OXOS with isopropylsulfinate magnesium chloride (3 equivalents) in NMP (5 volumes) at 180 °C allows the formation of SUL and ALC in assay yields of 77% and 18%, respectively, after 6 minutes. After 80 minutes, the assay yields of SUL and ALC are 90% and 5%, respectively (Figure 5). It is proposed that the magnesium salt acts as a Lewis acid to promote the formation of OXOS from ALC (Scheme 15). Isopropylsulfinate magnesium chloride lacks stability at high temperatures and can undergo 85% decomposition in 1 hour at 200 °C as a 1 M solution in NMP. 11 H NMR. As a result, the conversion was carried out utilizing 3 equivalents of isopropylsulfinate magnesium chloride.

[0156] Scheme 14. Preparation of SUL from OXOS using isopropylsulfinate magnesium chloride

[0157] [ka]

[0158] Scheme 15. Possible mechanism for the generation of ALC and its conversion to SUL

[0159] [ka]

[0160] The use of alternative isopropylsulfinate salts to modify the reactivity and increase the stability of isopropylsulfinate magnesium chloride was evaluated. Treatment of 1 equivalent of the in situ prepared reagent with 1 equivalent of zinc chloride yielded promising results. A commercially available zinc chloride solution in tetrahydrofuran (0.5 M) was added to a solution of isopropylsulfinate magnesium chloride prepared as previously described (Scheme 12). The new species formed was 1H NMR revealed distinct isopropylsulfinate magnesium chloride and zinc isopropylsulfinate (Figure 6), and the structure was assumed to be that of isopropylsulfinate zinc chloride, with magnesium chloride produced as a by-product. A solvent exchange to NMP (5 volumes) was performed, and OXOS was added to the reaction mixture (Scheme 16). Using this mixed reagent, the conversion of ALC to SUL was efficient at a productive rate at 120 °C. Therefore, the conversion can be carried out at 120 °C and under very strict moisture-free conditions, while avoiding decomposition of the reagents and reaction intermediates (Figure 7). Furthermore, there is no evidence of an alternative mechanism for producing ALC without the involvement of water using this mixed magnesium-zinc reagent; all ALC produced during the process can be contributed by the incoming reagents or solvent. This does not mean that this mixed salt cannot be used at 180°C (Figure 8), but rather a temperature of 140°C was chosen to carry out the process, allowing for good reaction rates and a 90% yield of SUL with 7% ALC in 7 hours while using limited equivalents (1.5 equivalents) of magnesium-zinc species. 1 H NMR experiments showed it to be stable at 150 °C for 16 hours.

[0161] Scheme 16. Preparation of SUL from OXOS using isopropylsulfinate magnesium chloride-zinc chloride

[0162] [ka]

[0163] The robustness of this process was assessed using a 20% excess of sulfur dioxide during the formation of isopropylsulfinate magnesium chloride, followed by a solvent exchange to NMP after 24 hours of stirring of the mixed salts at 20°C. Only 40% ( 1It was observed that 1H NMR (by OXOS) remained and 60% of the material was incompatible for forming the sulfone SSO (Scheme 15). Use of this mixture to convert OXOS to SUL using 1.5 equivalents of reagent resulted in the formation of only 62 LC area % SUL, with 33 LC area % of OXOS unreacted, demonstrating the lack of robustness of this process, as controlling sulfur dioxide dosage during plant operation can be problematic. Thus, the use of CALID to prepare SUL represents an advantageous aspect of a commercial process for producing compound A.

[0164] [Example 5] <Development of a commercial method for preparing compound A> Ozonolysis of the alkene group of SUL, followed by oxidation of the resulting aldehyde to the corresponding carboxylic acid group of compound A using sodium chlorite, represents a more environmentally friendly alternative to the ruthenium oxide / sodium periodate method used for the initial preparation of compound A. Additionally, the ozonolysis route simplifies product isolation, as it potentially eliminates the formation of several undesired dimeric impurities that are difficult to remove via crystallization.

[0165] In developing safe reaction conditions for ozonolysis, aqueous mixtures were utilized (Scheme 17). Under these conditions, the high-energy ozonide intermediate (OZO) is hydrolyzed, thus avoiding its accumulation and making the process safe. The LC area % of accumulated OZO was measured versus the volumetric percentage of water used in the acetonitrile / water reaction mixture, and the results are reported in Figure 9. Using 10% water, the total energy release for the ozonolysis mixture (20 volumes of solvent) was 92 J / g, with a decomposition temperature of 240 °C, representing no safety concern. Another parameter requiring control to ensure safety during ozonolysis is the gas-phase concentration of oxygen in the vessel during the reaction. The limiting oxygen concentration (LOC) for combustion of the mixture was measured at 10.75 vol.%, and the ozonolysis process was performed at half the LOC (approximately 5 vol.% oxygen) to ensure a margin of safety to avoid possible combustion.

[0166] Scheme 17. Preparation of Compound A from SUL using an Ozonolysis-Pynic Tandem Process

[0167] [ka]

[0168] Two different modes of processing were implemented in a GMP setting for this conversion: (i) semi-batch ozonolysis using ozone sparging in a batch vessel, and (ii) continuous stirred-tank reactor processing. Initially, continuous processing seemed attractive to mitigate the common safety concerns associated with using ozonolysis reactions in commercial processes. A schematic and photograph of the continuous ozonolysis apparatus utilized in one embodiment are presented in Figures 10 and 11, respectively. A CFS-3 ozone generator model, commercially available from Ozonia, was utilized to process 4.8 kg of SUL while producing approximately 0.9 mol of ozone per hour. Ozone was generated from an air supply and introduced through a valve located at the bottom of the continuous stirred-tank reactor (CSTR) as shown in Figure 10. The starting material solution was introduced at a flow rate of 60 mL / min using a dip tube with the outlet located above a glass frit located at the bottom of the vessel (0.9 L volume). Vigorous agitation of the mixture was important for proper gas dispersion; an example can be seen in Figure 11. A nitrogen headspace purge with a flow rate 3X that of the air stream introduced at the bottom is maintained to ensure that less than 5% by volume of oxygen / ozone is present in the gas phase. The reaction mixture is maintained at 20°C using jacket control. A Raman probe is used at the CSTR outlet to measure the level of residual SUL.

[0169] To minimize risk in the use of continuous ozonolysis, accumulated reaction mixture aliquots were sampled and reaction completion was verified by HPLC. An aliquot was placed in a 2 M aqueous solution of sodium chlorite (4 equiv.), and the resulting mixture was stirred at 20° C. for 16 h. Due to the low solubility of process intermediates (ALD, PAC) in aqueous solution, the addition of an aqueous solution of sodium chlorite to the ozonolysis reaction stream is the preferred option used to avoid initial precipitation of those intermediates and represents the mode of addition used for the semi-batch ozonolysis process described below.

[0170] The semi-batch approach to carrying out the ozonolysis of SUL has the advantage of operating with excess alkene starting material for most of the conversion and employing a simpler manufacturing footprint. While a maximum of 0.4 LC area % of the impurity DHCA was observed to be formed using this processing mode, a reliable approach to monitoring reaction completion and safe processing conditions is required. As noted above, safe processing conditions for this reaction manifold are provided by using an aqueous medium and maintaining an oxygen concentration below 5% by volume. The air / ozone gas stream is diluted with nitrogen downstream of the ozone generator but upstream of the introduction of the reaction gases through a dip tube into the reaction vessel, as seen in Figure 12. The nitrogen gas flow is used four times as much as the air flow to ensure that the gas entering the vessel contains less than 5% by volume of oxygen / ozone. A headspace ozone detector can be utilized to monitor reaction completion for ozonolysis by detecting an increase in the outlet gas concentration. However, this technique has been found to be difficult to implement, given that the measured changes in outlet ozone concentration can be subtle. On the other hand, consumption of starting materials was found to be linear for this conversion (Figure 13), thus allowing HPLC analysis of a few samples during the conversion, combined with knowledge of the ozone generator output, to accurately predict the time for reaction completion. A 23 kg batch of SUL was processed using the CFS-14 ozone generator model commercially available from Ozonia, which allows a maximum output of approximately 540 g of ozone / hour. However, because maintaining a stable ozone output is easier with a machine at less than maximum capacity, an output of 285 g of ozone / hour was utilized for production, achieving a power of 6080 W (80% of capacity) and 3.1 SCFM (30% of maximum air flow). The concentration of ozone in the generator output gas was approximately 4.3 wt% using these settings, and this gas stream was mixed with nitrogen (12.5 SCFM) before entering the processing vessel. The predicted time to reaction completion using the ozone gas output (7.4 h) was exceeded by 10% (actually 8.2 h).Ozone is known to react with water to produce hydroxy groups, and a portion of the ozone is consumed in this manner, which explains why excess ozone must be available.

[0171] For this semi-batch process, ozone was introduced near the bottom of the reaction vessel via a dip tube. The first device evaluated for delivering gas to the reaction system was a standard ozone sparging unit with a 100 μm pore size and a total surface area of ​​0.32 square feet. Using this sparger to deliver a combined gas flow (air, ozone, and nitrogen) of 15.6 SCFM resulted in a pressure drop that caused the sparger surface to cool. This drop in temperature was accompanied by the crystallization of the starting material SUL and the product ALD, followed by blockage of the sparger pores with the crystallized material. Because the solubilities of SUL and ALD in acetonitrile / water (9 / 1 volume ratio) at 10 °C are 25 mg / mL and 21 mg / mL, respectively, only approximately 50% of either material could be solubilized at that temperature in the 20-volume solvent mixture used. When the sparger became blocked by starting material or product crystals, the available surface for gas transfer decreased, exacerbating the situation and requiring process interruption and sparger repair. A different ozone sparger was designed to address this issue. The alternative sparger incorporated 1 / 8-inch diameter holes (37 holes) drilled into a C22 Hastelloy tubing. Both spargers are shown in Figure 14. The impact of using either sparger on the reaction mixture and sparger surface temperatures at a typical gas flow (15 SFCM) was measured, and the results are shown in Table 3. As detailed in the table, there was a significant difference between the sparger surface temperature and the reaction mixture temperature for the 100 μm pore sparger (e.g., 30 °C vs. 11 °C), causing the problems detailed above. The alternative sparger alleviates these concerns. To avoid any precipitation of SUL and ALD during the ozonolysis process, the conversion was carried out at 30 °C.

[0172] [Table 4]

[0173] After completion of the ozone decomposition treatment, the addition of a 2 M aqueous sodium chlorite solution was observed to enable the formation of Compound A. The mixture was treated with 2 M aqueous sodium bisulfite to eliminate all oxidants for further processing. Phase separation occurred following the addition of isopropyl acetate, and two washes of the organic layer were performed with 2 M aqueous sodium phosphate (pH 6). Finally, the organic phase was washed with 1.1 M aqueous sodium chloride to prepare a solution of Compound A with an assay yield of >95% and a purity of >98 LC area%.

[0174] [Example 6] <Isolation of Compound A as the DABCO Salt> Considering that the drug substance control points enabling robust removal of impurities were not available for the free acid, several salts of Compound A were prepared and tested to find candidates effective for enabling facile removal of impurities. Evaluating over 30 organic salts and over 5 inorganic salts of Compound A for that purpose promoted the identification of the hemi-DABCO salt and the hemi-calcium salt as promising competitors. However, while the hemi-calcium salt showed only moderate removal of starting material SUL or impurity HAC, the use of hemi-DABCO isopropyl acetate (232-DAB) enabled efficient removal of both species (IPAC = isopropyl acetate).

[0175] [[ID=十六]] [Chemical formula]

[0176] 232-DAB [[ID=二十六]]5 LC area% of SUL and 1 LC area% of HAC were at significantly higher levels than observed in a typical ozone decomposition-pinacolic oxidation reaction mixture and could be completely removed during isolation of the latter salt. 232-DAB was subjected to TGA and 1A stable crystalline monosolvate (isopropyl acetate) hemi-DABCO salt was observed by H NMR. A single polymorph of the material was identified. The polymorphic form and isopropyl acetate level of the material did not change with dynamic vapor sorption experiments performed at 0-90% relative humidity. Additionally, a robust crystallization protocol based on temperature and the use of an antisolvent could be designed for hemi-DABCO isopropyl acetate using isopropyl acetate and heptane.

[0177] A solubility curve showing values ​​at different time intervals during the crystallization process is shown in Figure 15. Addition of DABCO (0.5 equivalents) to a solution of Compound A in 4 volumes of isopropyl acetate at 55°C seeded the solution with 232-DAB, allowing for supersaturation release and crystallization of approximately 20% of the material. Cooling to 20°C over 2 hours prompted the crystallization of another 60% of the material. Four volumes of heptane were then added to reduce the supernatant concentration to approximately 5 mg / mL in preparation for batch filtration. Use of this crystallization procedure allowed for the isolation of 232-DAB in 83% yield and >99.9 LC area% purity (scale up to 23.2 kg).

[0178] [Example 7] <Isolation of Compound A> Considering that crystallization 232-DAB was performed in organic solvents, an aqueous crystallization protocol was desired to isolate compound A via an orthogonal purification process. Given the general instability of compound A in alcoholic solvents at temperatures above 20-30 °C due to the inability to remove the ester impurity via Fischer esterification and crystallization, alcohol-water solvent mixtures were not feasible for crystallization. Other aqueous mixtures with water-miscible solvents exhibited steep solubility curves that were not conducive to crystallization design. In contrast, acetic acid and water were suitable for crystallizing the material, with good growth characteristics without the drawbacks detailed above. A robust temperature- and antisolvent-based crystallization was designed using this solvent mixture and was implemented after salt cleavage in an aqueous hydrochloric acid (2 equivalents) / isopropyl acetate mixture, two subsequent washes of the organic layer with 2 M aqueous sodium phosphate (pH 6), a wash with 1.1 M aqueous sodium chloride, and a solvent exchange from isopropyl acetate to acetic acid.

[0179] Figure 16 shows a curve showing the solubility values ​​at different points in the crystallization process. A solution of compound A in acetic acid (6.6 volumes of acetic acid) was warmed to 55-60°C and 4.4 volumes of water were added. The solution was seeded with compound A to allow for approximately 30% supersaturated release and crystallization of compound A. The crystallization was cooled to 20°C over 10 hours, resulting in another 55% crystallization of the material. One volume of water was then added to reduce the supernatant concentration to approximately 5 mg / mL in preparation for rapid batch filtration. Three water washes (3 x 10 volumes) were performed to minimize the presence of residual acetic acid in the isolated material. Compound A (up to 18.0 kg) was isolated using this protocol in >92% isolated yield (100% by weight) and >99.9 LC area% purity with <200 ppm residual water and <200 ppm residual acetic acid.

[0180] The material was milled using a Pallman Universal Mill (wing beater, scale up to 16 kg) and the results are summarized in Table 4. The target d50 for Compound A was set to <35 μm based on oral absorption modeling (GastroPlus v 9.0) for the range of doses (60-480 mg) evaluated to provide complete absorption at a fasted gastric pH of 1.3.

[0181] [Table 5]

[0182] A robust and efficient method suitable for commercial production of drug substance (Compound A) with high purity has been developed. Notable aspects of the method include: (i) the use of the bench-stable Vilsmeier reagent, methoxymethylene-N,N-dimethyliminium methyl sulfate, for the selective in situ activation of the primary alcohol intermediate; (ii) the isolation of the intermediate DHO in crystalline form, which enhances processability for impurity removal; (iii) the use of a new stable isopropyl calcium sulfinate reagent in crystalline form to ensure robust preparation of the sulfone intermediate; (iv) the development of a safe ozonolysis protocol carried out in an aqueous solvent mixture suitable for either batch or continuous manufacturing modes; and (v) enhanced purity control of Compound A through the formation of a salt of Compound A, which allows for effective removal of impurities. Demonstration of the new method provided 18 kg of pure Compound A (99.9 LC area%) from DLAC in 49.8% overall yield, representing a significant improvement over the previously described FIH method, which only resulted in a 32% overall yield.

Claims

1. compound 【Chemistry 1】 1. A method for preparing 【Chemistry 2】 with methoxymethylene-N,N-dimethyliminium methylsulfate.

2. The method of claim 1 , wherein the reaction is carried out in the presence of a base.

3. The base is KOAc, NaOAc, LiOAc, or K ２ CO ３ The method of claim 2, wherein

4. 3. The method of claim 2, wherein the base is NaOAc.

5. The process according to any one of claims 1 to 3, wherein the reaction is carried out in a solvent.

6. 6. The method of claim 5, wherein the solvent is toluene.

7. compound 【Transformation 3】 1. A method for preparing 【Chemistry 4】 with isopropylsulfinate zinc chloride.

8. 8. The process of claim 7, wherein the reaction is carried out in the presence of a magnesium salt.

9. Magnesium salt is MgCl ２ The method of claim 8, wherein

10. 8. The method of claim 7, wherein the isopropyl sulfinate zinc chloride is generated in situ from isopropyl magnesium chloride.

11. 8. The method of claim 7, wherein the reaction is carried out in the range of 100°C to 150°C.

12. 8. The method of claim 7, wherein the reaction is carried out in the range of 150°C to 200°C.

13. 1. A crystalline form of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4-methylhept-6-en-1-ol (DHO), characterized by a reflection X-ray powder diffraction pattern containing peaks at 7.3°±0.2° 2θ, 14.5°±0.2° 2θ, 15.8°±0.2° 2θ, 15.9°±0.2° 2θ, and 23.1°±0.2° 2θ.

14. 14. The crystalline form of claim 13, wherein the reflection X-ray powder diffraction pattern further comprises peaks at 8.5°±0.2° 2θ, 10.0°±0.2° 2θ, 11.0°±0.2° 2θ, 13.4°±0.2° 2θ, 18.8°±0.2° 2θ, and 22.0°±0.2° 2θ.

15. The reflection X-ray powder diffraction pattern was 6.3°±0.2° 2θ, 10.5°±0.2° 2θ, 11.5°±0.2° 2θ, 12.8°±0.2° 2θ, 14.8°±0.2° 2θ, 15.2°±0.2° 2θ, 17.0°±0.2° 2θ, 17.5°±0.2° 2θ, 17.8°±0. 2° 2θ, 18.4° ± 0.2° 2θ, 19.0° ± 0.2° 2θ, 19.7° ± 0.2° 2θ, 19.9° ± 0.2° 2θ, 20.7° ± 0.2° 2θ, 21.2° ± 0.2° 2θ, 21.3° ± 0.2° 2θ, 22.4° ± 0.2° 2θ, 23.6° ± 0.2° 2θ, 24. 2°±0.2° 2θ, 24.9°±0.2° 2θ, 25.7°±0.2° 2θ, 26.3°±0.2° 2θ, 27.0°±0.2° 2θ, 28.3°±0.2° 2θ, 28.7°±0.2° 2θ, 29.3°±0.2° 2θ, 29.7°±0.2° 2θ, 30.8°±0.2° 15. The crystalline form of claim 14, further comprising one or more peaks at 31.4°±0.2°2θ, 31.8°±0.2°2θ, 33.0°±0.2°2θ, 34.2°±0.2°2θ, 35.8°±0.2°2θ, 37.0°±0.2°2θ, and 37.5°±0.2°2θ.

16. 14. The crystalline form of claim 13, wherein the crystalline form is a crystalline anhydrate.

17. 17. The crystalline form of any one of claims 13 to 16, wherein the peaks are present when reflection X-ray powder diffraction is performed using Cu-Kα radiation.

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