EA043674B1 - METHODS FOR OBTAINING MDM2 INHIBITOR - Google Patents

Description

Field of technology to which the invention relates

The present invention provides methods for preparing 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutane-2- yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (compound A) and its intermediates.

BACKGROUND OF THE INVENTION p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of multiple genes involved in cell cycle termination, apoptosis, aging, and DNA repair. Unlike normal cells, which rarely induce p53 activation, tumor cells are subject to constant cellular stress as a result of various damaging factors, including hypoxia and proapoptotic oncogene activation. Thus, there is a significant selective advantage for inactivation of the p53 pathway in tumors, and it has been suggested that ablation of p53 function may be a prerequisite for tumor survival. In support of this approach, three groups of researchers used mouse models to demonstrate that the absence of p53 function is a persistent requirement for the maintenance of established tumors. When the researchers restored p53 function in tumors with inactivated p53, the tumors regressed.

In 50% of solid tumors and 10% of liquid tumors, p53 is inactivated by mutation and/or lost. In cancer, other major members of the p53 pathway are also altered genetically or epigenetically. MDM2, an oncoprotein, inhibits p53 function and is activated by gene amplification with an incidence reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, p14ARF. It has been suggested that downstream changes in p53 may be responsible, at least in part, for inactivation of the p53 pathway in p53 ^WT tumors. In support of this concept, some p53 ^WT tumors appear to have a reduced apoptotic capacity, although their ability to undergo cell cycle termination remains unaffected. One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity through three mechanisms: 1) acting as an E3 ubiquitin ligase, promoting p53 degradation; 2) binding and blocking the transcription activation domain of p53 and 3) export of p53 from the nucleus to the cytoplasm. All three mechanisms can be blocked by neutralizing the MDM2-p53 interaction. In particular, this therapeutic strategy can be applied to tumors that are p53 ^WT , and studies with small molecule MDM2 inhibitors have shown promise in reducing tumor growth both in vitro and in vivo. Additionally, in patients with p53-inactivated tumors, stabilization of wild-type p53 in normal tissues by MDM2 inhibition may provide selective protection of normal tissues from mitotic poisons.

The present invention provides a compound capable of inhibiting the interaction between p53 and MDM2 and activating downstream p53 effector genes. The compound of the present invention as such can be used in the treatment of malignant tumors, bacterial infections, viral infections, ulcers and inflammations. In particular, the compound of the present invention is useful for the treatment of solid tumors such as breast, colon, lung and prostate tumors and liquid tissue tumors such as lymphomas and leukemias. As used herein, MDM2 refers to the human MDM2 protein, and p53 refers to the human p53 protein. Human MDM2 may also be called HDM2 or hMDM2.

Compound, 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3methylbutan-2-yl)-3 α-Methyl-2-oxopiperidin-3-yl)acetic acid (also referred to herein as Compound A) is an MDM2 inhibitor and has the following chemical structure.

H ₃ C^ ^sn z

Compound A

Compound A is described in PCT Published Application No. WO 2011/153509 (Example No. 362) and is being studied in human clinical studies for the treatment of various types of malignant tumors. The present invention provides improved methods for preparing compound A, as well as its intermediates.

- 1 043674

The essence of the invention

In one embodiment, the present invention provides a method for producing the following compound (DHO):

process involving connection interaction (ABA)

with methoxymethylene-N,N-dimethyliminium methyl sulfate.

In one embodiment, this interaction occurs in the presence of a base. In a particular embodiment, the base is an alkali metal salt or an alkaline earth metal salt, such as, for example, KOAc, NaOAc, LiOAc, CaCO ₃ and K2CO3. In one embodiment, the reaction is carried out in a solvent. In a specific embodiment, 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 mixtures thereof.

In one embodiment, the present invention provides a method for producing a compound (SUL)

comprising reacting the compound with an isopropylating agent, such as, but not limited to, zinc chloride isopropyl sulfinate.

In one embodiment, the reaction is carried out in the presence of an alkaline earth metal salt. In a specific embodiment, the alkaline earth metal salt is a magnesium salt, such as, but not limited to, MgBr2 or MgCl2. In a specific embodiment, the isopropylating agent is produced in situ from isopropyl magnesium chloride. In one embodiment, the reaction is carried out at a temperature of from 100 to 200°C, for example from 100 to 150°C, for example at 120°C, or from 150 to 200°C, for example at 180°C.

In one embodiment, the present invention provides a crystalline form of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazole -2-yl)-4-methylhept-6-en-1-ol (DHO), characterized by a powder X-ray diffraction pattern with reflections containing intensity peaks at angles of 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 X-ray powder diffraction pattern of the DHO crystal additionally contains peaks at angles of 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 X-ray powder diffraction pattern of the DHO crystal additionally contains one or more peaks at angles of 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,

- 2 043674

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° 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, X-ray powder diffraction of crystalline DHO reflections is performed using Cu-Ka radiation.

Brief description of drawings

The following figures represent specific embodiments of the invention described and are not intended to limit the scope of the present invention in any other way.

In fig. Figure 1 shows the rate of conversion of (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3methyltetrahydro-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.

In fig. Figure 2 shows the rate of conversion of DLAC to ABA over time at 115°C.

In fig. Figure 3 shows the solubility of (1R,2R,4S)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl4,5-dihydrooxazol-2-yl)-4-methylhept -6-en-1-ol (DHO) during crystallization at 25°C.

In fig. Figure 4 shows the yield of (3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutane-2- yl)-3-methylpiperidin-2-one (SUL) over time using magnesium chloride isopropylsulfinate at 120°C (14 mol% water (relative to (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]pyridine-4ium-naphthalene-1-sulfonate, toluene hemisolvate (OXOS)) in the reaction mixture).

In fig. 5 shows the yield of SUL over time using magnesium chloride isopropyl sulfinate at 180°C (11 mol% water (relative to OXOS) in the reaction mixture).

In fig. Figure 6 shows 1H NMR analysis of various forms of isopropyl sulfinate in THF-d ₈ .

In fig. 7 shows the SUL yield over time using magnesium sulfinate-ZnCl2 at 120°C (17 mol% water (relative to OXOS) in the reaction mixture).

In fig. 8 shows the SUL yield over time using magnesium sulfinate-ZnCl2 at 180°C (17 mol% water (relative to OXOS) in the reaction mixture).

In fig. Figure 9 shows the content of (3R,5R,6S)-3-((1,2,4-trioxolan-3-yl)methyl)-5-(3-chlorophenyl)-6-(4chlorophenyl)-1-((S) -1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one (OZO), expressed as % LC area, relative to the water content, expressed as % by weight, in the reaction mixture at 20°C.

In fig. Figure 10 shows a diagram of the apparatus for the continuous mode of ozonolysis and Pinnik oxidation.

In fig. Figure 11 shows an image of a continuous ozonolysis apparatus.

In fig. Figure 12 shows a diagram of an apparatus for semi-continuous ozonolysis and Pinnick oxidation.

In fig. Figure 13 shows the SUL flow rate for ozonolysis in semi-continuous mode.

In fig. 14 shows the modification of the bubbling device for the development of ozonolysis technology.

In fig. 15 shows the solubility of 232-DAB during crystallization.

In fig. 16 shows the solubility of compound A during crystallization.

In fig. 17 shows a powder X-ray diffraction pattern of crystalline DHO determined in reflection mode.

In fig. 18 shows a powder X-ray diffraction pattern of crystalline DHO determined in reflection mode with a ruler to indicate the positions of the peaks.

In fig. 19 shows a thermogram obtained by differential scanning calorimetry (DSC) of crystalline DHO.

Detailed Description of the Invention

The present invention relates to methods for preparing 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4chlorophenyl)-1 -((S)-1 -(isopropylsulfonyl)-3-methylbutane-2- yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (compound A), as well as its intermediates and methods for preparing these intermediates.

In one aspect, the present invention provides a method for producing Compound A in high purity.

In another aspect, the present invention uses a laboratory-stable Vilsmeyer reagent, methoxymethylene-K,K-dimethyliminium methyl sulfate (Corbett, M.T.; Caille, S., Synlett, 2007, 28, 2845), to achieve selective in situ activation of the intermediate primary alcohol when preparing compound A.

In another aspect, the present disclosure uses a laboratory-stable crystalline isopropylating agent, calcium isopropyl sulfinate, to produce a high-yield sulfone intermediate in the preparation of Compound A.

- 3 043674

In another aspect, the present disclosure uses a safe ozonolysis reaction carried out in a mixture of aqueous solvents in a batch or continuous production mode in the process of producing Compound A.

In another aspect, the present invention provides a crystalline form of (1R,2R,4S)-2-(3chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl )-4-methylhept-6-en-1-ol (DHO), characterized by a powder X-ray diffraction pattern with reflections, including peaks at angles of 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 X-ray powder diffraction pattern of the DHO crystal additionally contains peaks at angles of 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 X-ray powder diffraction pattern of the DHO crystal additionally contains one or more peaks at angles of 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° 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, crystalline DHO X-ray powder diffraction is performed using Cu-Ka radiation.

In another aspect, the present disclosure provides control of the purity of Compound A by crystallizing its 1,4-diazabicyclo[2,2,2]octane (DABCO) salt, which can be effectively purified.

In one embodiment, the present invention provides a method suitable for scaling up the production of compound A (99.9% LC area) with an overall yield of 49.8% of the starting material DLAC.

The term containing implies an open end (expandability) including the specified component, but not excluding other elements.

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

The terms patient and subject can be used interchangeably and refer to animals such as dogs, cats, cows, horses, sheep and humans. Specific patients are mammals. The term patient includes men and women.

The term pharmaceutically acceptable means that the specified substance, such as a compound of the present invention or a salt of the compound, or a preparation containing the compound, or a particular excipient, is suitable for administration to a patient.

Terms treatment, treat or treatment, etc. include preventive (eg prophylactic) and palliative treatment.

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

The compound(s) of the present invention can be administered to a patient in a therapeutically effective amount. The compound(s) may be administered alone or as part of a pharmaceutically acceptable composition or preparation. In addition, the compound(s) or compositions can be administered once, for example, by bolus injection, repeatedly, for example, using a series of tablets, or administered substantially continuously over a period of time, for example, using transdermal delivery. It should also be noted that the dosage of the compound(s) may vary over time.

The compound(s) 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 should be noted that the additional pharmaceutically active compounds/agents may be a conventional small organic chemical molecule or may be a macromolecule such as proteins, antibodies, peptibodies, DNA, RNA, or fragments of such macromolecules.

In the event that a patient is to receive or is receiving multiple pharmaceutically active compounds, these compounds can be administered simultaneously or sequentially. For example, in the case of tablets, the active compounds may be contained in a single tablet or in separate tablets that can be administered simultaneously or sequentially in any order. In addition, it should be recognized that compositions can take different forms. For example, one or more compounds may be delivered as a tablet and the other may be administered by injection or orally as a syrup. All combinations, delivery methods and sequences of administration are contemplated.

The term malignant tumor refers to a physiological condition in mammals that is characterized by uncontrolled cell growth. Common classes of malignant tumors include carcinomas, lymphomas, sarcomas, and blastomas.

The compound(s) of the present invention can be used for the treatment of cancer. Methods of treating cancer include administering to a patient in need of such treatment a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof.

The compound(s) of the present invention can be used for the treatment of tumors. Methods of treating a tumor include administering to a patient in need of such treatment a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof.

The invention also relates to the use of the compound(s) of the present invention in the manufacture of a medicament for the treatment of a condition such as cancer.

Malignant tumors that can be treated with the compound(s) of the present invention include, but are not limited to, carcinomas such as bladder, breast, colon, rectal, kidney, liver, lung (small cell lung cancer and non-small cell lung cancer ), esophagus, gall bladder, ovaries, pancreas, stomach, cervix, thyroid, prostate and skin (including squamous cell carcinoma); hematopoietic tumors of the lymphoid cell line (including leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, villous cell lymphoma and Burkett's lymphoma); tumors of hematopoietic tissue of myeloid origin (including acute and chronic myelogenous leukemia, myelodysplastic syndrome and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma, as well as other sarcomas, such as soft tissue and bone); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma and schwannomas) and other tumors (including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderma pigmentosum, keratoctantoma, follicular thyroid cancer and Kaposi's sarcoma). Other types of cancer that can be treated with the compound(s) of the present invention include endometrial cancer, head and neck cancer, glioblastoma, malignant ascites and malignant tumors of hematopoietic tissue.

Specific types of malignant tumors that can be treated with the compound(s) of the present invention include soft tissue sarcomas, malignant bone tumors such as osteosarcoma, breast tumors, bladder cancer, Li-Fraumeni syndrome, brain tumors, rhabdomyosarcoma , adrenal cortical carcinoma, colon malignancy, non-small cell lung cancer and acute granulocytic leukemia (AML).

In a specific embodiment of the invention that relates to the treatment of cancer, the cancer is identified as p53 wild type (p53 ^WT ). In another specific embodiment, the cancer is identified as p53 and CDKN2A mutant. In another aspect, the present invention provides a diagnostic for determining which patients should be administered a compound of the present invention. For example, to determine the p53 and/or CDKN2A status of cancer cells, a sample of a patient's cancer cells may be collected and analyzed. In one aspect, a patient with a cancer that is p53 ^WT will be selected for treatment over patients with a cancer that is p53 mutated. In another aspect, a patient with a cancer that is both p53 and has a mutant CDNK2A protein is selected over a patient who does not have these characteristics. The process of collecting malignant cells for analysis is well known to those skilled in the art.

The term p53 ^WT refers to the protein encoded by genomic DNA sequence No. NC 000017, version 9 (7512445, ..., 7531642) (genetic database); the protein encoded by cDNA sequence No. NM 000546 (genetic database); or a protein having sequence No. NP 000537.3 (genetic database).

The term CDNK2A mutant means a CDNK2A protein that is not wild type.

The term wild-type CDKN2A refers to the protein encoded by genomic DNA sequence no. 9:21957751-21984490 (Ensembl ID); the protein encoded by cDNA sequence No. NM 000077 (genetic database) or NM 058195 9 (genetic database); or a protein having sequence No. NP 000068 or NP 478102 (genetic database).

In another aspect, the present invention relates to the use of the compound(s) of the present invention in combination with one or more pharmaceutical agents that are an inhibitor of a protein in the phosphatidylinositol 3-kinase (PI3K) pathway. Combination of the compound(s) of the present invention with inhibitors of PI3K pathway proteins showed synergism in malignant cell growth assays, including enhanced apoptosis and cell killing. Examples of PI3K pathway proteins include PI3K, mTOR, and PKB (also known as Akt). The PI3K protein exists in several isoforms, including α, β, δ, or γ. A PI3K inhibitor that can be used in combination with

- 5 043674 compound of the present invention may be selective for one or more isoforms. By selective is meant that the compound(s) inhibit one or more isoforms to a greater extent than other isoforms. Selectivity is a concept well known to those skilled in the art and can be measured by well known activity in in vitro or cellular studies. Preferred selectivity includes greater than 2-fold, preferably 10-fold, or more preferably 100-fold greater selectivity for one or more isoforms over other isoforms. In one aspect, PI3K inhibitors that can be used in combination with the compound(s) of the present invention are a selective PI3Ka inhibitor. In another aspect, the compound is a selective PI3K inhibitor.

Examples of PI3K inhibitors that can be used in combination with the compound(s) of the present invention include those described, for example, in WO 2010/151791; WO 2010/151737; WO 2010/151735; WO 2010/151740; WO 2008/118455; WO 2008/118454; WO 2008/118468; US 20100331293; US 20100331306; US 20090023761; US 20090030002; US 20090137581; US 20090054405; US 20090163489; US 20100273764; US 20110092504 or WO 2010/108074.

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

mTOR is a protein in the PI3K pathway. Another aspect of the present invention is the use of an mTOR inhibitor in combination with the compound(s) of the present invention. Suitable mTOR inhibitors that can be used in combination with the compound(s) of the present invention include those disclosed, for example, in WO 2010/132598 and WO 2010/096314.

PKB (Akt) is also a protein in the PI3K pathway. Another aspect of the present invention is the use of an mTOR inhibitor in combination with the compound(s) of the present invention. PKB inhibitors that can be used in combination with the compound(s) of the present invention include those disclosed, for example, in US 7354944; US 7700636; US 7919514; US 7514566; US 20090270445 A1; US 7919504; US 7897619 and WO 2010/083246.

The combinations of the present invention can also be used in conjunction with radiation therapy, hormonal therapy, surgery and immunotherapy, which are well known to those skilled in the art.

Since one aspect of the present invention involves the treatment of disease/conditions through a combination of pharmaceutically active compounds that can be administered separately, the invention further relates to the combination of separate pharmaceutical compositions in the form of a kit. The kit includes two separate pharmaceutical compositions: a compound of the present invention and a second pharmaceutical compound. The kit includes a container for individual compositions, such as a divided vessel or a divided foil pouch. Additional examples of containers include syringes, boxes and bags. Typically the kit includes instructions for using the individual components. The kit form is particularly suitable when the individual components are preferably administered in different dosage forms (eg, orally and parenterally), administered at different dosing intervals, or when the individual components of the combination need to be titrated by the attending physician or veterinarian.

An example of such a set is the so-called blister packaging. Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms (tablets, capsules, etc.). Blister packs typically consist of a backing of relatively rigid material covered with a film, preferably of a transparent polymeric material. During the packaging process, depressions/pockets are formed in the plastic film. The pockets are the size and shape of the tablets or capsules being packaged. The tablets or capsules are then placed into the pockets and a backing of relatively rigid material is soldered to the polymer film on the side of the film opposite the direction in which the pockets were formed. As a result, the tablets or capsules are hermetically sealed in pockets between the polymer film and the substrate. Preferably, the strength of the support is such that the tablets or capsules can be removed from the blister pack by manually applying pressure to the pockets, resulting in a hole being formed in the support at the location of the recess. The tablet or capsule can then be removed through the designated opening.

The kit may desirably be provided with a reminder, for example, in the form of numbers next to the tablets or capsules, the numbers corresponding to the dosage days on which the tablets or capsules should be taken orally. Another example of such a reminder is a calendar printed on a card, for example: First week, Monday, Tuesday, ... etc., Second week, Monday, Tuesday, ... etc. etc. Other reminder options will be obvious. The daily dose may be one tablet or capsule or several tablets or capsules that must be taken on a specific day. In addition, a daily dose of a compound of the present invention may consist of a single tablet or capsule, while a daily dose of a second compound may consist of several tablets or capsules, and vice versa. The leaflet should contain this information and facilitate the correct administration of active substances.

Another specific embodiment of the invention relates to a dispenser designed to dispense daily doses individually in the order of their intended use. It is preferable that the dispenser be provided with a reminder to further facilitate patient compliance with the drug regimen. An example of such a reminder is a mechanical counter that shows the number of daily doses dispensed. Another example of such a reminder is a battery-powered memory microchip associated with an LCD display or an audible reminder that, for example, reads out the date of the last daily dose and/or reminds when the next dose is due.

The compound(s) of the present invention and other pharmaceutically active compounds, if desired, can be administered to a patient orally, rectally, parenterally (eg, intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, topically (eg, powders, ointments, or drops) or as a buccal or nasal spray. All methods that are used by those skilled in the art to administer a pharmaceutically active substance are contemplated.

Compositions suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for the preparation of sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerin, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and organic esters for injection, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of contamination of compositions by microorganisms can be achieved by using various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic substances such as sugars, sodium chloride and the like. Prolonged absorption of pharmaceutical compositions for injection can be achieved through the use of absorption retardants, such as aluminum monostearate and gelatin.

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) excipients or diluents such as starches, lactose, sucrose, mannitol and silicic acid; (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum acacia; (c) humectants, for example glycerin; (d) disintegrants such as, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) dissolution retarders, such as paraffin; (f) absorption accelerators, for example quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin and bentonite; and (i) lubricants such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, 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 can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms, such as tablets, dragees, capsules, pills and granules, can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may also contain an opacifying agent and may be formulated to release the active compound or compounds in a specific part of the intestinal tract in a delayed manner. Examples of potting compositions that can be used are polymeric substances and waxes. The active compound may also be in microencapsulated form, if desired, with one or more of the above excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil,

- 7 043674 corn germ oil, olive oil, castor oil and sesame oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, or mixtures of these substances, etc.

In addition to such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents. Suspensions, in addition to the active compound(s), may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agaragar and tragacanth or mixtures of these substances and etc.

Compositions for rectal administration are preferred suppositories, which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at normal room temperature but liquid at bodies and therefore melts in the rectum or vaginal cavity and releases the active component.

Dosage forms for topical administration of the compound(s) of the present invention include ointments, powders, sprays and inhalation dosage forms. The active ingredients are mixed with physiologically acceptable carriers and any preservatives, buffers or, if necessary, propellants under sterile conditions. Ophthalmic compositions, ophthalmic ointments, powders and solutions are also considered to be included within the scope of the present invention.

The compound(s) of the present invention can be administered to a patient in doses of from about 0.1 to about 3000 mg per day. For a normal adult weighing about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kg of body weight is usually sufficient. The specific dosage and dosage range that can be used depends on a number of factors, including the needs of the patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound administered. Determination of dosage ranges and optimal dosages for a particular patient is within the competence of one of ordinary skill in the art.

The compound(s) of the present invention can be administered in the form of pharmaceutically acceptable salts, esters, amides or prodrugs.

The term salts refers to inorganic and organic salts of the compounds of the present invention. The salts may be prepared in situ during the final isolation and purification of the compound or by separately reacting the purified compound in free base or acid form with a suitable organic or inorganic base or acid and isolating the salt thus formed.

Examples of pharmaceutically acceptable esters of the compound(s) of the present invention include C ₁ -C8 alkyl esters. Suitable esters also include C ₅ -C ₇ cycloalkyl esters as well as arylalkyl esters such as benzyl. Typically C ₁ -C4 alkyl esters are used. Esters of the compound(s) of the present invention can be prepared by methods well known in the art.

Examples of pharmaceutically acceptable amides of the compound(s) of the present invention include those derived from ammonia, primary C ₁ -C ₈ -alkylamines and secondary C ₁ -C ₈ -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. Commonly used amides are those derived from ammonia, C ₁ -C ₃ primary alkyl amines and C ₁ -C ₂ secondary dialkyl amines. The amides of the compound(s) of the present invention can be prepared by methods well known to those skilled in the art.

The term prodrug refers to compounds that are transformed in vivo to form a compound of the present invention. Transformation can occur through various mechanisms, for example, through hydrolysis in the blood. A review of the use of prodrugs is provided by T. Higuchi and W. Stella, Prodrugs as New Delivery Systems, Vol. 14, A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

By way of illustration, since the compound(s) of the invention contains a carboxylic acid functionality, the prodrug may include an ester formed by replacing the hydrogen atom of the carboxylic acid group with a group such as C ₁ -C ₈ -alkyl, (C ₂ -C ₁₂ )alkanoyloxymethyl , 1-(alkanoyloxy)ethyl containing from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl containing from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl containing from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy) ethyl containing from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl containing from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl containing from 3 to 9 carbon atoms, 1-(N-( alkoxycarbonyl)aminomethyl containing from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C ₁ -C ₂ )alkylamino(C ₂ -C ₃ )alkyl (for example, β-dimethylaminoethyl), carbamoyl-(C ₁ -C ₂ )alkyl, N,N-di(C ₁ -C ₂ )alkylcarbamoyl(C ₁ -C ₂ )alkyl and

- 8 043674 piperidino-, pyrrolidino- or morpholino(C ₂ -C ₃ )alkyl.

The compound(s) of the present invention may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereoisomeric forms of the compound(s), as well as mixtures thereof, including racemic mixtures, are intended to form part of the present invention. In addition, the present invention provides for all geometric and positional isomers. For example, if a compound contains a double bond, both cis and trans forms (denoted Z and E, respectively), as well as mixtures thereof, are contemplated.

Mixtures of stereoisomers, eg mixtures of diastereomers, depending on their physicochemical differences, can be separated into their individual stereochemical components by known methods such as chromatography and/or fractional crystallization. Enantiomers can also be separated by converting an enantiomeric mixture into a diastereomeric mixture by reacting with a suitable optically active compound (eg, alcohol), separating the resulting diastereomers, and then converting (eg, by hydrolysis) the individual diastereomers into the corresponding pure enantiomers.

The compound(s) of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water (hydrate), ethanol, and the like. The present invention contemplates and covers both solvated and non-solvated forms as defined herein.

It is also possible that the compound(s) of the present invention may exist in different tautomeric forms. All tautomers of the compound(s) 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 compound(s) are included in the present invention.

Those skilled in the art will appreciate that the compound names and structures contained herein may be based on the specific tautomer of the compound. Although it is possible to use the name or structure only for a particular tautomer, all tautomers are intended to be covered by the present invention unless otherwise noted.

It is also intended that the present invention covers compounds that are synthesized in vitro by laboratory methods, for example, methods well known to synthetic chemists; or synthesized by in vivo methods such as metabolism, fermentation, biological processing, etc. It is also contemplated that the compound(s) of the present invention can be synthesized using a combination of in vitro and in vivo methods.

The present invention also includes isotopically labeled compounds that are identical to the compounds that are described herein, except for the fact that one or more atoms are replaced by an atom whose atomic mass or mass number differs from the atomic mass or mass number typically found in nature. Examples of isotopes that may be included in the compound(s) of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁶ O , ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ⁴⁸ F and ³⁶ Cl. In one aspect, the present invention relates to compounds in which one or more hydrogen atoms are replaced by deuterium atoms ( ^2H ).

The compound(s) of the present invention that contain the above isotopes and/or other isotopes of other atoms are included within the scope of the present invention. Some isotopically labeled compounds of the present invention, for example compounds into which radioactive isotopes such as ³ H and ¹⁴ C are included, are used in tissue distribution assays of drugs and/or substrates. Tritium-labeled isotopes, i.e. ³ H, and carbon-14, i.e. ¹⁴ C are preferred, in particular, due to the ease of their preparation and detection. In addition, substitution with heavier isotopes such as deuterium, e.g. 2H may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some cases. The isotopically labeled compounds of the present invention can generally be prepared by replacing a readily available isotopically labeled reagent with a non-isotopically labeled reagent.

The compound(s) of the present invention may exist in various solid states, including crystalline states, as well as in an amorphous state. Various crystalline states (also called polymorphs) and amorphous state of the compound(s) of the present invention are included within the scope of the present invention as provided herein. When synthesizing the compound(s) of the present invention, it may be desirable to use certain leaving groups.

The term leaving groups (LG) generally refers to groups that can be substituted 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 ₃ ), N-hydroxysuccinimide, N-hydroxybenzotriazole, etc. P. Examples of nucleophiles include, but are not limited to, amines, thiols, alcohols, Grignard reagents, anionic species (eg, alkoxides, amides, carbanions), and the like.

All patents, published patent applications, and other publications cited herein are incorporated herein by reference.

The specific experimental examples presented herein illustrate specific embodiments of the present invention. These examples are for illustrative purposes and are not intended to limit the scope of the claims in any way.

Ή-NMR spectra were routinely acquired on a Bruker Avance III 500 spectrometric system (Bruker, Billerica, MA) operating at 1H frequency 500.13 MHz, equipped with a Bruker PABBI 5 mm z-gradient probe; or on a Bruker Avance II or Avance III 400 spectrometer operating at 1H frequency 400.23 MHz, equipped with a Bruker PABBO 5 mm z-gradient probe. For NMR analysis, samples were routinely dissolved in 500 μl of DMSO-d ₆ or CD ₃ OD. The 1H chemical shifts refer to the residual solvent signals from DMSO-d ₆ at δ 2.50 and CD ₃ OD at δ 3.30.

Significant peaks were tabulated and standardly included the number of protons, multiplicity (s - singlet; d - doublet; dd - doublet of doublets; t - triplet; q - quartet; m - multiplet; br.s - broadened singlet) and constant(s) interactions in hertz (Hz). Electron ionization (EI) mass spectra were routinely recorded on an Agilent Technologies 6140 Quadrupole LC/MS mass spectrometer (Agilent Technologies, Englewood, CO). Mass spectrometry results are presented as mass-to-charge ratios, sometimes with the relative intensity of each ion indicated (in parentheses). The starting materials in the examples below are routinely obtained from either commercial sources, such as Sigma-Aldrich, St. Louis, MO, or by methods published in the literature.

X-ray powder diffraction (XRPD) data were acquired using a Bruker D8 Discover X-ray diffraction system (Bruker, Billerica, MA) equipped with a Brown detector and a Cu-Ka radiation source operating in Bragg-Brentano reflectance geometry. The 2Θ values were generally determined with an accuracy of ±0.2°. The samples were generally prepared without any special treatment other than applying slight pressure to obtain a flat surface. Samples were evaluated uncoated 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°.

Differential scanning calorimetry (DSC) was performed on a Perkin Elmer DSC-7 instrument or a TA Instruments Q2000 instrument. Samples were obtained in a closed gold sample cell at a temperature ramp rate of 5°C/min from 20 to about 350°C. The DSC thermogram of crystalline DHO is shown in FIG. 19 with a melting point of 73.86°C.

Examples

Example 1: Method for preparing selected intermediates

Step A. 2-(3-Chlorophenyl)-1-(4-chlorophenyl)ethanone.

Sodium bis-(Trimethylsilyl)amide (1 M in tetrahydrofuran, 117 ml) was slowly added to a solution of 2-(3-chlorophenyl)acetic acid (10 g, 58.6 mmol) at -78°C in tetrahydrofuran (58 ml) in for 1 hour. After stirring at -78°C for 40 minutes, a solution of methyl 4-chlorobenzoate (10 g, 58.6 mmol) in tetrahydrofuran (35 ml) was added over 10 minutes. The reaction mixture was stirred at -78°C for 3 hours and then allowed to warm to 25°C. After 2 hours at 25°C, the reaction was quenched with saturated aqueous ammonium chloride and most of the tetrahydrofuran was removed under reduced pressure. The residue was extracted with ethyl acetate (2x100 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.

An alternative method for the preparation of 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone.

Thionyl chloride (39.1 kg, 329 mol) for 30 min. The mixture was heated 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 1.5 hours. The mixture was heated to 20°C and stirred for 15 hours. Water (200 L) and ethanol (200 L) were added to the mixture and the two-phase mixture was stirred for 2 hours. The phases were separated and the organic phase was washed twice with an aqueous solution of tetrasodium ethylenediaminetetraacetic acid (3 wt.%, 200 L) and once with water ( 200 l). Heptane (1600 L) was added to the organic phase over 15 min. The suspension was stirred for 30 minutes, cooled to -5°C and filtered. The filtered substance was dried at 40°C for 20 hours. 2-(3-chlorophenyl)-1(4-chlorophenyl)ethanone was isolated with a yield of 83.6% (67.4 kg).

Ή NMR (500 MHz, DMSO-d ₆ ) δ ppm: 8.05 (m, 2H), 7.62 (m, 2H), 7.33 (m, 3H), 7.21 (br. d, J=7.3 Hz, 1H), 4.45 (s, 2H);

MS (ESI) = 265.1 [M+H]+.

Step B. Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

Methyl methacrylate (12.65 ml, 119 mmol) was added to a solution of 2-(3-chlorophenyl)-1-(4chlorophenyl)ethanone (30 g, 113 mmol) (prepared in step A) in tetrahydrofuran (283 ml). Potassium tert-butoxide (1.27 g, 11.3 mmol) was then added and the reaction mixture was stirred at room temperature for 2 days. Then the solvent was removed in vacuo and replaced with ethyl acetate in an amount of 300 ml. The organic phase was washed with brine (50 ml), water (3x50 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-5oxopentanoate as a mixture of diastereomers with a ratio of about 1:1.

1H NMR (400 MHz, CDCl3) δ ppm: 7.87 (m, 2H), 7.38 (m, 2H), 7.27-7.14 (m series, 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, 5.5 Hz, 0.5H), 1.96 (ddd, J=13.7, 9.0, 4.3 Hz, 0.5H), 1.22 (d, J=7.0 Hz, 1.5H), 1.16 (d, J=7.0 Hz, 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 o o

Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate (40 g, 104.0 mmol) (obtained in step B) was dissolved in 200 ml anhydrous toluene and concentrated in vacuo. The residue was placed under high vacuum for 2 hours before use. The compound was divided into 2 batches of 20 g and worked up 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 apparatus. RuCl ₂ (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 left to stir at room temperature. If necessary, additional hydrogen was added to the reaction mixture. After 3 days, the reaction mixtures were combined and partitioned between 50% saturated ammonium chloride 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.

The crude product (mainly (4R,5R)-isopropyl-4-(3-chlorophenyl)-5-(4-chlorophenyl)-5hydroxy-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. Aqueous solution 1N. hydrochloric acid was added with stirring until the pH of the aqueous layer was about 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 mixture was refluxed with a Dean-Stark fitting until the secoacid was consumed (about 2 hours). The reaction mixture was cooled to room temperature and washed with 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;

- 11 043674 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-pyran2-one as a white solid with an enantiomeric ratio of approximately 94:6 and a mixture of methyl diastereomers with a ratio of 7:3.

1H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.22-6.98 (m series, 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, 0.7N), 3.67 (dt, J=6.6, 4.3 Hz, 0.3n), 3.55 (td, J=7, 8, 4.7 Hz, 0.7H), 2.96 (d 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 (dd, J=14.1, 12.5, 7.4 Hz, 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]+;

[a]D (22°C, s=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

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) (prepared in step C) and allyl bromide (3.48 ml, 40.3 mmol) in tetrahydrofuran (22 ml) at -35°C (acetonitrile/dry ice bath) were treated with a solution of lithium bis-(trimethylsilyl)amide in tetrahydrofuran (1.0 M, 17.45 ml, 17.45 mmol). The reaction mixture was allowed to warm to -5°C for 1 hour and then quenched with 50% saturated ammonium chloride. The reaction mixture was diluted with 100 ml ethyl acetate and the layers were separated. The organic phase was washed with brine, dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound as a white solid on standing in vacuo. Chiral supercritical fluid chromatography (SFC) (92% CO ₂ , 8% methanol (20 mM ammonia), 5 ml/min, Phenomenex Lux-2 column (Phenomenex, Torrance, CA), 100 bar (10000 kPa), 40°C , 5-minute method) was used to determine that the compound had 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-(3chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one was recrystallized by adding to heptane (4.7 g suspension in 40 ml) at reflux followed by dropwise addition of 1.5 ml of toluene to dissolve. The solution was cooled to 0°C. The resulting white solid was filtered and washed with 20 ml of cold heptane to obtain a white powder. Chiral SFC (92% CO ₂ , 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 (400 MHz, CDCl3) δ ppm: 7.24 (ddd, J=8.0, 2.0, 1.2 Hz, 1H), 7.20-7.15 (m series, 3H), 6.91 (t, J=2.0 Hz, 1H), 6.78 (bd, J=7.6 Hz, 1H), 6.60 (m, 2H), 5.84 ( ddt, J=17.6, 10.2, 7.4 Hz, 1H), 5.70 (d, J=5.3 Hz, 1H), 5.21-5.13 (m series, 2H), 3.82 (dt, J=11.7, 4.5 Hz, 1H), 2.62 (ABX Jab=13.7 Hz, Jax=7.6 Hz, 1H), 2.53 (ABX, JAB= 13.9 Hz, J _BX =7.2 Hz, 1H), 1.99 (dd, J=14.1, 11.9 Hz, 1H), 1.92 (ddd, J=13.9, 3, 9, 1.2 Hz, 1H);

¹³ C NMR (CDCl ₃ , 100 MHz, 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=1730 cm ^-1 . [a] _D (24°C, s=1.0, CH ₂ Cl ₂ )=-191°;

m.p. 111-114°C.

An alternative procedure for the preparation of (3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

Step 1. Isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

A solution of 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone (Step A) (67.4 kg, 255 mol) in THF (325 L) s.b.

- 12 043674 was sewn azeotropically to achieve a water content determined by the Karl Fischer method of 0.05% by weight. 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 30 min and the mixture was stirred for 6 h. The mixture was then cooled to 10°C and an aqueous monohydrate solution was added over 5 min citric acid (20 wt.%, 35 l). 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 separated. The organic phase was distilled under reduced pressure to give a distillate of 560 L, while isopropanol (350 L) was added to give a solution of methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5oxopentanoate in isopropanol ( 54 wt.%, total mass of solution 140 kg). The water content in the solution was 0.01 wt.%, determined by the Karl Fischer method. Additional isopropanol (420 L) and sulfuric acid (53 kg, 535 mol) were added to the solution. The mixture was refluxed and stirred for 12 hours, during which time 200 L of the 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 30 min. 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 with water (200 L) four times. The organic phase was distilled under reduced pressure to give a distillate of 500 L, while isopropanol (50 L) was added to give a solution of isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate in isopropanol (60 wt.%, 134 kg of total solution mass). The water content in the solution was 0.02 wt.%, determined by the Karl Fischer method. Isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate was obtained in a total yield of 81% as a mixture of diastereoisomers with a ratio of about 1:1.

1H NMR (400 MHz, CDCl3, δ ppm): 7.70-7.80 (m, 2H), 7.22-7.28 (m, 2H), 7.00-7.18 (series m, 4H), 4.784.96 (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

To a degassed solution of isopropyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5oxopentanoate (from step 1) in isopropanol (60 wt.%, 252 kg total mass of solution, 151 kg of starting material per isopropyl ether base, 385 mol) degassed isopropanol (900 L) and potassium tert-butoxide (13 kg, 116 mol) were added. Separately, a degassed solution of (S)-RUCY®-XylBINAP (also known as RuCl[(S)-diapene][(S)-xylbinap]) (230 g, 0.2 mol, catalyst, Takasago International Corporation, Rockleigh, NJ) was prepared ) in isopropanol (25 l). The mixture was purged with hydrogen four times at 5 bar (500 kPa) and stirred at 20°C for 5.5 hours. The hydrogen injection was stopped and the mixture was degassed with nitrogen. Tetrahydrofuran (460 L) was added to the mixture. A solution of lithium hydroxide (24 kg, 576 mol) in water (305 L) was added to the reaction mixture over 40 minutes and the resulting mixture was stirred at 20°C for 24 hours. A solution of concentrated hydrochloric acid (79.3 kg, 11.4 M, 740 mol) in water (690 L) for 2 hours. Toluene (580 L) was added, then the mixture was stirred for 30 minutes and the phases were separated. The aqueous phase was extracted with toluene (700 L). The combined organic layers were washed with aqueous sodium chloride (25 wt.%, 700 kg). The organic phase was subjected to distillation at atmospheric pressure and a temperature of 100°C to obtain a distillate with a volume of 2700 l while adding toluene (800 l). After this solvent change, less than 0.05 wt.% isopropanol or water (content determined by the Karl Fischer method) remained in the mixture. Carbonyldiimidazole (59 kg, 365 mol) was added to the toluene solution over 2 hours and the mixture was stirred at 20°C for another 2 hours. The mixture was then cooled to 10°C and a solution of orthophosphoric acid (72 kg, 545 mol) in water was added (400 L) for 1 hour, maintaining the temperature of the mixture below 20°C. The mixture was stirred for 30 minutes, the phases were separated and the organic layer was washed with aqueous sodium chloride (25 wt.%, 484 kg). Toluene (400 L) was distilled at atmospheric pressure and a temperature of 110°C. After cooling the solution to 20°C, tetrahydrofuran (500 L) was added and the water content measured by the Karl Fischer method was 0.03 wt%. 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 in toluene (255 kg, 26 wt.%, 492 mol) was added over 6 hours and the mixture was stirred at -10°C for 1 hour. The mixture was warmed to 0°C and an aqueous solution of phosphoric acid (40 wt. .%, 400 mol) for 3 hours. The mixture was heated to 20°C. Water (200 L) and dichloromethane (400 L) were added. The mixture was stirred for 15 minutes and the phases were separated. The solution was distilled at atmospheric pressure and 100°C to obtain a distillate with a volume of 1350 L, and the residual toluene in the mixture was measured 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 hours, cooled to 30°C and filtered. The filtered material was washed three times

- 13 043674 heptane (80 l). The solids were dried at 55°C for 48 hours to obtain 90.1 kg of (3S,5R,6R)-3allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran -2-she with a total yield of 63%. Chiral HPLC showed an enantiomeric ratio of 99.95:0.05.

Example 2. Differences between the first-in-human process and the industrial process for producing compound A.

The synthesis of DLAC on a gram scale has been previously reported. 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 illustrated in Scheme 1. For this synthesis, the OXOS intermediate was used as the regulatory starting material. DLAC ring opening using excess L-valinol (3 equivalents) at elevated temperature was provided by ABA amide, which was extracted in dichloromethane. Excess L-valinol was removed using an aqueous hydrochloric acid wash and the product solution was used in the next step without purification. Reducing the amount of L-valinol used in this conversion was identified as a further development goal due to the high cost of this starting material. Notably, the preparation of ABA, ABA1 or ABA2 analogues, from the corresponding DLAC, DLAC1 or DLAC2 analogues, which bear side chains containing a group with the same oxidation state as the carboxylic acid of compound A, was not successful due to the formation of undesirable succinimides, SUC1 or SUC2. Considering the similar rates observed for the formation of the target 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.

Scheme 1

First-in-human production process for compound A

Scheme 2

An attempt to obtain ABA analogues (k1 » k2)

- 14 043674

Oxoiminium salt OXOS was prepared from ABA by double activation with two equivalents of toluenesulfonic anhydride and 2,6-lutidine at elevated temperature. The cation thus obtained was isolated as a 2-naphthylsulfonic acid salt, which had satisfactory impurity removal properties. Identification of an alternative reagent for toluenesulfonic anhydride (TsO ₂ ) was considered for several reasons, including the need to eliminate the persistent tosylate intermediate DHO-OT, which underwent slow conversion to OXOS at elevated temperature (120°C). This intermediate (DHO-OT) is an alkylating agent and therefore a potentially mutagenic impurity. One possible alternative was to isolate the crystalline intermediate DHO (Scheme 3) with increased control over efficient impurity removal for the manufacturing sequence. However, this required the use of a reagent that provides selective chemoselective activation of the primary alcohol ABA in the presence of a secondary benzyl alcohol. Reagents based on sulfonic anhydrides do not have this effect.

Scheme 3

Intermediates in the preparation of OXOS from ABA using Ts ₂ O

120 °C I h |

OXOS

Preparation of SUL from OXOS was accomplished by treating OXOS with isopropylsulfinic acid in the presence of sodium tert-butoxide. This transformation proceeds through the reversible formation of a diastereomeric pair of sulfinate intermediates (SULFI) and subsequent rearrangement into the thermodynamic product SUL, which crystallizes from acetonitrile and water (Scheme 4). The byproduct ALC is irreversibly formed under these conditions in the presence of water. Isopropyl sulfinic acid, an oil at 20°C, was prepared from isopropyl magnesium chloride and isolated after aqueous treatment. Before use in the formation of SUL, azeotropic drying of this reagent was necessary to avoid the formation of the unwanted ALC byproduct in large quantities. However, decomposition of isopropyl sulfinate due to disproportionation during drying was observed and thus this operation was excluded. Consequently, efforts were made to discover a stable crystalline salt of isopropyl sulfinic acid that was stable under drying conditions and that could be called a commercial regulatory starting material. As an alternative, a method for in situ preparation of the sulfinic acid salt from isopropylmagnesium chloride without aqueous treatment and further reaction with OXOS was considered.

Scheme 4

Intermediates and by-products formed during the production of SUL from OXOS

Oxidation of the alkene group of SUL was carried out by treatment with catalytic ruthenium chloride (2 mol.%) and an excess amount of sodium periodate (5 eq.). The crude product was isolated as

- 15 043674 crystalline ethanol solvate. It has been observed that some features of this stage are undesirable. First, the heavy metal used in this stage had to be removed, which was achieved using DARCO-G resin for first-in-human delivery. In addition, several equivalents of sodium periodate were needed to carry out this process, and the reagent had to be loaded into the reaction vessel in batches to minimize the formation of impurities. A complex post-processing protocol (extraction and filtration) was required to remove the large quantities of salts used for the conversion. Also, at this stage of transformation, many dimeric impurities were formed, which made it difficult to control the purity of the drug substance. Using the ethanol solvate of Compound A as a crystalline control point was problematic and was only moderately effective in removing impurities present in the mixture. In addition, the crystallization process had to be carried out as an evaporation process due to the low ethanol concentration (5% v/v) which was necessary to reduce the high loss of mother liquor during the filtration process. It has also been noted that the use of ethanol in the crystallization process reduces the reliability of the process due to the undesirable formation of the corresponding ethyl ester at temperatures above 30°C and the difficulty encountered in removing the ethyl ester from the desired ethanol solvate. Likewise, when methanol was used in the crystallization of compound A, the formation of the corresponding methyl ester at elevated temperatures, which was also difficult to separate from the drug substance, significantly reduced the efficiency of this crystallization method, particularly when operating on a multigram scale. Therefore, the development of a more consistent and environmentally benign oxidation route to obtain Compound A from SUL, as well as the establishment of a robust drug isolation strategy that demonstrates effective control of critical parameters, is required as part of a commercially viable process.

Table 1

Modifications for the FIH process in the industrial _______method for preparing compound A_______

CP1 stage process FIH process Implementation industrial method ABA from DLAC Three equivalents of L-valinol used Decrease in quantity L-valinol equivalents up to two OXOS from ABA DHO not allocated Replacement of Ts ₂ O with a chemoselective reagent to ensure the release of DHO OXOS from ABA PMI and persistent DHOOT intermediates are formed Replacement of Ts ₂ O with a reagent that provides the formation of an intermediate product that quickly turns into OXOS SUL from OXOS Isopropylsulfinic acid is liquid at 20°C and is unstable under conditions azeotropic drying Detection of crystalline salt isopropylsulfinic acid, stable under drying conditions Compound A from At the last stage Replacing reagents with SUL ruthenium is used catalyst last stage Compound A from At the last stage Replacing reagents with SUL excess is used amount (5 eq.) sodium periodate last stage Selection Release is only moderate Salt detection connections A V effective in removing connections A, good form impurities and poorly matched corresponding scheme ethanol solvate crystallization scheme crystallization and

- 16 043674

Selection connections A Separation is not very effective in removing the unwanted corresponding methyl ester that may form in the crystallization system used (MeOH/ _H2O ) with excellent removal properties impurities Development of a new crystallization process for the drug substance, compound A, which does not contain impurities

Example 3. Development of an industrial method for producing the intermediate product OXOS.

Thermal amidation of DLAC to ABA with L-valinol was accomplished through a multistep mechanism using an EST ester intermediate (Scheme 5). The initial transesterification of DLAC to EST was determined to be a reversible process (k1>k_1 with 2 equiv of L-valinol), resulting in the accumulation of EST before rearrangement into the amide product ABA. When the reaction is carried out at 60°C, a rapid conversion of DLAC to EST is observed at the beginning of the reaction, followed by a slow conversion of EST to ABA over several days (k1> _k2 ) (Fig. 1). At elevated temperatures (115°C) (Figure 2), the rearrangement of EST into the more stable ABA occurs more rapidly, resulting in an increase in the overall rate of interaction due to increased concentration of EST.

Scheme 5

Kinetics of thermal amidation of DLAC to ABA

The FIH process used a thermal melt with 3 eq. L-valinol to ensure rapid conversion of DLAC to ABA at 110°C. Consistent with the mechanistic understanding of this conversion, decreasing the L-valinol loading (from 3 to 2 eq) resulted in a decrease in the overall rate of interaction, as the conversion of DLAC to EST (k1) directly affects the relative concentration of EST. When using 2 eq. L-valinol, the reaction was observed to require 72 hours to achieve conversion at 115°C, using toluene (1 volume) to ensure homogeneity of the reaction. However, this longer processing time may be justified based on the significant cost/cost savings. Removal of excess L-valinol was achieved by adding toluene (4 volumes) and subsequent washing of the organic mixture with an aqueous solution of hydrochloric acid. The resulting organic solution was azeotropically dried and filtered through a final filter to obtain ABA in 91% analytical yield as a 28 wt% solution in toluene containing 2.7 LC area % DHO, 1.0 LC area % DLAC starting material and 1 .0% LC area EST. Thermolysis of ABA to produce DHO directly at higher temperatures resulted in complex mixtures of products.

Isolation of the DHO intermediate provided an additional opportunity to remove impurities from the process stream and enhance the overall control strategy for drug delivery to market. Primary to this strategy was the identification of conditions that would couple the dehydrative double cyclization of ABA with OXOS into two separate monocyclization reactions by designing a chemoselective activation of the primary alcohol ABA (condition A), which would allow the release of DHO in crystalline form (Scheme 6).

Scheme 6

Sequential dehydration of ABA to OXOS

- 17 043674

The reagents sulfonyl chloride and sulfonic anhydride were found to be non-selective in distinguishing between primary and secondary alcohols ABA, and were difficult to obtain as anhydrous reagents. In addition, the use of acid catalysts also provides complex mixtures of products. However, the Vilsmeyer salt reagent, methoxymethylene-N,N-dimethyliminium methyl sulfate, successfully achieved the desired selectivity. This reagent was easily prepared without specific precautions to exclude moisture and can be stored at 20°C for several months without loss of titer. In addition, it exhibited more moderate reactivity and improved chemoselectivity compared to the conventional Vilsmeyer salt halide derivative, chloromethylene-N,N-dimethyliminium chloride, and also eliminated the formation of alkyl halide by-products. The formation of DHO from ABA using methoxymethylene-N,N-dimethyliminium methyl sulfate in toluene was assessed in the presence of various weak inorganic bases at 25°C and the conversion to DHO was recorded (Scheme 7). It was observed that the reaction was best achieved with KOAc, but NaOAc was preferred given its low hygroscopicity and cost.

Scheme 7

Evaluation of different bases in the monocyclization of ABA to DHO

The desired chemoselectivity of this transformation was achieved due to the unique ability of methoxymethylene-N,N-dimethyliminium methyl sulfate to undergo dynamic transesterification with alcohols through the formation of labile intermediate imidates. This reversibility was examined by activating 4-chlorobenzyl alcohol (CHA) with the deuterated reagent DEU to form 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 exists in low concentrations during the reaction and undergoes rapid intramolecular displacement by the pendant amide to form the oxazoline DHO (Scheme 8). Any imidate formed by derivatization of the secondary alcohol group in the ABA group does not undergo further cyclization to OXOS at the operating reaction temperature (30°C).

Scheme 8

Reversible activation of alcohols with Vilsmeyer reagents

Equilibrium solubility measurements were obtained for DHO in various solvents. It was observed that all values obtained were greater than 20 mg/mL at 20°C (including heptane), with the exception of water (<0.1 mg/mL), which was selected as the antisolvent. Acetonitrile was chosen as the crystallization solvent because it provided easy removal of impurities when combined with water. A curve showing solubility values at various times during crystallization is shown in FIG. 3. Using this protocol, crystalline DHO was isolated with

- 18 043674 yield 88% from DLAC with >98% LC area (Scheme 9).

A Bruker D8 powder X-ray diffractometer was used to obtain powder X-ray diffraction reflectance patterns of crystalline DHO (Fig. 17) and was equipped with a Brown detector and a Cu-Ka radiation source operating in Bragg-Brentano reflectance geometry. The resulting 2-theta (2θ) values were generally accurate to within ±0.2°. The samples were usually obtained without any special treatment other than applying slight pressure to obtain a flat surface. Unless otherwise stated, samples were measured without coating. 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. The sample was static during the measurement.

The peaks presented in the table. 2 were identified from the X-ray diffraction pattern of crystalline DHO.

Table 2 ___________XRD peaks for crystalline DHO____________

Peak Corner d value Intensity Relative intensity 1 6.3 14.12344 753 14.90% 2 7.3 12.15811 3080 61.10% 3 8.5 10.43913 2521 50.00% 4 10.0 8.86434 871 17.30% 5 10.5 8.42517 569 11.30% 6 11.0 8.03156 934 18.50% 7 11.5 7.6782 448 8.90% 8 12.8 6.9125 618 12.30% 9 13.4 6.61879 2598 51.50% 10 14.5 6.11557 4427 87.80% eleven 14.8 5.98483 474 9.40% 12 15.2 5.84027 331 6.60% 13 15.9 5.57383 5042 100.00% 14 15.8 5.59708 4060 80.50% 15 17.0 5.22058 757 15.00% 16 17.5 5.0574 749 14.80% 17 17.8 4.97509 613 12.20% 18 18.4 4.82391 662 13.10% 19 18.8 4.72167 937 18.60% 20 19.0 4.67741 969 19.20% 21 19.7 4.49805 165 3.30% 22 19.9 4.46033 169 3.30%

- 19 043674

Industrial method for obtaining DHO from DLAC

1.HaOAc(1.2eq.) o

Ct

DLAC ii.Toluene .(4.vol.) Ig.Promvvka 1nHC1

IV. Forest ^opyaaya. drying...Qi

Toluene (Hob.)

115^0/72 i'

91% ii. BosHrfNt^CI .£11.3amema 'raoiasfitylya ia acetomiga?r»ig iv. ^fyetavyeadaya (ad "¥oa" rizh/water1

Toluene -(5όβ.ϊ 30 °C, 2 h

Ci

>98%. according to short circuit and area

The abandonment of the double dehydrative cyclization of ABA to OXOS required the development of a method for converting DHO to OXOS. Methanesulfonic anhydride ( _Ms2O ) was found to provide faster conversion to OXOS compared to _Ts2O because the potentially mutagenic mesylate intermediate DHO-OM was completely consumed at 75°C within 10 hours. This improvement is likely due to with a decrease in steric isolation occurring in the transition state leading from DHO-OM to OXOS compared to that associated with the cyclization of DHO-OT to OXOS. The conversion was found to proceed well with 2,6-lutidine as the base in toluene. Nucleophilic organic bases and inorganic bases undesirably provided complex mixtures of products. The mesylate salt OXOS formed during the transformation is poorly soluble in toluo

- 20 043674 le and forms a separate liquid layer during the reaction. To allow further processing, it is necessary to dilute the reaction mixture with dichloromethane (8 vol) before removing the mesylate salts using aqueous sulfuric acid washes. Metathesis of the salt with aqueous sodium 1-naphthalene sulfonate was followed by distillation of dichloromethane, which crystallized the OXOS salt of the toluene hemisolvate 1-naphthalene sulfonate in 90% yield, 99.5% LC area, and 99.7 wt% from DHO (Scheme 10).

Scheme 10

Industrial method for obtaining OXOS from DHO

The following experimental procedures illustrate the production of OXOS.

1.DMF (1.2 eq.) ~

О О bo°с, 2h Me OSO ₃ Me ?sC -------------* ©N=\ ^Me0 ° ^Me Me ⁷ ОМе

DMS Vilsmeyer salt

X _f O eq. DMF solution

Adduct of N,N-dimethylformamide and dimethyl sulfate.

A 500 mL Atlas reactor connected to a reflux condenser and equipped with an overhead stirrer was charged with dimethyl sulfate (200.0 mL, 2.11 mol, 1.0 eq.) under a nitrogen atmosphere. The reactor contents were heated to 60°C. DMF (200.0 mL, 2.56 mol, 1.2 eq) was added dropwise over 60 min (3.3 mL/min). After addition was complete, the reaction mixture was stirred for 2 hours at 60°C. After completion of the reaction, the reaction mixture was cooled to room temperature to obtain the N,N-dimethylformamide-dimethyl sulfate adduct as a solution in residual DMF (402.6 g, 2.02 mol, 95.8% analytical yield, 82.8 wt.% in DMF).

1.0 eq. toluene solution (S)-2-((2R,3R)-2-(3-Chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N-((S)-1-hydroxy-3methylbutane-2 -yl)-2-methylpent-4-enamide (ABA).

(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2Hpyran was loaded into a 5 L ChemGlass reactor equipped with a reflux condenser and an overhead stirrer -2-one (DLAC) (201.8 g, 0.53 mol, 98.6 wt%, 1.0 eq), L-valinol (110.8 g, 1.06 mol, 2.0 eq .) and toluene (205 ml, 1 ml/g) under nitrogen atmosphere. The reactor contents were refluxed (115°C) with constant stirring for 72 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and diluted with toluene (800 ml, 5 ml/g). The reaction mixture was quenched by adding 1N in portions. HCl (1000 ml, 5 ml/g). The phases were separated and then the organic layer was washed twice with brine (2x400 ml, 2 ml/g). The organic phase was dried over magnesium sulfate, filtered through a final filter (high porosity) while washing with toluene and concentrated to a volume of about 800 ml to obtain ABA as a solution in toluene (229.4 g, 0.48 mol, 90.5% analytical yield, 27.9 wt.% in toluene).

1H 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), 2.97 (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, 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 C 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,

- 21 043674

19.40, 18.72.

ABA 1.0 eq.

i. NaOAc (1.2 eq.) Me /~~*O Vilsmeyer salt (1.5 eq.) Toluene (5 vol.) 30 °C, 2h

ϋ Water treatment iii. MeCN (8 vol.) _C |

Deionized water (8 vol.) 25°C, 7h

DHO (1R,2R,4S)-2-(3-Chlorophenyl)-1-(4-chlorophenyl)-4-((S)-4-isopropyl-4,5-dihydrooxazol-2-yl)-4methylhept-6 -en-1-ol (DHO).

(S)-2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl) was charged into a 5 L ChemGlass reactor equipped with a reflux condenser and an overhead stirrer. -N-((S)-1hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide (ABA) (229.4 g, 0.48 mol, 27.9 wt% in toluene, 1 .0 eq.) and toluene (1145 ml, 5 ml/g) under nitrogen atmosphere. (Note: Since ABA was prepared as a stock solution in toluene containing 685 ml of residual toluene, the amount of additional toluene required is 460 ml). The reactor contents were heated to 30°C. NaOAc (48.3 g, 0.59 mol, 1.2 eq.) and N,N-dimethylformamide-dimethyl sulfate adduct (174.1 g, 0.72 mol, 82.8 wt.%, 1.5 eq. ) were successively added to the reaction mixture. After stirring at 30°C for 2 hours, the reaction mixture was cooled to room temperature. The reaction was quenched with a saturated aqueous solution of NH ₄ Cl (750 ml, 3 ml/g) and H2O (500 ml, 2 ml/g). The phases were separated and then the organic layer was washed twice with brine (2x750 ml, 3 ml/g). The organic phase was dried over magnesium sulfate, filtered through a final filter (large porosity) while washing with toluene and concentrated in vacuo. The crude residue was recrystallized from MeCN:H ₂ O (50:50) to give DHO as a white crystalline solid (206.5 g, 0.45 mol, 87.7% yield over two steps, adjusted to wt%. ).

¹ H 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), 2.072.12 (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 C 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.

i. Ms ₂ O <1.1 eq.)

2,6-lutidine (2.0 eq.) Toluene (5 vol.) 75 ⁱⁿ C, 1" h

jj, Water treatment iii. Na 1-NpS0 ₃ (1.5 eq.)

Cl

DHO OXOS

1.0 e".

Toluene hemisolvate of naphthalene-1-sulfonate (3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6 ,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium (OXOS).

((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 eq .) and toluene (1000 ml, 5 ml/g) under nitrogen atmosphere.Methanesulfonic acid anhydride (88.2 g, 0.49 mol, 1.2 equiv.) and 2,6-lutidine (95 .0 ml, 0.82 mol, 2.0 eq.) The reactor contents were heated to 75°C with constant stirring for 16 hours. After 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 H ₂ SO ₄ (45.0 ml, 0.82 mol, 2.0 eq.) in H ₂ O (955 ml, 5 ml/g).The phases were separated, and then the organic layer washed twice with an aqueous solution of sodium 1-naphthalene sulfonate (2 x 72.5 g, 0.31 mol, 0.75 eq.) in H2O (2 x 800 ml, 4 ml/g) The organic phase was dried over sodium 1-naphthalene sulfonate (10.0 g , 0.04 mol, 0.1 eq.), filtered through a final filter (large porosity) while washing with dichloromethane and concentrated in vacuo. The crude residue was recrystallized from toluene to give OXOS as a cream-colored crystalline solid (260.1 g, 0.37 mol, 90.0% wt% adjusted yield).

- 22 043674

1H NMR (400 MHz, chloroform-d) δ ppm: 9.14 (d, J=8.50 Hz, 1H), 8.35 (dd, J=7.26, 1.24 Hz, 1H ), 7.86 (t, J=8.71 Hz, 2H), 7.57 (t, J=7.70 Hz, 1H), 7.43-7.50 (m, 2H), 7.13 -7.39 (m, 7.5H), 7.03-7.10 (m, 3H), 6.07 (d, J=11.20 Hz, 1H), 5.80 (ddt, J=17 ,00, 9.90, 7.39, 7.39 Hz, 1H), 5.51 (t, J=9.74 Hz, 1H), 5.26-5.34 (m, 2H), 4, 76 (dd, J=10.37, 4.66, 2.18 Hz, 1H), 4.62 (dd, J=9.12, 4.77 Hz, 1H), 3.51-3.60 ( m, 1H), 2.86 (t, J=13.68 Hz, 1H), 2.65-2.71 (m, 1H), 2.55-2.60 (m, 1H), 2.35 (s, 1.5H), 1.95 (dd, J=13.89, 3.52 Hz, 1H), 1.52 (s, 3H), 0.54-0.67 (m, 7H);

¹³ C NMR (101 MHz, chloroform-d) δ ppm: 183.28, 142.16, 140.01, 137.71, 135.89, 134.15, 134.12, 133.28, 132 ,15, 130.38, 130.30, 129.95, 129.62, 129.43, 129.06, 128.90, 128.34, 128.09, 127.92, 127.66, 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, intermediate DHO-OMs can be split and purified before being converted to OXOS.

/—o i-Pr—< I

MsOZ _/ Η °Cl

DHO-OM

1H 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 (dd wide, 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 C 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.

Example 4. Development of an industrial method for obtaining the pre-final intermediate SUL.

Treatment of OXOS with the isopropyl sulfinate salt at elevated temperatures results in the formation of the diastereomeric sulfinate ester pair SULFI (Scheme 4), which is converted to the more thermodynamically stable product SUL. This type of sulfinate-sulfonic rearrangement has been described in conventional processes as occurring through ion pair formation and a recombination mechanism for benzohydryl sulfinate esters. However, in the present process, the results of the crossover experiment shown in Scheme 11 indicate that the rearrangement involves dissociated ions. Given that OXOS reacts quantitatively with water at temperatures above 70°C, there is ample opportunity for the formation of alcoholic ALC. If OXOS cannot be recovered from ALC, water must be strictly excluded from the reaction mixture. One way to achieve this goal is to produce an isopropyl sulfinic acid salt that is stable under azeotropic drying conditions, can be isolated with high purity, and reacts efficiently with OXOS to form SUL.

Scheme 11

Intermediates and by-products generated during the production of SUL from OXOS

After evaluating several isopropyl sufic acid salt variants to achieve this goal, including lithium, sodium, potassium, magnesium and ammonium salts, the calcium sulfinate dihydrate salt was isolated as a stable and crystalline species. This salt was obtained by reacting isopropylmagnesium chloride with sulfur dioxide (Scheme 12), leading to the formation of isopropylsulfinic acid after quenching with a solution of hydrochloric acid. This substance was treated with calcium acetate, which allowed the isolation of calcium isopropyl sulfinate dihydrate (CALID) by reactive crystallization. Although the hydrate is not optimal for use in the preparation of SUL, given the sensitivity of the process described above to water, the calcium salt was chemically stable (i.e., no disproportionation product was observed within 48 h) after azeotropic drying in toluene at high

- 23 043674 high temperature (up to 110°C). Thus, drying of the reactant slurry can be included as part of the process before adding OXOS and producing SUL. Using X-ray powder diffraction analysis of oven-dehydrated samples, CALID was found to undergo polymorphic changes after complete drying (<100 ppm water) at <15% relative humidity. However, CALID is stable as a dehydrated material and converts back to its original dihydrate form upon re-adsorption of water at >20% relative humidity. The SUL manufacturing process involves drying CALID and OXOS toluene suspensions by azeotropic distillation under reduced pressure to obtain mixtures containing less than 100 ppm. water. Then the dry suspensions were combined and the solvent was replaced with dimethylacetamide (Scheme 13). The resulting solution was heated to 120°C and stirred for a maximum of 20 hours. During this time, sulfinic acid esters formed during the first hour at 120°C rearranged to form SUL. Typical levels of ALC impurity formed under these conditions are 3% LC area. SUL was isolated in 82% and >99.5% LC area yield after aqueous treatment and crystallization from acetonitrile and water (scale to 23.3 kg).

Scheme 12

Preparation of calcium isopropyl sulfinate dihydrate (CALID)

SO ₂ Ca(OAc)g Mec _Me

Me tHF Me HCl Me EtOH/H ₂ O / ^-S n x ^C % x ^S— \

У-MgCI ---->-SO ₂ MgCI ---* >-SO ₂ H -------------► Me ^{7 0 0} Me

Me stage i Me Me _{Stage 2} (2x H ₂ O)

Yield 75%

Scheme 13

Retrieving SUL using CALID

Isopropylmagnesium chloride was prepared in situ as a dry reagent and used directly in the production of SUL, offering an alternative strategy for the production of SUL from OXOS. As shown in Scheme 14, a solution of isopropyl magnesium chloride in tetrahydrofuran was treated with sulfur dioxide to produce isopropyl magnesium chloride sulfinate. In-situ FTIR (peak at 1325 cm ^-1 ) was used to check the total consumption of sulfur dioxide. The monomer-fibrin complex reaction was used to confirm the absence of alkyl Grignard reagent before subsequent processing. After changing the solvent to N-methylpyrrolidinone (NMP), OXOS was added to obtain SUL at 120°C or 180°C (Scheme 14). Removing unwanted moisture and/or magnesium chloride hydroxide from the reaction mixture thus obtained is a difficult task. For example, when using three equivalents of magnesium chloride isopropyl sulfinate relative to OXOS, about 5 mole percent magnesium chloride hydroxide (relative to OXOS) is present in the original Grignard solution. NMP (5 vol.) and OXOS contain 5-10 mol.% water (relative to OXOS). Thus, it is difficult to avoid the formation of a minimum of 15 mol% ALC when using this reagent. Notably, the ALC levels observed during SUL formation under these conditions exceed the measured amount of water or hydroxide contained in the reaction mixture by a margin that depends on the operating temperature. A second mechanism, other than direct opening of OXOS by water or hydroxide salt (Scheme 4), must be applied to account for the formation of ALC. This postulated mechanism involves the reaction of SULFI with magnesium chloride isopropyl sulfinate to form ALC (Scheme 15).

There is an efficient mechanism to explain the conversion of ALC to SUL at elevated temperatures (eg, 180°C), but it does not occur at an industrially acceptable rate at 120°C (Figure 4). Treatment of OXOS with magnesium chloride isopropyl sulfinate (3 eq) in NMP (5 vol) at 180°C produced SUL and ALC with analytical yields of 77 and 18%, respectively, after 6 min. After 80 min, the analytical yields of SUL and ALC were 90 and 5%, respectively (Figure 5). Magnesium salts are proposed to act as Lewis acids, promoting the formation of OXOS from ALC (Scheme 15). Isopropyl sulfinate magnesium chloride has poor stability at elevated temperatures and, as observed by 1H NMR, decomposes by 85% within 1 hour at 200°C as a 1 M solution in

NMP. Therefore, 3 eq. was used to carry out the conversion. isopropyl sulfinate magnesium chloride.

Scheme 14

Preparation of SUL from OXOS using Magnesium Chloride Isopropyl Sulfinate

Scheme 15

Possible mechanisms of ALC formation and conversion of ALC to SUL

The use of alternative isopropyl sulfinate salts to modify the reactivity and increase the stability of magnesium chloride isopropyl sulfinate was evaluated. Treatment of one equivalent of the reagent prepared in-situ with one equivalent of zinc chloride gave promising results. A commercially available tetrahydrofuran zinc chloride solution (0.5 M) was added to the magnesium chloride isopropyl sulfinate solution prepared according to the method described above (Scheme 12). The new species formed was found to be different from magnesium chloride isopropyl sulfinate and zinc isopropyl sulfinate by 1H NMR (Figure 6), and its structure was postulated to be that of zinc chloride isopropyl sulfinate with magnesium chloride formed as a by-product. The solvent was replaced with NMP (5 vol.) and OXOS was added to the reaction mixture (Scheme 16). Using this mixed reagent, the conversion of ALC to SUL occurred at high throughput at 120°C. Thus, the transformation can be carried out at 120°C, avoiding the decomposition of reactants and reaction intermediates (Fig. 7) and under very harsh anhydrous conditions. Additionally, there is no evidence for an alternative mechanism to provide water-free ALC with this mixed magnesium-zinc reagent, and all ALC generated during the process may be accounted for by the incoming reagents or solvent. This does not mean that this mixed salt cannot be used at 180°C (Fig. 8), but a temperature of 140°C was chosen for the process, which provides a good reaction rate when using limited equivalents (1.5 equivalents) of magnesium-zinc compounds and providing 90% yield of SUL with 7% ALC for 7 hours. According to 1H NMR experiments, mixed magnesium-zinc compounds showed stability at 150°C for 16 hours.

- 25 043674

Scheme 16

Preparation of SUL from OXOS using Magnesium Chloride-Zinc Chloride Isopropyl Sulfinate

Me ^MgCI Me „I so.

Absence confirmation

THF

SO ₂ and alkyl reagent|

H^inyara f 5—SO ₂ MgCI

Me

i. ZnCI ₂ / THF ii. Replacement with NMR

The stability of this process was assessed using 20% excess sulfur dioxide during the formation of magnesium chloride isopropyl sulfinate and performing a solvent exchange to NMP after stirring the mixed salt for 24 hours at 20°C. It was observed that only 40% (by 1H NMR) of the sulfinate reagent remained and that 60% of the material had disproportionated to form the sulfone SSO (Scheme 15). Using this mixture to convert OXOS to SUL with 1.5 eq. reagent produced SUL on only 62% of the LC area and left 33% of the LC area of OXOS unreacted, demonstrating a lack of robustness for this process as controlling sulfur dioxide dosing during production operation may be problematic. Thus, the use of CALID to produce SUL represents an advantageous aspect of the industrial process for producing Compound A.

Example 5. Development of an industrial method for producing 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 with sodium chlorite provides a more environmentally friendly alternative to the ruthenium oxide/sodium periodate method used to initially produce Compound A. In addition, the ozonolysis process is likely to eliminate the formation of several undesirable dimeric impurities that are difficult to remove by crystallization, and thus will simplify product isolation.

When identifying safe reaction conditions for ozonolysis, an aqueous mixture was used (Scheme 17). Under these conditions, the high-energy ozonide (OZO) intermediate is hydrolyzed, avoiding its accumulation and making the process safe. The LC area % of accumulated OZO was measured relative to the volume percentage of water used in the acetonitrile/water reaction mixture, with the results presented in FIG. 9. The total energy release for the ozonolysis mixture (20 volumes of solvent) with 10% water is 92 J/g at a decomposition temperature of 240°C, which does not pose a safety threat. Another parameter that requires control to ensure safety during ozonolysis corresponds to the concentration of the oxygen gas phase in the vessel during the reaction. The limiting oxygen concentration (LOC) for combustion of the mixture was determined to be 10.75 vol.%, while the ozonolysis process was carried out at half the LOC (~5 vol.% oxygen) to ensure a sufficient level of safety to avoid possible fire.

- 26 043674

Scheme 17

Preparation of compound A from SUL using tandem ozonolysis-Pinnick oxidation process

In the GMP scheme, two different processing modes are used in practice for this transformation: (i) semi-continuous ozonolysis using ozone sparging in a batch tank and (ii) a continuous process in a stirred tank reactor. Initially, the continuous process appears to be effective in eliminating the general safety concerns associated with the use of the ozonolysis reaction in an industrial process. A diagram and illustration of a continuous ozonolysis apparatus used in one embodiment is shown in FIG. 10 and 11 respectively. The ozone generator model CFS-3, sold by Ozonia, was used to treat 4.8 kg of SUL, producing approximately 0.9 mol ozone/h. Ozone is generated from the supply air and introduced through a valve located at the bottom of the continuous stirred tank reactor (CSTR), as shown in FIG. 10. The starting material solution was injected at a rate of 60 ml/min using a dip tube with an outlet over a glass frit located at the bottom of the vessel (capacity 0.9 L). Vigorous stirring of the mixture is important for proper gas dispersion and an example can be seen in FIG. 11. Maintain a nitrogen purge of the free volume at 3 times the flow rate of the air introduced from below, thus ensuring the presence of a gas phase of less than 5 vol.% oxygen/ozone. The temperature of the reaction mixture, controlled by the reactor jacket, was maintained at 20°C. A Raman probe was used at the CSTR output to measure residual SUL levels.

To minimize the risk when using continuous ozonolysis, samples were taken from the accumulated portions of the reaction mixture and the completion of the reaction was checked using HPLC. The portions were loaded into a 2 M aqueous solution of sodium chlorite (4 eq.) and the resulting mixture was stirred for 16 hours at 20°C. Due to the low solubility of the intermediates (ALD, PAC) in aqueous solutions, the addition of an aqueous sodium chlorite solution to the ozonolysis reaction stream is preferred over the option used to prevent the initial precipitation of these intermediates and is the addition method used for the semi-continuous ozonolysis process described below.

SUL's semi-continuous ozonolysis process has the advantages of processing with excess alkene feedstock for most of the conversion and using simpler production sites. Formation of no more than 0.4% LC area of DHCA impurity has been observed using this processing regimen, but a reliable method is required to monitor reaction completion and safe processing conditions. As described above, safe processing conditions for this reaction manifold are achieved by using an aqueous environment and maintaining an oxygen concentration below 5 vol.%. The air/ozone gas stream was diluted with nitrogen after the ozone generator but before introducing the reactant gas through the dip tube into the reaction vessel, as shown in FIG. 12. The nitrogen gas flow was used four times the air flow so that the gas entering the vessel contained no more than 5 vol.% oxygen/ozone. A headspace ozone detector can be used to monitor the completion of the ozonolysis reaction by measuring the increase in outlet gas concentration. However, this method has proven difficult to implement because the measured changes in ozone output concentration may be negligible. On the other hand, feed material consumption was found to be linear for this conversion (Figure 13), allowing HPLC analysis of multiple samples during the conversion in combination with generator outlet ozone data to accurately predict reaction completion time. A CFS-14 ozone generator model sold by Ozonia was used to process a 23 kg batch of SUL, which provides a maximum output of approximately 540 g ozone per hour. One

- 27 043674 ko stable ozone output is easier to maintain if the device is operated at less than maximum power, and thus a production rate of 285 g ozone/h was used and a power of 6080 W (80% power) and 3.1 standard cubic ft/min (approx. 87.8 L/min) (30% of maximum air flow). At these settings, the ozone concentration in the gas at the generator outlet was approximately 4.3 wt.% and this gas stream was mixed with nitrogen (12.5 scfm (about 353.8 l/min) before entering the process vessel. The predicted time to completion of the reaction using the ozone gas yield (7.4 hours) was exceeded by 10% (actually 8.2 hours).It is known that ozone reacts with water to form a hydroxyl radical and thus part of the ozone is consumed, which explains the excess ozone that needs to be used.

For this semi-continuous treatment, ozone was introduced to the bottom of the reaction vessel through a dip tube. The first device characterized for introducing gas into the reaction system was a standard ozone sparging unit with a pore size of 100 microns and a total area of 0.32 square feet (0.03 m ² ). When this bubbler was used to deliver 15.6 scfm (approx. 441.5 L/min) of combined gas flow (air, ozone, and nitrogen), a drop in pressure occurred, causing the surface of the bubbler to cool. This decrease in temperature was accompanied by crystallization of the SUL feed and ALD product, followed by plugging of the bubbler pores with the crystallized material. The solubility of SUL and ALD at 10°C in acetonitrile/water (9/1 volume ratio) is 25 and 21 mg/ml, respectively, and thus only about 50% of any material can be dissolved at this temperature in 20 volumes of used solvent mixtures. Once the sparger becomes blocked by feed or product crystals, the available surface area for gas transfer is reduced and the situation becomes more difficult, requiring the process to be interrupted and the sparger to be restored. To solve this problem, another ozone bubbler was developed. The alternative bubbler had 1/8 inch (0.32 cm) diameter holes perforated in C22 Hastelloy tubing (37 holes). Both bubblers are shown in Fig. 14. The effect of the use of either bubbler on the reaction mixture and bubbler surface temperature at a typical gas flow (15 standard cubic feet per minute (about 424.5 L/min)) was measured and the results are shown in Table 1. 3. As indicated in the table, there is a significant difference (30°C versus 11°C, for example) between the surface temperature of the bubbler and the reaction mixture temperature for a bubbler with 100 µm diameter holes, which causes the problems described above. An alternative bubbler solves these problems. To avoid precipitation of SUL and ALD during ozonolysis, the conversion was carried out at 30 °C.

Table 3

Bubbler surface temperature control using different bubblers

Gas flow (standard cubic feet per minute) Bubbler pore size Reaction temperature (°C) Bubbler surface temperature (°C) 15 (425 l/min) 100 12 2 15 (425 l/min) 100 thirty eleven 15 (425 l/min) 1/8 inch (0.32cm) 13 eleven 15 (425 l/min) 1/8 inch 25 22

After completion of the ozonolysis process, 2 M aqueous sodium chlorite was added to form Compound A. The mixture was treated with 2 M aqueous sodium bisulfite to remove all oxidizing agents for further processing. Phase separation was followed by the addition of isopropyl acetate and two washes of the organic layer with 2 M aqueous sodium phosphate (pH 6). Finally, the organic phase was washed with 1.1 M aqueous sodium chloride to obtain a solution of compound A with >95% analytical yield and >98% LC area purity.

Example 6 Isolation of Compound A as DABCO Salt.

Considering that a drug breakpoint to reliably remove impurities was not available for the free acid, several salts of Compound A were prepared and analyzed to find an effective option for easy removal of impurities. To this end, more than 30 organic salts and 5 inorganic salts of Compound A were investigated, facilitating the identification of the hemi-DABCO salt and the hemi-calcium salt as potential candidates. However, the hemi-calcium salt demonstrated only moderate removal of SUL parent material or HAC impurity, whereas the use of the hemi-DABCO isopropyl acetate salt (232-DAB) was effective in removing both species (IPAC = isopropyl acetate).

- 28 043674

5% LC area of SUL and 1% LC area of HAC can be completely removed during the final salt release, levels that are significantly higher than those observed in a typical Pinnick ozonolysis-oxidation reaction mixture. 232-DAB is a stable crystalline semi-DABCO monosolvate (isopropyl acetate) salt obtained by TGA and 1H NMR. A single polymorph of the material has been identified. The polymorphic form and level of isopropyl acetate in the material did not change in the dynamic vapor sorption experiment conducted at relative humidity from 0 to 90%. Additionally, a robust crystallization protocol based on temperature and antisolvent use can be developed for the hemi-DABCO isopropyl acetate salt using isopropyl acetate and heptane.

A solubility curve showing values at various time intervals during crystallization is shown in FIG. 15. After adding DABCO (0.5 eq.) to a solution of compound A in 4 volumes of isopropyl acetate at 55°C, the solution was seeded with 232-DAB, which ensured desupersaturation and crystallization of approximately 20% of the material. Cooling to 20°C for 2 hours caused an additional 60% of the material to crystallize. Four volumes of heptane were then added to reduce the supernatant concentration to about 5 mg/mL in preparation for batch filtration. Use of this crystallization procedure provided isolation of 232-DAB in 83% yield and >99.9% LC area purity (scale to 23.2 kg).

Example 7. Isolation of compound A.

An aqueous crystallization protocol was desirable to isolate Compound A through an orthogonal purification process, given that the crystallization of 232-DAB was carried out in organic solvents. Mixtures of hydroalcoholic solvents could not be used for crystallization, given the general instability of Compound A in alcoholic solvents above 2030°C due to Fischer esterification and the inability to remove ester impurities by crystallization. Other aqueous mixtures with water-miscible solvents showed steep solubility curves that were not conducive to the crystallization plan. In contrast, acetic acid and water resulted in crystallization of the material without the disadvantages detailed above and with good growth characteristics. Using this solvent mixture, steady-state temperature and antisolvent crystallization were calculated and carried out after salt cleavage in aqueous hydrochloric acid (2 eq)/isopropyl acetate, two subsequent washes of the organic layer with 2 M aqueous sodium phosphate (pH 6), rinsing 1.1 M aqueous solution of sodium chloride and replacing the solvent of isopropylacetate with acetic acid.

A curve showing solubility values at various times during crystallization is shown in FIG. 16. A solution of compound A in acetic acid (6.6 volumes of acetic acid) was heated to 55-60°C and 4.4 volumes of water was added. The solution was seeded with compound A, which ensured the creation of supersaturation and crystallization of about 30% of compound A. The crystallization mixture was cooled to 20°C for 10 hours, which ensured the crystallization of an additional 55% of the material. One volume of water was then added to reduce the supernatant concentration to about 5 mg/mL in preparation for rapid batch filtration. Three washes with water (3x10 volumes) were performed to minimize the presence of residual acetic acid in the recovered material. Compound A (up to 18.0 kg) was isolated using this protocol in >92% (100 wt%) yield and >99.9% LC area purity with <200 ppm. residual water and <200 ppm residual acetic acid.

The material was ground using a Pallman Universal Mill (impact mill, material weight up to 16 kg), and the results are presented in table. 4. The d ₅₀ target for Compound A was set to <35 µM based on oral absorption modeling (GastroPlus v 9.0) for the dose range evaluated (60 to 480 mg) to ensure complete absorption at a fasting gastric pH of 1 ,3.

- 29 043674

Table 4

Particle size distribution of compound A before and after dry grinding

Material diO (µm) d50 (µm) d90 (µm) Unground compound A 14.6 42.5 102 Ground compound A 3.2 19.1 43.7

A reliable and efficient method suitable for the industrial production of the drug substance (compound A) of high purity has been developed. Significant aspects of the process include (i) the use of a laboratory-stable Vilsmeyer reagent, methoxymethyl en-N,Ndimethyliminium methyl sulfate, for selective in situ activation of the primary alcohol intermediate; (ii) isolating intermediate DHO in crystalline form, which increases the ability of the process to remove impurities; (iii) use of a new and stable reagent based on calcium isopropyl sulfinate in crystalline form to ensure reliable preparation of the sulfone intermediate; (iv) development of a safe ozonolysis protocol carried out in an aqueous solvent mixture, which corresponds to either a batch or continuous production mode; (v) more effective control of the purity of Compound A due to the formation of a salt of Compound A, which allows for effective removal of impurities. The new process was demonstrated to provide 18 kg of pure compound A (99.9% LC area) with an overall yield of 49.8% of DLAC, representing a significant improvement over the reported FIH process, which provided an overall yield of only 32 %.

Claims (6)

1. Method of obtaining a compound involving interaction with methoxymethylene-N,N-dimethyliminium methyl sulfate. 2. The method according to claim 1, characterized in that the interaction is carried out in the presence of a base. 3. Method according to claim 2, characterized in that the base is KOAc, NaOAc, LiOAc or K2CO3. 4. Method according to claim 2, characterized in that the base is NaOAc. 5. Method according to any one of claims 1-3, characterized in that the interaction is carried out in a solvent. 6. The method according to claim 5, characterized in that the solvent is toluene.