Dihydroartemisinin derivatives and treatment of fibrotic diseases

Abstract

Compounds of the formulae 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, pharmaceutical formulations containing them, and the use of these compounds for the treatment of fibrotic diseases.

Description

Technical Field

[0001] This invention relates to compounds derived from dihydroartemisinin and the use of these compounds in the treatment of fibrotic diseases. Background Technology

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

[0003] Artemisinin, (1) R 4 S 5 R 8 S 9 R ,12 S ,13 R )-1,5,9-trimethyl-11,14,15,16-tetraoxatetracyclo[10.3.1.0] 4,13 .0 8,13 Hexadecane-10-one, (3 R ,5a S 6 R ,8a S 9 R ,12 S ,12a R )-Octahydro-3,6,9-trimethyl-3,12-epoxy-12 H -pyrano[4,3- j ]-1,2-Benzadioxane-10(3 H Artemisinin-based ketones are a well-known antimalarial agent, originally extracted from the herb Artemisia annua. It was discovered in China in 1972. Artemisinin-based combination therapy (ACT, a therapy using artemisinin or one of its derivatives) is currently the standard treatment for malaria worldwide. Dihydroartemisinin, DHA, (1 R 4 S 5 R 8 S 9 R 10 S ,12 R ,13 R )-1,5,9-trimethyl-11,14,15,16-tetraoxatetracyclo[10.3.1.0] 4,13 .0 8,13 Hexadecane-10-ol, (3) R ,5a S 6 R ,8a S 9 R ,12 S ,12a R )-Decahydro-3,6,9-trimethyl-3,12-epoxy-12 H -pyrano[4,3-j 1,2-Benzadioxane-10-ol, as the name suggests, is a hydrogenated derivative of artemisinin. Dihydroartemisinin is the active metabolite of all artemisinin compounds (artemisinin, artesunate, artemether, artesyl ether, etc.) and can also be used 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., “Dihydroartemisinin: A Potential Drug for the Treatment of Malignancies and Inflammatory Diseases”) Front. Oncol. , 11, 722331 (2021) summarized recent research and reported that dihydroartemisinin has anticancer and anti-inflammatory effects.

[0004] Artesunate is a hemisuccinate of dihydroartemisinin. Unlike artemisinin, dihydroartemisinin, or artemether / artesinyl ether (the methyl / ethyl ether of dihydroartemisinin), only artesunate has sufficient water solubility for intravenous administration. In fact, in the United States, artesunate is only available in powder form for reconstitution for intravenous administration, but elsewhere it can be readily administered orally in tablet or suspension form. Artesunate is used to treat malaria, but is not recommended for prophylaxis due to its short biological half-life. In the United States, artesunate is being tested for the topical treatment of vulvar, vaginal, and anal intraepithelial neoplasia; and it has even been reported to be tested for the treatment of COVID-19.

[0005] The structural formulas of artemisinin, dihydroartemisinin, and artesunate are as follows:

[0006] Artemisinin dihydroartemisinin artesunate.

[0007] For further information on artemisinin, dihydroartemisinin, and artesunate and dihydroartemisinin, please see the Wikipedia entries https: / / en.wikipedia.org / wiki / Artemisinin, https: / / en.wikipedia.org / wiki / Dihydroartemisinin, and https: / / en.wikipedia.org / wiki / Artesunate, and the documents cited therein. For a pharmacokinetic review of dihydroartemisinin and artesunate, see 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).

[0008] fibrosis

[0009] Rosenbloom et al., “Human Fibrotic Diseases: Current Challenges in Fibrosis Research,” Fibrosis , 1627, 1-23 (2017) stated: “Human fibrosis constitutes a major global health problem due to the large number of individuals affected, the incomplete understanding of the pathogenesis of the fibrotic process, the significant heterogeneity of its etiology and clinical manifestations, the lack of appropriate and well-validated biomarkers, and, most importantly, the current lack of effective disease-modifying treatments.” Rosenbloom et al. listed the following diseases in Table 1 under the category of systemic fibrosis: systemic sclerosis, multifocal fibrosis (IgG4-associated fibrosis), renal systemic fibrosis, and scleroderma graft-versus-host disease; and under the category of organ-specific fibrosis: cardiac fibrosis, including hypertension-associated cardiac fibrosis, post-myocardial infarction fibrosis, and Chagas disease-induced myocardial fibrosis; and renal fibrosis, including diabetic and hypertensive nephropathy, urinary tract obstruction-induced renal fibrosis, inflammation / autoimmune-induced renal fibrosis, aristolochic acid nephropathy, and polycystic ovary syndrome. Kidney disease; pulmonary fibrosis, including idiopathic pulmonary fibrosis, silica-induced pneumoconiosis (siliconeosis), asbestos-induced pulmonary fibrosis (asbestosis), and chemotherapy-induced pulmonary fibrosis; hepatic and portal vein fibrosis, including alcoholic and non-alcoholic liver fibrosis, hepatitis C-induced liver fibrosis, primary biliary cholangitis, and parasite-induced liver fibrosis (schistosomiasis); and other organ-specific fibrotic diseases, including radiation-induced fibrosis (various organs), bladder fibrosis, intestinal fibrosis, peritoneal sclerosis, diffuse fasciitis, localized scleroderma, keloids, Dupuytren's disease, Peroni disease, myelofibrosis, and oral submucosal fibrosis. Other fibrotic diseases include cardiomyopathy, such as diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, and radiation-induced cardiomyopathy; Hermansky-Pudler syndrome; pancreatic fibrosis; lung complications of COVID-19 infection; and Duchenne muscular dystrophy.

[0010] Rosenbloom et al. continued, regarding fibrotic diseases: "Although their pathogenic mechanisms are quite diverse and remain elusive in many cases, these diseases share a common feature: the uncontrolled, gradual accumulation of fibrotic tissue in the affected organs, leading to organ dysfunction and eventual failure. Despite significant heterogeneity in the etiologies and clinical manifestations of fibrotic diseases, numerous studies have identified activated myofibroblasts as the common cellular component ultimately leading to the replacement of normal tissue with nonfunctional fibrotic tissue," and "Despite substantial progress in understanding the pathogenesis of fibrotic diseases, they remain a significant challenge, not only because of the diversity and multiplicity of initiation events, but also because of the large number of pro-fibrotic mediators involved. While TGF-β (transforming growth factor-β) is considered a major fibrotic effector, many other cytokines and signaling molecules also participate 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 pointed out: "Currently, treatment interventions for fibrotic diseases are quite limited. For example, only two drugs, pirfenidone and nintedanib, are currently approved for the treatment of IPF, and no disease-modifying drugs are approved for SSc [systemic sclerosis] or other fibrotic diseases."

[0011] Artesunate has been shown to be active as an anti-fibrotic agent in a bleomycin-induced rat model of pulmonary fibrosis: 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 theNotch signaling pathway”, Exp. Ther. Med. , 14, 561-1566 (2017). However, the short biological half-life of artesunate makes it unsuitable as a long-term treatment for fibrotic diseases, especially as an oral medication.

[0012] The goal is to develop novel dihydroartemisinin derivatives as a drug treatment for fibrotic diseases. Summary of the Invention

[0013] In a first aspect, the present invention is a compound of any one 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 its salts, especially its pharmaceutically acceptable salts.

[0014] These compounds are derivatives of dihydroartemisinin, either carboxylic esters of dihydroartemisinin (PT-1, PT-2, PT-4) or carbamates of dihydroartemisinin (PT-13, PT-14, PT-20, PT-23 to PT-25 and PT-27 to PT-30).

[0015] In a second aspect, the present invention is a pharmaceutical preparation for treating fibrotic diseases, comprising the compounds of the first aspect of the present invention.

[0016] In a third aspect, the present invention relates to the use of the compounds of the first aspect of the present invention or the pharmaceutical preparations of the second aspect of the present invention for the treatment of fibrotic diseases.

[0017] Compounds of any one 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 useful for the treatment of fibrotic diseases, because studies of induced pluripotent stem cell (iPSC)-derived cardiac fibroblasts activated with transforming growth factor-β (TGF-β) have shown that they lead to a dose-dependent decrease in the levels of fibrosis markers, and because some of them have been shown to inhibit the MD2 signaling pathway, demonstrating antifibrotic efficacy in primary lung fibroblasts by measuring their effects on acta2, col1a1, and CTGF; demonstrating antifibrotic efficacy in a mouse model of CCl4-induced liver injury by oral administration; and demonstrating antifibrotic efficacy in a mouse model of bleomycin-induced skin injury by topical administration; and because artesunate has been shown to have antifibrotic activity in a rat model of bleomycin-induced pulmonary fibrosis. Furthermore, because these dihydroartemisinin derivatives have a longer biological half-life than artesunate, they are expected to be more effective than artesunate in treating these fibrotic diseases.

[0018] Compounds of any one 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 a variety of routes as an effective treatment for fibrotic diseases mentioned in the section entitled "Fibrotic Diseases" in the background art.

[0019] The preferred embodiments of the present invention are characterized by the description of this application and the features of claims 1 to 16. Detailed Implementation

[0020] definition

[0021] The section titled "Fibrotic Diseases" in the background section describes fibrotic diseases and their treatment.

[0022] The "therapeutic effective amount" of the compound of the first aspect of the present invention refers to an amount that is sufficient to effectively treat the fibrotic disease when administered to a subject (i.e., a human).

[0023] "Treatment" or "treatment" of fibrotic disease in subjects includes one or more of the following: (1) Prevent or reduce the risk of fibrotic disease, i.e., no clinical symptoms of fibrotic disease in subjects who are susceptible to fibrotic disease but have not yet developed or have shown symptoms of fibrotic disease (i.e., prevention). (2) Inhibit fibrotic diseases, that is, prevent or reduce the development of fibrotic diseases or their clinical symptoms; and (3) Alleviate fibrotic diseases, that is, promote the regression, reversal or improvement of fibrotic diseases, or reduce the number, frequency, duration or severity of their clinical symptoms.

[0024] "Treatment" does not necessarily mean "cure" or complete cure, such as treating all clinical symptoms of fibrotic disease, although "treatment" can include "cure." Rather, "treatment" means that administering the compound provides clinical benefit compared to not administering it; and treatment can also be evaluated by improvements in biomarkers of the treated fibrotic disease.

[0025] For a given subject, the effective therapeutic dose varies depending on the subject's health and physical condition, the nature and extent of fibrosis, assessment of medical conditions, and other relevant factors. The effective therapeutic dose is expected to fall within a relatively wide range and can be determined through routine trials.

[0026] The words “comprising” or “containing” and their grammatical variations are inclusive terms and not restrictive terms. They are intended to indicate the presence of the stated components, groups, steps, etc., but do not preclude the presence or addition of other components, groups, steps, etc. Therefore, “comprising” does not mean “composed of,” “essentially composed of,” or “composed solely of”; and for example, a formulation “comprising” a compound must contain that compound, but may also contain other active ingredients, prodrugs, and / or excipients. Unless the context requires otherwise, the singular forms “a,” “an,” and “the” include plural references.

[0027] compound

[0028] 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, particularly their pharmaceutically acceptable salts, can be prepared by conventional methods. For PT-1, PT-2, and PT-4, this involves the esterification of dihydroartemisinin with a suitable acid (usually activated) to obtain the desired carboxylic acid ester side chain. For PT-13, PT-14, PT-20, PT-23 to PT-25 and PT-27 to PT-30, this involves the esterification of dihydroartemisinin with 1,1-carbonyldiimidazole (CDI), followed by the substitution of the imidazole ring with a suitable amine such as aziridine-3-ol (for PT-13), 3-methylaziridine-3-ol (for PT-14), or 2,6-diazaspiro[3.4]octane-5-one (for PT-30) to obtain the desired carbamate side chain.

[0029] When the carboxylic acid or carbamate to be added to dihydroartemisinin is reactive to the reaction conditions, such as when it contains an amino or hydroxyl group, these groups are usually protected with a protecting group before the reaction to form the carboxylic acid ester or carbamate. The protecting group is, for example, an acid-labile 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, and is removed after the reaction.

[0030] Therefore, for example, the preparation of PT-1 (i.e., 3-hydroxy-2-methylpropionate of DHA) can be achieved by: in a polar aprotic solvent such as dichloromethane, in a solvent such as DCC (… N , N In the presence of coupling agents such as '-dicyclohexylcarbodiimide' or similar compounds, and in the presence of organic bases such as DMAP (4-dimethylaminopyridine), dihydroartemisinin is coupled with... O-TBDPS-3-hydroxy-2-methylpropionate esterification, followed by deprotection of the side-chain hydroxyl groups in dimethylformamide with a reagent such as cesium fluoride, as follows:

[0031] The same method can be used, using appropriately protected acids, such as O -TBDPS-3-hydroxy-2,2-dimethylpropionic acid (for PT-2) and O -TBDPS-3-hydroxy-3,3-dimethylpropionic acid (for PT-4), a compound of formulas PT-2 and PT-4. PT-4 can also be prepared in one step using unprotected 3-hydroxy-3,3-dimethylpropionic acid.

[0032] Compounds such as PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30 can be prepared by coordinating dihydroartemisinin with 1,1-carbonyldiimidazole (CDI) in a polar aprotic solvent such as dichloromethane to form 1 H -Imidazole-1-carboxylic acid esters, subsequently in the presence of an organic base such as triethylamine (TEA) in a polar aprotic solvent such as dichloromethane, by reacting with a suitable amine such as aziridine-3-ol (for the preparation of PT-13), 3-methylaziridine-3-ol (for PT-14), 2,6-diazaspiro[3.4]octane-5-one (for PT-30) to replace imidazole, as follows:

[0033] This invention includes salts (e.g., pharmaceutically acceptable salts) of compounds of any one 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 can be used in the methods described in this application. These salts are preferably formed using pharmaceutically acceptable acids or bases. For a broad discussion of pharmaceutical salts, their selection, preparation, and uses, see, for example, “Handbook of Pharmaceutically Acceptable Salts”, Stahl and Wermuth, eds., Verlag Helvetica Chimica Acta, Zürich, Switzerland. Unless the context otherwise requires, 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.

[0034] Since PT-23 and PT-24 each contain an amino group, they can form acid addition salts when the amino group reacts with inorganic acids such as hydrochloric acid or organic acids such as maleic acid. Typically, the compounds are treated with an excess of acid in a protic solvent such as water or lower alkanols or combinations thereof, and the solution is thoroughly removed to allow the resulting acid addition salts to crystallize.

[0035] Preparation and administration

[0036] Compounds of any one 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, may be administered to the treated subject via any suitable route, depending on the nature of the subject's condition. Routes of administration include injection (including intravenous, intraperitoneal, intramuscular, and subcutaneous injection), transdermal or mucosal delivery, administration by topical application, nasal spray, suppository, etc., or oral administration. Formulations may optionally be liposomal, emulsion, formulations intended for administration via mucous membranes, or transdermal formulations. For example, formulations suitable for each of these methods of administration can be found in "Remington: The Science and Practice of Pharmacy", 20th ed., Gennaro, ed., Lippincott Williams & Wilkins, Philadelphia, Pa., USA. If the compound is orally available, the typical formulation is oral, and the typical dosage form is a tablet or capsule for oral administration. Intravenous formulations may be particularly suitable for administration to patients with acute illnesses, such as those subjects who may require hospitalization.

[0037] Depending on the intended administration method, the pharmaceutical composition may be in the form of a solid, semi-solid, or liquid dosage form, preferably a single-dose dosage form suitable for a precise single-dose administration. In addition to an effective amount of artesunate or dihydroartemisinin, the composition may also contain suitable pharmaceutically acceptable excipients, including adjuvants that facilitate the formulation of the active compound into a pharmaceutically usable formulation. "Pharmaceutically acceptable excipients" refers to excipients or mixtures of excipients that do not interfere with the effectiveness of the biological activity of the active compound and are non-toxic or have no other adverse effects on the recipient.

[0038] For solid compositions, common excipients include, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For instance, the active compound described herein and optional adjuvants can be dissolved or dispersed in water or an aqueous excipient (such as, for example, water, physiological saline, dextran aqueous solution, etc.) to form a solution or suspension, thereby preparing a liquid, pharmacologically administerable composition. If desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary excipients, such as wetting agents or emulsifiers, pH buffers, etc., for example, sodium acetate, sorbitol monolaurate, sodium triethanolamine acetate, triethanolamine oleate, etc.

[0039] For oral administration, the composition is typically in the form of tablets or capsules; or, particularly for use in children, it may be an aqueous or non-aqueous solution, a suspension, or a syrup. Tablets and capsules are preferred forms for oral administration. Orally administered tablets and capsules typically contain one or more commonly used fillers, such as lactose and corn starch. Lubricants (such as magnesium stearate) and binders (such as carbomer) are also usually added. When using a liquid suspension, the active ingredient may be combined with emulsifiers and suspending agents. Flavoring agents, coloring agents, and / or sweeteners may also be added if desired. Other optional excipients for incorporation into oral formulations include preservatives, suspending agents, thickeners, etc.

[0040] Typically, a pharmaceutical composition of one of the 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 for use or both indicating the purpose of the pharmaceutical composition or kit in the treatment of fibrotic diseases.

[0041] Those skilled in the art of pharmaceutical formulation can prepare suitable pharmaceutical compositions of the compounds of this invention without extensive experimentation, relying solely on their personal knowledge and the disclosure of this application, by selecting appropriate dosage forms, excipients, packaging, etc., to achieve therapeutically effective formulations.

[0042] The appropriate (i.e., therapeutically effective) dose of any 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, for systemic administration is expected to be at least 10 mg / day and not more than 600 mg / day; for example, for adult subjects, at least 30 mg / day and not more than 400 mg / day, depending on the nature, extent, and severity of the fibrotic disease and factors such as liver and kidney function. For pediatric subjects (depending on additional factors such as age and weight) and subjects with severe liver or kidney impairment (depending on the extent of the impairment), the dose may be appropriately reduced to the lower end of the aforementioned outer range. These doses represent average daily doses and are not necessarily doses given in a single administration. Dosing frequency may be more than once / day (where the dose or daily dose is spread across the number of administrations per day), but more commonly once / day (where the dose is given in a single administration). Optionally, especially in cases of severe liver damage, the frequency of administration may be less than once a day, such as once a week and every other day, for example once a week, twice a week (especially every three days), three times a week (especially every two days) or every other day.

[0043] For a specific subject and the nature, extent, and severity of fibrosis, a person skilled in the art of treating fibrosis can determine, based solely on personal knowledge and the disclosure of this application, the therapeutically effective amount 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 and their salts, to achieve a therapeutically effective amount, without conducting extensive experiments.

[0044] preparation

[0045] Abbreviations: BOC: tert-butyloxycarbonyl; 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; EtOAc: Ethyl acetate; HATU: Azabenzotriazole tetramethylureonium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazole[4,5-b]pyridinium 3-oxide hexafluorophosphate; LCMS: liquid chromatography-mass spectrometry; TBDPSCl: tert-butyldiphenylsilyl oxide; TEA: triethylamine; TEMPO: (2,2,6,6-tetramethylpiperidin-1-yl)oxy; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TLC: thin-layer chromatography.

[0046] Preparation 1: Preparation of PT-1, DHA 3-hydroxy-2-methylpropionate

[0047] Step 1: 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropane-1-ol (1A-2)

[0048] At 0 °C, NaH (2.90 g, 73.2 mmol, 60% purity, 1.1 equivalent) was added fractionally to a solution of 2-methylpropane-1,3-diol (6.00 g, 66.6 mmol, 5.90 mL, 1.0 equivalent) in THF (90 mL), and the mixture was stirred at 0 °C for 10 min. TBDPSCl (20.1 g, 73.2 mmol, 18.7 mL, 1.1 equivalent) was added fractionally to the reaction mixture, and the mixture was stirred at 20 °C for 12 h. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was cooled to 0 °C, poured into a saturated aqueous solution of 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 the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 1 / 9. 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropane-1-ol (13.0 g, 39.6 mmol) was given 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).

[0049] Step 2: 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropionic acid (1A-3)

[0050] To a solution of 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropane-1-ol (5.50 g, 16.7 mmol, 1.0 equivalent) in DCM (330 mL), TEMPO (527 mg, 3.40 mmol, 0.2 equivalent), H₂O (15.1 g, 837 mmol, 15.1 mL, 50.0 equivalent), and PhI(OAc)₂ (13.5 g, 41.9 mmol, 2.5 equivalent) were added. The mixture was stirred at 20 °C for 12 h. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was poured into a saturated aqueous solution of Na₂S₂O₃ (60 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine (60 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 3 / 7. 3-( ( tert-Butyldiphenylsilyl)oxy)-2-methylpropionic acid (2.50 g, 7.30 mmol) is 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).

[0051] Step 3: DHA 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropionate (1A-4)

[0052] Add 3-( (tert-Butyldiphenylsilyl)oxy)-2-methylpropionic acid (1.30 g, 3.8 mmol, 1.0 equivalent), DCC (1.60 g, 7.60 mmol, 1.5 mL, 2.0 equivalent), and DMAP (46.4 mg, 379 μmol, 0.1 equivalent) were added, and the resulting mixture was stirred at 20 °C for 12 h. LCMS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was filtered and concentrated under vacuum to give the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 1 / 9. DHA 3-((tert-Butyldiphenylsilyl)oxy)-2-methylpropionic acid ester (1.4 g, crude) was given 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 1.37 – 1.14 (m, 6H), 1.04 (s, 9H), 0.97 (d, J = 6.0 Hz, 3H), 0.87 – 0.83 (m,3H).

[0053] Step 4: DHA 3-hydroxy-2-methylpropionate (PT-1)

[0054] CsF (973 mg, 6.40 mmol, 237 μL, 3.0 equivalent) was added to a solution of DHA 3-((tert-butyldiphenylsilyl)oxy)-2-methylpropionate (1.30 g, 2.10 mmol, 1.0 equivalent) in DMF (26 mL). The mixture was stirred at 20 °C for 13 h. LCMS showed that the starting material was completely consumed. The reaction mixture was poured into H2O (50 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to give the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 3 / 7. DHA 3-hydroxy-2-methylpropionate (60.0 mg) was given as a white solid. M + Na = 393.2 (LCMS); 1 H NMR (400MHz, 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).

[0055] Preparation 2: Preparation of PT-2,DHA 3-hydroxy-2,2-dimethylpropionate

[0056] Step 1: 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionic acid (1A-2)

[0057] At 0°C, TBDPSCl (5.12 g, 18.6 mmol, 1.0 equivalent) was added to a solution of 3-hydroxy-2,2-dimethylpropionic 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), and the resulting mixture was stirred at 20°C for 1 h. TLC (EtOAc / petroleum ether 1 / 5, R f =0.69) indicates that the starting material was completely consumed. The reaction mixture was poured into HCl solution (1 M aqueous solution, 30 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered, and concentrated under vacuum to give the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 1 / 10. 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionic acid (5.00 g, 14.0 mmol, 83% yield) was given 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).

[0058] Step 2: DHA 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionate (1A-3)

[0059] Add (3 g) to a solution of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionic acid (2.51 g, 7.03 mmol, 2.0 equivalents) in DCM (60 mL) R ,5a S 6 R ,8a S 9 R 10 S ,12 R ,12a R )-3,6,9-trimethyldecahydro-12 H -3,12-epoxy[1,2]dioxane-heptane[4,3-] iIsochre-10-ol (1.00 g, 3.52 mmol, 1.0 equivalent) and DMAP (430 mg, 3.52 mmol, 1.0 equivalent), EDCI (1.35 g, 7.03 mmol, 2.0 equivalent), and the resulting mixture was stirred at 20 °C for 12 h. TLC (EtOAc / petroleum ether = 1 / 5, R f = 0.64) indicates that the starting material was completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0. DHA 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionate (1.90 g, 3.05 mmol, 87% yield) was given 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, 13.7Hz, 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).

[0060] Step 3: DHA 3-hydroxy-2,2-dimethylpropionate (PT-2)

[0061] At 20 °C, CsF (366 mg, 2.41 mmol, 88.9 μL, 3.0 equivalent) was added to a solution of DHA 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionate (500 mg, 803 μmol, 1.0 equivalent) in DMF (20 mL), and the resulting mixture was stirred at 20 °C for 12 h. TLC (EtOAc / petroleum ether = 1 / 1, R f = 0.24) indicates that some of the starting material was retained, and the aforementioned spot was detected. The reaction mixture was poured into H2O (30 mL) and extracted with EtOAc (20 mL × 5). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, filtered, and concentrated under vacuum to give the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient of 0 / 1 to 1 / 1. DHA 3-hydroxy-2,2-dimethylpropionate (52.0 mg, 135 μmol, 6% yield) was given 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 = 7.1 Hz, 3H).

[0062] Preparation 3: Preparation of PT-4, DHA 3-hydroxy-3-methylbutyrate

[0063] DHA (2.41 g, 8.47 mmol, 2.0 equivalent) and DCC (1.75 g, 8.47 mmol, 1.71 mL, 2.0 equivalent), DMAP (51.7 mg, 423 μmol, 0.1 equivalent), were added to a solution of 3-hydroxy-3-methylbutyric acid (500 mg, 4.23 mmol, 1.0 equivalent) in DCM (30 mL). The resulting mixture was stirred at 20 °C for 12 h. LCMS showed that the starting material had been 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 rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0. The solution was then purified by preparative HPLC (Phenomenex Luna C18 column (75 × 30 mm, 3 μm); flow rate: 25 mL / min; gradient: 45% – 70% B over 8 min; mobile phase A: 0.04% HCl aqueous solution, mobile phase B: acetonitrile). DHA 3-hydroxy-3-methylbutyrate (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).

[0064] Preparation 4: Preparation of PT-13, DHA 3-hydroxyazacyclobutane-1-carboxylic acid ester

[0065] DHA (1.00 g, 3.52 mmol, 1.0 equivalent) was added to a solution of CDI (684 mg, 4.22 mmol, 1.2 equivalent) in DCM (100 mL). The reaction mixture was stirred at 20 °C for 10 min, and then aziridine-3-ol (385 mg, 3.52 mmol, 1.0 equivalent, HCl salt) and TEA (427 mg, 4.22 mmol, 587 μL, 1.2 equivalent) were added. The resulting mixture was stirred at 20 °C for 12 h. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0. DHA 3-hydroxyaziridine-1-carboxylic acid ester (270 mg, 698 μmol, 20% yield) was given as a white solid. M – H - = 382.2 (LCMS); 1 H NMR (400 MHz, CDCl3) δ 5.66 (d, J = 9.8Hz, 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).

[0066] Preparation 5: PT-14, DHA 3-hydroxyazacyclobutane-1-carboxylic acid ester, (3 R ,5a S 6 R ,8a S 9 R ,12 S ,12a R )-Decahydro-3,6,9-trimethyl-3,12-epoxy-12 H- Pyrano[4,3-j Preparation of 1,2-benzodioxane-10-yl-3-hydroxyazacyclobutane-1-carboxylic acid ester

[0067] DHA (10.0 g, 35.2 mmol, 1.0 equivalent) was added to a solution of CDI (6.84 g, 42.2 mmol, 1.2 equivalent) in DCM (400 mL), and the reaction mixture was stirred at 25 °C for 3 h. Then, 3-methylazacyclobutane-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 equivalent) were added, and the resulting mixture was stirred at 25 °C for 12 h. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using a 0 / 1 to 1 / 1 EtOAc / petroleum ether gradient. DHA 3-hydroxy-3-methylazacyclobutane-1-carboxylic acid ester (5.14 g, 12.91 mmol, 36.7% yield) 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).

[0068] PT-20, DHA(2-hydroxyethyl)(methyl)carbamate, was prepared similarly using 2-methylaminoethane-1-ol instead of 3-methylazacyclobutane-3-ol. After purification, DHA(2-hydroxyethyl)(methyl)carbamate (640 mg, 1.66 mmol, 50% yield) 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).

[0069] Similarly, PT-24, DHA 3-amino-3-methylazacyclobutane-1-carboxylic acid ester was prepared by replacing 3-methylazacyclobutane-3-ol with 3-methylazacyclobutane-3-amine. After purification, DHA 3-amino-3-methylazacyclobutane-1-carboxylic acid ester (300 mg, 756 μmol, 43% yield) was obtained as a white solid. M + H + = 397.2 (LCMS); 1 H NMR (400 MHz, DMSO-) d 6 ) 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 = 6.3Hz, 3H), 0.83 – 0.74 (m, 3H).

[0070] Similarly, PT-25, DHA 3-carboxy-3-methylazacyclobutane-1-carboxylic acid ester was prepared by replacing 3-methylazacyclobutane-3-ol with 3-methylazacyclobutane-3-carboxylic acid. After purification, DHA 3-carboxy-3-methylazacyclobutane-1-carboxylic acid ester (200 mg, 467 μmol, 9% yield) was obtained as a white solid. M + H + = 426.2 (LCMS); 1 H NMR (400 MHz, DMSO-) d 6 ) δ 13.13 – 12.66 (m, 1H), 5.54 (s, 1H), 5.50 (d, J = 9.8Hz, 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).

[0071] Preparation 6: Preparation of PT-28, DHA 3-methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid ester

[0072] Step 1: 3-Methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid tert-butyl ester (1A-2)

[0073] HATU (1.32 g, 3.48 mmol, 1.5 equivalent) was added to a solution of 1-(tert-butoxycarbonyl)-3-methylazacyclobutane-3-carboxylic acid (500 mg, 2.32 mmol, 1.0 equivalent), methylamine (235 mg, 3.48 mmol, 1.5 equivalent, HCl salt), and DIEA (1.20 g, 9.29 mmol, 1.60 mL, 4.0 equivalent) in DMF (5.0 mL). The mixture was stirred at 30 °C for 12 h. LCMS showed that the starting material had been 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 EtOAc (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0. 3-Methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid tert-butyl ester (500 mg, 2.19 mmol, 94% yield) was obtained as a yellow oil. M + H + =229.2 (LCMS); 1 H NMR (400 MHz, CDCl3) δ4.11 (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).

[0074] Step 2: N, 3-Dimethylazacyclobutane-3-carboxamide (1A-3)

[0075] A mixture of 3-methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid tert-butyl ester (500 mg, 2.19 mmol, 1.0 equivalent) in DCM (3.0 mL) and TFA (1.0 mL) was stirred for 1 h at 25°C. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain N3-Dimethylazacyclobutane-3-carboxamide (530 mg, crude product, TFA salt) is a yellow oily substance. M + H + = 129.1 (LCMS).

[0076] Step 3: DHA 3-methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid ester (PT-28)

[0077] Add DHA (480 mg, 1.69 mmol, 1.0 equivalent) to a solution of CDI (328 mg, 2.03 mmol, 1.2 equivalents) in DCM (6.0 mL). Stir the mixture at 30 °C for 0.5 h. Then add DHA (480 mg, 1.69 mmol, 1.0 equivalents) to the above solution in DCM (2.0 mL). N 3-Dimethylazacyclobutane-3-carboxamide (523 mg, 2.16 mmol, 1.2 equivalents, TFA salt) and TEA (256 mg, 2.53 mmol, 352 μL, 1.5 equivalents) were mixed. The mixture was stirred at 30 °C for 0.5 h. LCMS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0, followed by preparative HPLC (Phenomenex Luna C18 column (78 × 30 mm, 3 μm); flow rate: 25 mL / min; gradient: 45% - 75% B over 9 min; mobile phase A: 10 mm NH4HCO3 aqueous solution, mobile phase B: acetonitrile). DHA 3-methyl-3-(methylcarbamoyl)azacyclobutane-1-carboxylic acid ester (180 mg, 400 μmol, 23% yield) was obtained as a white solid. M + H + =439.2 (LCMS); 1 H 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).

[0078] 3-Methylazacyclobutane-3-carboxamide [prepared from 1-(tert-butoxycarbonyl)-3-methylazacyclobutane-3-carboxylic acid by reaction with ammonium acetate / DIEA / HATU in DMF, followed by deprotection] was used instead of N, PT-27, DHA 3-methyl-3-carbamoylaziridine-1-carboxylic acid ester, was similarly prepared using 3-dimethylaziridine-3-carboxamide. After purification, DHA 3-methyl-3-carbamoylaziridine-1-carboxylic acid ester (190 mg, 447 μmol, 18% yield) was obtained as a white solid. M + H + = 425.2 (LCMS); 1 H NMR (400 MHz, DMSO- d 6 ) 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).

[0079] 3-Methylazacyclobutane-3-dimethylformamide [prepared from 1-(tert-butoxycarbonyl)-3-methylazacyclobutane-3-carboxylic acid by reaction with dimethylamine / DIEA / HATU in DMF followed by deprotection] was used instead of N PT-29, DHA 3-methyl-3-(dimethylcarbamoyl)azacyclobutane-1-carboxylic acid ester, was similarly prepared using 3-dimethylazacyclobutane-3-carboxamide. After purification, DHA 3-methyl-3-(dimethylcarbamoyl)azacyclobutane-1-carboxylic acid ester (110 mg, 240 μmol, 15% yield) was obtained as a white solid. M + H + = 453.3 (LCMS); 1 H 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).

[0080] PT-23, DHA 3-aminoazacyclobutane-1-carboxylic acid ester was prepared using a similar method, but requiring amine protection and deprotection. 3-Aminoazacyclobutane-1-carboxylic acid benzyl ester was converted to 3-(2,2,2-trifluoroacetamyl)azacyclobutane-1-carboxylic acid benzyl ester in DCM by reaction at 25°C with 1.5 equivalents of trifluoroacetic anhydride, 3.0 equivalents of TEA, and 0.1 equivalents of DMAP for 1 h, followed by acidification with 1M hydrochloric acid for post-treatment, extraction into ethyl acetate, and concentration and purification by rapid chromatography. 3-(2,2,2-trifluoroacetamyl)azacyclobutane-1-carboxylic acid benzyl ester was debenzylated by reduction in ethyl acetate with hydrogen (1 atm) and 10% Pd / C, filtered, and concentrated under vacuum to obtain… N -(azacyclobutane-3-yl)-2,2,2-trifluoroacetamide. This compound was reacted with DHA / CDLTEA in DCM in the same manner as PT-28, concentrated under vacuum, and purified by rapid silica gel column chromatography to give DHA 3-(2,2,2-trifluoroacetamido)azacyclobutane-1-carboxylic acid ester as a white solid. This compound was then reacted with 1.5 equivalents of sodium hydroxide in ethanol / water, filtered, and purified by preparative HPLC to give DHA 3-aminoazacyclobutane-1-carboxylic acid ester (30.0 mg, 70.6 μmol, 6% yield, 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).

[0081] Preparation 7: PT-30, DHA 5-oxo-2,6-diazaspiro[3,4]octane-2-carboxylic acid ester, (3 R ,5a S 6 R ,8aS 9 R ,12 S ,12a R )-Decahydro-3,6,9-trimethyl-3,12-epoxy-12 H -pyrano[4,3- j Preparation of 1,2-benzodioxane-10-yl-5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylic acid ester

[0082] 2,6-diazaspiro[3,4]octane-5-one N -BOC-azacyclobutane-3-carboxylic acid methyl ester was prepared by a five-step process. The process involved setting the methyl ester at 25 °C... N -BOC-azacyclobutane-3-carboxylic acid methyl ester reacted with 1.1 equivalents of each allyl bromide and hexamethyldisilazane lithium in THF for 2 h to give N -BOC-3-allylazacyclobutane-3-carboxylic acid methyl ester; and it was oxidized in 1:1 THF / water at 25 °C with 2.5 equivalents of sodium periodate and 0.2 equivalents of potassium osmium tetroxide for 2 h to obtain N -BOC-3-(2-oxoethyl)azacyclobutane-3-carboxylic acid methyl ester. This compound was reacted with 1.5 equivalents of hydroxylamine hydrochloride in methanol at 25-80 °C for 2 h to give... N -BOC-3-(2-(hydroxyimino)oxoethyl)azacyclobutane-3-carboxylic acid methyl ester; then cyclized with hydrogen over 10% Ru / SiO2 using flow chemistry, to obtain N 2 -BOC-2,6-diazaspiro[3,4]octane-5-one. Finally, N 2 -BOC-2,6-diazaspiro[3.4]octane-5-one was deprotected in acetonitrile at 80 °C with 1.2 equivalents of 4-toluenesulfonic acid for 16 h to give 2,6-diazaspiro[3.4]octane-5-one, which was then purified to hydrochloride.

[0083] CDI (5.13 g, 31.7 mmol, 1.2 equivalent) was added to a solution of DHA (7.5 g, 26.4 mmol, 1.0 equivalent) in DCM (300 mL). The mixture was stirred at 25 °C for 2 h. Then, 2,6-diazaspiro[3.4]octane-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 equivalent) were added to the above solution. The mixture was stirred at 25 °C for 12 h. LCMS showed that the starting material had been completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain the residue, which was purified by rapid silica gel chromatography using an EtOAc / petroleum ether gradient from 0 / 1 to 1 / 0. DHA 5-oxo-2,6-diazaspiro[3,4]octane-2-carboxylate (5.02 g, 11.5 mmol, 43.6% yield) was obtained as a white solid. M + H + = 437.2 (LCMS); 1 H NMR (400MHz, DMSO- d 6 ) 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 = 7.0 Hz, 3H).

[0084] PT-30, DHA 5-oxo-2,6-diazaspiro[3,4]octane-2-carboxylic acid ester, (3 R ,5a S 6 R ,8a S 9 R ,12 S ,12a R )-Decahydro-3,6,9-trimethyl-3,12-epoxy-12 H- Pyrano[4,3- j 1,2-Benzadioxane-10-yl-5-oxo-2,6-diazaspiro[3.4]octane-2-carboxylic acid ester, prepared as a white crystalline solid. Differential scanning calorimetry showed a broad peak starting at about 150 °C, peaking at about 174 °C (melting point not observed before decomposition), while thermogravimetric analysis showed a weight loss of about 0.9% at 150 °C. X-ray powder diffraction using Cu Kα radiation (λ = 1.5408 Å) showed sharp peaks characteristic of crystallinity, with the five largest peaks listed below in °2θ (±0.2°), with approximate intensities in parentheses: 4.5 (70), 9.0 (100), 9.7 (46), 12.4 (46), and 13.6 (27). PT-30 exhibits low solubility (<0.1 mg / mL) in aqueous acidic conditions and simulated gastric fluid, and approximately 0.7 mg / mL in simulated intestinal fluid under both fasting and feeding conditions. It is soluble in common organic solvents, with the highest solubility in methanol and tetrahydrofuran. It is chemically stable after one week of exposure to 25°C / 60%RH and 40°C / 75%RH, with no change in crystalline morphology, and is slightly hygroscopic (absorbing 0.5% water from 40% to 95%RH).

[0085] Example

[0086] Example 1: Antifibrotic effect in cardiac fibroblasts

[0087] Cardiac fibroblasts derived from induced pluripotent stem cells were constructed, containing a fusion of α-smooth muscle actin and a green fluorescent protein (ACTA2-GFP) construct. ACTA2 is a myofibroblast marker that causes fibroblasts to fluoresce, with the intensity depending on the degree of fibrosis. When these fibroblasts were activated with TGF-β and simultaneously treated with various concentrations of PT compounds or the comparative compounds dihydroartemisinin or artesunate for 48 h and fluorescence was measured, they showed a dose-dependent decrease in fluorescence, with the IC50 of the PT compounds being relatively high. 50 The following (unit: M): 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 antifibrotic efficacy in this model.

[0088] Example 2: Pharmacokinetics in Mice

[0089] The pharmacokinetics of the PT compound were measured in intravenous (IV) and oral (PO) administration in 6–10-week-old male C57BL / 6J mice using three mice / groups. The test compound was prepared in a suitable aqueous medium to obtain a clear solution for IV administration, or a clear solution or homogeneous suspension for PO administration. Animals were administered the drug within 4 h of formulation preparation; the dose volume was determined by measuring animal body weight on the morning of the administration day. Blood samples (approximately 25 μL at each time point) were collected at 5 min, 15 min, 1 h, 3 h, and 5 h post-administration into pre-chilled EDTA-K2 tubes and placed on wet ice until centrifugation. The tubes were centrifuged at approximately 4°C at 3200 × g for 10 min, and plasma was analyzed by LC-MS / MS. The results are shown in the table below.

[0090]

[0091] Example 3: Plasma protein binding

[0092] Using artesunate, DHA, and warfarin as comparisons, three plasma protein binding assays were performed on compounds PT-2, PT-14, PT-28, and PT-30. The results are shown in the table below.

[0093]

[0094] NC: Not calculated; The percentage of unbound values ​​may be unreliable due to low recovery rates.

[0095] Example 4: HEK Blue-TLR4 assay (MD2 pathway)

[0096] HEK Blue-TLR4 cells were seeded in assay medium and pre-incubated for 1 h with the medium or PT-2, PT-14, or PT-30 dissolved in the medium (using artesunate, DHA, and artemisinin as a comparative). Then, they were co-stimulated with 10 ng / mL lipopolysaccharide. After 20 h, reporter activity was read at 655 nm using a spectrophotometer. The PT compounds were active in this assay, indicating that they inhibited the MD2 signaling pathway.

[0097] Example 5: Other determinations of PT-30

[0098] PT-30 exhibited antifibrotic efficacy in primary lung fibroblasts, with its effects on acta2, col1a1, and CTGF measured. It also demonstrated antifibrotic efficacy in a mouse model of CCl4-induced liver injury via oral administration and in a mouse model of bleomycin-induced skin injury via topical administration (50 μL, 100 μM in 0.1% DMSO / PBS).

[0099] Example 6: Prophetic Human Cases Using PT-30 in PBC

[0100] The trial participants were adult men or women diagnosed with primary biliary cholangitis (PBC) who met at least two of the following three criteria: (a) a history of alkaline phosphatase (ALP) levels above the upper limit of normal (ULN) for at least six months; (b) a positive antimitochondrial antibody titer > 1 / 40 by immunofluorescence, or a positive M2 assay by enzyme-linked immunosorbent assay (ELISA), or a positive PBC-specific antinuclear antibody; and (c) a documented liver biopsy consistent with PBC, use of a stable and recommended dose of UDCA in the past twelve months, or intolerance to UDCA, and ALP ≥ 1.67. ULN. Exclusion criteria include AST or ALT ≥ 3. ULN (Ultra-Limited Nerve), Total Bilirubin (TBIL) ≥ 2 ULN (Ultra-Low Noise), history of autoimmune hepatitis or chronic viral hepatitis, PSC (Percutaneous Coronary Syndrome), currently using fibrates or simvastatin, having used colchicine, methotrexate, azathioprine, or systemic steroids within the past two months, undergoing experimental treatment for PBC, and using experimental or unapproved immunosuppressants. The primary endpoint is ALP reduction, and the secondary endpoint is ALP < 1.67. Response rates were defined as ULN (ultra-low bilirubin) with total bilirubin within the normal range and ALP reduction > 15%. Additional secondary endpoints included changes in GGT, TBIL, and 5'-nucleotidase, all other recognized biomarkers for PBC. Subjects were randomized to receive placebo, PT-30 tablets at doses of 10 mg / day, 30 mg / day, 50 mg / day, 100 mg / day, 200 mg / day, or 400 mg / day, orally once daily for 12 weeks. Subjects showed a dose-dependent decrease in ALP, indicating that PT-30 administration has a therapeutic effect on PBC fibrosis.

Claims

1. A compound of any one of formulae PT-1, PT-2, PT-4, PT-13, PT-14, PT-20, PT-23 to PT-25, and PT-27 to PT-30: PT-1 PT-2 PT-4 PT-13 PT-14 PT-20 PT-23 PT-24 PT-25 PT-27 PT-28 PT-29 PT-30 or a salt, in particular a pharmaceutically acceptable salt thereof. ， 2. The compound according to claim 1, which is a compound of any one of formulae PT-1, PT-2, and PT-4, or a salt, in particular a pharmaceutically acceptable salt thereof.

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

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

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

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

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

8. The compound according to claim 5, which is a compound of formula PT-30, or a salt, 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 preparation for use in the treatment of a fibrotic disease in a human subject, comprising a therapeutically effective amount of a compound of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

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

12. The method of claim 11, wherein, The systemic fibrotic disease is systemic sclerosis, multifocal fibrosclerosis (IgG4-related fibrosis), nephrogenic systemic fibrosis, or sclerodermatous graft-versus-host disease.

13. The method of claim 12, wherein, The fibrotic disease is an organ-specific fibrotic disease.

14. The method of claim 11, wherein, The organ-specific fibrotic disease is cardiac fibrosis, renal fibrosis, pulmonary fibrosis, liver and portal vein fibrosis, radiation-induced fibrosis, bladder fibrosis, intestinal fibrosis, pancreatic fibrosis, peritoneal sclerosis, diffuse fasciitis, localized scleroderma, keloid, palmar fibromatosis, Peyronie’s disease, myelofibrosis, or oral submucous fibrosis.

15. The method of claim 14, wherein, The amount of the compound administered is 10 mg / day to 600 mg / day, for example 30 mg / day to 400 mg / day, such as 10 mg / day, 30 mg / day, 50 mg / day, 100 mg / day, 200 mg / day, or 400 mg / day.

16. The method of any one of claims 11 to 15, wherein, ​

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