Methods to treat bladder cancer

Abstract

A method for treating bladder cancer is provided at this institution.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] Not applicable Background Technology

[0003] STING (an interferon gene stimulator) is a signaling molecule involved in the innate response to dsDNA in the cytosol. STING deletions have been reported in numerous human cancers. Furthermore, deregulation of STING signaling in human cancers has also been reported in melanoma (Xia T, et al., "Recurrent Loss of STING Signaling in Melanoma Correlates with Susceptibility to Viral Oncolysis" Cancer Res. 2016) and colorectal cancer (Xia T, et al., "Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis" Cell Rep. 2016;14:282-97). Interestingly, genomic analysis results in these studies suggested that the loss of STING expression was due to epigenetic changes rather than gene deletions or mutations (Xia, Cancer Res. 2016; Xia, Cell Rep. 2016). The anticancer activity of STING is also supported by evidence obtained from mouse model studies. STING knockout mice exhibited defective tumor control. (Woo SR, et al. "STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors" Immunity 2014;41:830-42).

[0004] Furthermore, the role of STING in tumorigenesis defense has been demonstrated in various natural mouse models, including glioma (Ohkuri T, et al., "Protective role of STING against gliomagenesis: Rational use of STING agonist in anti-glioma immunotherapy" Oncoimmunology. 2015;4:e999523) and colorectal cancer (Zhu Q, et al., "Cutting edge: STING mediates protection against colorectal tumorigenesis by governing the magnitude of intestinal inflammation" J. Immunol. 2014;193:4779-82). This antitumor effect may be due to its ability to counteract the overactivation of NF-kB and STAT3 (Okihuri 2015). Activation of the STING pathway has also shown potent activity in preclinical mouse tumor models. (Woo 2014; Chandra D, et al. "STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer" Cancer Immunol Res. 2014;2:901-10; Corrales L, et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity" Cell Rep. 2015;11:1018-30; Curran E, et al. “STING Pathway Activation Stimulates Potent Immunity against Acute Myeloid Leukemia” Cell Rep. 2016;15:2357-66;"Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells" Cancer Res. 2016;76:2137-52). This antitumor activity may be due to the induction of an adaptive immune response following the disruption of the tumor vascular system (Corrales L, et al., "The host STING pathway at the interface of cancer and immunity" J. Clin. Invest. 2016;126:2404-11). Therefore, direct or intratumoral stimulation of STING by agonists in the tumor microenvironment may represent a novel approach for treating cancer.

[0005] Furthermore, bladder cancer is the fourth and eleventh most common cancer in men and women, respectively, and has the only route of drug administration for patients with early-stage cancer (Kamat et al., "What is new in non-muscle-invasive bladder cancer in 2016?" Turk J Urol 2017;43(1):9-13). In 2018, approximately 81,190 new cases of bladder cancer were estimated in the United States, and about 17,240 deaths due to bladder cancer were expected (American Association for Cancer Research website). Administration of BCG (Bacillus calmette-Guérin) is a frontline treatment option for high-risk non-muscle-invasive bladder cancer (NMIBC). Unfortunately, up to 40% of patients will not respond to BCG treatment, and many of these patients will undergo radical cystectomy, requiring the removal of the entire bladder (Zlotta, AR, et al., "The management of BCG failure in non-muscle-invasive bladder cancer: an update," Can Urol Assoc J 2009;3(Suppl4):S199-205), resulting in a poorer quality of life after surgery. Therefore, there is an important unmet medical need for alternative bladder cancer therapies to avoid or delay radical cystectomy.

[0006] An embodiment may provide a method for treating bladder cancer in a patient, comprising administering compound 1 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer:

[0007]

[0008] In some embodiments, the pharmaceutically acceptable salt administered according to the embodiments as reported herein is a diammonium salt or a triethylamine (TEA) salt. Further embodiments may provide treatment for bladder cancer by administering a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient to a patient requiring treatment for bladder cancer.

[0009] Further embodiments may provide a method for treating bladder cancer in a patient, comprising administering compound 2 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer:

[0010]

[0011] Further embodiments may provide for the use of compound 1, compound 2, or a pharmaceutically acceptable salt thereof as reported herein for the manufacture of a pharmaceutical composition for treating bladder cancer.

[0012] An embodiment may provide for the use of a pharmaceutical composition comprising compound 1, compound 2, a pharmaceutically acceptable salt thereof, or compound 1, compound 2, or a pharmaceutically acceptable salt thereof in the treatment of bladder cancer.

[0013] An embodiment may provide a method for treating bladder cancer comprising: identifying an individual having bladder cancer treatable by compound 1, compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1, compound 2, or a pharmaceutically acceptable salt thereof; and administering a therapeutically effective amount of compound 1, compound 2, a pharmaceutically acceptable salt, or a pharmaceutical composition to the individual identified as having bladder cancer treatable.

[0014] In some embodiments, an individual is identified as having bladder cancer treatable by compound 1, compound 2, its pharmaceutically acceptable salt, or by a pharmaceutical composition comprising compound 1, compound 2, or its pharmaceutically acceptable salt, due to the presence of a REF STING variant allele in the patient.

[0015] Some embodiments provide a method for treating bladder cancer in a patient having a REF STING allele, comprising administering compound 1, compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1, compound 2, or a pharmaceutically acceptable salt thereof to said patient.

[0016] Some embodiments provide a method for treating cancer in a patient having a WT STING allele, comprising administering a pharmaceutical composition comprising compound 1, compound 2, a pharmaceutically acceptable salt thereof, or compound 1 or a pharmaceutically acceptable salt thereof to the patient.

[0017] Some embodiments provide a method for treating cancer in a patient, comprising administering to a patient having the AQ STING allele compound 1, compound 2, or a pharmaceutically acceptable salt of compound 1 or compound 2, or a pharmaceutical composition comprising compound 1, compound 2, or a pharmaceutically acceptable salt thereof.

[0018] Some embodiments provide a method for treating cancer in a patient having an HAQ STING allele, comprising administering to the patient compound 1, compound 2, or a pharmaceutically acceptable salt of compound 1 or compound 2, or a pharmaceutically acceptable salt thereof compound 1, compound 2, or said pharmaceutically acceptable salt.

[0019] In some embodiments, compound 1 is provided as a free acid. In some embodiments, the compound is provided as an NH4 salt or as a triethylamine (TEA) salt.

[0020] An embodiment may provide a method for treating bladder cancer in a patient, comprising administering a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer as described above.

[0021] An embodiment may provide a method for treating bladder cancer in a patient, comprising administering compound 1, compound 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a patient requiring treatment for bladder cancer as reported above. The bladder cancer treated as reported herein may be a urothelial carcinoma. Brief explanation of the drawing

[0022] FIG. 1 illustrates the synthesis of compound 1a. This synthesis is also reported in U.S. Patent Application No. 15 / 898,533 filed on February 17, 2018, which is incorporated herein by reference. FIGS. 2a and 2b show alternative syntheses of compound 1 and compound 1a. The alternative syntheses are also shown in FIGS. 2c to 2e. Figure 3 is for compound 1 1 Shows an H NMR spectroscopic image. Figures 4a, 4b, and 4c show the X-ray crystallographic results (ORTEP figures) for the asymmetric crystal of compound 1, the first molecule from the asymmetric crystal, and the second molecule from the asymmetric crystal, respectively. Figure 5 shows a diagram of the X-ray crystal structure of human WT STING as a complex with compound 1. Figure 6 shows the human REF STING C-terminal domain as a complex with compound 1. Figure 7 shows the tumor volume from days 20–21 quantified by MRI. Figure 8 shows the survival curves for all groups in bladder cancer treatment example 4. Figure 9 shows the expression vector map for WT STING (pLenti-WT human STING-Puro). Figures 10 and 11 illustrate Example 108 and show the healing activity of compound 1a in a CT26 double tumor model. Figure 12 shows the tumor volume plot and survival curve for the treated tumor accompanying Example 109. Figure 13 shows the tumor volume plot and survival curve for the treated tumor accompanying Example 110. Specific details for implementing the invention

[0023] A compound (Compound 1) and a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition, may be useful for treating bladder cancer. Compound 2 or a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition comprising Compound 2 or a pharmaceutically acceptable salt thereof, may also be useful for treating bladder cancer. The compound may activate the interferon gene stimulator (STING).

[0024] This is compound 1:

[0025]

[0026] This is compound 2:

[0027]

[0028] In some embodiments, compound 1 is provided as a free acid. In some embodiments, the compound is provided, for example, as an NH4 salt. References to "compound 1a" will refer to the diammonium salt of compound 1.

[0029] An embodiment may provide a method for treating bladder cancer in a patient, comprising administering a therapeutically effective amount of compound 1, compound 2, or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer.

[0030] A pharmaceutical composition for treating bladder cancer may also be provided, comprising the compound reported herein or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable excipient. An embodiment as reported herein may be used to treat bladder cancer or to manufacture a medicine useful for the treatment of bladder cancer.

[0031] A person skilled in the art will recognize that when substituents bonded to phosphorus atoms (P1, P2) have both single and double bonds, they may be tautomeric. For example, compounds may be tautomeric at equilibrium. An example is given below:

[0032]

[0033] These tautomers shall be deemed to be included within the scope of the claims. The structural representation of any one tautomer for a given compound will represent the same compound.

[0034] Treatment methods

[0035] An embodiment may provide a method for treating bladder cancer in a patient, comprising administering a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer.

[0036] In some embodiments, compound 1 is provided as its free acid or a pharmaceutically acceptable salt. In some embodiments, the compound to be administered is provided as its NH4 salt, free acid, or a pharmaceutically acceptable salt. In some embodiments, the compound is provided as the NH4 salt.

[0037] Some embodiments provide a method for treating bladder cancer comprising administering a therapeutically effective amount of a STING agonist to a patient requiring treatment for bladder cancer. In these embodiments, administration may be intravesicular, but is not required. Examples of potential STING agonists that may be used to treat bladder cancer and may be administered intravesicularly, though not required, are U.S. Patent Application No. 15 / 898,533 filed February 17, 2018; PCT Application No. PCT / US2018 / 018556 filed February 17, 2018; PCT Application No. PCT / US2018 / 018561 filed February 17, 2018; US 2014 / 0205653 A1; US ​​2014 / 0329889 A1; US ​​2014 / 0341976 A1; US 2007 / 0149462 A1; WO 2018 / 098203 A1; WO 2018 / 198076 A1; WO 2018 / 198084 A1; WO 2018 / 140831 A2; US 2014 / 0341976 A1; WO 2015 / 185565 A1; US 7,709,458 B2; US 7,592,326; US 7,569,555 B2; US 2014 / 0205653 A1; US 2014 / 0341976 A1; US 2015 / 0056224 A1; US 2016 / 0362441 A1; US 2017 / 0158724 A1; US 2017 / 044206 A1; US 5,547,941; US 7,569,555 B2; US 7,592,326 B2; US 7,709,458 B2; US 9,549,944 B2; WO 2009 / 133560 A1; WO 2015 / 074145 A1; WO 2015 / 077354 A1; WO 2015 / 185565 A1; WO 2016 / 100261 A1; WO 2016 / 120305 A1; WO 2016 / 145102 A1; WO 2017 / 027645 A1; WO 2017 / 027646 A1; WO 2017 / 075477 A1; WO 2019 / 043634 A2;WO 2019 / 046496 A1; WO 2019 / 046498 A1; WO 2019 / 046500 A1; WO 2019 / 046511 A1; WO 2017 / 093933 A1; WO 2017 / 123657 A1; WO 2017 / 175156 A1; EP 1740,192 B1; CN 102199183 A; Fu, J., et al., “STING Agonist Formulated Cancer Vaccines Can Cure Established Tumors Resistant to PD-1 Blockade,” Sci. Translational Med., 7(283): 283ra52, (April 15, 2015); Corrales, L. et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports, 11: 1018-1030 (2015); and Lioux, T. et al., "Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING)," J. Med. Chem., 59: 10253-10267 (2016). All such literature, including compounds within it, is incorporated herein by reference; and in the event that any component of any of such literature contradicts or otherwise inconsistent with any part of this specification, this specification shall prevail.

[0038] Dosage

[0039] The optimal dose for the treatment of bladder cancer can be determined empirically for each individual using known methods and will depend on various factors including the activity of the agent; the individual's age, body weight, overall health, sex, and diet; the time and route of administration; and other medications the individual is taking. The optimal dose can be established using commercially available tests and procedures widely known in the relevant art. The administration of the compound may be by any suitable route.

[0040] As used herein, “pharmaceutical acceptable salt” refers to an acid-added salt or a base-added salt of a compound. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not impart any excessively harmful or undesirable effects to the subject receiving it and in the context of its administration. Pharmaceutically acceptable salts include, but are not limited to, salts of both inorganic acids and carboxylic acids and metal complexes. Pharmaceutically acceptable salts also include metal salts, such as aluminum, calcium, iron, magnesium, manganese, and complex salts. In addition, pharmaceutically permissible salts are acid salts, e.g., acetic acid, aspartic acid, alkylsulfonic acid, arylsulfonic acid, acetyl, benzenesulfonic acid, benzoic acid, carbonic acid, disulfuric acid, ditartaric acid, butyric acid, calcium edetate, camsylic acid, carbonic acid, chlorobenzoic acid, citric acid, edetic acid, edicilic acid, estolic acid, esyl, esyl acid, formic acid, fumaric acid, gluteptic acid, gluconic acid, gluconic acid, glutamic acid, glycolic acid, glycolyl arsanilic acid, hexamic acid, hexylresorbic acid, hydrabamic acid, hydrobromide, hydrochloric acid, hydrochloride, hydroiochloride, hydroiodide, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, Methyl nitric acid, methyl sulfate, mucinic acid, muconic acid, naphsylic acid, nitric acid, oxalic acid, p-nitromethanesulfonic acid, pamosic acid, pantothenic acid, phosphoric acid, monohydrogenated phosphate, dihydrogenated phosphate, phthalic acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfonic acid, sulfuric acid, tannic acid, tartaric acid, teoclic acid, toluenesulfonic acid, etc., are included but not limited thereto. Sodium salts and potassium salts may also be prepared.

[0041] Embodiments may be diammonium salts. Pharmaceutically acceptable salts may be derived from amino acids including, but not limited to, cysteine. Methods for preparing compounds as salts are known to those skilled in the art (see, for example, [Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66: 1, 1977]).

[0042] The "effective dose" or "therapeutic effective dose" of the therapeutic agent is an amount sufficient to provide an observable therapeutic benefit compared to the untreated bladder cancer remaining in the subject or patient.

[0043] Active agents as reported herein may be combined with pharmaceutically acceptable carriers to provide pharmaceutical formulations thereof. The specific choice of carrier and formulation will depend on the specific route of administration for which the composition is intended.

[0044] "Pharmaceutical acceptable carriers" as used herein refer to non-toxic carriers, ajuvants, or vehicles that do not impair the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, ajuvants, or vehicles that may be used in the compositions of the present invention include, but are not limited to, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol, and wool fats.

[0045] The composition of the present invention may be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, intravesical, vaginal, or implantable administration, etc. In some embodiments, the formulation comprises components from natural or non-natural sources. In some embodiments, the formulation or carrier may be provided in a sterile form. Non-limiting examples of sterile carriers include endotoxin-free or pyrogen-free waters. The composition may be administered via intravesical administration.

[0046] As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In certain embodiments, the compound is administered by intravenous, oral, subcutaneous, or intramuscular administration. The sterile injectable form of the composition of the present invention may be an aqueous or oil suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be used are water, Ringer’s solution, and isotonic sodium chloride solution. Additionally, sterile fixing oils are commonly used as solvents or suspension media. In some embodiments, the composition is administered into the bladder.

[0047] For this purpose, any non-irritating fixing oil, including synthetic mono- or di-glycerides, may be used. Fatty acids and their glyceride derivatives are useful for the preparation of injectables, and naturally pharmaceutically acceptable oils, such as olive oil or castor oil, are also useful, particularly in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers, which are commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0048] For oral administration, the compound or salt may be provided in acceptable oral administration forms, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricants, such as magnesium stearate, may also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. If an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavorings, or coloring agents may also be added. Preservatives may also be added. Suitable examples of pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents, such as solvents, e.g., ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (e.g., methyl paraben, ethyl paraben, propyl paraben, etc.).

[0049] “Immediate-release” means including a conventional release in which the release of the drug begins immediately after administration. As used herein, the term “immediate-release” includes a form of administration that releases the drug from the gastric contents without the intention of delaying or prolonging the release or absorption of the drug. The purpose is to ensure that the drug is rapidly released after administration, for example, to be able to release at least 80% of the drug within approximately 30 minutes after the onset of release in a dissolution test.

[0050] "Sustained-release" or "extended-release" includes a dosage form selected to achieve therapeutic or convenience purposes for which the drug-release characteristics over time and / or location are not provided by conventional dosage forms, such as solutions or immediate-release dosage forms.

[0051] The term "steady state" means that plasma levels for a given activator are achieved and maintained at a level above the minimum effective therapeutic level and below the minimum toxic plasma level for a given activator with subsequent doses of the activator.

[0052] As used herein, the term "dosage range" refers to the upper and lower limits of the permissible variation in the amount of a specified agent. Typically, any dose of the agent within the specified range may be administered to a patient receiving treatment.

[0053] The term "treat" is used herein to mean alleviating, reducing, or mitigating at least one symptom of the disease in a subject. For example, with respect to bladder cancer, the term "treat" may mean preventing or delaying the onset (i.e., the period prior to clinical signs of the disease or symptoms of bladder cancer) and / or reducing the risk of progression or worsening of symptoms of bladder cancer. The term "protect" is used herein to mean preventing, delaying, or treating, or all of the progression, persistence, or worsening of symptoms of bladder cancer in a subject, appropriately.

[0054] The terms “subject” or “patient” are intended to include animals that have bladder cancer or may suffer from it. Examples of subjects or patients include mammals, e.g., humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human who has bladder cancer, is at risk of having it, or is potentially likely to have it.

[0055] The term "about" or "approximately" typically means within 20% of a given value or range, more preferably within 10%, and most preferably within 5%. Alternatively, particularly in biological systems, the term "about" means within the logarithm (i.e., 10 times) of a given value, preferably within 2 times.

[0056] In the context of describing the invention (particularly in the context of the following claims), the use of singular and similar designations shall be interpreted as including both singular and plural forms, unless otherwise indicated herein or clearly contradictory in the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be interpreted as non-restrictive terms (i.e., meaning “comprising, but not limited thereto).” Unless otherwise indicated herein, references to ranges of values ​​are intended to be provided merely as abbreviations for each individual value falling within said range, and each individual value is included in this specification as individually enumerated herein.

[0057] Exemplary proliferative disorders that can be treated using one or more of the compounds disclosed herein include, but are not limited to, cancer, precancerous or precancerous conditions, and metastatic lesions in tissues and organs of the body. Proliferative disorders may include hyperplasia, metaplasia, and dysplasia.

[0058] The compounds disclosed herein or their pharmaceutically acceptable salts may be used to treat or prevent proliferative disorders in subjects having an increased risk of developing cancer compared to the general population, or to treat or prevent cancer, or to identify suitable candidates for such purposes.

[0059] Pharmaceutical Formulations and Routes of Administration

[0060] A pharmaceutical formulation comprising compound 1 or a pharmaceutically acceptable salt thereof for the treatment of bladder cancer is provided herein. The pharmaceutical formulation may additionally comprise a carrier or excipient, a stabilizer, a flavoring agent and / or a coloring agent.

[0061] Compound 1 or its pharmaceutically acceptable salt may be administered using various routes of administration known to those skilled in the art. Routes of administration include oral administration and intravesical administration. In certain embodiments, pharmaceutical formulations comprising the compound or its pharmaceutically acceptable salt may be taken orally in the form of a liquid, syrup, tablet, capsule, powder, sprinkle, chewable, or dissolvable disc. Alternatively, pharmaceutical formulations of the present invention may be administered intravenously or transdermally. Additional routes of administration are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Gennaro AR, Ed., 20th sup.th Edition, Mack Publishing Co., Easton, Pa.).

[0062] In some embodiments, the compound or pharmaceutically acceptable salt is formulated as a paste, jelly, or suspension. For example, the drug is dissolved, captured, or suspended in a gelatin solution or in the form of semi-solid drug particles, microencapsulated particles, or drug-polymer particles. An advantage of oral jelly formulations is that it is easier to administer the drug to patients who have difficulty swallowing tablets, capsules, or pills. In certain embodiments, the compound is thoroughly mixed and suspended in a suitable medium to form a paste or gel. Additional agents may be optionally mixed to provide flavor during oral administration. Sweeteners and raspberry-flavored peanut butter or alginate are examples of many suitable flavor maskers. In various embodiments, the paste or jelly may also be formulated with suitable binders or excipients known in the relevant art for topical administration.

[0063] Methods for manufacturing sustained-release formulations in the form of tablets, capsules, or pills are known in the relevant art. In some embodiments, sustained-release formulations are prepared by coating the active ingredient of a drug with a polymer, preferably a water-insoluble polymer. For example, water-insoluble polymers are used in the pharmaceutical field as sustained-release coating agents, enteric coating agents, or gastric coating agents. Water-insoluble polymers may include, for example, ethyl cellulose, purified shellac, white shellac, aminoalkyl methacrylate copolymer RS, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethyl-cellulose, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, aminoalkyl methacrylate copolymer E, or polyvinyl acetal diethylaminoacetate.

[0064] The type, degree of substitution, and molecular weight of the water-insoluble polymer may depend on the solubility of the active ingredient in water or alcohol, the desired sustained release level, etc. The water-insoluble polymer may be used alone or in combination. Hydrogenated oil, stearic acid, or cetanol may be additionally incorporated as a coating aid, and medium-chain triglycerides, triacetin, triethyl citrate, or cetanol may be additionally incorporated as a plasticizer.

[0065] In some embodiments, the sustained-release formulation is a matrix-type tablet or granule. The active ingredient may be coated with up to three different types of polymers. Among these three different types of polymers, these may include: 1) a water-insoluble polymer, e.g., ethylcellulose; 2) a pH-independent gelling polymer, e.g., hydroxypropylmethylcellulose; and 3) a pH-dependent gelling polymer, e.g., sodium alginate. These three different types of polymers may be used together to attenuate the release rate of the drug.

[0066] Dosage Form: Release Characteristics

[0067] Sustained-release formulations can achieve a certain degree of sustained effect. However, the exposure and / or bioavailability of the active ingredient may vary based on various factors, such as the absorption window, the carrier or excipient used in the formulation, the mode of delivery, and / or the time of passage of the active ingredient through the patient's gastrointestinal tract.

[0068] The regimen may contain at least one sustained-release portion for performing a sustained-release function and one immediate-release portion for performing an immediate-release function. In certain embodiments, where the regimen is in a single-dose form, it may be in the form of a tablet formed from a mixture of sustained-release granules constituting the sustained-release portion and immediate-release granules constituting the immediate-release portion, a capsule formulation obtained by filling a capsule with sustained-release granules and immediate-release granules, or a press-coated tablet in which an outer layer constituting the immediate-release portion is formed on an inner core constituting the sustained-release portion. However, there are no limitations to the above embodiments.

[0069] Furthermore, there are no specific restrictions on the encapsulation state of the drug in the composition, immediate-release portion, or sustained-release portion; the compound may be uniformly dispersed in the composition, immediate-release portion, or sustained-release portion, or may be contained only in a portion of the composition, immediate-release portion, or sustained-release portion, but may be contained such that a concentration gradient exists.

[0070] The sustained-release portion of the composition according to the present invention may contain at least one non-pH-dependent polymer material or pH-dependent polymer material for controlling drug release.

[0071] The non-pH-dependent polymeric material used herein may comprise a polymeric material that exhibits little change in charge state under pH conditions typically found in the gastrointestinal tract, specifically at pH 1 to pH 8. This refers to a polymeric material that does not possess functional groups whose charge state changes with pH, ​​such as basic functional groups like amino groups or acidic functional groups like carboxylic acid groups. It is noted that the non-pH-dependent polymeric material may be included to provide a sustained-release function to the composition according to the present invention, but may also be included for other purposes. Furthermore, the non-pH-dependent polymeric material used in the present invention may be water-insoluble, swell in water, or dissolve in water to form a gel.

[0072] Examples of water-insoluble non-pH-dependent polymeric materials include, but are not limited to, cellulose ethers, cellulose esters, and methacrylic acid-acrylic acid copolymers (trademark Eudragit, manufactured by Rohm GmbH & Co. KG (Darmstadt, Germany)). Examples include, but are not limited to, cellulose alkyl ethers such as ethylcellulose (trademark Ethocel, manufactured by Dow Chemical Company (USA)), ethyl methylcellulose, ethyl propylcellulose or isopropylcellulose, and butylcellulose; cellulose aralkyl ethers such as benzyl cellulose; cellulose cyanoalkyl ethers such as cyanoethylcellulose; cellulose organic acid esters such as cellulose acetate butyrate, cellulose acetate, cellulose propionate or cellulose butyrate; and cellulose acetate propionate; ethyl acrylate-methyl methacrylate copolymers (trademark Eudragit NE, manufactured by Rom GmbH & Co. Kage (Darmstadt, Germany)); and aminoalkyl methacrylate copolymers RS (trademark Eudragit RL, Eudragit RS). There are no specific limitations on the average particle size of the water-insoluble polymer used in the present invention, but generally, the smaller the average particle size, the better the performance. The average particle size is preferably 0.1 to 100 μm, more preferably 1 to 50 μm, particularly preferably 3 to 15 μm, and most preferably 5 to 15 μm.In addition, examples of water-soluble or water-swelling non-pH-dependent polymeric materials include polyethylene oxide (trademark Polyox, manufactured by Dow Chemical Company, molecular weight 100,000 to 7,000,000), low-substituted hydroxypropyl cellulose (trademark L-HPC, manufactured by Shin-Etsu Chemical (Japan)), hydroxypropyl cellulose (trademark HPC, manufactured by Nippon Soda, Co., Ltd. (Japan)), hydroxypropyl methylcellulose (trademark Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical (Japan)), and methylcellulose (trademark Metolose SM, manufactured by Shin-Etsu Chemical (Japan)). Includes, but is not limited to.

[0073] In some embodiments, a single non-pH-dependent polymer material may be contained in the composition, or a plurality of non-pH-dependent polymer materials may be contained. When used in the embodiments reported herein, the non-pH-dependent polymer material may be a water-insoluble polymer material, more preferably ethylcellulose, an ethyl acrylate-methyl methacrylate copolymer (trademark Eudrajit NE), or an aminoalkyl methacrylate copolymer RS ​​(trademark Eudrajit RL, Eudrajit RS). Particularly preferably, it is at least one of ethylcellulose and an aminoalkyl methacrylate copolymer RS. Most preferably, it is ethylcellulose. There are no particular limitations on the amount of non-pH-dependent polymer material contained in the composition; this amount may be appropriately adjusted, for example, for the purpose of controlling sustained drug release.

[0074] The pH-dependent polymeric material that may be used in the embodiments reported herein may be a polymeric material whose charge state changes under pH conditions generally found in the gastrointestinal tract, specifically at pH 1 to pH 8. This refers to a polymeric material having functional groups whose charge state changes according to pH, such as basic functional groups like amino groups or acidic functional groups like carboxylic acid groups, for example. The pH-dependent functional group of the pH-dependent polymeric material is preferably an acidic functional group, and the pH-dependent polymeric material most preferably has a carboxylic acid group.

[0075] The pH-dependent polymer material used in the present invention may be water-insoluble, swell in water, or dissolve in water to form a gel. Examples of the pH-dependent polymer material used in the present invention include, but are not limited to, enteric polymer materials. Examples of enteric polymeric materials include methacrylate-methyl methacrylate copolymer (Eudrazit L100, Eudragit S100, manufactured by Rohm GmbH & Co. Kage (Darmstadt, Germany)), methacrylate-ethyl acrylate copolymer (Eudrazit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co. Kage (Darmstadt, Germany)), hydroxypropyl methylcellulose phthalate (HP-55, HP-50, manufactured by Shin-Etsu Chemical (Japan)), hydroxypropyl methylcellulose acetate succinate (AQOAT, manufactured by Shin-Etsu Chemical (Japan)), carboxymethyl ethylcellulose (CMEC, manufactured by Freund Corporation (Japan)), and cellulose acetate phthalate, but thereof It is not limited.

[0076] Examples of pH-dependent polymeric materials that swell in water or dissolve in water to form a gel include, but are not limited to, alginic acid, pectin, carboxyvinyl polymers, and carboxymethyl cellulose. In the present invention, a single pH-dependent polymeric material may be contained in the composition, or a plurality of pH-dependent polymeric materials may be contained. The pH-dependent polymeric material used in the present invention is preferably an enteric polymeric material, more preferably a methacrylic acid-ethyl acrylate copolymer, a methacrylic acid-methyl methacrylate copolymer, hydroxypropyl methylcellulose phthalate, or hydroxypropyl methylcellulose acetate succinate, particularly preferably a methacrylic acid-ethyl acrylate copolymer.

[0077] When using a pH-dependent polymer material in the manufacturing process of the composition according to the present invention, commercially available products in powder, granule, or suspension form in which the pH-dependent polymer material is pre-dispersed in a solvent may be used as is, or such commercially available products may be dispersed in water or an organic solvent. Performance improves as the particle size of the pH-dependent polymer material decreases, and the pH-dependent polymer material is preferably in powder form. In the case of a methacrylic acid-ethyl acrylate copolymer, an example is Eudragit L100-55. There are no specific limitations on the average particle size of the pH-dependent polymer material used in the present invention, but the average particle size is preferably 0.05 to 100 μm, more preferably 0.05 to 70 μm, and most preferably 0.05 to 50 μm. In addition, there are no specific limitations on the amount of pH-dependent polymer material, and for example, in the case of enteric polymer material, the amount is generally 0.1 to 90 parts by weight, preferably 1 to 70 parts by weight, more preferably 5 to 60 parts by weight, and particularly preferably 10 to 50 parts by weight, based on 100 parts by weight of the composition.

[0078] The formulation according to the embodiments reported herein may additionally contain, if necessary, any one of various additives, such as any one of various pharmaceutically acceptable carriers, such as diluents, lubricants, binders, and disintegrants, as well as preservatives, coloring agents, sweeteners, plasticizers, film coating agents, etc. Examples of diluents include, but are not limited to, lactose, mannitol, dibasic calcium phosphate, starch, pre-gelatinized starch, crystalline cellulose, hard silica anhydride, synthetic aluminum silicate, magnesium aluminate metasilicate, etc. Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate, etc. Examples of binders include, but are not limited to, hydroxypropyl cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include, but are not limited to, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, etc. Examples of preservatives include, but are not limited to, parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Preferred examples of coloring agents include, but are not limited to, water-insoluble lake pigments, natural pigments (e.g., beta-carotene, chlorophyll, red ferric oxide), yellow ferric oxide, red ferric oxide, black ferric oxide, etc. Preferred examples of sweeteners include, but are not limited to, sodium saccharin, dipotassium glycyrrhizate, aspartame, stevia, etc. Examples of plasticizers include, but are not limited to, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, etc. Examples of film coating agents include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, etc.

[0079] manufacturing method

[0080] To produce embodiments as reported herein, a single conventional method or a combination of conventional methods may be used. For example, when producing drug-containing granules as a sustained-release or immediate-release portion, granulation is the main operation, but this may be combined with other operations such as mixing, drying, sieving, and sorting. As a granulation method, for example, a wet granulation method in which a binder and a solvent are added to the powder and granulation is performed, a dry granulation method in which the powder is compressed and granulation is performed, a melt granulation method in which a binder that melts upon heating is added and heating and granulation are performed, etc. may be used.

[0081] In addition, depending on the granulation method, a mixing granulation method using a planetary mixer, screw mixer, etc., a high-speed mixing granulation method using a Henschel mixer, super mixer, etc., an extrusion granulation method using a cylindrical granulator, rotary granulator, screw extrusion granulator, pellet mill type granulator, etc., a wet high-shear granulation method, a fluidized bed granulation method, a compression granulation method, a grinding granulation method, or a spray granulation method may be used. After granulation, drying, cracking, and sieving using a dryer, fluidized bed, etc., may be performed to obtain granules or fine granules for use. In addition, a granulation solvent may be used when preparing the composition according to the present invention. There are no specific restrictions on such granulation solvents, and they may be water or various organic solvents, for example, water, lower alcohols such as methanol or ethanol, ketones such as acetone or methyl ethyl ketone, methylene chloride, or any mixture thereof.

[0082] In the case of sustained-release granules contained in the embodiment, at least one drug and at least one selected from non-pH-dependent polymer materials and pH-dependent polymer materials are mixed together, a diluent and a binder are added as needed, and granulation is performed to obtain granules. The obtained granules can be dried using a tray dryer, a fluidized bed dryer, etc., and sieved using a mill or an oscillator to obtain sustained-release granules. Alternatively, as a method for manufacturing sustained-release granules in the present invention, it is possible to add at least one drug, at least one selected from non-pH-dependent polymer materials and pH-dependent polymer materials, and as needed, a diluent and a binder using a dry compactor, such as a roller compactor or a slug tablet press, and then perform compression molding while mixing, and then perform granulation by grinding to an appropriate size. Granules produced using such a granulator can be used as is as granules or fine granules according to the present invention, or they can be further ground and sieved using a power mill, a roll granulator, a rotor speed mill, etc., to obtain continuous-release granules. It should be noted that immediate-release granules can also be produced for continuous-release granules.

[0083] Compression-molded articles may be produced as a drug-containing sustained-release or immediate-release portion, or as a composition reported herein using a single conventional method or a combination of conventional methods. For example, compression-molded articles may be obtained by using at least one drug, at least one selected from non-pH-dependent polymeric material and pH-dependent polymeric material, a diluent such as mannitol or lactose, a binder such as polyvinylpyrrolidone or crystalline cellulose, a disintegrant such as sodium carmellose or crospovidone, and a lubricant such as magnesium stearate or talc, and by performing tableting using a conventional method. In this case, tableting is the main operation in the method of producing compression-molded articles, but it may be combined with other operations, such as mixing, drying, forming a sugar coating, and coating.

[0084] Examples of tableting methods include, but are not limited to, direct compression molding, in which at least one drug and a pharmacologically acceptable additive are mixed together and then the mixture is directly compressed into a tablet using a tablet press, and dry granulation or wet granulation, in which a sustained-release granule or immediate-release granule according to the present invention is applied to the compression mold after adding a lubricant or disintegrant as needed. There are no particular restrictions on the tablet press used for compression molding; for example, a single-punch tablet press, a rotary tablet press, or a press-coating tablet press may be used.

[0085] The drug-containing sustained-release granules or immediate-release granules, or compression-molded articles according to the embodiments of the present invention, may be used as a composition in the form of granules or tablets as they are, but may also be applied to further processing to produce a composition. For example, a film coating may be provided on the compression-molded article or granules using a film substrate material such as ethylcellulose, casein, methylcellulose, hydroxypropyl methylcellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac, etc., or a sugar coating may be provided using a sugar coating liquid containing saccharose, sugar alcohol, gum arabic powder, talc, etc., to produce a film-coated tablet or a sugar-coated tablet. One solvent in this coating technique may be purified water, but organic solvents such as alcohols, ketones, ethers, or chlorinated hydrocarbons, or mixtures thereof may also be used. For example, ethanol, acetone, methylene chloride, etc., may be used as organic solvents. In addition, as a coating device, a device commonly used in coating technology for manufacturing pharmaceuticals may be used, examples include a spray coating device in which coating is performed by spraying a coating liquid, etc., and a lamination rotor fluid bed granulator.

[0086] When manufacturing capsule formulations, the capsule formulations may be produced by using an automatic capsule filler to fill hard gelatin capsules or HPMC capsules with mini-tablets, or with sustained-release granules or immediate-release granules as described above. Alternatively, in the case of dry syrups used by mixing with water or the like upon administration, or formulations for administration per tube, the sustained-release granules or immediate-release granules as described above may be mixed with a thickener or a dispersant to disperse these granules, and then the mixture may be formed into granules or tablets. Furthermore, liquids or jellies may be prepared using water and substances selected from dispersants, emulsifiers, thickeners, preservatives, pH adjusters, sweeteners, flavoring agents, flavorings, etc. However, there are no limitations on other manufacturing methods as described above.

[0087] To enable a more complete understanding of the embodiments described herein, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the invention.

[0088] Examples

[0089] The following abbreviations may be used throughout the examples.

[0090] All: Allil

[0091] DMT: 4,4'-Dimethoxytrityl

[0092] (DMTO-:

[0093] )

[0094] Bz: Benzoyl

[0095] Finnig base: i-Pr2NEt (diisopropylethylamine)

[0096] AllylOH: allyl alcohol

[0097] OAll: -OCH2CHCH2

[0098] ACN: Acetonitrile

[0099] All: -CH2CHCH2

[0100] 2-NitroBnBr: 2-nitrobenzyl bromide

[0101] Bz: Benzoyl

[0102] i-Pr: Isopropyl

[0103] CE: Cyanoethyl

[0104] (-OCE: )

[0105] DEAD: Diethyl azodicarboxylate

[0106] DIAD: Diisopropyl azodicarboxylate

[0107] DCM: Dichloromethane

[0108] DDTT: N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazole-5-yl)formimidoamide

[0109]

[0110] DMOCP: 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinan 2-oxide

[0111]

[0112] TBS: t-butyldimethylsilyl

[0113] 3H-Benzo[c][1,2]Dithiol-3-one:

[0114]

[0115] Example 1 -- Synthesis of Compound 1a

[0116] The overall reaction scheme for this synthesis is available in Fig. 1.

[0117] Step A

[0118]

[0119] (2R,3R,4R,5R)-5-(6-benzamido-9H-purine-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (Compound 100) (mixture of phosphorus diastereomers; 80.0 g, 91.332 mmol, 1 equivalent, ChemGenes Corporation Catalog # ANP-9151), in a mixture of allyl alcohol (9.63 ml, 142 mmol, 1.55 equivalents) and triphenylphosphine (38.3 g, 146 mmol, 1.60 equivalents) at ambient temperature DEAD (40 wt% solution in toluene; 54.2 ml, 137 mmol (1.5 equivalents) was added. Stirring was continued at ambient temperature, and the reaction was monitored by LC / MS. At completion (19 h), the mixture was concentrated under vacuum (35°C), and the resulting mixture was purified by silica gel column chromatography (800 g x 2 columns, 40 to 60% EtOAc in n-heptane buffered with 0.5% triethylamine) to obtain compound 101 as a white foam (84.2 g, quantitative yield, mixture of phosphorus diastereomers).

[0120] 1¹H NMR (3:2 mixture of phosphorus diastereomers, 400 MHz, CDCl₃) δ 1.14 - 1.21 (m, 12 H) 2.40 (t, J=6.2 Hz, 1.2 H) 2.59 (t, J=6.2 Hz, 0.8 H) 3.27 (d, J=8.6 Hz, 1 H) 3.52 - 3.66 (m, 5 H) 3.78 (s 2.4 H) 3.79 (s 3.6 H) 4.28 - 4.34 (m, 1 H) 4.84 - 4.96 (m, 0.4 H) 4.99 (d, J=5.5 Hz, 2 H) 4.95 - 5.10 (m, 0.6 H) 5.05 (d, J=10.9 Hz, 1 H) 5.22 (br d, J=17.6 Hz, 1 H) 5.64 (br d, J=53.2 Hz, 0.6 H) 5.70 (br d, J=51.6 Hz, 0.4 H) 5.96 - 6.75 (m, 1 H) 6.20 (d, J=16.0 Hz, 0.6 H) 6.24 (d, J=17.2Hz, 0.4 H) 6.74 - 6.79 (m, 4 H) 7.02 - 7.06 (m, 2H) 7.17 - 7.24 (m, 8 H) 7.32 - 7.34 (m, 2 H) 7.41 - 7.44 (m, 2 H) 8.11 (s, 1H) 8.52 (s, 0.4 H) 8.54 (s, 0.6 H).

[0121] Step B

[0122]

[0123] Water (0.118 ml, 6.55 mmol, 2.0 equivalents) and pyridine trifluoroacetate salt (0.759 g, 3.93 mmol, 1.2 equivalents) were added to a solution of compound 101 (3.00 g, 3.28 mmol, 1 equivalent) in acetonitrile (30 ml). After stirring for 1 minute at ambient temperature, tert-butylamine (14.5 g, 21.0 ml, 0.20 mol, 60 equivalents) was added. Upon complete decomposition of the cyanoethyl group (monitored by LC / MS), the reaction mixture was concentrated under vacuum and azeotropically mixed with acetonitrile twice. The crude mixture was dissolved in DCM (45.0 ml) and treated with water (0.118 ml, 6.55 mmol, 2.0 equivalents) and NaHSO4-SiO2 (1.18 g, 6.55 mmol, 2 equivalents) at ambient temperature. Upon complete decomposition of the DMT group (monitored by LC / MS, approximately 1 hour), the reaction mixture was filtered and washed twice with DCM / MeOH (9 / 1, 20 ml). The combined filtrate was concentrated under vacuum and treated with a 1:1 mixture of n-heptane / toluene (~30 ml). The upper layer was removed by gradient separation. The same process was repeated once more using n-heptane / toluene (1 / 1, 30 ml), and the lower layer was azeotropically mixed twice with acetonitrile to obtain Compound 102 (assuming a theoretical yield of 100%). The product was used in subsequent steps without further purification.

[0124] Step C

[0125]

[0126] Pyridine trifluoroacetate salt (azeozoically dried with pyridine; 0.760 g, 3.94 mmol, 1.25 eq) was added to a mixture of Compound 102 (1.56 g, 3.27 mmol, 1 equivalent) and Compound 101 (3.00 g, 3.28 mmol, 1 equivalent) in acetonitrile (30 ml). After 5 minutes, DDTT (0.840 g, 4.09 mmol, 1.30 equivalents, ChemGenes Corporation Catalog # RN-1588) was added, and upon complete sulfidation (monitored by LC / MS), the reaction mixture was concentrated under vacuum. The residue was dissolved in DCM (30 ml) and treated with water (0.57 ml, 32 mmol, 10 equivalents) and 6% dichloroacetic acid (1.56 ml, 18.9 mmol, 6.0 equivalents) in DCM (30 ml). After 20 minutes, the reaction mixture was quenched with pyridine (20 ml) and concentrated under vacuum. The residue was azeotropically mixed with pyridine to obtain compound 103 (3.22 g, assumed to be a 100% theoretical yield). The product was used in the next step without further purification.

[0127] Step D

[0128]

[0129] DMOCP (1.45 g, 7.88 mmol, 2.50 equivalents) was added at ambient temperature to a solution of compound 103 (3.22 g, 3.15 mmol, 1 equivalent) in pyridine (100 ml). Upon complete macrocyclization (monitored by LC / MS), water (1.7 ml, 94.5 mmol, 10 times the amount of DMOCP) was added, followed by the addition of 3H-benzo[c][1,2]dithiol-3-one (0.795 g, 4.73 mmol, 1.5 equivalents). Upon complete sulfidation (approximately 40 minutes), the reaction mixture was partially concentrated to approximately 15 ml under vacuum and poured into a mixture of saturated aqueous NaHCO3 (50 ml) and water (30 ml). After stirring at ambient temperature for 10 minutes, the mixture was extracted with a 1:1 mixture of EtOAc / MTBE (60 ml x 3 times). The organic layers were combined, washed with brine (25 ml), dried over MgSO4, and concentrated under vacuum. The residue was purified by silica gel column chromatography (0-20% MeOH in DCM) to obtain compound 104 (3.31 g, 3.20 mmol, assumed to be a 100% theoretical yield) as a brown oil. The product was used in subsequent steps without further purification.

[0130] Step E

[0131]

[0132] 2-nitrobenzyl bromide (2.42 g, 11.2 mmol, 3.50 equivalents) and triethylamine (1.78 ml, 12.8 mmol, 4.00 equivalents) were added to a solution of compound 104 (3.31 g, 3.20 mmol, 1 equivalent) in acetonitrile (66.2 ml). Upon complete reaction (monitored by LC / MS, for approximately 20 hours at ambient temperature), the reaction mixture was concentrated under vacuum and purified by silica gel column chromatography (100% ethyl acetate in 60% ethyl acetate / n-heptane) to obtain 0.568 g of product as a mixture of phosphorus diastereomers. Compound 105 (SR isomer; 0.225 g, 0.180 mmol, 5.6% total yield from compound 101) and compound 106 (RR isomer; 0.187 g, 0.149 mmol, 4.7% total yield from compound 1) were obtained by HPLC separation for the purification of diastereomers.

[0133] Compound 105 (SpRp)

[0134] 1 H NMR (400 MHz, CDCl3) δ = 8.63 (s, 1H), δ = 8.61 (s, 1H), 8.04 - 8.00 (m, 2H), 7.99 (s, 1H), 7.90 (s, 1H), 7.65 - 7.44 (m, 8H), 7.40 - 7.31 (m, 4H), 7.25 - 7.21 (m, 4H), 6.15 - 5.89 (m, 5H), 5.61 (dd, J = 52.0, 5.1 Hz, 1H), 5.55 (ddd, J = 51.2, 4.7, 2.7 Hz, 1H) 5.51 - 5.42 (m, 1H), 5.31 - 5.22 (m, 2H), 5.11 (dd, J = 3.9, 9.8 Hz, 2H), 5.04 - 4.95 (m, 4H), 4.55 - 4.37 (m, 7H), 4.29 - 4.12 (m, 3H)

[0135] Compound 106 (RpRp)

[0136] 1 1H NMR (400 MHz, CDCl3) δ = 8.65 (s, 2H), 8.06 (dd, J = 1.4, 8.0 Hz, 2H), 7.98 (s, 2H), 7.57 - 7.52 (m, 6H), 7.47 - 7.32 (m, 6H), 7.25 - 7.21 (m, 4H), 6.15 (d, J = 18.7 Hz, 2H), 6.09 - 5.99 (m, 2H), 5.82-5.76 (m, 2H), 5.60 (dd, J = 51.8, 4.9 Hz, 2H), 5.27 (dd, J = 1.2, 17.2 Hz, 2H), 5.12 (dd, J = 1.0, 10.4 Hz, 2H), 5.06 - 4.96 (m, 4H), 4.55 - 4.40 (m, 4H), 4.36 - 4.24 (m, 4H), 4.21 - 4.02 (m, 2H)

[0137] 정제용 HPLC 조건:

[0138]

[0139] More F

[0140]

[0141] Hobbeda-Grubbs Catalyst™ Generation 2 (1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium; available from SIGMA-ALDRITCH® catalog number 569755; CAS 301224-40-8; 91 mg, 0.15 mmol, 0.35 equivalents) and quinone (0.102 ml, 1.243 mmol, 3.0 equivalents) were added to a heated (90°C) solution of Compound 105 (519 mg, 0.414 mmol, 1 equivalent) in toluene (519 ml). The mixture was heated under reflux, and the progress of the reaction was monitored by LC / MS. After 3 hours, additional catalyst (91 mg, 0.15 mmol, 0.35 equivalents) was added, and the reaction was continued for an additional 3 hours. After cooling, the mixture was treated with DMSO (0.59 ml, 8.3 mmol, 20 equivalents) at ambient temperature for 15 hours, concentrated under vacuum, and purified by silica gel column chromatography (25 g SiO2, 100% ethyl acetate in 66% ethyl acetate in n-heptane) to obtain compound 107 (200 mg, 0.163 mmol, 39% yield) as a brown dry foam.

[0142] 1H NMR(400 MHz, CDCl3) δ = 8.19 (s, 1H), 8.12 (dd, J = 7.8 Hz, 1.9 Hz, 1H), 8.10 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.89 (s, 1H), 7.63 (br d, J = 7.0 Hz, 1H), 7.53 - 7.41 (m, 10H), 7.35 - 7.30 (m, 2H), 7.25 - 7.20 (m, 4H), 6.23 (d, J = 17.6 Hz, 1H), 6.14 (d, J = 18.8 Hz, 1H), 5.86 - 5.75 (m, 1H), 5.75 (dt, J = 15.3, 5.0 Hz, 1H), 5.67 (dt, J = 15.3, 4.7 Hz, 1H), 5.60 (dd, J = 52.0, 3.9 Hz. 1H), 5.48 (dd, J = 50.4, 3.9 Hz. 1H)5.50 - 5.39 (m, 1H), 4.91 - 4.64 (m, 4H), 4.57 - 4.25 (m, 9H), 4.15 (d, J = 7.03 Hz, 1H), 4.11 (d, J = 7.03 Hz, 1H).

[0143] 단계 G

[0144]

[0145] Thiophenol (0.88 mL, 8.55 mmol, 119 equivalents) and triethylamine (0.88 mL, 6.31 mmol, 88 equivalents) were added to a solution of compound 107 (88 mg, 0.072 mmol, 1 equivalent) in 1,4-dioxane (1.76 ml). The resulting mixture was stirred at ambient temperature. At the end of the reaction (monitored by LC / MS, 13 hours), methanol (5.28 ml) and 28% ammonium hydroxide (3.52 ml) were added, and the resulting mixture was heated to 50°C. At the end of the reaction (monitored by LC / MS, 5 hours), the mixture was cooled to ambient temperature, the resulting brownish slurry was filtered, and washed with water (15 ml). The filtrate was filtered again to remove additional solids. The final filtrate was extracted twice with a 1:1 mixture of toluene and heptane (30 ml). The aqueous layer was concentrated under vacuum and then resuspended in water (6 ml). The resulting solid was filtered, and the filtrate was treated with HPLC for purification to obtain compound 1 diammonium salt (also referred to as compound 1a) (39 mg, 0.050 mmol, 70% yield) as a white solid.

[0146] Compound 1a (SpRp, trans)

[0147] 1 H NMR (400 MHz, CD3OD) δ = 9.05 (s, 1H), 8.33 (s, 1H), 8.25 (s, 1H), 8.12 (s, 1H), 6.34 (br s, 2H), 5.88 (br s, 2H), 5.66 (br d, J = 51.6 Hz, 1H), 5.59 (br d, J = 52.2 Hz, 1H) 5.01 (br s, 2H), 4.68 - 4.34 (m, 6H), 4.07 - 3.82 (m, 2H), 3.79 - 3.55 (m, 2H);

[0148] 31 P NMR (162 MHz, CD3OD) δ = 55.48 (s, 1P), 55.16 (s, 1P).

[0149] HPLC conditions for the purification of Compound 1a:

[0150]

[0151] Example 1.1 for Compound 1a -- Alternative Synthesis

[0152] An alternative synthesis route for compound 1a is presented in FIGS. 2a and 2b, as well as FIG. 2c, and is reported below.

[0153] Step 1

[0154]

[0155] Compound 129 (570 g, 1.53 mol, 1 wt, 1 vol, 1 equivalent) was dissolved in pyridine (2.85 L, 35.2 mol, 4.89 wt, 5.0 vols, 23 equivalents). The mixture was cooled to 2.6°C and treated with 4,4'-dimethoxytrityl chloride (DMTCl; 543 g, 1.60 mol, 0.953 wt, 1.05 equivalents). The mixture was stirred at 0 to 5°C for 2 hours, then heated to ambient temperature. The reaction was monitored by LC / MS, and complete conversion was confirmed after stirring overnight. The reaction mixture was cooled to below 5°C and quenched by treatment with MeOH (124 ml, 3.05 mol, 0.172 wt, 0.217 vol, 2.0 equivalents) for 15 minutes. The mixture was co-evaporated with toluene (2.00 L, 3.04 wt, 3.51 vol) under vacuum and then diluted with a mixture of EtOAc (2.850 L, 4.5 wt, 5.0 vol) and n-heptane (2.85 L, 3.42 wt, 5.0 vol). The organic layer was washed with saturated NaHCO3 (9 wt% solution in water; 2.0 L, 3.5 vol). Additional EtOAc (2.85 L, 4.5 wt, 5.0 vol) was added to completely dissolve the crude product. After stirring for 5 minutes, the two layers were separated. The organic layer was washed with water (2.0 L, 3.5 wt, 3.5 vol). The solid was slowly precipitated from the organic layer. The aqueous layer was separated. Subsequently, the organic layer was concentrated to approximately 1 vol. The crude product was slurried with a mixture of n-heptane (2.00 L, 2.40 wt, 3.51 vol) and toluene (0.50 L, 0.76 wt, 0.88 vol). After stirring for 15 minutes, the pale yellow solid was collected by vacuum filtration. The filter cake was washed sequentially as follows: (1) n-heptane (0.60 L, 0.72 wt, 1.05 vol) and toluene (0.30 L, 0.46 wt, 0.(2) n-heptane (3.00 L, 3.6 wt, 5.26 vol) was added to the mixture of 53 vol) and then dried without heat for 30 minutes, then transferred to a tray and dried overnight in a vacuum oven at 50°C to obtain compound 130 (996.7 g, 1.47 mol, 1.75 wt, 97% yield) as a pale yellow solid.

[0156] 1 H NMR (400 MHz, chloroform-d) δ = 8.99 (s, 1H), 8.76 (s, 1H), 8.21 (s, 1H), 8.04 - 8.00 (m, 2H), 7.64 - 7.59 (m, 1H), 7.57 - 7.50 (m, 2H), 7.41 - 7.36 (m, 2H), 7.32 - 7.15 (m, 7H), 6.83 - 6.76 (m, 4H), 6.31 (dd, J = 2.5, 17.0 Hz, 1H), 5.68 (ddd, J = 2.3, 4.7, 52.7 Hz, 1H), 4.88 - 4.77 (m, 1H), 4.26 - 4.21 (m, 1H), 3.77 (s, 6H), 3.57 (dd, J = 3.1, 10.9 Hz, 1H), 3.43 (dd, J = 4.1, 10.7 Hz, 1H), 2.60 (br s, 1H)

[0157] Step 1'

[0158]

[0159] Compound 129 (430 g, 1.15 mol, 1 wt, 1 vol, 1 equivalent) and imidazole (118 g, 1.73 mol, 0.274 wt, 1.50 equivalents) were dissolved in DMF (1.72 L, 3.78 wt, 4.0 vol), and the resulting mixture was cooled to 5°C. TBS-Cl (191 g, 1.27 mol, 0.444 wt, 1.10 equivalents) was added. The mixture was stirred at 0 to 11°C for 2 hours and then slowly heated to ambient temperature (progress was monitored by LCMS). The reaction was completed 6 hours after the addition of TBS-Cl and stirred for an additional 20 hours while still at ambient temperature. The mixture was cooled to 2°C and treated with methanol (93 ml, 74 g, 2.3 mol, 0.17 wt, 0.22 wt, 2.0 equivalents) for 10 minutes. The reaction mixture was diluted with a mixture of MTBE (1.72 L, 1.23 kg, 2.96 wt, 4.0 vol) and EtOAc (1.72 L, 1.55 kg, 3.60 wt, 4.0 vol), and then diluted with saturated NH4Cl (28 wt% solution in water; 2.15 L, 5.0 vol). Solid began to slowly precipitate from the solution. The mixture was heated to 24°C, and water (1.08 L, 1.08 kg, 2.5 wt, 2.5 vol) was added (T-internal = 22°C). Additional solid began to precipitate from the mixture. Additional water (1.08 L, 1.08 kg, 2.5 wt, 2.5 vol) and MTBE (1.40 L, 1.04 kg, 2.4 wt, 3.3 vol) were added to the mixture. The grayish-white solid was collected by vacuum filtration. The reactor was washed with water (320 ml, 0.74 vol) followed by MTBE (1.80 L, 1.33 kg, 3.10 wt, 4.19 vol) to transfer any remaining solid to a filter. The filter cake was washed sequentially as follows: (1) water (1.80 L, 1.80 kg, 4.2 wt, 4.(2 vol), (2) water (1.80 L, 1.80 kg, 4.2 wt, 4.2 vol), (3) a mixture of MTBE (0.90 L, 0.67 kg, 1.5 wt, 2.1 vol) and n-heptane (0.90 L, 0.62 kg, 1.4 wt, 2.1 vol), and (4) a mixture of MTBE (0.90 L, 0.67 kg, 1.5 wt, 2.1 vol) and n-heptane (0.90 L, 0.62 kg, 1.4 wt, 2.1 vol). The recovered solid was dried under vacuum at 40°C for 2 days to obtain compound 133 as a white solid (483 g, 0.991 mol, 1.12 wt, 86% yield).

[0160] 1 H NMR (400 MHz, chloroform-d) δ = 8.97 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 8.04 - 8.00 (m, 2H), 7.64 - 7.58 (m, 1H), 7.56 - 7.51 (m, 2H), 6.40 (dd, J = 2.3, 16.0 Hz, 1H), 5.45 (ddd, J = 2.7, 4.3, 53.1 Hz, 1H), 4.75 - 4.66 (m, 1H), 4.22 - 4.17 (m, 1H), 4.07 (dd, J = 2.3, 11.7 Hz, 1H), 3.91 (dd, J = 2.7, 11.7 Hz, 1H), 2.38 (dd, J = 2.7, 7.0 Hz, 1H), 0.92 (s, 9H), 0.11 (s, 3H), 0.11 (s, 3H).

[0161] Step 2

[0162]

[0163] Compound 130 (993 g, 1.47 mol, 1 wt, 1 vol, 1 equivalent) and imidazole (150 g, 2.20 mol, 0.151 wt, 1.5 equivalents) were dissolved in DMF (3.48 L, 3.28 kg, 3.3 wt, 3.5 vol), and the mixture was cooled to 5°C. TBS-Cl (244 g, 1.62 mol, 0.245 wt, 1.10 equivalents) was added. The reaction mixture was stirred at 0 to 5°C for 2 hours, slowly heated to ambient temperature, and monitored by LCMS. After 17 hours, additional imidazole (100 g, 1.47 mol, 0.10 wt, 1.0 equivalent) and TBS-Cl (111 g, 735 mmol, 0.112 wt, 0.50 equivalent) were added, and stirring was continued for 2 hours at ambient temperature and for 2 hours at 35°C. The resulting mixture was cooled to 13.6°C and treated with MeOH (119 ml, 2.94 mol, 2 equivalents) for 10 minutes. In separate reactors, ice (5 kg, 5 wt in water; 5.0 L, 5 vol) and saturated NH4Cl (28 wt% solution) were added. The reaction mixture was added to the ice / NH4Cl mixture. A grayish-white solid immediately began to precipitate from the solution. An additional 2 kg of ice (2 kg, 2 wt) and water (3.0 L, 3 vol) were added to the mixture. The reaction flask was washed with water (0.50 L, 0.5 vol), and the washing solution was added to the mixture. n-heptane (2.00 L, 2 vol) was added to the mixture, and stirring was continued for 10 minutes. The grayish-white solid was collected by vacuum filtration. The filter cake was washed with: (1) water (4.0 L, 4.0 vol), (2) water (4.0 L, 4.0 vol), (3) n-heptane (4.0 L, 4.0 vol), and (4) n-heptane (4.0 L, 4.0 vol). The recovered solid was dried under vacuum at 45°C for 4 days to obtain Compound 131 as a grayish-white solid (1.095 kg, 1.39 mol, 1.It was obtained as 10 wt, 94% yield.

[0164] 1 H NMR (400 MHz, chloroform-d) δ = 9.09 (s, 1H), 8.78 (s, 1H), 8.28 (s, 1H), 8.02 (d, J = 7.4 Hz, 2H), 7.63 - 7.59 (m, 1H), 7.55 - 7.50 (m, 2H), 7.37 (d, J = 7.1 Hz, 2H), 7.29 - 7.17 (m, 7H), 6.79 (d, J = 7.9 Hz, 4H), 6.29 (dd, J = 2.9, 16.2 Hz, 1H), 5.60 (ddd, J = 2.7, 3.9, 53.1 Hz, 1H), 4.78 (ddd, J = 4. 7, 6.4, 15.8 Hz, 1H), 4.26 - 4.22 (m, 1H), 3.77 (s, 6H), 3.58 (dd, J = 3.1, 10.9 Hz, 1H), 3.26 (dd, J = 3.7, 10.7 Hz, 1H), 0.85 (s, 9H), 0.10 (s, 3H), 0.02 (s, 3H)

[0165] Step 3

[0166]

[0167] Compound 131 (1000 g, 1.27 mol, 1 wt, 1 vol, 1 equivalent) and trans-2-butene-1,4-diol ( 1Olefin geometry was confirmed by H NMR; 335 g, 3.80 mol (0.335 wt, 3.0 eq) was azeotropically mixed twice with THF (3.0 L, 3.0 vol). The residue was dissolved in a mixture of THF (10 L, 10 vol) and toluene (15 L, 15 vol). Triphenylphosphine (432 g, 1.65 mol, 0.432 wt, 1.3 equivalents) was added, and the reaction mixture was cooled to -5°C. DIAD (0.320 L, 1.65 mol, 333 g, 0.333 wt, 0.320 vol, 1.3 equivalents) was slowly added over 20 minutes while maintaining the T-internal temperature below 5°C. The reaction mixture was stirred at 0–5°C for 1 hour and monitored by LCMS. The ice bath was removed, and the mixture was allowed to warm to room temperature. After stirring overnight (17 hours), triphenylphosphine (83 g, 0.32 mol, 0.083 wt, 0.25 equivalents) and DIAD (62 ml, 0.32 mol, 64 g, 0.064 wt, 0.062 vol, 0.25 equivalents) were added. After an additional 1 hour, at room temperature, the reaction mixture was diluted with MTBE (10 L, 10 vol), washed twice with semi-saturated NaCl (18 wt% solution in water; 2 x 4 L), and concentrated under vacuum to obtain a concentrated oil. The mixture was redissolved in a mixture of MTBE (4.00 L, 4 vol) and n-heptane (0.50 L, 0.5 vol), and then cooled to 0°C. Seed crystals of triphenylphosphine oxide were added to the solution. The solid began to slowly precipitate from the solution, and the mixture was stirred overnight. The white solid was collected by vacuum filtration and washed with MTBE (2 L, 2 vol) to isolate 540 g of triphenylphosphine oxide.The filtrate was concentrated and purified by biotage 150 L KP-Sil (SiO2 5 kg; pretreated with 1% TEA in Hep / EtOAc; eluent: heptane / EtOAc (48 L of 33% EtOAc with 1% TEA, 24 L of 50% EtOAc with 1% TEA, 24 L of 66% EtOAc with 1% TEA) → 100% EtOAc with 1% TEA). The column was monitored by TLC (2:1 EtOAc / n-heptane). The clear product fractions were combined and concentrated under vacuum to obtain compound 132 as a pale white foamy solid (634 g, containing 14 wt% DIAD-induced co-product, 545 g of pure, 0.63 mol, 50% adjusted yield). The mixture fractions were combined and concentrated under vacuum to obtain a pale yellow foamed solid (750 g), which was then applied to a Biotage 150 M HP-sphere (2.5 kg SiO2; pretreated with 1% TEA in Hep / EtOAc; sample loaded with toluene eluent: Hep / EtOAc / 1% TEA (12 L of 50% EtOAc with 1% TEA, 16 L of 66% EtOAc with 1% TEA) → EtOAc with 1% TEA). The column was monitored by TLC (2 / 1 / 0.03 EtOAc / n-hep / TEA). The clear product fractions were combined and concentrated under vacuum to obtain an additional compound 132 as a pale white foamed solid (206 g, 0.24 mol, 18% yield).

[0168] 1H NMR(400 MHz, 클로로포름-d) δ = 8.58 (s, 1H), 8.10 (s, 1H), 7.43 - 7.37 (m, 2H), 7.32 - 7.28 (m, 2H), 7.24 - 7.15 (m, 8H), 7.03 - 6.98 (m, 2H), 6.78 - 6.73 (m, 4H), 6.18 (dd, J = 2.7, 17.2 Hz, 1H), 5.88 (td, J = 5.5, 15.6 Hz, 1H), 5.77 (td, J = 5.1, 15.6 Hz, 1H), 5.60 (ddd, J = 2.7, 4.3, 53.1 Hz, 1H), 5.03 - 4.96 (m, 2H), 4.91 (ddd, J = 4.5, 6.6, 16.6 Hz, 1H), 4.18 - 4.14 (m, 1H), 3.88 - 3.82 (m, 2H), 3.78 (s, 6H), 3.52 (dd, J = 2.7, 10.9 Hz, 1H), 3.14 (dd, J = 3.5, 10.9 Hz, 1H), 0.85 (s, 9H), 0.10 (s, 3H), 0.01 (s, 3H).

[0169] 단계 4

[0170]

[0171] Compound 132 (800 g, 0.930 mol, 1 wt, 1 vol, 1 equivalent) and Compound 133 (522 g, 1.07 mol, 0.652 wt, 1.15 equivalents) were azeotropically dried in THF (2 x 3 L, 2 x 3.8 vol) and redissolved in THF (9.60 L, 8.45 kg, 12.0 vol) at room temperature. Triphenylphosphine (317 g, 1.21 mol, 0.396 wt, 1.30 equivalents) was added, and the mixture was cooled to below -5°C. DIAD (226 ml, 1.16 mol, 235 g, 0.294 wt, 0.283 vol, 1.25 equivalents) was added within T-at below 7°C. The reaction mixture was allowed to be slowly heated to room temperature. The reaction mixture was monitored by LCMS. After 21 hours, the reaction mixture was concentrated with a thick oil under vacuum, azeotropically mixed with n-heptane (2.00, 1.37 kg, 1.71 wt, 2.50 vol), and then redissolved in a mixture of MTBE (2.40 L, 1.78 kg, 2.2 wt, 3.0 vol) and n-heptane (800 ml, 547 g, 0.68 wt, 1.0 vol). The solution was seeded with triphenylphosphine oxide, cooled to 5°C, diluted with n-heptane (400 ml, 274 g, 0.34 wt, 0.50 vol), and stirred at 5°C for 30 minutes. The white solid precipitate was collected by vacuum filtration and washed with a 2:1 (v / v) mixture of MTBE and n-heptane (1.8 L) to obtain triphenylphosphine oxide (455 g). The filtrate was concentrated under vacuum and purified through 150 L of biotage KP-Sil (SiO2 5 kg; pretreated with 1% TEA; loaded samples dissolved in toluene eluent: 9:1 heptane / EtOAc (16 L) and 15 TEA, 3.6:1 (46 L), 2:1 (20 L) and 1% TEA, 1:1 (30 L) and 1% TEA, and 100% EtOAc (16 L) and 1% TEA).The combined clear product fraction was concentrated under vacuum to obtain Compound 134 as a grayish-white solid foam (662.2 g). The mixture fractions were combined and concentrated under vacuum (480 g). Before loading onto a 150 L biotage, the white insoluble solid formed by dilution with toluene (300 ml) was removed by vacuum filtration. This substance, which is soluble in toluene, was purified using a 150 M biotage HP-sphere (2.5 kg SiO2 (pretreated with 1% TEA); sample loaded with toluene; eluent: 2:1 heptane / EtOAc (26 L) w / 1% TEA, 1:1 (25 L) w / 1% TEA, 1:4 (34 L) w / 1% TEA). The column was monitored by TLC (1:1 heptane / EtOAc). The combined clean product fraction was concentrated under vacuum to obtain additional compound 134 as a grayish-white solid foam (165.5 g. Total 662.2 + 165.5 g = 827.7 g, 930 mmol, 1.03 wt, 67% yield).

[0172] 1H NMR(400 MHz, 클로로포름-d) δ = 8.47 (s, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.38 - 7.31 (m, 5H), 7.27 - 7.19 (m, 6H), 7.14 - 7.06 (m, 3H), 6.93 - 6.87 (m, 2H), 6.76 (d, J = 8.6 Hz, 4H), 6.26 (dd, J = 2.0, 16.0 Hz, 1H), 6.15 (dd, J = 2.7, 17.2 Hz, 1H), 5.86 (dd, J = 4.7, 15.2 Hz, 1H), 5.80 (dd, J = 4.7, 15.2 Hz, 1H), 5.51 (ddd, J = 2.7, 4.3, 52.8 Hz, 1H), 5.31 (ddd, J = 2.0, 4.3, 52.8 Hz, 1H), 4.87 (d, J = 4.7 Hz, 2H), 4.85 - 4.81 (m, 1H), 4.79 (d, J = 4.3 Hz, 2H), 4.71 - 4.59 (m, 1H), 4.20 - 4.13 (m, 2H), 4.06 (dd, J = 2.7, 11.3 Hz, 1H), 3.90 (dd, J = 2.7, 11.7 Hz, 1H), 3.77 (s, 6H), 3.52 (dd, J = 3.1, 10.9 Hz, 1H), 3.18 (dd, J = 3.9, 10.9 Hz, 1H), 0.92 (s, 9H), 0.84 (s, 9H), 0.10 (s, 3H), 0.09 (s, 6H), 0.07 (s, 3H)

[0173] 단계 5-6

[0174]

[0175] Diphenyl phosphite (90 ml, 109 g, 0.46 mol, 0.26 wt, 0.22 vol, 1.5 equivalents) was added to a solution of compound 134 (410.7 g, 309 mmol, 1 wt, 1 vol, 1 equivalent) in pyridine (1.23 L, 1.21 kg, 15.2 mol, 2.9 wt, 3.0 vol, 49 equivalents). The reaction mixture was stirred at room temperature and monitored by LCMS. After 2 hours (80% conversion), additional diphenyl phosphite (29.9 ml, 36.2 g, 155 mmol, 0.088 wt, 0.073 vol, 0.50 equivalents) was added. After an additional 1 hour, additional diphenyl phosphate (6.0 ml, 7.2 g, 31 mmol, 0.018 wt, 0.015 vol, 0.10 equivalents) was added, and the reaction mixture was continued for an additional 0.5 hours (98% conversion). The reaction mixture was added to a mixture of saturated NaHCO3 (9 wt% solution in water; 2.1 L, 5 vol) and water (1.0 L ml, 2.5 vol), and the temperature was maintained between 4.7 and 12°C. The reactor was washed with a small amount of EtOAc. Stirring was continued at room temperature for 30 minutes, and the reaction was monitored by LCMS (100% conversion). The reaction mixture was extracted twice with a 1:1 mixture of EtOAc and MTBE (2 x 8.2 L, 2 x 20 vol). The combined organic layer was washed with water (4.1 L, 10 vol), concentrated under vacuum, and azeotropically mixed with toluene (3 x 4.1 L, 3 x 10 vol; continuous feed) to remove pyridine to obtain compound 135 (0.55 equivalents of pyridine residue).

[0176] Step 6 - Crude compound 135 was dissolved in dichloromethane (3.08 L, 4.07 kg, 9.9 wt, 7.5 vol) at ambient temperature. Water (55.7 ml, 0.136 vol, 10 equivalents) was added, followed by the addition of a solution of dichloroacetic acid (77 ml, 120 g, 0.93 mol, 0.29 wt, 0.19 vol, 3.0 equivalents) in DCM (3.08 L, 7.5 vol), while maintaining an internal T below 25°C (turned into an orange solution). After 30 minutes, triethylsilane (Et3SiH; 494 ml, 359 g, 3.09 mol, 0.875 wt, 1.20 vol, 10.0 equivalents) was added (T-internal proceeded from 18.2°C to 17°C), and stirring was continued for 20 minutes. Triethylamine (431 ml, 313 g, 3.09 mol, 0.762 wt, 1.05 vol, 10.0 equivalents) was added (T-internal proceeded from 17.8°C to 22°C). The mixture was concentrated to 1.55 kg (3.8 wt), redissolved in EtOAc (6.2 L, 5.5 kg, 14 wt, 15 vol), and washed sequentially with: (1) water (1.0 L, 2.5 vol) and saturated NaHCO3 (9 wt% solution in water; 0.82 L, 2.0 vol). The crude product EtOAc solution was stored overnight at -20°C, and the next day, the solution was concentrated under vacuum at 25°C. The obtained crude mixture (654 g) was then softened with: (1) n-heptane (3.01 L, 7.5 vol), and (2) a mixture of n-heptane (2.46 L, 6.0 vol) and toluene (0.82 L, 2.0 vol). The solution portion (supernatant) was carefully decanted, and the solid remaining at the bottom was dissolved in acetonitrile (4.1 L, 10 vol). The mixture was concentrated under vacuum at 25°C and azeotropically mixed twice with acetonitrile to obtain Compound 136. The product was used in subsequent steps without purification (a theoretical yield of 100% was assumed).

[0177] Step 7

[0178]

[0179] Step 7a Compound 136 (337 g, 309 mmol, 1 wt, 1 vol, 1 equivalent) was dissolved in pyridine anhydrous (13.5 L, 13.2 kg, 39 wt, 40 vol) at room temperature. Triethylamine (129 ml, 927 mmol, 94 g, 0.28 wt, 0.38 vol, 3.0 equivalents) was added, followed by the addition of 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinan 2-oxide (DMOCP; 103 g, 556 mmol, 0.31 wt, 1.80 equivalents). The resulting mixture was stirred at ambient temperature for 30 minutes and monitored by LCMS (100% conversion rate) to obtain Compound 137.

[0180] Step 7b TEA (129 ml, 927 mmol, 94 g, 0.28 wt, 0.38 vol, 3.0 equivalents), water (100 ml, 5.56 mol, 0.30 wt, 0.30 wt, 18 equivalents), and sulfur (34.7 g, 1.08 mol, 0.10 wt, 3.5 equivalents) were added to the above mixture of compound 137. After 90 minutes (100% conversion), NaHCO3 (9 wt% solution in water; 3.37 L, 10 vol) was added, and the T-inside was maintained below 30°C (16.6°C to 27°C). The resulting mixture was filtered to remove salts. The filtrate was concentrated into a mixture under vacuum, diluted with MTBE (5.1 L, 15 vol), and washed twice with NaCl (30 wt% solution in water; 2 x 1.35 L, 2 x 4 vol). Insoluble solids were filtered, the filtrate was concentrated under vacuum, and azeotropically mixed with toluene (4.0 L, 12 vol). The resulting solid was removed by filtration, the crude mixture was dissolved in toluene, and purified using a 150 L KP-Sil biotage (SiO2 5 kg; pretreated with Hep / EtOAc / TEA (1.5 / 1.5 / 0.03 CV); eluted with EtOAc / TEA (3 / 0.03 CV), EtOAc / MeOH / TEA (4 / 0.2 / 0.04 CV), EtOAc / MeOH / TEA (2 / 0.2 / 0.02 CV). The column was monitored by TLC (EtOAc / MeOH / TEA=9 / 1 / 0.1). The fractions containing the Sp isomer were combined and concentrated under vacuum to obtain compound 138 as a pale pink foamy solid (Sp isomer; 154 g, 128 mmol, 0.46 wt, 41.3% yield). The fractions containing the Rp isomer were combined and concentrated under vacuum to obtain compound 240 as a pale pink foamed solid (Rp isomer; 64 g, 53 mmol, 0.19 wt, 17% yield).

[0181] Compound 138 (Sp isomer):

[0182] 1 H NMR(400 MHz, 클로로포름-d) δ = 8.51 (s, 1H), 8.50 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 7.49 - 7.44 (m, 2H), 7.38 - 7.27 (m, 4H), 7.25 - 7.21 (m, 2H), 7.14 (t, J = 7.1 Hz, 2H), 6.44 (dd, J = 2.5, 13.9 Hz, 1H), 6.18 (d, J = 15.2 Hz, 1H), 5.78 (td, J = 6.3, 15.6 Hz, 1H), 5.69 (td, J = 4.7, 15.6 Hz, 1H), 5.56 (dd, J = 3.9, 50.8 Hz, 1H), 5.20 - 5.06 (m, 1H), 4.95 - 4.79 (m, 4H), 4.69 (dd, J = 4.3, 16.0 Hz, 1H), 4.54 - 4.38 (m, 3H), 4.35 (d, J = 5.5 Hz, 1H), 4.32 - 4.29 (m, 1H), 4.05 (dd, J = 1.6, 11.7 Hz, 1H), 3.91 (dd, J = 3.1, 11.7 Hz, 1H), 3.14 - 3.06 (m, 6H), 1.30 (t, J = 7.4 Hz, 9H), 0.91 (s, 9H), 0.90 (s, 9H), 0.12 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H)

[0183] 화합물 240 (Rp 이성질체):

[0184] 1H NMR(400 MHz, 클로로포름-d) δ = 8.54 (s, 1H), 8.38 (s, 1H), 8.33 (s, 1H), 8.01 (s, 1H), 7.39 - 7.09 (m, 10H), 6.39 (dd, J = 2.3, 14.1 Hz, 1H), 6.13 (d, J = 17.2 Hz, 1H), 5.72 (d, J = 3.1 Hz, 2H), 5.68 (dd, J = 4.3, 51.2 Hz, 1H), 5.43 - 5.29 (m, 1H), 5.10 - 4.96 (m, 3H), 4.90 - 4.83 (m, 2H), 4.78 - 4.72 (m, 1H), 4.52 (ddd, J = 3.9, 6.6, 17.2 Hz, 1H), 4.44 - 4.35 (m, 2H), 4.31 - 4.26 (m, 1H), 4.20 - 4.12 (m, 2H), 3.87 (dd, J = 3.5, 11.7 Hz, 1H), 3.79 - 3.77 (m, 1H), 3.15 - 3.09 (m, 6H), 1.33 (t, J = 7.4 Hz, 9H), 0.94 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H)

[0185] 단계 8

[0186]

[0187] Compound 138 (221 g, 183 mmol, 1 wt, 1 vol, 1 equivalent) was dissolved in a mixture of pyridine (530 ml, 6.56 mol, 519 g, 2.3 wt, 2.4 vol) and TEA (2.65 L, 19.0 mol, 1.93 kg, 8.7 wt, 12 vol, 104 equivalents). Triethylamine trihydrofluoride (1.62 mol, 262 g, 1.2 wt, 1.2 vol, 8.9 equivalents as a complex, 27 equivalents of HF 264 ml) was added, and the conversion rate was monitored by LCMS while stirring the mixture at room temperature. After 3 hours (97% conversion rate), methoxytrimethylsilane (TMSOMe; 1.40 L, 10.2 mol, 1.06 kg, 4.8 wt, 6.3 vol, 55 equivalents) was added, and stirring was continued for 30 minutes. The adhesive solid was coated onto the reactor. The solution portion (supernatant) was gently drained. The solid was softened twice with toluene (2 x 2.2 L, 2 x 10 vol; supernatant was gently drained). The crude solid remaining in the reactor was dissolved in dichloromethane (2.2 L, 10 vol) and washed with NH4Cl (28 wt% solution in water; 2.2 L, 10 vol). The aqueous layer was back-extracted with dichloromethane (2.2 L, 10 vol). The combined organic layer was washed with a mixture of NaCl (36 wt% solution in water; 1.1 L, 5 vol) and water (1.1 L, 5 vol), and then concentrated under vacuum to obtain compound 139 as a yellowish-brown dry foam (152 g, 155 mmol, 0.70 wt, 85% yield). The crude product was used in subsequent steps without purification.

[0188] Step 9

[0189]

[0190] Compound 139 (150 g, 153 mmol, 1 wt, 1 vol, 1 equivalent) was azeotropically mixed with acetonitrile (4 L, 27 vol), and then the acetonitrile (1.05 L, 0.83 kg, 5.5 wt, 7.0 vol) was redissolved at room temperature. 2-nitrobenzyl bromide (44.4 g, 205 mmol, 0.30 wt, 1.34 equivalents) was added at room temperature, and the reaction products were monitored by LCMS. After 23 hours (100% conversion), EtOAc (1.50 L, 10 vol), NH4Cl (28 wt% solution in water; 300 ml, 2 vol), and water (300 ml, 2 vol) were added (pH = 6), and the resulting mixture was partially concentrated to a weight of 1.11 kg under vacuum at 25°C. EtOAc (2.25 L, 15 vol) was added, and the mixture was stirred for 5 minutes. Two layers were separated. The aqueous layer was extracted with ethyl acetate (750 ml, 5 vol). The combined organic layer was washed sequentially with: (1) a mixture of NaCl (36 wt% solution in water; 300 ml, 2 vol) and water (300 ml, 2 vol) and (2) water (600 ml, 4 vol). The organic layer was concentrated under vacuum and azeotropically mixed with n-heptane (1.50 L, 10 vol). MTBE (0.95 L, 6.3 vol) was added to the crude solid, and the mixture was heated at 40°C. The mixture was diluted with EtOAc (300 ml, 2 vol) and slowly cooled to 0°C. The dense solid was allowed to settle, and the supernatant was pumped through a filter frit tube. The solid was washed twice with MTBE (2 x 300 ml, 2 x 2 vol; the supernatant was pumped through a filter frit tube for each wash) and dried overnight at 40°C to obtain Compound 140 as a pale yellow solid (156 g). The filtrate was concentrated under vacuum to obtain a brown oil (17.8 g) was obtained, and this was subjected to purification using 340 g of Biotage Snap-Ultra (eluent in EtOAc: 0 to 5% MeOH) to obtain an additional compound 140 as a pale yellow solid (5.8 g). Total 156 g + 5.8 g = 161.8 g (pure 152 mmol, 95% purity, 99% yield).

[0191] 1 H NMR (400 MHz, chloroform-d) δ = 8.46 (s, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 8.09 - 8.06 (m, 1H), 7.89 (s, 1H), 7.54 - 7.51 (m, 1H), 7.49 - 7.45 (m, 4H), 7.37 - 7.28 (m, 3H), 7.24 - 7.19 (m, 3H), 7.16 - 7.11 (m, 2H), 6.22 (d, J = 16.8 Hz, 1H), 6.14 (dd, J = 2.7, 17.2 Hz, 1H), 5.83 - 5.61 (m, 3H), 5.60 - 5.48 (m, 1H), 5.07 (dd, J = 3.5, 51.6 Hz, 1H), 5.06 - 4.96 (m, 1H), 4.79 (dd, J = 4.9, 15.8 Hz, 1H), 4.69 (d, J = 5.9 Hz, 2H), 4.67 - 4.56 (m, 1H), 4.48 - 4.40 (m, 3H), 4.37 - 4.30 (m, 1H), 4.27 (d, J = 5.9 Hz, 2H), 4.19 - 4.13 (m, 1H), 3.93 - 3.85 (m, 1H), 3.85 - 3.78 (m, 1H)

[0192] Steps 10-11

[0193]

[0194] Step 10 Compound 140 (95% purity, pure 73.2 g, 72.3 mmol, 1 wt, 1 vol, 1 equivalent) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (25.3 ml, 79.5 mmol, 0.33 wt, 0.35 vol, 1.10 equivalents) were azeotropically mixed three times with anhydrous acetonitrile (3 x 2 L), redissolved in dichloromethane (0.73 L, 10 vol), and cooled to 0-5°C. Diisopropylammonium tetrazolide (6.19 g, 36.1 mmol, 0.085 wt, 0.50 equivalents) was added. The resulting reaction mixture was stirred at 0°C for 10 hours, heated to 10°C over 2 hours, maintained at 10°C for 10 hours, and heated to room temperature over 2 hours. The reaction mixture was monitored by LCMS and TLC (EtOAc with 0.5% TEA). After 18 hours, acetonitrile anhydrous (0.73 L, 10 vol) was added, and the mixture was stored at -20°C for 3 days.

[0195] Step 11a The mixture from Step 10 was heated to ambient temperature, and a mixture of pyridine trifluoroacetate salt (twice azeotropically mixed with pyridine; 41.9 g, 217 mmol, 0.57 wt, 3.0 equivalents) and acetonitrile (5.85 L, 80 vol) was added in several portions (100 mL every 30 minutes over 9 hours) via a dropping funnel. The reaction mixture was monitored by LCMS. After 13 hours, a solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (5.8 mL, 18 mmol, 0.25 equivalents) in acetonitrile (24 mL) was added over 4 hours. The amount of additional reagent was determined based on the remaining compound 140 (~30% based on LCMS). The additional conversion rate of Dior was observed after 6 hours.

[0196] Step 11b ((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazolin-3-thione (DDTT; 20.8 g, 101 mmol, 0.28 wt, 1.4 equivalents) was added, and stirring was continued for 1 hour. The reaction mixture was partially concentrated to ~800 mL and diluted with MTBE (1.46 L, 20 vol), NaHCO3 (9 wt% solution in water; 1.1 L, 15 vol), and water (0.37 L, 5 vol). pH = 8. The layers were separated, and the aqueous layer was extracted with a mixture of MTBE (1.46 L, 20 vol) and EtOAc (1.10 L, 15 vol). The combined organic layer was washed twice with 30% aqueous NaCl (2 x 0.73 L, 2 x 10 vol), concentrated at 35°C under vacuum, and azeotropically mixed with toluene (1.46 L, 20 vol). LCMS and TLC (EtOAc) indicated Compound 143 (SpRp, target): Compound 241 (SpSp) = 5:1

[0197] The crude product was purified using biotage 150M KP-Sil, (SiO2 2.5 kg; eluent: EtOAc / Hep: 2:1 (4 CV), 3:1 (2.5 CV), 4:1 (2.5 CV), 100% EA (3 CV), EA 4 CV with 5-10% MeOH) to obtain compound 143 (36 g, 31.5 mmol, 44% yield).

[0198] Compound 143 (SpRp):

[0199] 1H NMR(400 MHz, 클로로포름-d) δ = 8.59 (s, 1H), 8.10 (s, 1H), 8.03 - 7.99 (m, 1H), 7.91 (s, 1H), 7.56 - 7.53 (m, 2H), 7.49 - 7.40 (m, 5H), 7.35 - 7.28 (m, 2H), 7.24 - 7.16 (m, 4H), 6.92 (s, 1H), 6.29 (d, J = 14.9 Hz, 1H), 6.08 (d, J = 20.7 Hz, 1H), 5.97 - 5.83 (m, 1H), 5.76 (td, J = 4.7, 15.6 Hz, 1H), 5.61 - 5.51 (m, 2H), 5.40 (d, J = 4.3 Hz, 1H), 5.29 - 5.17 (m, 1H), 4.91 (dd, J = 7.4, 14.9 Hz, 1H), 4.86 - 4.75 (m, 3H), 4.63 (dd, J = 3.7, 9.2 Hz, 1H), 4.58 - 4.43 (m, 5H), 4.34 - 4.19 (m, 4H), 2.79 (td, J = 5.9, 16.8 Hz, 1H), 2.66 (td, J = 6.3, 16.8 Hz, 1H).

[0200] 화합물 241 (SpSp)

[0201] 1 H NMR(400 MHz, 클로로포름-d) δ = 8.11 (s, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.94 (s, 1H), 7.90 (s, 1H), 7.61 (s, 1H), 7.56 - 7.40 (m, 7H), 7.33 - 7.28 (m, 2H), 7.23 - 7.17 (m, 4H), 6.22 (d, J = 17.6 Hz, 1H), 6.15 (d, J = 18.8 Hz, 1H), 5.85 (dd, J = 3.5, 51.2 Hz, 1H), 5.75 - 5.45 (m, 5H), 4.95 - 4.23 (m, 14H), 2.82 (t, J = 6.1 Hz, 2H).

[0202] 단계 12

[0203]

[0204] Compound 143 (71.6 g, 62.6 mmol, 1 wt, 1 vol, 1 equivalent) was dissolved in 1,4-dioxane (0.43 L, 6 vol). Thiophenol (215 ml, 2.09 mol, 230 g, 3.2 wt, 3 vol, >30 equivalents) was added, followed by the addition of triethylamine (215 ml, 1.54 mol, 156 g, 2.2 wt, 3 vol). A slight exothermic reaction was observed (T-internal increased to ~7°C), and therefore, cooling was performed using a water / ice bath to control the T-internal temperature below 27°C. The reaction products were monitored by LCMS. After 2 hours, MeOH (0.57 L, 8 vol) and NH4OH (28 wt%; 15 mol, 0.57 L, 8 vol, >200 equivalents) were added. The resulting mixture was heated at 50°C for 5 hours, cooled to room temperature, and stirred overnight. After 14 hours, water (0.72 L, 10 vol) was added (no solids were observed), and the mixture was extracted three times with a 1:1 (v / v) mixture of n-heptane and toluene (3 x 0.86 L, 3 x 12 vol), followed by extraction with toluene (0.57 L, 8 vol). The aqueous layer was concentrated under vacuum at 40-50°C and diluted with water (1.07 L, 15 vol). The resulting slurry was kept at room temperature overnight. The resulting solid was filtered and washed with water (0.36 L, 5 vol). The filtrate was still turbid and was filtered through Celite and Cuno filters. Turbidity was still present. HCl (1.0 M solution in water; 132 ml, 132 mmol, 2.1 equivalents) was added over 1 hour, and the pH was checked (pH < 2). Stirring was continued at room temperature for 1 hour, and the mixture was filtered. The filter cake was washed with water (8 x 0.20 L), dried in a vacuum oven at 35°C for 2 days, and then left unheated for 1 day to obtain Compound 1 as a pale orange solid (44.88 g, 60.1 mmol, 0.It was obtained as 63 wt, 96% yield.

[0205] Step 13

[0206]

[0207] Ammonia (2.0 M solution in MeOH; 220 ml, 440 mmol, 10 vol, 15 equivalents) was added to free acid compound 1 (22.42 g, 30.03 mmol, 1 wt, 1 vol, 1 equivalent). EtOH (55 ml, 2.5 vol) was added, and the resulting solution was filtered through a Kuno filter (0.45 micrometers; PTFE) and washed with a 1:1 (v / v) mixture of MeOH and EtOH (90 mL, 4 vol). The filtrate was concentrated under vacuum at 30°C to obtain a grayish-white solid, which was dried overnight at room temperature, ground with a spatula (for easy crushing), and further dried under vacuum at room temperature. Subsequently, the isolated solid was suspended in toluene (250 ml) and stirred at room temperature for 30 minutes. Next, the solid was collected by vacuum filtration and washed twice with toluene (2 x 50 ml). Then, the solid was dried under vacuum in a vacuum oven to obtain 22.4 g of compound 1a (the di-ammonium salt of compound 1).

[0208] Recrystallization: Compound 1a (22.14 g, 28.36 mmol, 1 wt, 1 vol, 1 equivalent) was dissolved in a mixture of water (664 ml, 30 vol) and ammonium hydroxide (28 wt%; 2.5 ml, 18 mmol, 0.63 equivalents) (pH = 9-10), and extracted with toluene three times (3 x 300 ml, 3 x 14 vol), EtOAc three times (3 x 200 ml, 3 x 9 vol), and toluene three times (3 x 300 ml, 3 x 14 vol). The resulting aqueous layer was treated with HCl (1.0 M solution in water; 90 ml, 90 mmol, 3.2 equivalents) for a period of 3.5 hours (pH ≤ 2). The mixture was stirred for 30 minutes, and then the solid precipitate was collected by vacuum filtration. The filter cake was washed three times with water (3 x 200 ml, 3 x 9 vol) and dried overnight under vacuum. Ammonia (2.0 M solution in MeOH; 250 ml, 500 mmol, 17.6 equivalents) and ethanol (100 ml) were added to the solid, and the resulting mixture was concentrated under vacuum until crystals appeared (~100 ml); at this point, concentration was stopped, and the mixture was stirred for 20 minutes. Ethanol (45 mL) was added, and the mixture was partially concentrated (45 mL removed). The same operation was repeated more than twice, then the mixture was cooled to 0°C and stirred for 3.5 hours. The white solid was collected by vacuum filtration and washed with cold ethanol (20 ml) followed by ethyl acetate (2 x 50 mL). The white solid was dried under vacuum at room temperature for 3 days to obtain compound 1a as a white solid (16.6 g, 21.3 mmol, 0.75 wt, 75% yield). The filtrate was concentrated under vacuum and dried under vacuum at room temperature for 3 days to obtain compound 1a as an off-white solid (4.16 g, 5.3 mmol, 18% yield).

[0209] Example 1.2 - Compound 1 1H NMR analysis

[0210] of compound 1a 1 The 1H NMR spectrograph is shown in Fig. 3. The generated spectra are as follows:

[0211] 1 ¹H NMR spectrum (400 MHz, DMSO-d6, δH 2.49 ppm, 80 ℃)

[0212] δ(ppm):3.05-3.13(4H, m), 3.70(1H, dd, J=13, 5 Hz), 3.78(1H, dd, J=12, 4Hz), 4.21-4.24(2H, m), 4.28(1H, m), 4.38(1H, m), 4.53-4.68(2H, m), 5.22(1H, m), 5.76(2H, s), 5.78(1H, m), 6.26(1H, m), 6.29(1H, m), 8.13(1H, s), 8.14(1H, s), 8.36(1H, brs), 8.59(1H, brs).

[0213] Example 1.3 - X-ray analysis of Compound 1

[0214] About 2 mg of Compound 1 was dissolved in 600 µL of water. 120 µL of this solution was placed in another glass vial, and then this vial was stored in a fixed container containing 3 mL of MeCN at room temperature for one week. This is the H2O / MeCN vapor diffusion method for sample preparation.

[0215] A colorless block single crystal (0.1 x 0.1 x 0.1 mm) identified in the crystallization solution was dispersed in liquid Parava 10312 and mounted on Dual-Thickness MicroMounts™ (MiTeGen). Diffraction data were collected at -160°C on XtaLAB Pro P200 MM007HF (Rigaku) ​​by the ω-axis vibration method using multilayer mirror monochromatic copper-Kα radiation.

[0216] Figure 4a shows the ORTEP diagram of a molecule of compound 1 among the asymmetric units, along with numerous disordered water molecules. Figure 4b shows the crystal structure of one molecule of compound 1 from Figure 4a. Figure 4c shows the crystal structure of another molecule of compound 1 shown in Figure 4a.

[0217] The crystal structure of Compound 1 was interpreted with a final R-factor of 0.1354. The flack parameter was nearly 0 (0.083 (17)), indicating that the absolute coordination of Compound 1 is (R, S). The crystal structure analysis also indicated that many water molecules are present in the large channels of Compound 1, suggesting that water molecules can easily exit the channels. The analysis also confirmed that the stereotypes of both crystallographically independent molecules of the asymmetric unit are nearly identical.

[0218] Additional parameters for X-ray analysis are presented below:

[0219]

[0220] Example 2 - X-ray structure confirming the complex with WT STING

[0221] To further understand the target-binding mechanism of the novel compound, the X-ray crystal structure of WT STING as a complex with the compound was determined.

[0222] A. Expression and purification of the WT-STING C-terminal domain (residues 155-341)

[0223] A DNA sequence encoding a human WT STING protein of amino acids 155 to 341 (SEQ ID No.: 4) was cloned into the pET21b vector following a His-TEV-Sumo tag at its N-terminus (SEQ ID No.: 5). The sequence of pET21b was entrusted to Addgene and is available at addgene.org / vector-database / 2550 / , the sequence of which is incorporated herein by reference.

[0224] E. E. coli BL21 (DE3) codon-plus cells were transformed with the above plasmid, and expression of the recombinant protein was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed by sumo protease and separated from tag-free WT STING 155-341 using a second Ni-NTA affinity column. The protein was further purified by anion-exchange and size-exclusion chromatography and stored in a buffer containing 20 mM Tris·HCl pH 7.5 and 150 mM NaCl at a concentration of 35 mg / ml.

[0225] B. Crystallization and structural determination of the WT STING C-terminal domain as a complex with Compound 1

[0226] To co-crystallize Compound 1 and WT STING 155-341, WT STING protein was diluted to 10 mg / ml using stock buffer (20 mM Tris·HCl pH 7.5 and 150 mM NaCl) and mixed with Compound 1 (100 mM stock in DMSO) at a molar ratio of 1:5. The mixture was incubated at 4°C for 4 hours and crystallized after centrifugation at 13,000 rpm for 20 minutes. Crystallization screen trays were set up at 18°C ​​using the hanging-drop vapor diffusion method. Crystals were grown by mixing 1 μL of the WT STING / Compound 1 solution with an equal volume of a well solution containing 100 mM HEPES pH 7.5, 200 mM CaCl2, and 15% (wt / vol) PEG 8000. 20% (wt / vol) PEG 400 was used as a cryoprotective reagent when crystals were flash-frozen in liquid nitrogen. Diffraction datasets were collected using a Pilatus detector at the SSRF BL19U1 beamline and processed by the HKL3000 and program SCALEPACK2MTZ in the CCP4 software suite.

[0227] The structure of WT STING 155-341 combined with Compound 1 was determined by molecular substitution using a programmed phaser (maximum likelihood molecular substitution) with PDB ID 4F9E as the initial search model. The presence of Compound 1 between the dimeric interfaces of WT STING was confirmed in the Fo-Fc difference map calculated for the model. The model was constructed and completed manually using the Coot program, and refined using the Refmac5 program in the CCP4 software suite. The final refined structure was reported with a resolution of 2.38 Å in space group P212121, with a unit cell measured at a=33.820, b=78.110, c=132.212, α=90.00, β=90.00, and γ=90.00. Two copies of WT STING 155-341 were identified as each asymmetric unit bound to one molecule of compound 1 at the dimer interface.

[0228] C. Interaction between WT STING and Compound 1 observed in X-ray crystal structure

[0229] FIG. 5 shows a diagram of the X-ray crystal structure of human WT STING as a complex with Compound 1. The inventors examined the X-ray crystal structure of human WT STING as a complex with Compound 1, co-crystallized from a sample of Compound 1a. The compound binds in an interfacial pocket formed by the dimer of the WT STING protein. Both sides of the adenine base of the compound form π-π stacking interactions with the guanidine groups of Tyr240 and Arg238, respectively. The transolefin linker forms van der Waals interactions with the aliphatic portion of the side chain of Arg238. Fluorine substituents at the C2' position of the ribose group of the compound are placed within hydrophobic holes defined by Thr263, Pro264, and Tyr163. The negatively charged thiophosphate group of the compound forms salt crosslinking with Arg238 and H-bond interactions with Ser162 and Thr267, respectively. In addition, the thiophosphate group also forms electrostatic interactions with the guanidine group of Arg 232. The LID loop region of WT STING, consisting of residues 226 to 243, wraps around two base groups and a transolefin linker.

[0230] Example 3 - Determination of the X-ray crystal structure of REF STING as a complex with Compound 1.

[0231] A. Expression and purification of the REF STING C-terminal domain (residues 155-341, sequence identification number: 6).

[0232] A DNA sequence encoding a human REF STING protein from amino acids 155 to 341 (SEQ ID: 6) was cloned into the pET21b vector following a His-TEV-Sumo tag at its N-terminus (SEQ ID: 7). The sequence of pET21b was entrusted to Addgene and is available at addgene.org / vector-database / 2550 / , the sequence of which is incorporated herein by reference.

[0233] E. E. coli BL21 (DE3) codon-plus cells were transformed with the above plasmid, and expression of the recombinant protein was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate by Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed by sumo protease and separated from tag-free REF STING_155-341 using a second Ni-NTA affinity column. The protein was further purified by anion-exchange and size-exclusion chromatography and stored in a buffer containing 20 mM Tris·HCl pH 7.5 and 150 mM NaCl at a concentration of 24 mg / ml.

[0234] B. Crystallization and structural determination of the REF STING C-terminal domain as a complex with Compound 1

[0235] To co-crystallize Compound 1 and REF STING_155-341, REF STING protein was diluted to 10 mg / ml using stock buffer (20 mM Tris·HCl pH 7.5 and 150 mM NaCl) and mixed with Compound 1 (100 mM stock in DMSO) at a molar ratio of 1:5. The mixture was incubated at 4°C for 4 hours and crystallized after centrifugation at 13,000 rpm for 20 minutes. Crystallization screen trays were set up at 18°C ​​using the hanging-drop vapor diffusion method. Crystals were grown by mixing 1 μL of the REF STING / Compound 1 solution with an equal volume of a well solution containing 100 mM HEPES pH 7.5, 200 mM CaCl2, and 15% (wt / vol) PEG 8000. 20% (wt / vol) PEG 400 was used as a cryoprotective reagent when the crystals were flash-frozen in liquid nitrogen. Diffraction datasets were collected using a Pilatus detector at the SSRF BL18U1 beamline and processed by the HKL3000 and program SCALEPACK2MTZ in the CCP4 software suite. The structure is shown in Fig. 6.

[0236] The structure of REF STING_155-341 combined with Compound 1 was determined by molecular substitution using a program phasor (maximum likelihood molecular substitution), using the previously determined WT STING 155-341 structure (described above) as the initial search model. The presence of Compound 1 between the dimer interfaces of REF STING was confirmed in the Fo-Fc difference map calculated for the model. The model was constructed and completed manually with the Coot program and refined with the Refmac5 program in the CCP4 software suite. The final refined structure was reported with a resolution of 2.76 Å in space group P212121, with a unit cell measured at a = 33.733, b = 77.831, c = 131.689, α = 90.00, β = 90.00, γ = 90.00. Two copies of REF STING 155-341 were identified as asymmetric units each bound to one molecule of compound 1 at the dimer interface.

[0237] C. Interaction between REF STING and Compound 1 observed in X-ray crystal structure

[0238] Figure 6 shows the X-ray crystal structure of human REF STING as a complex with Compound 1, co-crystallized from a sample of Compound 1a. The compound binds in an interfacial pocket formed by the dimer of the STING protein. Both sides of the adenine base of the compound form π-π stacking interactions with the guanidine groups of Tyr240 and Arg238, respectively. The transolefin linker forms van der Waals interactions with the aliphatic portion of the Arg238 side chain, while the guanidine portion of the Arg238 side chain forms π-π stacking interactions with the imidazole group of the His232 side chain from the outside. The olefin linker is in contact with the interaction pairs of the Arg238 and His232 side chains. A fluorine substituent at the C2' position of the ribose group of the compound is placed within the hydrophobic holes defined by Thr263, Pro264, and Tyr163. The negatively charged thiophosphate group of the compound forms a salt crosslink with Arg238 and H-bond interactions with Ser162 and Thr267, respectively. The LID loop region of REF STING, consisting of residues 226 to 243, surrounds two base groups and a transolefin linker.

[0239] Example 4 - In vivo evaluation of Compound 1a in a murine bladder cancer model using an intravesical administration route

[0240] The MBT-2 orthotopic model of murine bladder cancer was established and characterized by Lee et al. in 2012 ("Tumor Establishment Features of Orthotopic Murine Bladder Cancer Models," Urological Oncology 2012;53: 396-400) and exhibited histology similar to human bladder cancer. Therefore, this model was selected to evaluate the anti-bladder cancer activity of Compound 1. The model was established by applying a protocol with an HCl pre-treatment procedure described by Lee et al. Briefly, 30 μL of 0.1 N HCl solution was introduced into the mouse bladder via a catheter and allowed to remain in the bladder for 15 seconds. Subsequently, the HCl solution was replaced with 30 μL of 0.1 N NaOH solution, followed by a single flushing step with 1 X PBS (pH 7.4). The subsequent tumor cell transplantation procedure was modified from the original literature. Briefly, after the flushing step, MBT-2 tumor cells (2 x 10⁶ in 50 μL of RPMI1640 medium) 6 Canine cells) were injected into the bladder and allowed to remain for 45 minutes. At the end of this 45-minute period, the bladder was emptied.

[0241] Three days after tumor cell transplantation, mice were treated with various doses of Compound 1a, BCG (OncoTICE®, Merck Canada Inc.), anti-mouse PD-1 antibody (clone # RMP1-14, Bioxcell), and combinations of Compound 1a and PD-1 antibody according to the schedule summarized in Table 1. Both Compound 1a and BCG were administered via the intravesical route, while the PD1 antibody was administered via intraperitoneal injection.

[0242] On each administration day, BCG was diluted with 0.9% NaCl (Lavoisier, France) to achieve a final concentration of 16.875 mg / mL for administration. Any remaining administration solution was discarded after use. For the anti-PD-1 antibody, 1 mg / mL working solution for administration was prepared using 1 x PBS (Lonza, France) to dilute the stock solution. For Compound 1a, a 10 mg / mL stock solution was first prepared by dissolving the dried powder in 1 x PBS. Subsequently, Compound 1a at various concentrations, including 5 mg / mL (400 μg / mouse group), 2.5 mg / mL (200 μg / mouse group), and 1.25 mg / mL (100 μg / mouse group), was prepared by further diluting the stock solution with 1 x PBS.

[0243] Table 1. Treatment and Administration Schedule Planning

[0244]

[0245] 1. IVe, intravesical insertion

[0246] 2. IP, intraperitoneal injection

[0247] From days 20–21, tumor growth within the bladder was quantified by MRI (Magnetic Resonance Imaging), and the tumor sizes of all mice were graphed as shown in Fig. 7. Fig. 7 shows the quantification of tumor volume by MRI from days 20–21 for all study animals. As shown in Fig. 7, all Compound 1a treatment groups (Groups 4 to 7) exhibited much smaller tumor volumes than the vehicle group (Group 1), BCG group (Group 2), and PD1 antibody group (Group 3).

[0248] Compound 1a also showed a statistically significant survival benefit compared to vehicle-treated animals, as shown in Fig. 8.

[0249] Neither BCG nor anti-PD1 antibody showed significant anticancer activity in this orthotopic mouse bladder cancer model, which suggested that the model used was a BCG / PD1 antibody resistant / refractory model.

[0250] Example 103 - HAQ STING Agonist Active Reporter Test

[0251] THP1-Dual™ cells (InvivoGen, Cat# thpd-nfis) EC 50 It was applied to the determination. THP1 Dual™ cells were characterized by the vendor Invivogen to possess the HAQ STING genotype (Insight 201402-1). Cells were grown and maintained under conditions in accordance with the manufacturer's recommendations. EC 50 For determination, the induction of the Interferon Regulatory Factor (IRF) pathway described in the manufacturer's manual was followed. Briefly, cells were seeded and treated with various concentrations of compounds for 20 hours while incubating at 37°C and 5% CO2. Cells were resuspended, and QUANTI-Luc™ solution (Cat. #: rep-qlc1) was added. The generated luminescence was measured using a luminometer (Envision, Perkin Elmer). The acquired signals were plotted to determine EC₀. 50 It was calculated using GraphPad Prism 7 software.

[0252] Human STING EC for Compound 1a 50 (μM) values ​​are reported in Table 2 below.

[0253] Example 104 - STING variant-specific reporter assay

[0254] Human STING has four major variants, including WT, HAQ, REF, and AQ variants. REF-STING, also referred to as R232H, occurs in, for example, about 14% of the human population. Compared to the wild-type allele, R232H showed a reduced response to bacterial and metazoan cyclic dinucleotides. Details regarding these four major variants, as well as other rare variants, are reported in the literature [Yi G, et al., "Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides" PLoS One 2013; 8:e77846]. STING variant-specific reporter cell lines were established using THP1-Dual™ KO-STING cells (Invivogen, Cat# thpd-kostg) and three STING variant protein expression vectors. The expression vector map for WT STING is presented in Figure 9. For the other two expression vectors, different STING variant sequences were used in the corresponding vectors by replacing WT STING with an appropriate nucleotide sequence.

[0255] STING variant-expression vectors for WT-STING, REF-STING, and AQ-STING were prepared and stably transfected into THP1-Dual™ KO-STING cells to prepare STING variant-specific reporter assays for WT-STING, REF-STING, and AQ-STING, respectively. EC 50The values ​​were determined as described above in Example 103 for the HAQ STING agonist activity reporter assay. The results are presented in Table 2 below. The DNA sequences used for these STING variants are presented in Sequence Identification No. 1 (nucleotide sequence of WT human STING), Sequence Identification No. 2 (nucleotide sequence of REF human STING), and Sequence Identification No. 3 (nucleotide sequence of AQ human STING).

[0256]

[0257]

[0258] Example 105 -- Mouse STING Agonist Activity Reporter Test

[0259] RAW-Lucia™ ISG cells (Invivogen, Cat# rawl-isg) were used for the mouse STING agonist reporter assay. EC 50 The values ​​were determined as described above in Example 103 for the HAQ STING agonist activity reporter assay. The results are presented in Table 2 below.

[0260] Example 106 -- Differential Scanning Fluorescence (DSF) Calibration

[0261] The DSF assay was used to measure the physical interaction between the compound and the recombinant STING protein. The truncated recombinant STING protein (aa155-341) (sequence identification number: 4) was expressed in E. coli as described below and isolated for the assay. The assay matrix was prepared in 384-well plates with a final volume of 10 μL per well, consisting of 1 μM recombinant STING protein (aa155-341) (sequence identification number: 4), 100 mM PBS pH 7.4 supplemented with 100 mM KCl, 5X SYPRO orange dye, and 50 μM of the compound (final DMSO concentration 0-1%). The assay was performed on a QuantStudio 12K flex real-time PCR system using excitation and emission filters at 470 and 586 nm, respectively, and a temperature gradient from 25°C to 95°C at a rate of 0.05°C / min. The thermal melting point (Tm) and the difference in thermal melting point (dTm D) of unbound and ligand-bound recombinant STING proteins were calculated according to the fluorescence derivative curve assigned by Applied Biosystems® Protein Thermal Shift software (algorithm version 1.3).

[0262] Generally, compounds with a ΔTm value greater than 0 are considered to have a physical interaction with the test protein, and the value of ΔTm indicates a positive correlation with the binding affinity of the compound. Here, compound 1a showed a ΔTm of 17.6 (Table 2), which implies a physical interaction with the STING protein.

[0263] Table 2 In vitro characterization of Compound 1a

[0264]

[0265] Example 107 -- In Vivo Human PBMC Stimulation Test

[0266] Human blood from five healthy donors was collected using 10.0 mL BD Vacutainer sodium heparin tubes (cat# 367874). Peripheral blood mononuclear cell (PBMC) isolation was performed using SIGMA ACCUSPIN 50 mL tubes (cat# A2055) and SIGMA ACCUSPIN System-HISTOPAQUE-1077 (cat# A7054) according to the manufacturer's protocol. The PBMC layer was collected and washed with 1x phosphate-buffered saline (PBS) as suggested by SIGMA. The PBMCs were counted and finally suspended at 1x10e6 / ml in RPMI (Corning cat# 10-041-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco cat# 20140.79). 1 ml of cells (1 x 10⁶) were transferred to a 5 ml round-bottom polypropylene test tube (cat#352063) and stimulated at different concentrations (0, 0.1, 1, 10 μM) for 24 hours in a 5% CO₂ incubator at 37°C.

[0267] After 24 hours of incubation, the tubes were centrifuged at 1400 rpm for 5 minutes, and the supernatant was collected. The supernatant was stored at -80°C for subsequent IFNβ measurements. IFNβ measurements were performed using a human IFN-β base kit (Meso Scale Diagnostics cat# K151ADA) and the protocol provided by the manufacturer. IFN-β estimation was performed by reading the calibration plate on a MESO SECTOR Imager 2400 and using the MSD Discovery Workbench 4.0 program. IFNβ protein was analyzed after 24 hours. The results suggested that compound 1a could induce primary human PBMC IFNβ protein production in a dose-dependent manner.

[0268] The results presented in Table 3 reflect the average of measurements performed using five different donors.

[0269] Table 3 In vitro human PBMC stimulation assay

[0270]

[0271] For IFNβ mRNA quantification, total RNA was isolated using the RN Easy Small Kit (Qiagen, Germany) according to the manufacturer's protocol. IFNβ mRNA was quantified by qPCR assay. Briefly, total RNA (400 ng to 1000 ng) was converted to cDNA in a 60-μl reaction volume using SuperScript VILO MasterMix (Life Technologies, USA). The acquired cDNA (10 ng) was subsequently amplified using the Applied Biosystems TacMAN expression assay with RNA-specific primers for IFNB1 (Hs01077958_s1) and GAPDH (Hs99999905_m1). qPCR analysis was performed using TaqMan Fast Advanced Master Mix (Life Technologies, USA) on an Applied Biosystems QuantStudio 12K Flex Real-time PCR System, consisting of 40 cycles of 95°C for 2 seconds, 95°C for 1 second, and 60°C for 20 seconds, followed by an initial 2-minute step at 50°C. Relative gene expression was calculated after normalization to the reference gene GAPDH using the 2-ΔΔCT method. Calculations were performed using Applied Biosystems QuantStudio 12K Flex software v1.2.2. The IFNβ mRNA fold change relative to vehicle-treated samples is summarized in Table 4. The results suggested that compound 1a can induce IFNβ mRNA in primary PBMCs in a dose- and time-dependent manner. Table 4 presents the mean calculated from five different donors.

[0272] Table 4 -- In Vivo Human PBMC 3-hr & 24-hr Stimulation Scheme (mRNA)

[0273]

[0274] Example 108 -- Anticancer effect of Compound 1a on CT26 dual tumor model

[0275] Compound 1a was tested for its anticancer activity in the CT26 dual tumor model, a mouse colon cancer model. Female 5–6 week old Balb / cJ mice (Jackson Labs, Bar Harbor, Maine) were injected with 10 lateral plates on each side of each animal. 5 CT26 tumor cells were injected subcutaneously at 100-cell levels. In Study A, the average tumor size was approximately 100 mm. 3 When [the threshold] was reached, treatment (1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg) was initiated 5 days after tumor transplantation. In Study B, the average tumor size was approximately 120 mm 3 When [the target was reached], treatment (0.6 mg / kg and 10 mg / kg) was started 8 days after tumor transplantation. The treatment plan is listed in Tables 5 and 6.

[0276] Table 5 Dosage plan for Study A

[0277]

[0278] IT is within the tumor.

[0279] Table 6 Dosage plan for Study B

[0280]

[0281] IT is within the tumor.

[0282] All mice in the study had two subcutaneous CT26 tumors. "Treated tumors" referred to tumors to which the compound was directly administered, whereas "untreated tumors" referred to tumors without direct administration of the compound. Tumor volume was tracked throughout the experiment. Tumor volume was measured twice weekly after the start of treatment. Tumor burden was calculated from caliper measurements using the equation (LxW) for the volume of the spherical ellipsoid. 2It is calculated by ) / 2, where L and W are the orthogonal length and width measurements (mm), respectively.

[0283] Compound 1a demonstrated potent and curative activity in the CT26 dual tumor model (Figs. 10 and 11). For treated tumors, a 20% cure rate was detected even at the lowest dose tested in the study (Fig. 8, 0.6 mg / kg dose). At the same time, the highest dose (10 mg / kg) achieved a 100% cure rate for the animals with the corresponding tumors at the end of the study. For untreated tumors, a dose-dependent antitumor effect was also evident. The upper dose group (10 mg / kg) demonstrated an 80% cure rate; all lower doses also demonstrated tumor growth inhibitory activity. Thus, a therapeutic range of 0.6 mg / kg to 10 mg / kg of Compound 1a was observed, and based on the effects at the uninjected distal tumor site, antitumor activity was manifested not only locally but also systemically. In conclusion, these results indicate that local administration of Compound 1a can induce both local and systemic (uninjected) anticancer activity.

[0284] Example 109 -- Anticancer effect of Compound 1a on CT26 liver metastatic model

[0285] Compound 1a was tested for its anticancer activity in a CT26 liver metastatic model. Luciferase-expressing CT26 tumor cells (5 x 10⁶ per mouse) were injected into anesthetized female 5–6 week old BALB / cJ mice (Jackson Labs, Bar Harbor, Maine). 5 Canine cells) were transplanted into the spleen. Tumor cells were allowed to circulate into the animal's liver through a subsequent 10-minute waiting period. Subsequently, the spleen was removed, and the animal was sutured to allow for recovery. After 3 days, CT26 tumor cells (10 per mouse) 5This time, the canine cells were re-implanted subcutaneously (sc) under the right forelimb to enable the development of a tumor mass for compound administration. Nine days after transplantation into the spleen, the compound (10 mg / kg) was administered once into the sc tumor.

[0286] The local anticancer effect of the compound was measured through its effect on sc tumors, while the abscopal effect of the compound was evaluated by the overall survival of treated mice compared to vehicle-treated control mice, based on the adverse effects on growing tumor masses within the mouse liver. Compound 1a demonstrated both potent activity against local sc tumors and therapeutic systemic activity in 9 out of 10 treated animals (Fig. 12). These results indicate that local administration of compound 1a can induce both local and systemic (abscopal) anticancer activity, including in deep lesions such as those in the liver.

[0287] Example 110 -- Anticancer effect of Compound 1a on GL261 brain orthotopic model

[0288] Compound 1a was tested for its anticancer activity in a GL261 brain orthotopic model. GL261 is a murine glioma cell line. Luciferase-expressing GL261 mouse glioma cells (2x10⁶ 4 Canine cells / mouse) were intracranially implanted into female 5-6 week old B6 albino mice (Jackson Labs, Bar Harbor, Maine). After 3 to 4 days, GL261 cells were implanted subcutaneously beneath the right forelimb to allow for the development of a tumor mass for compound administration (10 6Canine cells (mouse) were transplanted. Ten days after intracranial tumor cell transplantation, a single intratumoral dose of compound (10 mg / kg) was administered into the sc tumor. The local anticancer effect of the compound was measured by its effect on the sc tumor, while the non-injection effect of the compound was evaluated by the total survival of treated mice compared to vehicle-treated control mice, based on the adverse effects of the growing tumor mass within the mouse brain. Compound 1a demonstrated both potent activity against local sc tumors and therapeutic systemic activity in 5 out of 8 treated animals (Fig. 13). These results indicate that local administration of compound 1a can induce both local and systemic (non-injection) anticancer activity, including in deep lesions such as those in the brain.

[0289] Example 111 - Comparison

[0290] As reported in the literature incorporated herein by reference [Corrales, et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports (2015) 11:1018-1030], EC in human STING assays of WT STING, HAQ STING, AQ STING, and REF STING by head-to-head comparison using Compound 1a of the present disclosure, a natural STING ligand (2'3' cGAMP), and the putative STING agonist ML RR-S2 CDA, 50Values ​​were calculated. Tests were performed as described in the examples presented above. It should be noted that the test values ​​reported in Table 7 are limited to tests performed in head-to-head comparisons and may not reflect average values ​​determined over a larger number of tests as reported above. It should also be noted that "2'3' cGAMP" is the same as "ML cGAMP" as reported in the publication [Cell Reports].

[0291] Table 7

[0292]

[0293] Table 7 also reports the dissociation bond constants (Kd) for the binding of human WT STING to each of the three tested compounds as measured by isothermal titration calorimetry (ITC). ITC is a microcalorimetry technique that measures thermodynamic properties associated with intermolecular interactions. Based on these tests, Compound 1a appears to form the strongest bond with WT STING among the tested compounds.

[0294] substance

[0295] Recombinant human wild-type STING (aa, 139-379, H232R) protein was produced by expressing a construct encoding the cytosolic domain of human WT STING containing amino acids 139-379 in E. coli.

[0296] reagent

[0297] The suppliers of the reagents used in this study are listed below:

[0298]

[0299] Protein buffer preparation

[0300] STING protein was stored at -60°C as 90 μL and 100 μL aliquots at concentrations of 3.0 mg / ml and 20 mg / mL, respectively, in 5% glycerol-containing PBS, pH 7.5. On the day of analysis, the protein aliquots were thawed, diluted to 400 μL, and buffer-exchanged into PBS using an Amicon Ultra centrifuge filter unit (10k MW cutoff, 0.5 mL) while centrifuging at least four times for 10 minutes at 14,000 xg using an Eppendorf microcentrifuge, followed by final dilution to 20 μM to 30 μM (experiment-dependent) in 1X PBS. Protein concentrations were measured using a Nanodrop 2000 spectrophotometer and a 22140 (M -1 cm -1 It was determined using the protein absorption coefficient of ).

[0301] Sample manufacturing

[0302] 200 μL of 1 mM stock solutions of compounds 1a, 2'3' cGAMP and mL RR-S2CDA were fed into a 1.5 mL microcentrifuge tube. Before each experiment, the samples were diluted to concentrations ranging from 200 μM to 500 μM (experiment-dependent).

[0303] method

[0304] The assay was performed on an affinity ITC unit (TA Instruments no. 609003.901) equipped with an ITC cleaning accessory (TA Instruments no. 601800.901). Approximately 400 μL of a STING protein solution containing 20 μM to 30 μM STING protein was pipetted into an 185 μL calorimeter cell allowing for a slight allowable overload. The reference cell contained a milli-Q water equivalent. Incubation was performed at 25°C while injecting 20 x 2.5 μL of 100 μM to 300 μM compounds. The control software was ITC Run Ver. 3.3.0.0 (TA Instruments), which was used to obtain a thermographic plot consisting of multiple peaks of raw heat (μcal / sec) representing the exothermic rate at each injection. The analysis software was Nano Analysis Ver. Using 3.70 (TA Instruments), baseline-corrected values ​​were generated for blank or sample dilution heats (at saturation) and graphed by integrating the exothermic rate peaks. The generated isotherms were fitted to an independent model to derive thermodynamic parameters.

[0305] The values ​​of Kd and n (molar ratios at the curve inflection points) were derived and reported. Optimal conditions for the concentrations of the protein and ligand were determined from preliminary experiments.

[0306] result

[0307] The exothermic thermographs and their generated isotherms for the binding of test compounds to recombinant human wild-type STING (aa, 139-379, H232R) were determined. The binding of each compound to STING was endothermic, as indicated by a negative exothermic rate and an exothermic (positive direction) heat of dilution (observed after the compound achieved protein saturation). It was found that 2',3' cGAMPs elicited similar endothermic responses for various STING variants. Compound 1a provided a lowest Kd of 0.04 μM, followed by 2'3' cGAMP with a Kd of 0.07 μM and ML RR-S2 CDA with a Kd of 0.40 μM. All compounds provided n values ​​close to 0.5, suggesting that the STING protein exists as a dimer and that 1 mole of the compound binds to 2 moles of STING.

[0308] Example 112 - Identification of potential metabolites

[0309] To evaluate the formation of major metabolites, compound 1a was incubated in CD-1 mice, Sprague Dolly rats, Beagle dogs, Synomolgus monkeys, and human liver cells.

[0310] substance

[0311] Cryopreserved pooled hepatocytes were purchased from ThermoFisher Scientific (Waltham, Massachusetts), Xenotech, LLC (Kansas City, Kansas), and In Vitro ADMET Laboratories (Columbia, Maryland), and appropriate media were purchased from In Vitro ADMET Laboratories (Columbia, Maryland) and Life Technologies (Carlsbad, California). AOPI staining solution and phosphate buffer were purchased from Corning Life Sciences (Twixbury, Massachusetts) and Nexcelom Bioscience (Lawrence, Massachusetts), respectively. All chemicals, reagents, and solvents used in the analysis were analytical or HPLC grade.

[0312] Experimental Design and Procedure

[0313] Hepatocyte incubation

[0314] Compound 1a was weighed and dissolved in HPLC-water containing 0.12% formic acid PBS to make 1020 mmol / L. The solution was then individually diluted 2.5-fold to 4 mmol / L with Williams E medium containing 0.1% human serum albumin and 2 mmol / L L-glutamine, and then further diluted 21,000-fold to make a working stock solution with a concentration of 20 μmol / L.

[0315] Prior to incubation, cryopreserved hepatocytes were thawed in a 37°C water bath. One tube of cryopreserved hepatocytes was added to 50-mL conical tubes of cryopreserved hepatocyte recovery medium (UCRM) obtained in vitro from ADMET Laboratories (Columbia, Maryland). The cells were centrifuged on a Beckman centrifuge (Brea, California) at 740 rpm with a GH 3.8 rotor for 10 minutes at room temperature (4°C). The supernatant was removed, and the cells were resuspended in plating medium for counting. After resuspending the cells in the plating medium, 20 μL of the resuspended solution was transferred and mixed with 20 μL of AOPI staining solution. The solution was gently mixed, and the cells were counted using a cellometer (Nexcellom, Lawrence, Massachusetts). After counting, the cells were then resuspended in Williams E medium containing 2 mmol / L L-glutamine (pH 7.4) at 1 or 2 million viable cells / ml.

[0316] A hepatocyte suspension (50 μL / well) was added to a 48-well plate. The reaction was initiated by adding 50 microliters of working stock solution containing Compound 1a (20 μmol / L). The plates were placed in a tissue culture incubator (5% CO2 / 95% air humidification atmosphere and 37°C), and the reaction was stopped at 5, 30, 60, 120, 180, and 240 minutes with 200 μL of a stopping solution consisting of 100% methanol / acetonitrile (1 / 1, v / v) containing 2010 ng / mL furosemide and 0.2 μmol / L (R)-propranolol. The mixture was centrifuged, filtered, and the supernatant was collected for analysis. The final concentration of the cryopreserved hepatocytes was 1 x 10⁶ 6 It was dog cells / mL. The final incubation concentration of compound 1a was 10 μmol / L.

[0317] LC-MS / MS conditions for metabolite identification

[0318] The LC-MS / MS system was configured with a Shimadzu HPLC and an AB-SCIEX Triple TOF 5600 hybrid quadrupole and TOF mass spectrometer (Framingham, Massachusetts). The Shimadzu HPLC (Kyoto, Japan) consisted of a communication bus module (CBM-20A), an automated sampler (SIL-30AC) equipped with an attached rack changer (Rack Changer II) and two pumps (LC-30AD), and a column oven (CTO-30A). The mass spectrometer was calibrated using the AB-SCIEX APCI in both negative and positive calibration solutions (Framingham, Massachusetts). Samples obtained from incubation with hepatocytes were analyzed under both negative and positive scan modes. The basic analytical methods and instrument conditions are summarized below. Variations in spectrometer settings depend on the requirements of the analytes.

[0319] LC-MS / MS conditions:

[0320]

[0321] Data analysis of active data

[0322] Mass spectroscopic data were acquired using AB Science Analyst TF (version 1.5.1; Framingham, Massachusetts). Chromatograms and spectra were acquired using AB Science PeakView (version 2.2.0.1; Framingham, Massachusetts). Comparisons of relative peak areas for the extracted ion chromatograms were based on ± 0.0002 Da of the expected accurate mass-to-charge ratio (m / z) for each analyte of interest.

[0323] result

[0324] Metabolites were not detected during incubation with hepatocytes. Under the conditions of this analysis, Compound 1a exhibited a retention time of approximately 7.8 minutes. Under negative scan mode, Compound 1a deprotonated molecular ion m / z 745 (C 24 H 25F2N 10 O8P2S2 - ) and double deprotonated molecular ions m / z 372 (C 24 H 24 F2N 10 O8P2S2 2- ) represented. m / z 533 (C 19 H 19 FN 10 O4PS - ) and m / z 186 (C9H8N5 - Major MS / MS product ions containing ) were observed. Under positive scan mode, compound 1a was protonated molecular ion m / z 747 (C 24 H 27 F2N 10 O8P2S2 + ) and major MS / MS product ions m / z 651 (C 24 H 26 F2N 10 O6PS + ), m / z 252 (C 10 H 11 FN5O2 + ) and m / z 188 (C9H 10 N5 + ) was shown. MS and MS / MS data confirmed the structure of compound 1a.

[0325] Compound 1a was stable in incubation with mouse, rat, dog, monkey, and human liver cells. No apparent metabolites of compound 1a were identified in this study. In samples obtained from incubation with liver cells, only compound 1a itself could be detected and identified by fragmentation using tandem mass spectrometry (MS / MS).

[0326] All referenced literature in this disclosure is incorporated herein by reference, and in the event that any incorporated literature conflicts with the present specification, the present specification shall prevail. A person skilled in the art will recognize that various changes and modifications may be made to the materials provided herein and that such materials are within the scope and spirit of this disclosure.

[0327] Sequence list

[0328]

[0329]

[0330]

Claims

Claim 1 A pharmaceutical composition for use in a method of treating bladder cancer, comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer. Claim 2 A pharmaceutical composition according to claim 1, wherein the above-mentioned pharmaceutically permissible salt is a diammonium salt. Claim 3 A pharmaceutical composition for use in a method for treating bladder cancer, comprising compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt thereof to a patient requiring treatment for bladder cancer. Claim 4 A pharmaceutical composition in a method for treating bladder cancer, comprising one selected from the group consisting of compound 1, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising compound 1 and a pharmaceutically acceptable salt thereof. Claim 5 A pharmaceutical composition for use in a method for treating bladder cancer, comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises identifying an individual having bladder cancer treatable by compound 1 or a pharmaceutically acceptable salt of compound 1; and administering an effective amount of compound 1 or a pharmaceutically acceptable salt of compound 1 to the individual identified as having bladder cancer treatable. Claim 6 A pharmaceutical composition according to claim 5, wherein the individual is identified as having bladder cancer treatable by compound 1 or a pharmaceutically acceptable salt of compound 1 due to the presence of a REF STING variant allele in the patient. Claim 7 A pharmaceutical composition for use in a method of treating bladder cancer comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt of compound 1 to a patient having the REF STING allele. Claim 8 A pharmaceutical composition for use in a method of treating bladder cancer, comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt of compound 1 to a patient having the WT STING allele. Claim 9 A pharmaceutical composition for use in a method of treating bladder cancer comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt of compound 1 to a patient having the AQ STING allele. Claim 10 A pharmaceutical composition for use in a method of treating bladder cancer, comprising compound 1 or a pharmaceutically acceptable salt thereof, wherein the method comprises administering an effective amount of compound 1 or a pharmaceutically acceptable salt of compound 1 to a patient having the HAQ STING allele. Claim 11 A pharmaceutical composition according to any one of claims 1 to 10, wherein compound 1 or a pharmaceutically acceptable salt of compound 1 is administered intravesicularly into the bladder. Claim 12 A pharmaceutical composition according to any one of claims 1 to 10, wherein the bladder cancer is non-muscle invasive bladder cancer. Claim 13 A pharmaceutical composition according to claim 12, wherein compound 1 or a pharmaceutically acceptable salt of compound 1 is administered into the bladder. Claim 14 delete Claim 15 delete

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