Dihydroartemisinin derivatives and treatment of fibrous diseases

Abstract

Compounds of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, as well as their salts, pharmaceutical preparations containing them, and the use of these compounds for the treatment of fibrous diseases. TIFF2026524954000016.tif113164

Description

[Technical Field]

[0001] This invention relates to compounds that are dihydroartemisinin derivatives, and to the use of these compounds for the treatment of fibrous diseases. [Background technology]

[0002] Artemisinin, dihydroartemisinin (DHA), and artesunate

[0003] Artemisinin, (1R,4S,5R,8S,9R,12S,13R)-1,5,9-trimethyl-11,14,15,16-tetraoxa-tetracyclo[10.3.1.0 4 , 13 .0 8 , 13 Hexadecan-10-one, (3R,5aS,6R,8aS,9R,12S,12aR)-octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one is a well-known antimalarial agent originally extracted from the herb Artemisia annua. It was discovered in China in 1972. Artemisinin-based combination therapy (ACTs, therapies using artemisinin or one of its derivatives) is now the standard treatment for malaria worldwide. Dihydroartemisinin, DHA, (1R,4S,5R,8S,9R,10S,12R,13R)-1,5,9-trimethyl-11,14,15,16-tetraoxa-tetracyclo[10.3.1.0 4 , 13 .0 8 , 13Hexadecane-10-ol, (3R,5aS,6R,8aS,9R,12S,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-ol, as its name suggests, is a hydrogenated derivative of artemisinin. Dihydroartemisinin is the active metabolite of all artemisinin compounds (artemisinin, artesunate, artemether, arteether, etc.) and is also available as a drug itself. It is a semi-synthetic derivative of artemisinin and is widely used as an intermediate in the preparation of other artemisinin-derived antimalarial drugs. Yu et al.'s “Dihydroartemisinin: A Potential Drug for the Treatment of Malignancies and Inflammatory Diseases”, Front. Oncol., 11, 722331 (2021) summarizes bacterial studies and reports that dihydroartemisinin possesses both anticancer and anti-inflammatory effects.

[0004] Artesunate is a hemisuccinate ester of dihydroartemisinin. Unlike artemisinin, dihydroartemisinin, or artemethyl / arteether (methyl / ethyl ether of dihydroartemisinin), only artesunate has sufficient water solubility for intravenous administration. In fact, in the United States, artesunate is only available as a reconstituted powder for intravenous injection, while elsewhere it is readily available for oral administration in the form of tablets or suspensions. Artesunate is used to treat malaria, but is not recommended for prophylaxis due to its short biological half-life. In the United States, artesunate has been tested for the topical treatment of vulvar, vaginal, and anal intraepithelial neoplasia, and has even been reported to have been tested for the treatment of COVID-19.

[0005] The structures of artemisinin, dihydroartemisinin, and artesunate are as follows. [ka]

[0006] For further information on artemisinin, dihydroartemisinin, and artesunate and dihydroartemisinin, see the following Wikipedia article: See https: / / en.wikipedia.org / wiki / Artemisinin, https: / / en.wikipedia.org / wiki / Dihydroartemisinin, and https: / / en.wikipedia.org / wiki / Artesunate and the literature cited therein. A review of the pharmacokinetics of dihydroartemisinin and artesunate may be found in Morris et al., “Review of the clinical pharmacokinetics of artesunate and its active metabolite dihydroartemisinin following intravenous, intramuscular, oral or rectal administration”, Malaria J., 10, 263 (2011).

[0007] Fibrous disease

[0008] Rosenbloom et al., “Human Fibrotic Diseases: Current Challenges in Fibrosis Research”, Fibrosis, 1627, 1-23 (2017) states: “Human fibrotic diseases constitute a major health problem worldwide due to their large prevalence, incomplete understanding of the etiology of the fibrotic process, significant heterogeneity in their etiology and clinical manifestations, the lack of adequately and fully validated biomarkers, and, most importantly, the current lack of effective disease-modifying therapies.” Rosenbloom et al., in Table 1, categorize systemic fibrotic diseases as systemic sclerosis, multifocal fibrosclerosis (multifocal fibrosclerosis). The categories include fibrosclerosis (IgG4-related fibrosis), nephrogenic systemic fibrosis, and scleroderma-like graft-versus-host disease; under the category of organ-specific fibrotic diseases, cardiac fibrosis including hypertension-related cardiofibrosis, post-myocardial infarction and Chagas disease cardiofibrosis; renal fibrosis including diabetic and hypertensive nephropathy, renal fibrosis due to urinary tract obstruction, inflammatory / autoimmune renal fibrosis, aristolochic nephropathy, and polycystic kidney disease; idiopathic pulmonary fibrosis, silica-induced pneumoconiosis (silicosis), and asbestos-induced pulmonary fibrosis (lithiasis). The list includes pulmonary fibrosis, including pulmonary fibrosis (e.g., pulmonary fibrosis caused by chemotherapeutic agents); hepatic fibrosis and portal vein fibrosis, including alcoholic and non-alcoholic hepatic fibrosis, hepatic fibrosis due to hepatitis C, primary biliary cholangitis, and hepatic fibrosis caused by parasites (schistosomiasis); as well as other organ-specific fibrotic diseases, including radiation-induced fibrosis (of various organs), bladder fibrosis, intestinal fibrosis, peritoneal sclerosis, diffuse fasciitis, focal scleroderma, keloids, Dupuytren's contracture, Peyronie's disease, myelofibrosis, and oral submucosal fibrosis. Other fibrotic diseases include cardiomyopathy (e.g., diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and radiation-induced cardiomyopathy), Hermanski-Padlak syndrome, pancreatic fibrosis, pulmonary complications from COVID-19 infection, and Duchenne muscular dystrophy.

[0009] Rosenbloom et al. continue, regarding fibrous diseases: "While the mechanisms underlying them are highly diverse and remain unclear in some cases, these diseases share a common characteristic: the uncontrolled and progressive accumulation of fibrous tissue in the affected organs leads to their dysfunction and eventual failure. Despite the remarkable heterogeneity in the etiological mechanisms and clinical signs that cause fibrous diseases, numerous studies have identified activated myofibroblasts as a common cellular element responsible for the eventual replacement of normal tissue with non-functional fibrotic tissue." They also state, "While considerable progress has been made regarding the pathogenesis of fibrous diseases, they remain a major challenge due to the diverse and multiple initiation events and the involvement of numerous pro-fibrotic mediators. While TGF-β [transforming growth factor-β] is considered a major fibrotic effector, many other cytokines and signaling molecules are involved in the fibrotic response, creating a highly complex network of redundant signaling pathways that must be considered when attempting to develop effective anti-fibrotic therapies." They also point out: "Currently, therapeutic interventions for fibrous diseases are very limited. For example, only two drugs, pirfenidone and nintedanib, are approved for IPF, and no disease-modifying drugs are approved for SSc [systemic sclerosis] or other fibrous diseases."

[0010] Artesunate has been shown to be active as an anti-fibrotic agent in a bleomycin-induced pulmonary fibrosis model in rats: see Wang et al., "Anti-profibrotic effects of artesunate on bleomycin-induced pulmonary fibrosis in Sprague Dawley rats", Mol. Med. Rep., 12, 1291-1297 (2015), and Liu et al., "Artesunate ameliorates lung fibrosis via inhibiting the Notch signaling pathway", Exp. Ther. Med., 14, 561-1566 (2017). However, the short biological half-life of artesunate makes it unsuitable as a long-term drug, especially an oral drug, for the treatment of fibrotic diseases.

[0011] It is desirable to develop new dihydroartemisinin derivatives as pharmacological treatments for fibrotic diseases.

Summary of the Invention

[0012] In a first aspect, the present invention is any one compound of formula PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, and salts thereof, particularly pharmaceutically acceptable salts thereof.

[0013] These compounds are derivatives of dihydroartemisinin and are either carboxylate esters (PT-1, PT-2, PT-4) or carbamate esters (PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30) of dihydroartemisinin.

Chemical formula

[0014] <00001In a second aspect, the present invention is a pharmaceutical formulation for the treatment of fibrotic diseases, comprising a compound according to the first aspect of the present invention.

[0015] In a third aspect, the present invention relates to the use of a compound of the first aspect of the present invention or a pharmaceutical formulation of the second aspect of the present invention for the treatment of fibrotic diseases.

[0016] One of the compounds with formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23~PT-25, and PT-27~PT-30, and their salts, are expected to be useful in the treatment of fibrotic diseases. This is because studies in induced pluripotent stem cell (iPSC)-derived cardiac fibroblasts activated by transforming growth factor-β (TGF-β) have shown that they dose-dependently reduce the levels of fibrosis markers. Furthermore, some of them have been shown to inhibit the MD2 signaling pathway, demonstrating antifibrotic effects in primary lung fibroblasts by measuring their effects on acta2, col1a1, and CTGF; antifibrotic effects have been shown in a CCl4-injured mouse liver model by oral administration; and antifibrotic effects have been shown in a bleomycin-injured mouse skin model. Additionally, artesunate has been shown to have antifibrotic activity in a rat bleomycin-induced pulmonary fibrosis model. Furthermore, these dihydroartemisinin derivatives have a longer biological half-life than artesunate, and are expected to be more effective than artesunate in treating these fibrous diseases.

[0017] Any one of the compounds of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, and their salts, are expected to be administered via multiple routes of administration to serve as an effective therapy for fibrous diseases, as mentioned in the section titled "Fibrotic Diseases" in the background technology.

[0018] Preferred embodiments of the present invention are characterized by the features of the specification at the time of filing and of claims 1 to 16. [Modes for carrying out the invention]

[0019] definition

[0020] Fibrous diseases and their treatments are described in the section titled "Fibrous Diseases" in the background information.

[0021] In the first aspect of the present invention, the "therapeutic effective dose" of the compound means an amount sufficient to treat a fibrous disease when administered for the treatment of a fibrous disease in a subject (i.e., a human). "Treating" or "treating" fibrous disease in the subject includes one or more of the following: (1) To prevent or reduce the risk of developing fibrous disease; that is, to prevent the development of clinical symptoms of fibrous disease in subjects who may have a predisposition to fibrous disease but have not yet experienced or shown symptoms of fibrous disease (i.e., prophylaxis); (2) inhibiting fibrotic disease, that is, preventing or reducing the onset of fibrotic disease or its clinical symptoms; and (3) To alleviate fibrotic disorders, that is, to reduce, reverse or improve fibrotic disorders, or to reduce the number, frequency, duration or severity of their clinical symptoms. "Treatment" does not necessarily mean "cure" or complete cure (e.g., treatment of all clinical symptoms of fibrosis), although "treatment" may include "cure." Rather, "treatment" means the provision of clinical benefit by administering the compound compared to not administering the compound, and treatment may be evaluated by improvement in the biological markers of the treated fibrosis. The effective therapeutic dose for a particular target varies depending on the health and physical condition of the target, the nature and severity of the fibrous disease, the assessment of the medical situation, and other relevant factors. The effective therapeutic dose is expected to be within a relatively wide range, which can be determined through routine trials.

[0022] "Comprising" or "containing," and their grammatical variations, are words of inclusion, not limitation, meaning to specify the presence of a listed component, group, process, etc., but not to exclude the presence or addition of other components, groups, processes, etc. Therefore, "comprising" does not mean "consisting of," "consisting substantially of," or "consisting only of." For example, a formulation "comprising" a compound must contain that compound, but may also contain other active ingredients, prodrugs, and / or excipients. Unless context requires otherwise, the singular forms "a," "an," and "the" include multiple references.

[0023] compound

[0024] Compounds of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, as well as their salts, in particular their pharmaceutically acceptable salts, may be prepared by conventional methods. For PT-1, PT-2, and PT-4, this involves esterification of dihydroartemisinin using a suitable acid (generally activated) to obtain the desired carboxylate ester side chain. For PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, this involves esterification of dihydroartemisinin with 1,1-carbonyldiimidazole (CDI) to obtain the desired carbamate ester side chain, followed by substitution of the imidazole ring by reaction with a suitable amine (e.g., azetidine-3-ol (for PT-13), 3-methylazetidine-3-ol (for PT-14), 2,6-diazaspiro[3.4]octan-5-one (for PT-30), etc.).

[0025] When the carboxylic acid or carbamate-forming amine added to dihydroartemisinin is reactive to the reaction conditions, such as when it contains an amino group or a hydroxyl group, these groups are typically protected with an acid-unstable protecting group, such as tert-butoxycarbonyl (BOC) or a similar group for amino-containing reagents, or tert-butyldiphenylsilyl (TBDPS) or a similar group for hydroxyl-containing reagents, before the reaction to form a carboxylate or carbamate ester, and then removed after the reaction.

[0026] Therefore, for example, the preparation of PT-1, which is a 3-hydroxy-2-methylpropanoate ester of DHA, may be achieved as follows: esterification of dihydroartemisinin with O-TBDPS-3-hydroxy-2-methylpropanoic acid in a polar aprotic solvent such as dichloromethane, in the presence of an organic base such as DMAP (4-dimethylaminopyridine), and in the presence of a coupling agent such as DCC (N,N'-dicyclohexylcarbodiimide) or a similar compound, followed by deprotection of the side-chain hydroxyl with a reagent such as cesium fluoride in dimethylformamide. [ka]

[0027] Compounds of formulas PT-2 and PT-4 may be prepared by the same method using appropriately protected acids such as O-TBDPS-3-hydroxy-2,2-dimethylpropanoic acid (for PT-2) and O-TBDPS-3-hydroxy-3,3-dimethylpropanoic acid (for PT-4). PT-4 may be prepared in a single step using unprotected 3-hydroxy-3,3-dimethylpropanoic acid.

[0028] The preparation of compounds such as PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30 may be achieved as follows: by forming a 1H-imidazole-1-carboxylate ester by esterifying dihydroartemisinin with 1,1-carbonyldiimidazole (CDI) in a polar aprotic solvent such as dichloromethane, and subsequently substituting the imidazole with a suitable amine such as azetidine-3-ol (for the preparation of PT-13), 3-methylazetidine-3-ol (for PT-14), or 2,6-diazaspiro[3.4]octan-5-one (for PT-30) in a polar aprotic solvent such as dichloromethane in the presence of an organic base such as triethylamine (TEA). [ka]

[0029] Salts (e.g., pharmaceutically acceptable salts) of any one of the compounds of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30 are included in the present invention and are useful in the methods described herein. These salts are preferably formed with a pharmaceutically acceptable acid or base. For a broad discussion of pharmaceutically acceptable salts, their selection, preparation, and use, see, for example, "Handbook of Pharmaceutically Acceptable Salts," edited by Stahl and Wermuth, Verlag Helvetica Chimica Acta, Zurich, Switzerland. Unless otherwise required by context, references to PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, or any one of these compounds, refer to both the compound and its salts.

[0030] Since PT-23 and PT-24 each contain an amine group, the amine group may react with an inorganic acid such as hydrochloric acid or an organic acid such as maleic acid to form an acid addition salt. Typically, the compound is treated with an excess acid in a protic solvent such as water or a lower alkanol, or a combination thereof, to remove enough solvent to allow crystallization of the resulting acid addition salt.

[0031] Formulation and administration

[0032] Any one of the compounds of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, as well as their salts, may be administered by any route suitable for the subject being treated and the nature of the subject's condition. Routes of administration may include administration by injection (including intravenous, intraperitoneal, intramuscular, and subcutaneous injection), administration by mucosal or transdermal delivery, topical application, nasal spray, suppository, or oral administration. The formulation may be a liposomal formulation, emulsion, formulation designed for drug delivery via mucosa, or transdermal formulation, as appropriate. Formulations suitable for each of these methods of administration may be found, for example, in "Remington: The Science and Practice of Pharmacy," 20th edition, edited by Gennaro, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA. When the compound is available orally, the typical formulation is for oral use, and the typical dosage form is an orally administered tablet or capsule. Intravenous formulations may be particularly suitable for administration to patients with acute illnesses (for example, patients who may be hospitalized for treatment).

[0033] Depending on the intended mode of administration, the pharmaceutical composition may be in solid, semi-solid, or liquid dosage form, preferably a unit dosage form suitable for a single dose of a precise amount. In addition to an effective amount of artesunate or dihydroartemisinin, the composition may contain appropriate pharmaceutically acceptable excipients (including adjuvants that facilitate the processing of the active compound into a pharmaceutically usable preparation). "Pharmacologically acceptable excipients" means excipients or mixtures of excipients that do not interfere with the efficacy of the biological activity of the active compound and are not toxic or undesirable to the subject to which it is administered.

[0034] Conventional excipients for solid compositions include, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate. Pharmacologically administerable liquid compositions can be prepared, for example, by dissolving or dispersing the active compounds described herein and, as appropriate, pharmaceutical adjuvants in water or an aqueous excipient (e.g., water, saline solution, aqueous dextrose) to form a solution or suspension. If desired, the administered pharmaceutical composition may also contain small amounts of non-toxic auxiliary excipients such as wetting agents or emulsifiers, pH buffers (e.g., sodium acetate, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, etc.).

[0035] For oral administration, the composition generally takes the form of tablets or capsules. Alternatively, especially for use in children, it may be an aqueous or non-aqueous solution, suspension, or syrup. Tablets and capsules are preferred forms of oral administration. Tablets and capsules for oral use generally contain one or more commonly used fillers, such as lactose and corn starch. Lubricants such as magnesium stearate and binders such as carbopol are also typically added. When a liquid suspension is used, the activator may be combined with emulsifying and suspension excipients. Flavorings, colorings, and / or sweeteners may also be added, if desired. Other optional excipients for incorporation into oral formulations include preservatives, suspending agents, and thickening agents.

[0036] Typically, a single pharmaceutical composition of dihydroartemisinin derivatives of formulas PT-1 to PT-4, PT-7 to PT-20, PT-23 to PT-33, and PT-51 to PT-58, or a kit containing such a derivative, is packaged in a container with a label or instructions, or both, indicating the use of the pharmaceutical composition or kit in the treatment of fibrotic diseases.

[0037] Those skilled in the art of pharmaceutical formulations will be able to prepare suitable pharmaceutical compositions of the compounds of the present invention and achieve therapeutically effective formulations by selecting appropriate dosage forms, excipients, packaging, etc., based on their personal knowledge and the disclosures of this application, without conducting excessive experiments.

[0038] For systemic administration, the appropriate (i.e., therapeutically effective) dose of any one compound of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, as well as their salts, is expected to be at least 10 mg / day and no more than 600 mg / day for adult subjects, for example, at least 30 mg / day and no more than 400 mg / day, depending on factors such as the nature, degree, and severity of the fibrotic disease, as well as liver and kidney function. For pediatric subjects, depending on additional factors such as age and weight, and in subjects with significant liver or kidney impairment, the dose may be appropriately reduced to near or below the lower limit of the above external range, depending on the degree of impairment. These amounts represent the average daily dose and do not necessarily represent the amount given in a single dose. Administration may be more frequent than once daily (in which case the dose or daily dose is divided by the number of doses per day), but more typically it is once daily (in which case the dose is given as a single dose). In particular, in cases of severe hepatic impairment, administration may be less frequent than once daily, as appropriate, between once a week and every other day, for example, once a week, twice a week (especially with an interval of at least 3 days between doses), three times a week (especially with an interval of at least 2 days between doses), or every other day.

[0039] A person skilled in the art of treating fibrous diseases will be able to determine, without excessive experimentation and based on their personal knowledge and the disclosures of this application, the therapeutic effective dose of any one compound of formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, as well as the therapeutic effective dose of their salts, for a specific subject and the nature, degree, and severity of the fibrous disease.

[0040] preparation

[0041] Abbreviations: BOC: tert-butoxycarbonyl; CDI: 1,1'-carbonyldiimidazole; DCC: N,N'-di(cyclohexyl)carbodiimide; DCM: dichloromethane; DHA: dihydroartemisinin; DIEA: N,N-diisopropylethylamine; DMF: dimethylformamide; EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Depositphotos: ethyl acetate; HATU: hexafluorophosphate azabenzotriazole tetramethyluronium, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide Hexafluorophosphate; LCMS: Liquid chromatography-mass spectrometry; TBDPSCl: tert-butyldiphenylsilyl chloride; TEA: Triethylamine; TEMPO: (2,2,6,6-tetramethylpiperidine-1-yl)oxyl; TFA: Trifluoroacetic acid; THF: Tetrahydrofuran; TLC: Thin-layer chromatography.

[0042] Preparation 1: PT-1, DHA 3-hydroxy-2-methylpropionate [ka]

[0043] Step 1: 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropan-1-ol(1A-2) A solution of 2-methylpropane-1,3-diol (6.00 g, 66.6 mmol, 5.90 mL, 1.0 eq) in THF (90 mL) was added portionwise with NaH (2.90 g, 73.2 mmol, purity 60%, 1.1 eq) at 0 °C, and the mixture was stirred at 0 °C for 10 minutes. TBDPSCl (20.1 g, 73.2 mmol, 18.7 mL, 1.1 eq) was added portionwise to the reaction solution, and this was stirred at 20 °C for 12 hours. LCMS indicated that the starting material was completely consumed and the desired mass was detected. The reaction mixture was cooled to 0 °C, poured into saturated aqueous NH4Cl (100 mL), and extracted with EtOAc (100 mL × 3). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using a gradient of 0 / 1 to 1 / 9 EtOAc / petroleum ether. 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropan-1-ol (13.0 g, 39.6 mmol) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.70 (dd, J = 1.5, 7.8 Hz, 4H), 7.52 - 7.37 (m, 6H), 3.78 - 3.55 (m, 4H), 2.56 (br s, 1H), 2.05 - 1.93 (m, 1H), 1.08 (s, 9H), 0.85 (d, J = 6.9 Hz, 3H).

[0044] Step 2: 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoic acid (1A-3) To a solution of 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropan-1-ol (5.50 g, 16.7 mmol, 1.0 equivalent) in DCM (330 mL), TEMPO (527 mg, 3.40 mmol, 0.2 equivalents), H2O (15.1 g, 837 mmol, 15.1 mL, 50.0 equivalents) and PhI(OAc)2 (13.5 g, 41.9 mmol, 2.5 equivalents) were added. The mixture was stirred at 20°C for 12 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was poured into a saturated aqueous solution of Na2S2O3 (6 mL) and extracted with DCM (30 mL x 3). The combined organic layers were washed with saline solution (60 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 3 / 7. 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoic acid (2.50 g, 7.30 mmol) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.72 - 7.65 (m, 4H), 7.51 - 7.36 (m, 6H), 3.87 - 3.73 (m, 2H), 2.74 (dt, J = 5.6, 7.1 Hz, 1H), 1.19 (d, J = 7.0 Hz, 3H), 1.05 (s, 9H).

[0045] Step 3: DHA3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoate (1A-4) To a solution of DHA (2.20 g, 7.60 mmol, 2.0 equivalents) in DCM (50 mL), 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoic acid (1.30 g, 3.8 mmol, 1.0 equivalent), DCC (1.60 g, 7.60 mmol, 1.5 mL, 2.0 equivalents), and DMAP (46.4 mg, 379 μmol, 0.1 equivalent) were added, and the resulting mixture was stirred at 20°C for 12 hours. LC-MS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was filtered and concentrated under vacuum to obtain the residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 9. DHA3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoate (1.4 g, crude) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.75 - 7.60 (m, 4H), 7.48 - 7.34 (m, 6H), 5.88 - 5.77 (m, 1H), 5.45 (s, 1H), 3.96 - 3.71 (m, 2H), 2.84 - 2.71 (m, 1H), 2.65 - 2.53 (m, 1H), 2.38 (dt, J = 3.9, 14.0 Hz, 1H), 2.08 - 1.99 (m, 1H), 1.94 - 1.85 (m, 1H), 1.82 - 1.68 (m, 2H), 1.67 - 1.46 (m, 3H), 1.45 - 1.38 (m, 4H), 1.37 - 1.14 (m, 6H), 1.04 (s, 9H), 0.97 (d, J = 6.0 Hz, 3H), 0.87 - 0.83 (m, 3H).

[0046] Step 4: DHA 3-hydroxy-2-methylpropanoate (PT-1) To a solution of DHA3-((tert-butyldiphenylsilyl)oxy)-2-methylpropanoate (1.30 g, 2.10 mmol, 1.0 equivalent) in DMF (26 mL), CsF (973 mg, 6.40 mmol, 237 μL, 3.0 equivalents) was added. The mixture was stirred at 20°C for 13 hours. LC-MS showed that the starting material was completely consumed. The reaction mixture was poured into H2O (50 mL) and extracted with siRNA (30 mL x 3). The combined organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 3 / 7. DHA3-hydroxy-2-methylpropanoate (60.0 mg) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 5.87 - 5.78 (m, 1H), 5.50 - 5.42 (m, 1H), 3.86 - 3.68 (m, 2H), 2.86 - 2.72 (m, 1H), 2.67 - 2.54 (m, 1H), 2.39 (dt, J = 3.9, 13.9 Hz, 1H), 2.09 - 2.01 (m, 1H), 1.90 (ddd, J = 3.3, 6.4, 13.6 Hz, 1H), 1.84 - 1.71 (m, 3H), 1.64 (td, J = 4.5, 13.8 Hz, 1H), 1.52 - 1.42 (m, 4H), 1.39 - 1.20 (m, 6H), 1.10 - 0.96 (m, 4H), 0.91 - 0.85 (m, 3H).

[0047] Preparation 2: Preparation of PT-2, DHA 3-hydroxy-2,2-dimethylpropanoate [ka]

[0048] Step 1: 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoic acid (1A-2) To a solution of 3-hydroxy-2,2-dimethylpropanoic acid (2.00 g, 16.9 mmol, 1.0 equivalent) and imidazole (1.27 g, 18.6 mmol, 1.1 equivalent) in DCM (50 mL), TBDPSCl (5.12 g, 18.6 mmol, 4.77 mL, 1.1 equivalent) was added at 0°C, and the resulting mixture was stirred at 20°C for 1 hour. TLC (SiO / petroleum ether 1 / 5, R f A reading of 0.69 indicated that the starting material had been completely consumed. The reaction mixture was poured into HCl (1 M aqueous solution, 30 mL) and extracted with DCM (30 mL x 3). The combined organic layer was washed with saline solution (20 mL x 3), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 10. 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoic acid (5.00 g, 14.0 mmol, yield 83%) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.67 (dd, J = 1.4, 7.8 Hz, 4H), 7.45 - 7.36 (m, 6H), 3.66 (s, 2H), 1.23 (s, 6H), 1.06 (s, 9H).

[0049] Step 2: DHA3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoate (1A-3) To a solution of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoic acid (2.51 g, 7.03 mmol, 2.0 equivalents) in DCM (60 mL), (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-ol (1.00 g, 3.52 mmol, 1.0 equivalent), DMAP (430 mg, 3.52 mmol, 1.0 equivalent), and EDCI (1.35 g, 7.03 mmol, 2.0 equivalents) were added, and the resulting mixture was stirred at 20°C for 12 hours. TLC (siRNA / petroleum ether = 1 / 5, R fA ratio of 0.64 indicated that the starting material had been completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 0. DHA3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoate (1.90 g, 3.05 mmol, yield 87%) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.78 - 7.61 (m, 5H), 7.47 - 7.34 (m, 7H), 5.79 (d, J = 9.9 Hz, 1H), 5.44 (s, 1H), 3.75 (d, J = 9.5 Hz, 1H), 3.62 (d, J = 9.5 Hz, 1H), 2.67 - 2.53 (m, 1H), 2.37 (dt, J = 3.9, 14.0 Hz, 1H), 2.08 - 1.98 (m, 1H), 1.89 (quind, J = 3.3, 13.7 Hz, 1H), 1.81 - 1.69 (m, 2H), 1.66 - 1.58 (m, 1H), 1.54 - 1.16 (m, 10H), 1.10 - 1.02 (m, 11H), 0.97 (d, J = 6.0 Hz, 3H), 0.81 (d, J = 7.3 Hz, 3H).

[0050] Step 3: DHA 3-hydroxy-2,2-dimethylpropanoate (PT-2) To a solution of DHA3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoate (500 mg, 803 μmol, 1.0 equivalent) in DMF (20 mL), CsF (366 mg, 2.41 mmol, 88.9 μL, 3.0 equivalents) was added at 20°C, and the resulting mixture was stirred at 20°C for 12 hours. TLC (siRNA / petroleum ether = 1 / 1, R fA ratio of 0.24 (M + Na) indicated the presence of some residual starting material, and the desired spot was detected. The reaction mixture was poured into H2O (30 mL) and extracted with siRNA (20 mL x 5). The combined organic layer was washed with saline (10 mL x 3), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 1. DHA3-hydroxy-2,2-dimethylpropanoate (52.0 mg, 135 μmol, yield 6%) was obtained as a white solid. M + Na = 407.2; 1 H NMR (400 MHz, CDCl3) δ 5.78 (d, J = 9.9 Hz, 1H), 5.44 (s, 1H), 3.68 (d, J = 11.3 Hz, 1H), 3.53 (d, J = 11.3 Hz, 1H), 2.67 - 2.54 (m, 1H), 2.38 (dt, J = 3.9, 14.0 Hz, 1H), 2.09 - 1.85 (m, 3H), 1.83 - 1.58 (m, 4H), 1.56 - 1.15 (m, 11H), 1.20 - 0.94 (m, 5H), 0.87 (d, J = 7.1Hz, 3H).

[0051] Preparation 3: Preparation of PT-4 and DHA 3-hydroxy-3-methylbutanoate [ka]

[0052] To a solution of 3-hydroxy-3-methylbutanoic acid (500 mg, 4.23 mmol, 1.0 equivalent) in DCM (30 mL), DHA (2.41 g, 8.47 mmol, 2.0 equivalent), DCC (1.75 g, 8.47 mmol, 1.71 mL, 2.0 equivalent), and DMAP (51.7 mg, 423 μmol, 0.1 equivalent) were added, and the resulting mixture was stirred at 20°C for 12 hours. LC-MS showed that the starting materials were completely consumed and the desired mass was detected. The reaction mixture was filtered, and the filtrate was concentrated under vacuum to obtain the residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 0. The sample was then purified by preparative HPLC (Phenomenex Luna C18 column (75 × 30 mm, 3 μm); flow rate: 25 mL / min; gradient: 45% to 70% B over 8 minutes; mobile phase A: 0.04% HCl aqueous solution, mobile phase B: acetonitrile). DHA 3-hydroxy-3-methylbutanoate (242 mg, 623 μmol, 15% yield) was obtained as a white solid. M + Na = 407.2 (LCMS); 1 H NMR (400 MHz, CDCl3) δ 5.85 (d, J = 9.9 Hz, 1H), 5.47 (s, 1H), 2.65 - 2.52 (m, 3H), 2.40 (ddd, J = 3.9, 13.5, 14.5 Hz, 1H), 2.10 - 2.01 (m, 1H), 1.97 - 1.88 (m, 1H), 1.86 - 1.71 (m, 2H), 1.66 (td, J = 4.5, 13.7 Hz, 1H), 1.57 - 1.44 (m, 5H), 1.45 - 1.27 (m, 9H), 1.04 - 0.94 (m, 4H), 0.88 (d, J (= 7.1 Hz, 3H).

[0053] Preparation 4: Preparation of PT-13 and DHA3-hydroxyazetidine-1-carboxylate [ka]

[0054] To a solution of CDI (684 mg, 4.22 mmol, 1.2 equivalents) in DCM (100 mL), DHA (1.00 g, 3.52 mmol, 1.0 equivalent) was added. The reaction mixture was stirred at 20°C for 10 minutes, and then azetidine-3-ol (385 mg, 3.52 mmol, 1.0 equivalent, HCl salt) and TEA (427 mg, 4.22 mmol, 587 μL, 1.2 equivalents) were added. The resulting mixture was stirred at 20°C for 12 hours. LC-MS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an siRNA gradient from 0 / 1 to 1 / 0. DHA3-hydroxyazetidine-1-carboxylate (270 mg, 698 μmol, 20% yield) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) δ 5.66 (d, J = 9.8 Hz, 1H), 5.45 (s, 1H), 4.65 (br d, J = 4.6 Hz, 1H), 4.47 - 4.18 (m, 2H), 4.11 - 3.81 (m, 2H), 2.60 - 2.47 (m, 1H), 2.37 (dt, J = 3.9, 14.0 Hz, 1H), 2.22 (br s, 1H), 2.10 - 1.94 (m, 1H), 1.94 - 1.85 (m, 1H), 1.82 - 1.68 (m, 2H), 1.66 - 1.60 (m, 1H), 1.59 - 1.40 (m, 4H), 1.40 - 1.22 (m, 3H), 1.07 - 0.93 (m, 4H), 0.86 (d, J = 7.0 Hz, 3H).

[0055] Preparation 5: Preparation of PT-14, DHA 3-hydroxyazetidine-1-carboxylate, and (3R,5aS,6R,8aS,9R,12S,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-yl 3-hydroxyazetidine-1-carboxylate [ka]

[0056] To a solution of CDI (6.84 g, 42.2 mmol, 1.2 equivalents) in DCM (400 mL), DHA (10.0 g, 35.2 mmol, 1.0 equivalent) was added, and the reaction mixture was stirred at 25°C for 3 hours. Then, 3-methylazetidine-3-ol (4.35 g, 35.2 mmol, 1.0 equivalent, HCl salt) and TEA (4.27 g, 42.2 mmol, 5.87 mL, 1.2 equivalents) were added, and the resulting mixture was stirred at 25°C for 12 hours. LC-MS showed that the starting material was completely consumed, and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using a 0 / 1 to 1 / 1 siRNA / petroleum ether gradient. DHA 3-hydroxy-3-methylazetidine-1-carboxylate (5.14 g, 12.91 mmol, yield 36.7%) was obtained as a white solid. M - H - = 396.2 (LCMS); 1 H NMR (400 MHz, CDCl3) δ 5.67 (d, J = 9.8 Hz, 1H), 5.45 (s, 1H), 4.19 - 3.81 (m, 4H), 2.55 (ddd, J = 4.6, 7.2, 9.9 Hz, 1H), 2.38 (dt, J = 3.9, 14.0 Hz, 1H), 2.09 - 1.99 (m, 2H), 1.90 (ddd, J = 3.4, 6.4, 13.9 Hz, 1H), 1.81 - 1.69 (m, 2H), 1.66 - 1.59 (m, 1H), 1.54 (s, 3H), 1.53 - 1.23 (m, 7H), 1.06 - 0.93 (m, 4H), 0.87 (d, J = 7.1 Hz, 3H).

[0057] PT-20, DHA(2-hydroxyethyl)(methyl)carbamate was similarly prepared using 2-(methylamino)ethane-1-ol instead of 3-methylazetidine-3-ol, and after purification, DHA(2-hydroxyethyl)(methyl)carbamate (640 mg, 1.66 mmol, yield 50%) was obtained as a white solid. M + H + = 386.3 (LCMS); 1 H NMR (400 MHz, CDCl3) δ 5.62 (br t, J = 10.9 Hz, 1H), 5.37 (br d, J = 7.3 Hz, 1H), 3.76 - 3.65 (m, 2H), 3.54 - 3.29 (m, 2H), 2.95 (br d, J = 15.4 Hz, 3H), 2.59 - 2.15 (m, 3H), 2.03 - 1.91 (m, 1H), 1.88 - 1.77 (m, 1H), 1.75 - 1.61 (m, 2H), 1.60 - 1.49 (m, 1H), 1.48 - 1.30 (m, 5H), 1.29 - 1.15 (m, 2H), 0.99 - 0.86 (m, 4H), 0.81 (d, J = 7.1 Hz, 3H).

[0058] PT-24, DHA 3-amino-3-methylazetidine-1-carboxylate was similarly prepared using 3-methylazetidine-3-amine instead of 3-methylazetidine-3-ol, and after purification, DHA 3-amino-3-methylazetidine-1-carboxylate (300 mg, 756 μmol, yield 43%) was obtained as a white solid. M + H + = 397.2 (LCMS); 1H NMR (400 MHz, DMSO-d6) δ 5.53 (s, 1H), 5.49 (br d, J = 9.5 Hz, 1H), 3.80 - 3.60 (m, 4H), 2.31 - 2.13 (m, 4H), 2.04 - 1.96 (m, 1H), 1.87 - 1.76 (m, 1H), 1.67 - 1.51 (m, 3H), 1.49 - 1.39 (m, 2H), 1.35 (br d, J = 4.5 Hz, 1H), 1.29 (s, 6H), 1.17 (dt, J = 6.6, 11.3 Hz, 1H), 1.00 - 0.92 (m, 1H), 0.89 (d, J = 6.3 Hz, 3H), 0.83 - 0.74 (m, 3H).

[0059] PT-25, DHA 3-carboxy-3-methylazetidine-1-carboxylate was similarly prepared using 3-methylazetidine-3-carboxylic acid instead of 3-methylazetidine-3-ol, and after purification, DHA 3-carboxy-3-methylazetidine-1-carboxylate (200 mg, 467 μmol, yield 9%) was obtained as a white solid. M + H + = 426.2 (LCMS); 1H NMR (400 MHz, DMSO-d6) δ 13.13 - 12.66 (m, 1H), 5.54 (s, 1H), 5.50 (d, J = 9.8 Hz, 1H), 4.26 - 4.06 (m, 2H), 3.86 - 3.66 (m, 2H), 2.28 (ddd, J = 4.4, 6.9, 9.8 Hz, 1H), 2.19 (dt, J = 3.8, 14.0 Hz, 1H), 2.04 - 1.96 (m, 1H), 1.85 - 1.77 (m, 1H), 1.67 - 1.58 (m, 2H), 1.57 - 1.51 (m, 1H), 1.48 (br s, 1H), 1.46 (br s, 3H), 1.43 - 1.38 (m, 1H), 1.38 - 1.32 (m, 1H), 1.30 (s, 3H), 1.17 (dt, J = 6.7, 11.3 Hz, 1H), 1.01 - 0.92 (m, 1H), 0.89 (d, J = 6.3 Hz, 3H), 0.79 (d, J = 7.0 Hz, 3H).

[0060] Preparation 6: Preparation of PT-28 and DHA 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate [ka]

[0061] Step 1: tert-butyl 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate (1A-2) HATU (1.32 g, 3.48 mmol, 1.5 equivalent) was added to a solution of 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid (500 mg, 2.32 mmol, 1.0 equivalent), methanamine (235 mg, 3.48 mmol, 1.5 equivalents, HCl salt), and DIEA (1.20 g, 9.29 mmol, 1.60 mL, 4.0 equivalents) in DMF (5.0 mL). The mixture was stirred at 30°C for 12 hours. LC-MS showed that the starting materials were completely consumed and the desired mass was detected. The reaction mixture was poured into HCl (1 M aqueous solution, 10 mL) at 0°C and extracted with HCl (20 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an HCl / petroleum ether gradient from 0 / 1 to 1 / 0. tert-butyl 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate (500 mg, 2.19 mmol, yield 94%) was obtained as a yellow oily substance. M + H + =229.2 (LCMS); 1 H NMR (400 MHz, CDCl3) δ 4.11 (d, J = 8.5 Hz, 2H), 3.59 (d, J = 8.4 Hz, 2H), 2.89 - 2.82 (m, 3H), 2.78 (d, J = 4.8 Hz, 3H), 1.41 - 1.34 (m, 9H).

[0062] Step 2: N,3-Dimethylazetidine-3-Carboxamide (1A-3) A mixture of tert-butyl 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate (500 mg, 2.19 mmol, 1.0 equivalent) in DCM (3.0 mL) and TFA (1.0 mL) was stirred at 25°C for 1 hour. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain N,3-dimethylazetidine-3-carboxamide (530 mg, crude, TFA salt) as a yellow oil. M + H + = 129.1 (LCMS).

[0063] Step 3: DHA 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate (PT-28) DHA (480 mg, 1.69 mmol, 1.0 equivalent) was added to a solution of CDI (328 mg, 2.03 mmol, 1.2 equivalents) in DCM (6.0 mL). The mixture was stirred at 30°C for 0.5 hours. Then, N,3-dimethylazetidine-3-carboxamide (523 mg, 2.16 mmol, 1.2 equivalents, TFA salt) and TEA (256 mg, 2.53 mmol, 352 μL, 1.5 equivalents) in DCM (2.0 mL) were added to the above solution. The mixture was stirred at 30°C for 0.5 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 0. This residue was then further purified by preparative HPLC (Phenomenex Luna C18 column (78 × 30 mm, 3 μm); flow rate: 25 mL / min; gradient: 45%~75% B over 9 minutes; mobile phase A: 10 mM NH4HCO3 aqueous solution; mobile phase B: acetonitrile). DHA 3-methyl-3-(methylcarbamoyl)azetidine-1-carboxylate (180 mg, 400 μmol, yield 23%) was obtained as a white solid. M + H + = 439.2 (LCMS); 1H NMR (400 MHz, CDCl3) δ 5.67 (d, J = 9.8 Hz, 1H), 5.46 (s, 1H), 4.46 - 4.17 (m, 2H), 3.93 - 3.68 (m, 2H), 2.88 (d, J = 4.8 Hz, 3H), 2.63 - 2.49 (m, 1H), 2.39 (dt, J = 3.9, 14.0 Hz, 1H), 2.10 - 1.99 (m, 1H), 1.96 - 1.85 (m, 1H), 1.84 - 1.70 (m, 2H), 1.64 (s, 1H), 1.62 - 1.58 (m, 1H), 1.57 (s, 3H), 1.45 (s, 3H), 1.44 - 1.39 (m, 1H), 1.38 - 1.24 (m, 2H), 1.08 - 1.00 (m, 1H), 0.98 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H).

[0064] PT-27, DHA 3-methyl-3-carbamoylazetidine-1-carboxylate was similarly prepared using 3-methylazetidine-3-carboxamide [prepared from 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid by reaction with ammonium acetate / DIEA / HATU in DMF and subsequent deprotection] instead of N,3-dimethylazetidine-3-carboxamide, and after purification, DHA 3-methyl-3-carbamoylazetidine-1-carboxylate (190 mg, 447 μmol, yield 18%) was obtained as a white solid. M + H + = 425.2 (LCMS); 1H NMR (400 MHz, DMSO-d6) δ 7.43 (br s, 1H), 7.12 (br s, 1H), 5.53 (s, 1H), 5.49 (d, J = 9.6 Hz, 1H), 4.23 - 4.03 (m, 2H), 3.76 - 3.55 (m, 2H), 2.31 - 2.22 (m, 1H), 2.17 (br dd, J = 3.6, 13.8 Hz, 1H), 2.04 - 1.96 (m, 1H), 1.86 - 1.76 (m, 1H), 1.67 - 1.51 (m, 3H), 1.46 (br s, 4H), 1.42 - 1.32 (m, 2H), 1.30 (s, 3H), 1.21 - 1.13 (m, 1H), 0.99 - 0.92 (m, 1H), 0.89 (br d, J = 6.4 Hz, 3H), 0.82 - 0.76 (m, 3H).

[0065] PT-29, DHA 3-methyl-3-(dimethylcarbamoyl)azetidine-1-carboxylate was similarly prepared using 3-methylazetidine-3-dimethylcarboxamide [prepared from 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid by reaction with dimethylamine / DIEA / HATU in DMF and subsequent deprotection] instead of N,3-dimethylazetidine-3-carboxamide, and after purification, DHA 3-methyl-3-(dimethylcarbamoyl)azetidine-1-carboxylate (110 mg, 240 μmol, yield 15%) was obtained as a white solid. M + H + = 453.3 (LCMS); 1H NMR (400 MHz, CDCl3) δ 5.66 (br d, J = 8.8 Hz, 1H), 5.45 (s, 1H), 4.59 - 4.24 (m, 2H), 3.97 - 3.67 (m, 2H), 2.97 (s, 3H), 2.88 - 2.82 (m, 3H), 2.55 (ddd, J = 4.6, 6.9, 9.9 Hz, 1H), 2.38 (dt, J = 3.9, 14.0 Hz, 1H), 2.05 (td, J = 3.5, 14.6 Hz, 1H), 1.95 - 1.84 (m, 1H), 1.81 - 1.69 (m, 2H), 1.66 - 1.62 (m, 1H), 1.60 (s, 3H), 1.59 - 1.48 (m, 1H), 1.45 (s, 3H), 1.43 (s, 1H), 1.38 - 1.24 (m, 2H), 1.07 - 0.94 (m, 4H), 0.92 - 0.82 (m, 3H).

[0066] PT-23, DHA3-aminoazetidine-1-carboxylate was prepared by a similar method, but required protection and deprotection of the amine. Benzyl 3-aminoazetidine-1-carboxylate in DCM was converted to benzyl 3-(2,2,2-trifluoroacetamide)azetidine-1-carboxylate by reaction with 1.5 equivalents of trifluoroacetic anhydride, 3.0 equivalents of TEA, and 0.1 equivalents of DMAP at 25°C for 1 hour. The mixture was then acidified with 1M hydrochloric acid, extracted to ethyl acetate, and the extract was concentrated and purified by flash chromatography. Benzyl 3-(2,2,2-trifluoroacetamide)azetidine-1-carboxylate was debenzylated by reduction with hydrogen gas (1 atm) and 10% Pd / C in ethyl acetate, filtered, and concentrated under vacuum to obtain N-(azetidine-3-yl)-2,2,2-trifluoroacetamide. This was reacted with DHA / CDI / TEA in DCM in the same manner as PT-28, concentrated under vacuum, and purified by flash silica gel chromatography to obtain DHA3-(2,2,2-trifluoroacetamide)azetidine-1-carboxylate as a white solid. This was reacted with 1.5 equivalents of sodium hydroxide in ethanol / water, filtered, and purified by preparative HPLC to obtain DHA3-aminoazetidine-1-carboxylate (30.0 mg, 70.6 μmol, yield 6%, HCl salt) as a white solid. M + H + = 383.2 (LCMS); 1 H NMR (400 MHz, DMSO) δ 9.16 - 8.49 (m, 3H), 6.26 (d, J = 3.9 Hz, 1H), 5.43 (s, 1H), 5.01 - 4.95 (m, 1H), 4.18 - 4.04 (m, 4H), 2.38 - 2.26 (m, 1H), 2.18 (dt, J = 3.8, 13.9 Hz, 1H), 2.05 - 1.74 (m, 3H), 1.70 - 1.52 (m, 2H), 1.45 - 1.30 (m, 3H), 1.30 - 1.21 (m, 3H), 1.18 - 1.09 (m, 1H), 0.98 - 0.74 (m, 7H).

[0067] Preparation 7: PT-30, DHA 5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylate, (3R,5aS,6R,8aS,9R,12S,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-yl Preparation of 5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylate [ka]

[0068] 2,6-Diazaspiro[3.4]octan-5-one was prepared from methyl N-BOC-azetidine-3-carboxylate by a five-step process. Methyl N-BOC-azetidine-3-carboxylate was reacted with 1.1 equivalents each of allyl bromide and lithium hexamethyldisilazane in THF at 25°C for 2 hours to obtain methyl N-BOC-3-allylazetidine-3-carboxylate, which was then oxidized with 2.5 equivalents of sodium periodate and 0.2 equivalents of potassium osmite in 1:1 THF / water at 25°C for 2 hours to obtain methyl N-BOC-3-(2-oxoethyl)azetidine-3-carboxylate. This was reacted with 1.5 equivalents of hydroxylamine hydrochloride in methanol at 25-80°C for 2 hours to obtain methyl N-BOC-3-(2-(hydroxyimino)oxoethyl)azetidine-3-carboxylate, which was then cyclized with hydrogen on 10% Ru / SiO2 using flow chemistry to obtain N 2 -BOC-2,6-diazaspiro[3.4]octan-5-one was obtained. Finally, N 2 -BOC-2,6-diazaspiro[3.4]octan-5-one was deprotected in acetonitrile with 1.2 equivalents of 4-toluenesulfonic acid at 80°C for 16 hours to obtain 2,6-diazaspiro[3.4]octan-5-one, which was then purified as its hydrochloride salt.

[0069] To a solution of DHA (7.5 g, 26.4 mmol, 1.0 equivalent) in DCM (300 mL), CDI (5.13 g, 31.7 mmol, 1.2 equivalents) was added. The mixture was stirred at 25°C for 2 hours. Then, 2,6-diazaspiro[3.4]octan-5-one (4.29 g, 26.4 mmol, 1.0 equivalent, HCl salt) and TEA (3.20 g, 31.7 mmol, 4.41 mL, 1.2 equivalents) were added to the above solution. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain a residue, which was purified by flash silica gel chromatography using an siRNA / petroleum ether gradient from 0 / 1 to 1 / 0. DHA5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylate (5.02 g, 11.5 mmol, yield 43.6%) was obtained as a white solid. M + H + = 437.2 (LCMS); 1 H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 1H), 5.54 (s, 1H), 5.50 (br d, J = 9.6 Hz, 1H), 4.10 - 4.02 (m, 1H), 3.99 - 3.90 (m, 2H), 3.82 (br s, 1H), 3.16 (t, J = 6.7 Hz, 2H), 2.34 (br s, 2H), 2.29 - 2.14 (m, 2H), 2.00 (br d, J = 13.6 Hz, 1H), 1.86 - 1.78 (m, 1H), 1.67 - 1.58 (m, 2H), 1.53 (br t, J = 3.8 Hz, 1H), 1.45 (br d, J = 9.6 Hz, 2H), 1.30 (s, 4H), 1.22 - 1.13 (m, 1H), 1.01 - 0.93 (m, 1H), 0.89 (d, J = 6.3 Hz, 3H), 0.80 (br d, J = 7.0Hz, 3H).

[0070] PT-30, DHA 5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylate, (3R,5aS,6R,8aS,9R,12S,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-yl 5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylate was a white crystalline solid in its prepared state. Differential scanning calorimetry showed a broad peak starting at approximately 150°C and peaking at approximately 174°C (no melting point was observed before decomposition), while thermogravimetric analysis showed a weight loss of approximately 0.9% up to 150°C. X-ray powder diffraction using CuKα rays (λ=1.5408 Å) showed sharp peaks characteristic of crystallinity, with the top five peaks being as follows, in units of °2θ (±0.2°), with approximate intensity in parentheses: 4.5 (70), 9.0 (100), 9.7 (46), 12.4 (46), and 13.6 (27). PT-30 exhibited low solubility (<0.1 mg / mL) under acidic aqueous conditions and in artificial gastric fluid, and approximately 0.7 mg / mL in artificial intestinal fluid under fasting and postprandial conditions. It was soluble in common organic solvents, exhibiting maximum solubility in methanol and tetrahydrofuran. It was chemically stable with no change in crystalline form after 1 week of exposure at 25°C / 60%RH and 40°C / 75%RH, and was slightly hygroscopic (0.5% water absorption at 40%~95%RH).

[0071] Examples [Examples]

[0072] Example 1: Anti-fibrotic effect in cardiac fibroblasts

[0073] Induced pluripotent stem cell-derived cardiac fibroblasts were constructed to contain a fusion construct of α-smooth muscle actin and green fluorescent protein (ACTA2-GFP). ACTA2 is a myofibroblast marker, and as a result, fibroblasts emit fluorescence with an intensity dependent on the degree of fibrosis. These fibroblasts were activated with TGF-β and simultaneously treated with PT compounds, or the control dihydroartemisinin or artesunate, at various concentrations for 48 hours. When fluorescence was measured, they showed a dose-dependent decrease in fluorescence, and the IC50 of PT compounds was observed. 50 The concentrations in μM were as follows: PT-1, 2.3; PT-2, 0.88; PT-4, 5.6; PT-13, 2.6; PT-14, 0.57; and artesunate, 2.8. Other PT compounds, including PT-28 and PT-30, also showed anti-fibrotic efficacy in this model.

[0074] Example 2: Pharmacokinetics in Mice

[0075] The intravenous (IV) and oral (PO) pharmacokinetics of PT compounds were measured in 6-10 week old male C57BL / 6J mice, using 3 mice per group. The test compounds were formulated in a suitable water-based vehicle to obtain a clear solution for IV administration or a clear solution or homogeneous suspension for PO administration. Animals were administered the formulation within 4 hours of preparation, and the dose was determined by the animal's body weight measured on the morning of administration. Blood samples (approximately 25 μL per time point) were collected in pre-cooled EDTA-K2 tubes at 5 and 15 minutes after administration, and at 1, 3, and 5 hours, and kept on moist ice until centrifugation. The tubes were centrifuged at 3200 × g for 10 minutes at approximately 4°C, and the plasma was analyzed by LC-MS / MS. The results are shown in the table below. [Table 1]

[0076] Example 3: Plasma protein binding

[0077] Plasma protein binding assays were performed in triplicate for compounds PT-2, PT-14, PT-28, and PT-30, with artesunate, DHA, and warfarin as control groups. The results are shown in the table below. [Table 2] NC: Not calculated; * : The % unbound value may be unreliable due to low recovery rates.

[0078] Example 4: HEK Blue-TLR4 assay (MD2 pathway)

[0079] HEK Blue-TLR4 cells were seeded in detection medium and pre-incubated for 1 hour with a vehicle, or PT-2, PT-14, or PT-30 (artesunate, DHA, and artemisinin as control compounds) dissolved in the vehicle, followed by co-stimulation with 10 ng / mL lipopolysaccharide. After 20 hours, reporter activity was read at 655 nm using a spectrophotometer. The PT compounds were active in this assay, indicating that they inhibit the MD2 signaling pathway.

[0080] Example 5: Other assays of PT-30

[0081] PT-30 demonstrated antifibrotic efficacy in primary lung fibroblasts by measuring its effects on acta2, col1a1, and CTGF. Oral administration showed antifibrotic efficacy in a CCl4-injured mouse liver model, and topical administration (50 μL of 100 μM in 0.1% DMSO / PBS) showed antifibrotic efficacy in a bleomycin-injured mouse skin model.

[0082] Example 6: Expected human cases in PBC using PT-30

[0083] The study participants were adult men or women diagnosed with primary biliary cholangitis (PBC) based on at least two of the following three criteria: (a) a history of alkaline phosphatase (ALP) levels exceeding the upper limit of normal (ULN) for at least six months; (b) a positive anti-mitochondrial antibody titer greater than 1 / 40 by immunofluorescence, or M2-positive or positive PBC-specific antinuclear antibody by enzyme-linked immunosorbent assay; and (c) a documented liver biopsy result consistent with PBC, in which the patient was on a stable recommended dose of UDCA for the past 12 months or was intolerant to UDCA, and ALP ≥ 1.67 × ULN. Exclusion criteria include AST or ALT ≥ 3 × ULN, total bilirubin (TBIL) ≥ 2 × ULN, a history of autoimmune hepatitis or chronic viral hepatitis, PSC, current use of fibrates or simvastatin, use of colchicine, methotrexate, azathioprine, or systemic steroids in the past two months, use of experimental treatments for PBC, and use of experimental or unapproved immunosuppressants. The primary trial endpoint is a reduction in ALP, and secondary endpoints are ALP < 1.67 × ULN and total bilirubin within the normal range, as well as the response rate in subjects achieving a reduction in ALP greater than 15%. Additional secondary endpoints are changes in other recognized biochemical markers of PBC, namely GGT, TBIL, and 5' nucleotidase. Participants will be randomized to receive either placebo or PT-30 in tablet form once daily for 12 weeks at doses of 10 mg / day, 30 mg / day, 50 mg / day, 100 mg / day, 200 mg / day, or 400 mg / day. Participants will demonstrate a dose-dependent reduction in ALP, indicating a therapeutic effect of PT-30 administration on PBC fibrosis.

Claims

1. Formulas PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30: 【Chemistry 1】 Any one of the compounds, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, which is any one compound of formulas PT-1, PT-2, and PT-4, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, which is one of the compounds of formulas PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1, which is one of the compounds of formulas PT-2, PT-14, PT-28, and PT-30, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

5. A compound according to claim 1, which is a compound of formula PT-14 or PT-30, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

6. A compound of formula PT-14, the compound according to claim 5, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

7. The compound according to claim 6, which is a compound of formula PT-14.

8. A compound of formula PT-30, the compound according to claim 5, or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

9. The compound according to claim 8, which is a compound of formula PT-30.

10. A pharmaceutical formulation for the treatment of fibrous disease in a human subject, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

11. A method for treating a fibrous disease in a human subject, comprising administering to the human subject a therapeutically effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutical preparation according to claim 10.

12. The method according to claim 11, wherein the fibrous disease is a systemic fibrous disease.

13. The method according to claim 12, wherein the systemic fibrous disease is systemic sclerosis, multifocal fibrosclerosis (IgG4-related fibrosis), nephrogenic systemic fibrosis, or scleroderma-like graft-versus-host disease.

14. The method according to claim 11, wherein the fibrous disease is an organ-specific fibrous disease.

15. The method according to claim 14, wherein the organ-specific fibrotic disease is cardiac fibrosis, renal fibrosis, pulmonary fibrosis, hepatic and portal vein fibrosis, radiation-induced fibrosis, bladder fibrosis, intestinal fibrosis, pancreatic fibrosis, peritoneal sclerosis, diffuse fasciitis, focal scleroderma, keloid, Dupuytren's contracture, Peyronie's disease, myelofibrosis, or oral submucosal fibrosis.

16. The method according to any one of claims 11 to 15, wherein the amount of compound administered is 10 mg / day to 600 mg / day; for example, 30 mg / day to 400 mg / day, for example, 10 mg / day, 30 mg / day, 50 mg / day, 100 mg / day, 200 mg / day, or 400 mg / day.

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